35 veterinaria italiana 2022, 58 (1), 35-39. doi: 10.12834/vetit.1741.9188.2 accepted: 19.08.2019 | available on line: 16.11.2021 1department of clinical sciences, faculty of veterinary medicine, semnan university, semnan, iran. 2department of pathobiology, faculty of veterinary medicine, semnan university, semnan, iran. 3graduated from department of surgery and radiology, faculty of veterinary medicine, university of tehran, tehran, iran. *corresponding author at: department of clinical sciences, faculty of veterinary medicine, semnan university, semnan, iran. po box 35131-19111 semnan, iran. telefax: +98(23)33654215, e-mail: rezasani_vet@semnan.ac.ir. mona negadmonfared1, reza narenji sani1*, sahar ghaffari khaligh2 and farzad hayati3 keywords endometrial hyperplasia, endometrial thickness, isoflavone supplementation, ovariectomized cats. summary ovariectomy is identified as a standard treatment in different european countries. isoflavones, as nonsteroidal compounds in plants, are common constituents of soy and soy products. some available cat diets contain different concentrations of soy products. this study aimed to examine the effects of isoflavone supplementation on endometrial hyperplasia and endometrial thickness in ovariectomized cats. fifteen neutered adult cats were divided into control, estradiol, and isoflavone groups (five cats per group). subcutaneous injection of estradiol (0.5 μg) in sesame oil (100 μl) was done for 30 days in estradiol-treated cats. isoflavone-treated cats ingested a single oral tablet of soy extract for 30 days, while the controls received subcutaneous injections of the vehicle and oral placebo for 30 days. histopathological findings of hematoxylin and eosin-stained sections revealed a significant difference between the estradiol group and other groups in terms of hyperplastic epithelium and simple hyperplasia. thickness of myometrium was greater in the estradiol group compared to the isoflavone and control groups. higher concentrations of estrogen can affect the endometrium and myometrium, while 30-day ingestion of isoflavone didn't have any uterine effect. effects of isoflavone supplementation on endometrial thickness, endometrial hyperplasia, and cancer in ovariectomized cats in particular they are common constituents of soy and soy products (kurzer and xu 1997). isoflavones genistein and daidzein are known to cause changes in food intake, innate and acquired immunity, expression of steroid receptors, and lean body mass of domestic cats (bell et  al. 2008, cave et  al. 2007a, cave et al. 2007b, cave et al. 2007c), while soy diets lead to changes in the serum thyroid hormone (t4) (white et al. 2004). genistein and daidzein are the constituents of some available diets for sterile cats. the isoflavone content has been associated with the use of soy products in these diets (quaas et  al. 2013). since the effects of isoflavone supplementation on the endometrium of cats are still unknown, and ovariectomized cats may receive soy-containing diets, we investigated the effects of isoflavone supplementation on endometrial thickness, endometrial hyperplasia, and cancer in ovariectomized cats. introduction elective sterilization of female cats (felis catus) is a common procedure in veterinary practice. generally, population control, elimination of unwanted behaviors due to hormonal cycling, and prevention of reproductive diseases are among the advantages of sterilization. in sterilization of female cats, only ovaries or ovaries and uterus can be removed (ovariectomy vs. ovariohysterectomy). although in the united states and canada, ovariohysterectomy is preferred, in many european countries, ovariectomy has become the standard procedure (goethem et  al. 2006). ovariectomy has potential advantages such as improving the view of the ovarian pedicle, smaller incisions, and lower risk of complications due to surgical uterine manipulation (detora and mccarthy 2011). isoflavones are nonsteroidal compounds in plants. 36 veterinaria italiana 2022, 58 (1), 35-39. doi: 10.12834/vetit.1741.9188.2 isoflavone and endometrium in ovariectomized cats negadmonfared et al. on experimental day 0. on day 1, treatment inititiated. the experimental design was completely randomized and fifteen cats were randomly allocated to three experimental groups. the estradiol group received subcutaneous injections of estradiol (0.5  μg) in sesame oil (100  μl) for 30 days. the isoflavone group ingested an oral soy extract tablet. food was withheld for 18 hours before oral administration of the soy tablet containing 25 mg of genistein and 19 mg of daidzein (50 mg; soyagol, goldaru pharmaceutical co., iran) for 30 days. note that all cats received control treatments. the estradiol group received oral placebos, the isoflavone group was given vehicle injections, and the control group received oral placebos and subcutaneous vehicle injections. all treatments were performed every morning immediately before the fresh diet. uterine samples specimens were fixed in 10% nonbuffered formalin, and hematoxylin and eosin was employed to stain 4-μm tissue sections. a single pathologist (blind to regimens) interpreted and classified the results, based on standard criteria (emons et al. 2015). histopathological analysis was performed in luminal epithelial and glandular endometrium, while the histometry of myometrial thickness was examined in the horn and body of the uterus in all groups. histopathological findings in haematoxylin and eosin-stained sections were investigated in eight microscopic fields and captured by a camera, connected to an optic microscope in all samples. statistical analysis data were introduced in spss version 19.0. one-way anova and tukey's post-hoc tests were applied to examine myometrial thickness, while wilcoxon signed rank test was performed to compare changes in epithelial hyperplasia, endometrial hyperplasia, and endometrial stromal edema. post hoc pairwise comparisons were made using mann-whitney u test. results a significantly hyperplastic epithelium was found in the estradiol group compared to others (p < 0.05, table i, figure 1, a-c). according to the results, there was no atypical, or complex hyperplasia in endometrial glands in the groups, while typical hyperplasia was more frequently found in the estradiol group, compared to others (p < 0.05, table i, figure 2, a-c). also, no significant difference was found between the groups in terms of stromal edema in the endometrium (p > 0.05, table i, figure 3, a-c). myometrial thickness materials and methods animal husbandry all cats (12-16 months of age, 3.2  ±  0.3  kg in weight) evaluated in this study were from a specific pathogen-free colony. before the study, the animals were fed a commercial isoflavone-free diet for four weeks; the washout period was determined with respect to the half-life of conjugated genistein (22 hours) (cave et al. 2007d). the animals remained on the same balanced, extruded dry diet (whiskas with savory nuggets; original recipe, masterfoods usa, vernon, ca, usa). the animals' diet consisted of 10% crude fat, 31% (wt/wt) crude protein, 1.8% crude fiber, 8%  moisture, and 7% ash, without any soybean or by-products. the animals had free access to fresh food, which was prepared every morning; at the same time, the remaining food was weighed. once the animals were housed in semnan university pet care center, they were placed in individual cages with ad libitum access to water (unless otherwise stated) at a constant temperature in a 12:12  h light-dark cycle. the animals were used according to the animal welfare act, as well as the guide for care and use of laboratory animals (national research council 1996). the animal use and care committee of semnan university approved this study. note that this project was ethically approved by ethics committee and the code number for our research was 25875 received from this committee. surgical neutering after a 10-hour fasting period, water was withheld from the animals for two hours before inducing anesthesia. queens were pre-medicated with atropine 0.005 mg/kg body weight (bw), subcutaneously (s.c.). anaesthesia was induced with ketamine, 10 mg/kg bw, i.v., and diazepam, 0.2  mg/kg bw, intravenously (i.v.), and subsequently they were intubated and maintained on isoflurane (1.5%) in oxygen. afterwards, bilateral ovariectomy, using standard techniques, was applied. queens were maintained on lactated ringer solution for one day and were administered ceftriaxone, 25  mg/kg  bw,  i.v., q12 h and tramadol, 5 mg/kg bw, per os (p.o.), q12 h for three days. elizabethan collars were fitted to female cats and removed for suture removal after 10 days. after 30 days, the uteri were sampled from animals under general anesthesia after a hysterectomy. experimental design the animals were divided into control, estradiol, and isoflavone groups (five cats per group) 37veterinaria italiana 2022, 58 (1), 35-39. doi: 10.12834/vetit.1741.9188.2 negadmonfared et al. isoflavone and endometrium in ovariectomized cats table i. different histological classifications of uterine tissues in treated and control groups. classification estradiol isoflavone control p value epithelium hyperplasia n/n (%) 5/5 (100) * 0/5 (0) 0/5 (0) < 0.05 endometrial gland neoplastic hyperplasia n/n (%) 0/5 (0) 0/5 (0) 0/5 (0) > 0.05 endometrial gland atypical hyperplasia n/n (%) 0/5 (0) 0/5 (0) 0/5 (0) > 0.05 endometrial gland complex hyperplasia n/n (%) 0/5 (0) 0/5 (0) 0/5 (0) > 0.05 endometrial gland simple hyperplasia n/n (%) 5/5 (100) * 1/5 (20) 2/5 (40) < 0.05 endometrium stromal edema 5/5 (100) 2/5 (40) 3/5 (60) > 0.05 myometrium thickness (mm2) 16.5 * 3 6.4 < 0.05 *statistically significant (p < 0.05). was determined in scanned images of all samples across all groups. a significant difference was found between the estradiol group and others (p  <  0.05, table i, figure 4, a-c). discussion despite of the reported uterine effects related to genistein intake in humans (unfer et  al. 2004), rats (diel et al. 2004) and mice (jefferson et  al. 2002) so far, to the best of our knowledge, there were no studies testing the effect of genistein on the uterus in cats. evidences of uterine hyperplasia has been reported following acute, high dose (53 mg/kg/day) (rimoldi et al. 2007) and chronic, low dose (1.6 mg/ kg/day) genistein treatments (retana-márquez et al. 2012). our findings also indicated that 30 days treatment with 50 mg/day/cat did not determine endometrial hyperplasia, but treatment with estradiol resulted in endometrial hyperplasia. hyperplasia could lead to cancer development (salleh et  al. 2013). recently, genistein has been shown to promote the development of endometrial cancer in rats where administration of 150 mg/kg/ day of this compound orally stimulates excessive epithelial proliferation (kakehashi et  al. 2012). the longest randomized clinical trial was conducted to evaluate the endometrial effects of phytoestrogen administration. the results suggested that endometrial hyperplasia increased (unfer et  al. 2004), while studies with a shorter duration showed a reduction in endometrial hyperplasia (hale et  al. 2001, murray et al. 2003). in this study, we observed that isoflavone ingestion has no significant effects on glandular endometrium or histometry of myometrial thickness, compared to the control group. however, estrogen could affect uterine tissues, causing a significant increase in myometrial thickness. similarly, salleh and colleagues (salleh et al. 2013) as well as möller and colleagues (möller a b c figure 2. endometrial glands simple hyperplasia without any neoplastic, atypical and complex forms in groups of cats treated with estradiol (a, hematoxilin & eosin staining, 400×), isoflavone (b, hematoxilin & eosin staining, 100×) and placebos (control) (c, hematoxilin & eosin staining, 400×). a b c figure 1. the epithelium hyperplasia in groups of cats treated with estradiol (a), isoflavone (b) and placebos (control) (c) (hematoxilin & eosin staining, 400×). 38 veterinaria italiana 2022, 58 (1), 35-39. doi: 10.12834/vetit.1741.9188.2 isoflavone and endometrium in ovariectomized cats negadmonfared et al. conclusions in conclusion, consumption of 50 mg/day isoflavones in 30 days had not significant effect on uterine tissue in ovarectomized cats, though higher doses or longer intervals should be studied. acknowledgments the authors appreciate the contributions of dr. ebrahim shahrouzian, mr. gholami and the research and training pet hospital of the faculty of veterinary medicine, semnan university and spa pet clinic in semnan. et  al. 2012) reported that myometrial thickness was increased with genistein. they suggested that genistein stimulates myometrial hypertrophy. in contrast with long-term studies, our results indicated that longer treatments with isoflavone had no endometrial effects. however, long-term treatment with higher doses of estrogen (estradiol injection) had endometrial and myometrium effects. the cause of difference between our results and the longest trial might be the duration of treatment along with the dose of isoflavone and species. however, since higher concentrations of estrogen affected the endometrium and myometrium in our study, and a previous long-term study confirmed the endometrial effects of isoflavone (unfer et al. 2004), phytoestrogenic supplements can be effective in cats. however, longer experimental studies with isoflavones should be performed. a b c figure 3. stromal edema as an excess liquid in the interstitial (extra cellular) space in endothelium in groups of cats treated with estradiol (a), isoflavone (b) and placebos (control) (c) (hematoxilin & eosin staining, 400×). a b c figure 4. myometrium hyperplasia in groups of cats treated with estradiol (a), isoflavone (b) and placebos (control) (c) (hematoxilin & eosin staining, 50×). 39veterinaria italiana 2022, 58 (1), 35-39. doi: 10.12834/vetit.1741.9188.2 negadmonfared et al. isoflavone and endometrium in ovariectomized cats bell k., ugarte c., tucker l., roe w. & thomas d. 2008. assessment of reproductive histology and sex steroid receptor expression in the domestic cat (felis catus) following chronic exposure to phytoestrogens. reprod domest anim, 43, 126. cave n., backus r., marks s. & klasing k. 2007a. the bioavailability and disposition kinetics of genistein in cats. j vet pharmacol ther, 30 (4), 327-335. cave n., backus r., marks s. & klasing k. 2007b. oestradiol and genistein reduce food intake in male and female overweight cats after gonadectomy. n z vet j, 55 (3), 113-119. cave n., backus r., marks s. & klasing k. 2007c. oestradiol, but not genistein, inhibits the rise in food intake following gonadectomy in cats, but genistein is associated with an increase in lean body mass. j anim physiol anim nutr, 91 (9‐10), 400-410. cave n.j., backus r.c., marks s.l. & klasing k.c. 2007d. modulation of innate and acquired immunity by an estrogenic dose of genistein in gonadectomized cats. vet immunol immunopathol, 117 (1-2), 42-54. diel p., geis r.b., caldarelli a., schmidt s., leschowsky u.l., voss a. & vollmer g. 2004.the differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and proliferation in the rat is associated with a substance specific modulation of uterine gene expression. mol cell endocrinol, 221 (1-2), 21-32. detora m. & mccarthy r.j. 2011. ovariohysterectomy versus ovariectomy for elective sterilization of female dogs and cats: is removal of the uterus necessary? j am vet med assoc, 239 (11), 1409-1412. emons g., beckmann m., schmidt d., mallmann p. & uterus commission of the gynecological oncology working group (ago). 2015. new who classification of endometrial hyperplasias. geburtsh frauenheilk, 75 (2), 135-136. goethem b., schaefers‐okkens a. & kirpensteijn j. 2006. making a rational choice between ovariectomy and ovariohysterectomy in the dog: a discussion of the benefits of either technique. vet surg, 35 (2), 136-143. hale g.e., hughes c.l., robboy s.j., agarwal s.k. & bievre m. 2001. a double-blind randomized study on the effects of red clover isoflavones on the endometrium. menopause, 8 (5), 338-346. jefferson w.n., padilla-banks e., clark g. & newbold r.r. 2002. assessing estrogenic activity of phytochemicals using transcriptional activation and immature mouse uterotrophic responses. j chrom b, 777 (1-2), 179-189. references kakehashi a., tago y., yoshida m., sokuza y., wei m., fukushima s. & wanibuchi h. 2012. hormonally active doses of isoflavone aglycones promote mammary and endometrial carcinogenesis and alter the molecular tumor environment in donryu rats. toxicol sci, 126 (1), 39-51. kurzer m.s. & xu x. 1997. dietary phytoestrogens. ann rev nutr, 17 (1), 353-381. murray m.j., meyer w.r., lessey b.a., oi r.h., dewire r.e. & fritz m.a. 2003. soy protein isolate with isoflavones does not prevent estradiol-induced endometrial hyperplasia in postmenopausal women: a pilot trial. menopause, 10 (5), 456-464. möller f.j., ledwig c., zierau o., hertrampf t., degen g.h., diel p. & vollmer g. 2012. the rat prepubertal uterine myometrium and not the luminal epithelium is predominantly affected by a chronic dietary genistein exposure. arch toxicol, 86 (12), 1899-1910. national research council (us). 1996 . guide for the care and use of laboratory animals institute for laboratory animal research. national academies press, washington dc. quaas a.m., kono n., mack w.j., hodis h.n., felix j.c., paulson r.j. & shoupe d. 2013. the effect of isoflavone soy protein supplementation on endometrial thickness, hyperplasia and endometrial cancer risk in postmenopausal women: a randomized controlled trial. menopause, 20 (8), 840. retana-márquez s., aguirre f.g., alcántara m., garcía-díaz e., muñoz-gutiérrez m., arteaga-silva m. & delgadillo j.a. 2012. mesquite pod extract modifies the reproductive physiology and behavior of the female rat. horm behav, 61 (4), 549-558. rimoldi g., christoffel j., seidlova-wuttke d., jarry h. & wuttke w. 2007. effects of chronic genistein treatment in mammary gland, uterus, and vagina. environ health perspect, 115 (suppl 1), 62. salleh n., baines d.l., naftalin r.j. & milligan s.r. 2005. the hormonal control of uterine luminal fluid secretion and absorption. j membrane biol, 206 (1), 17-28. unfer v., casini m.l., costabile l., mignosa m., gerli s. & di renzo g.c. 2004. endometrial effects of long-term treatment with phytoestrogens: a randomized, double-blind, placebo-controlled study. fertil steril, 82 (1), 145-148. white h.l., freeman l.m., mahony o., graham p.a., hao q. & court m.h. 2004. effect of dietary soy on serum thyroid hormone concentrations in healthy adult cats. am j vet res, 65 (5), 586-591. 103 parole chiave italia, pestivirus d, sequenziamento di nuova generazione, trasmissione, virus della border disease. riassunto il virus della border disease (bdv) è diffuso a livello mondiale nei piccoli ruminanti, domestici e selvatici. in questo lavoro si riportano la sequenza del genoma di bdv genotipo 8 da un camoscio (ceppo italy‑58987), ottenuta mediante tecniche di sequenziamento di nuova generazione (ngs), e il confronto della stessa con le sequenze genomiche di altri pestivirus. la sequenza di 12.245 bp è stata allineata a 22 sequenze genomiche di pestivirus e ha mostrato una identità di sequenza nt/aa del 81,3/89,4% con altri genotipi di bdv, e del 65,9/67,8% con gli altri pestivirus. la sequenza genomica del bdv‑8 rilevato nel camoscio ha mostrato una identità media nt/aa del 91,2/95,0% con le sequenze genomiche di 3 ceppi di bdv isolati in svizzera, strettamente correlati con le sequenze delle porzioni di 5’‑utr e npro di bdv‑8. l’identificazione di isolati divergenti di bdv‑8 nel nordovest dell’italia e in svizzera suggerisce che questo genotipo possa aver circolato in un’area più ampia di quella inizialmente ipotizzata, e che possa avere una elevata adattabilità a ospiti diversi. sequenza genomica del genotipo 8 del virus della border disease da un camoscio ottenuta mediante tecniche di sequenziamento di nuova generazione keywords border disease virus, italy, next generation sequencing, pestivirus d, wild‑domestic transmission. summary border disease virus (bdv) is widespread both in domestic small ruminants and wildlife. here we report the genome of bdv genotype 8 from chamois, strain italy‑58987, obtained by next generation sequencing and the comparison with other pestiviruses. the sequence of 12,245 bp long was aligned to 22 pestivirus genomes and it showed a nt/aa similarity of 81.3/89.4% with bdv genotypes, and 65.9/67.8% with the other pestiviruses. the genome showed a mean nt/aa similarity of 91.2/95.0% with three swiss genomes closely related to the bdv‑8 5’‑utr and npro sequences. the identification of divergent bdv‑8 isolates in north‑western italy and in switzerland suggests that this genotype may have been circulating in a wider area than previously supposed, and may have a high host adaptability. veterinaria italiana 2019, 55 (1), 103‑105. doi: 10.12834/vetit.1768.9338.1 accepted: 30.01.2019 | available on line: 21.02.2019 istituto zooprofilattico sperimentale del piemonte, liguria e valle d'aosta, via bologna 148, 10154 turin, italy *corresponding author at: istituto zooprofilattico sperimentale del piemonte, liguria e valle d'aosta, via bologna, 148, 10154 turin, italy. tel.: +39 011 2686245, e‑mail: simone.peletto@izsto.it. francesco cerutti, claudio caruso, paola modesto, riccardo orusa, loretta masoero, pier luigi acutis and simone peletto* the genome of border disease virus genotype 8 from chamois using next generation sequencing short communication the pestivirus d genome is about 12.3  kb long, coding for four structural proteins (c, erns, e1 and e2) and seven to eight non‑structural proteins (npro, p7, ns2‑3, ns4a, ns4b, ns5a, ns5b) flanked by 5’‑ and 3’‑untranslated regions (utr). based on phylogenetic analysis of 5’‑utr or npro sequences, bdv has been divided into at least seven groups (bdv‑1 to ‑7) (giammarioli et  al. 2011). our group identified a new genotype, named bdv‑8, in a goat kid showing bd‑like syndrome with typical “hairy shaker” symptoms in north‑western italy and, border disease virus (bdv) is a single‑stranded positive sense rna virus belonging to the genus pestivirus, family flaviviridae. it is the causative agent of border disease (bd), a worldwide infection of domestic small ruminants, causing huge economic losses, and wildlife. according to a new proposed taxonomy of the genus pestivirus, bdv should be referred to as pestivirus d (smith et  al. 2017). here we use both names to refer to border disease virus, since at the time of writing, the readers may not be comfortable with the new classification only. 104 veterinaria italiana 2019, 55 (1), 103‑105. doi: 10.12834/vetit.1768.9338.1 bdv‑8 from chamois cerutti et al. bdv-3 gifhorn (kf925348) bdv-3 jsls12/01 (kc963426) bdv-2 reindeer (af144618) bdv-5 aveyron (kf918753) bdv-4 chamois (gu270877) bdv switzerland bovine r9336-11 (mf102261) bdv switzerland sheep r4785-06 (mf102260) bdv-1 bd31 (u70263) bdv-1 x818 (nc_003679) bdv coos bay 5nc (kj463422) csfv (nc_002657) pestivirus aydin 04 tr (nc_018713) bvdv-1 (nc_001461) bvdv-2 (nc_002032) bvdv-3 (nc_012812) pestivirus gira�e (nc_003678) pronghorn antelope pestivirus (nc_024018) porcine pestivirus bungowannah (nc_023176) atypical porcine pestivirus 1 (nc_030653) bdv fnk2012/1 (ab897785) bdv switzerland porcine bd35-15 (mf102262) bdv-3 gifhorn clone pbelogif3 (gq902940) bdv-8 bdv-8_italy-58987 chamois (mg649392) 3.0 0.67 0.99 0.99 0.99 0.99 0.99 0.99 0.97 1 0.81 0.85 0.87 0.98 0.86 0.9 0.97 0.99 1 1 1 1 figure 2. phylogenetic tree of the pestivirus genus based on the genome nucleotide sequence, with the border disease virus (bdv) clade highlighted in grey, and the genotype 8 clade marked by a side bar. a total of 3,635,077 read pairs were obtained and filtered with trim galore v0.4.3. the first 10  bp were removed from all reads to avoid poor quality, and the reads were trimmed to remove <  q30 nucleotides (nt) and adapter sequences. reads were assembled with megahit v1.1.1‑2 (li et al. 2016) and contigs were classified with blastn and the ncbi nr nucleotide database. the contig corresponding to bdv‑8 (genbank acc. num. mg649392) was 12,245 bp long, from nt 70 to nt 12,333 of the reference genome bdv x818 (genbank acc. num. nc_003679), which was used for sequence annotation. the genome was sequenced with a 20753.8x coverage, with 21.01% reads corresponding to the viral genome. the sequence was then aligned to 22 pestivirus genomes, representative of known phylogenetic groups for which genome sequences were available at the time of writing, with muscle software for multiple comparison and phylogenetic analysis (edgar 2004). the novel genome showed a nucleotide/amino acid similarity of 81.3/89.4% with pestivirus d genotypes, and 65.9/67.8% with the other pestiviruses. bdv‑8 showed a mean nucleotide/amino acid similarity of 91.2/95.0% with the three swiss genomes. the nucleotide diversity (p‑distance) for each gene between bdv‑8 and other bdv genotypes and pestivirus reference sequences was lower in the 5’ and 3’ untranslated regions, and higher in the ns5a gene (figure 1). the best molecular substitution model gtr+i+g was identified by jmodeltest2, and used as a priori information for the bayesian phylogenetic inference implemented in mrbayes v3.2.6 (ronquist et  al. afterwards, in an alpine chamois found dead with poor body condition (rupicapra rupicapra) (peletto et al. 2016, caruso et al. 2017). recently, genomes of three swiss bdv isolates from a sheep, a cattle and a pig were published and showed to be related to bdv‑8, according to 5’‑utr and npro sequences (stalder et al. 2017). these findings demonstrate that this novel genotype may infect also non ruminant species (i.e. pigs). the aim of this work was to provide the genome sequence of bdv‑8 strain italy‑58987 by next generation sequencing and to carry out a comparison with genomes of other pestiviruses. the virus was successfully isolated as previously reported (caruso et  al. 2017), and 800  µl of supernatant were used for total rna purification with trizol reagent (invitrogen), adapting the manufacturer’s protocol to the input volume. the rna was then processed with the rna clean & concentrator (zymo research) to increase the rna concentration and purity, eluting down to 10 µl molecular‑grade h 2 o. to process the sample for the library preparation, rna underwent reverse transcription with the reverse transcription system (promega) and second strand synthesis with the nebnext mrna second strand synthesis module (new england biolabs). the final reaction was purified with agencourt ampure xp magnetic beads (beckman coulter). both rna and ds‑cdna were tested for the presence of bdv genome by an in‑house sybr green real‑time rt‑pcr using 324/326 primer pair (vilcek & paton 2000). dna was processed with the nextera xt dna library preparation kit (illumina) following manufacturer’s instructions. the final library was sequenced on a illumina miseq with miseq reagent kit v3‑600, in two different 2x300 bp paired‑end runs (sra accession prjna514412). 5’ -u tr n p ro c ap si d ern s e1 e2 p 7 n s2 -3 n s4 a n s4 b n s5 a n s5 b 3’ -u tr 0.6 0.4 0.2 0.0 bdv pestivirus genes m ea n p -d is ta n ce species figure 1. mean nucleotide p‑distance between sequences of border disease virus (bdv) including bdv‑8 italy‑58987. bdv-swiss and non-bdv pestivirus according to genes. 105veterinaria italiana 2019, 55 (1), 103‑105. doi: 10.12834/vetit.1768.9338.1 cerutti et al. bdv‑8 from chamois may have led to underestimate its real circulation, revealing itself a risk for uncontrolled and unknown spread in different species. further studies on the epidemiology of bdv‑8 are needed to clarify the impact of such novel strain on livestock and wild ungulates. importantly, the availability of bdv genomes from different host species may provide clues about the genetic basis of cross‑species transmission. grant support funding: this work was supported by the italian ministry of health [ricerca corrente 2011 – project izs plv 16/11 rc “filodinamica, filogeografia e caratterizzazione molecolare full genome di bovine viral diarrhoea virus (bvdv)” – leading institution: istituto zooprofilattico sperimentale del piemonte, liguria e valle d’aosta]. 2012). the novel strain is placed within the bdv clade with a strong support of the posterior probability of the ancestor nodes (figure 2). the identification of divergent bdv‑8 isolates in north‑western italy and in switzerland, with the first collection in 2006 in a white alpine sheep, suggests that the virus circulation may be geographically wider than previously supposed (caruso et  al. 2017). the poor epidemiological information on such novel strain does not allow to infer whether it might be spread in the livestock and occasionally infecting wild ungulates, or vice versa. considering the wide range of host species in which the virus has been identified so far (goat, chamois, sheep, cattle and pig), this novel genotype seems to have a high adaptability. moreover, the diagnostic issues previously reported by peletto and colleagues (peletto et al. 2016) (i.e., the widely used bdv‑specific nested assay published by vilcek and paton fail to amplify bdv‑8 because of primer mismatches) caruso c., peletto s., cerutti f., modesto p., robetto s., domenis l., masoero l. & acutis p.l. 2017. evidence of circulation of the novel border disease virus genotype 8 in chamois. arch virol, 162, 511‑515. edgar r.c. 2004. muscle: multiple sequence alignment with high accuracy and high throughput. nucleic acids res, 32, 1792‑1797. giammarioli m., la rocca s.a., steinbach f., casciari c. & de mia g.m. 2011. genetic and antigenic typing of border disease virus (bdv) isolates from italy reveals the existence of a novel bdv group. vet microbiol, 147, 231‑236. li d., luo r., liu c.m., leung c.m., ting h.f., sadakane k., yamashita h. & lam t.w. 2016. megahit v1.0: a fast and scalable metagenome assembler driven by advanced methodologies and community practices. methods, 102, 3‑11. peletto s., caruso c., cerutti f., modesto p., zoppi s., dondo a., acutis p.l. & masoero l. 2016. a new genotype of border disease virus with implications for molecular diagnostics. arch virol, 161, 471‑477. references ronquist f., teslenko m., van der mark p., ayres d.l., darling a., höhna s., larget b., liu l., suchard m.a. & huelsenbeck j.p. 2012. mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. syst biol, 61, 539‑542. smith d.b., meyers g., bukh j., gould e.a., monath t., scott muerhoff a., pletnev a., rico‑hesse r., stapleton j.t., simmonds p. & becher p. 2017. proposed revision to the taxonomy of the genus pestivirus, family flaviviridae. j gen virol, 98 (8), 2106‑2112. stalder h., marti s., flückiger f., renevey n., hofmann m.a. & schweizer m. 2017. complete genome sequences of three border disease virus strains of the same subgenotype, bdswiss, isolated from sheep, cattle, and pigs in switzerland. genome announc, 5, e01238‑17. vilcek s. & paton d.j. 2000. a rt‑pcr assay for the rapid recognition of border disease virus. vet res, 31, 437‑445. 301 anaplasma phagocytophilum is an obligate intracellular zoonotic bacterium transmitted by ixodid ticks which can cause high fever, neutropenia (due to unique tropism for granulocytes), reduced milk production and hemorrhagic diathesis in ruminants (woldechiwet 2006, giadinis et  al. 2011). in europe the ixodes ricinus ticks is the most important vector. in a recent study the presence of a.  phagocytophilum infection in a bovine herd was associated with depression, nasal and eye discharge, anorexia, coughing, diarrhea, recumbency, weakness, swelling of the hind limbs, decrease of milk production (aktas and ozubek 2015); co‑infection with other anaplasma spp. did not seem to worsen the symptoms. the disease in ruminants is called tick‑borne fever (tbf). reproductive disorders, such as impaired spermatogenesis and abortion have been observed in sheep but not in goats (jones and davies 1995, garcia‑perez et  al. 2003, chianini et  al. 2004, lillini et al. 2006, stuen 2007). in greece, a.  phagocytophilum has been detected by pcr in humans (psaroulaki et  al. 2008) and ticks (kachrimanidou et al. 2011, papa et al. 2017). by pcr a.  ovis has been also detected in small ruminants (giadinis et  al. 2012), while serological evidence of the presence of a.  phagocytophilum has been recorded in an ill ram (giadinis et al. 2011). this manuscript describes sporadic abortions in two goat herds in northern greece, in which a. phagocytophilum was the only pathogen detected in placenta or aborted fetuses. the 1st goat herd (herd 1) was located in chalkidiki prefecture (northern greece) and consisted of 800  dairy goats of local breeds that were reared under the semi‑intensive feeding system. in 2011, the farmer observed sporadic abortions in goats of different ages, and about 30 abortions were recorded during the last 45 days of pregnancy. two aborted fetuses (from different mothers) without fetal membranes were submitted to the farm animal clinic of the aristotle university in thessaloniki. both fetuses were normal in appearance and no specific lesions were found by post mortem investigation. whole blood from the mothers, brain tissue and stomach content from the fetuses were collected. the 2nd farm (herd 2) was situated in thessaloniki 1laboratory of clinical microbiology and microbial pathogenesis, school of medicine, university of crete, heraklion, crete, greece. 2faculty of veterinary medicine, aristotle university of thessaloniki, greece. *corresponding author at: clinic of farm animals, aristotle university of thessaloniki, greece. tel.: +30 2310 99 4509, fax: +30 2310 99 4463, e‑mail: ngiadini@vet.auth.gr. keywords anaplasma phagocytophilum, abortion, goats, treatment. summary anaplasma phagocytophilum, transmitted by ixodes ticks, is an intracellular pathogen of zoonotic interest. regarding animals of veterinary importance, infection by this agent has been linked mainly to high fever, neutropenia, reduced milk production, but hemorrhagic diathesis, abortion and impaired spermatogenesis have also sporadically been reported. in greece, a. phagocytophilum has been detected in dogs, ticks and humans, while so far only a. ovis had been detected in farm animals. following the occurrence of multiple abortions in two goat farms in northern greece, samples were collected from aborted animals. stomach contents and placental tissue from aborted animals tested positive for a.  phagocytophilum by molecular assays and negative for other infectious and parasitic agents. treatment with oxytetracycline la stopped the abortions. in tick risk areas clinicians should consider a. phagocytophilum as a cause of abortion in goats. dimosthenis chochlakis1, nektarios giadinis2*, evanthia petridou2, george filioussis2, yannis tselentis1, anna psaroulaki1, evi ioannidou2, vasiliki papanikolopoulou2 and harilaos karatzias2 molecular evidence of anaplasma phagocytophilum in aborted goat fetuses and placenta veterinaria italiana 2020, 56 (4), 301‑303. doi: 10.12834/vetit.1173.6516.2 accepted: 12.12.2016 | available on line: 31.12.2020 short communication 302 a. phagocitophila in goats chochlakis et al. veterinaria italiana 2020, 56 (4), 301‑303. doi: 10.12834/vetit.1173.6516.2 sequences revealed were 100% identical to each other and 100% identical to already published sequences (16s rrna: genbank accession number eu090186) and 99% identical to the 16s rrna (m73220) and groesl (af548385) of the already published variant known to cause a more severe form of the disease (stuen et al. 2003). in both herds, treatment with oxytetracycline la of the rest pregnant animals (two injections with three days a part at a dose of 20 mg/kg) following a.  phagocytophilum detection, was effective and no other cases of abortion were observed. according to the owners, the animals in both herds were grazing during large periods of the year, consequently tick infestation was not unusual. at the intervention time, the animals didn’t have ticks, because few days earlier they had been treated for ectoparasites. abortions, which often are determined by zoonotic agents, are a serious economic threat in goat herds, as they can reduce the meat and milk productions (smith and sherman 2009, giadinis et al. 2013). anaplasma phagocytophilum has been demonstrated as a possible cause of reproductive failures in sheep flocks in italy, norway, spain and uk (jones and davies 1995, garcia‑perez et  al. 2003, chianini et  al. 2004, lillini et al. 2006, giudice et al. 2011). however, this is the first time that a.  phagocytophilum has been associated to abortion cases in goats. as regards the pathophysiology of abortions caused by a.  phagocytophilum, they seem to occur due to placental dysfunction (chianini et  al. 2004). it has recently been found that transplacental transmission of a.  phagocytophilum can occur in sheep without abortion (reppert et  al. 2013). it is interesting, that in the previous study, a.  phagocytophilum was detected in many tissues of an infected lamb during pregnancy. in the present study, the pathogen was detected in the stomach contents of the goat kids of the 1st herd and in placental tissue from the 2nd herd, while it was not found in other tissues of the aborted goat kids. this suggests that abortion might be a consequence of placental dysfunction. unfortunately, histopathology was not conducted in the fetal membranes. in any case, it would be interesting to investigate the distribution of a. phagocytophilum in various tissues. since the number of examined herds and fetuses was low, these data should be treated with caution. however, in case of a. phagocytophilum infection in goat herds it is likely that abortion is a sporadic event (giadinis et  al. 2011, giadinis et  al. 2013). abortion storm with ewe mortality has been reported once in sheep in uk (jones and davies 1995). however, this outbreak was not thoroughly investigated and oxytetracycline administration was not effective. in prefecture (northern greece) and consisted of 300  dairy goats of local breeds that were reared under the semi‑intensive feeding system. in 2014, the farmer observed sporadic abortions in goats of different ages, and referred about 15 abortions at the 3rd‑4th month of pregnancy. two aborted fetuses (from different mothers) and fetal membranes were submitted to the farm animal clinic of the aristotle university in thessaloniki. both fetuses and fetal membranes were normal in appearance and no specific lesions were detected at autopsy. whole blood from the aborting mothers along with fetal spleen, brain, liver, placental tissues and stomach contents were collected. all samples were tested by pcr or cultures for chlamydia  spp., brucella  spp., campylobacter  spp., salmonella  spp., escherichia coli, listeria spp., mycoplasma  spp., toxoplasma spp., neospora spp., and schmallenberg virus at the aristotle university and they were found all negative for the above pathogens (giadinis et al. 2013). dna was extracted from the sampled tissues using a commercial kit (tissue kit, qiagen, hilden germany); portions of dna samples were sent at the laboratory of clinical bacteriology, parasitology, zoonoses and geographical medicine of the university of crete, where they were tested by pcr for babesia species (targeting 18s rrna) and by a multiplex real‑time pcr for coxiella burnetii (targeting com1), anaplasma species (targeting msp4 of a.  centrale, a.  marginale and a. ovis) and a. phagocytophilum (targeting msp4). dna from positive samples against the pathogens of interest was used as positive marker; sterile water was used as negative marker. all samples were negative for babesia species, anaplasma species and coxiella burnetii, while dna from the stomach contents of the 1st herd and placental tissue from the 2nd herd were tested positive for a.  phagocytophilum. also, blood samples from the examined mothers were found positive for a. phagocytophilum. positive samples were further amplified using primers ehr16sr and ehr16sd (targeting a 345 bp portion of the 16s rrna (parola et  al. 1998) and hs1 and hs6 (targeting a 1300‑1450 bp portion of groesl) (sumner et al. 1997). pcr amplicons (345 bp for 16s rrna and 1350 bp of groesl) were purified using a commercial kit (qia quick pcr purification spin kit, qiagen, germany). sequencing was performed twice using the above described primers at a ceq 8000 beckman coulter sequencer. the sequences revealed were processed using chromas v1.49 and lasergene ver.7.1 software for viewing the chromatograms and editing of the retrieved nucleotide sequences, and nucleotide blastn for investigating the homology of the obtained sequences compared to the ncbi databases. 303 chochlakis et al. a. phagocitophila in goats veterinaria italiana 2020, 56 (4), 301‑303. doi: 10.12834/vetit.1173.6516.2 veterinaria italiana 2020, 56 (4), xxx‑xxx. doi: 10.12834/vetit.1173.6516.2 (stuen et al. 2003, teglas and foley 2006, granquist et  al. 2010). anaplasma phagocytophilum infection may persist in different animal species, depending on individual variations, genetic variants and host species involved. however, persistent infection in goats has not been reported yet (stuen 2007, thomas et al. 2012). the present study, oxytetracycline administration was very effective in controlling the abortions. regarding the responsible strain, the sequences revealed a 99% similarity with those of a.  phagocytophilum variant 1 (stuen et  al. 2003). the incidence and severity of the disease depend on geographic area, species and host breed, as well as the variant of a.  phagocytophilum involved aktas m. & özübek s. 2015. bovine anaplasmosis in turkey: first laboratory confirmed clinical cases caused by anaplasma phagocytophilum. vet microbiol, 178 (3‑4), 246‑251. chianini f., adams c. & buxton d. 2004. neuropathological changes in ovine fetuses caused by tick‑borne fever. vet rec, 155, 805‑806. garcia‑perez a.l., barandika j., oporto b., povedano j. & juste r.a. 2003. anaplasma phagocytophila as an abortifacient agent in sheep farms from northern spain. ann n y acad sci, 990, 429‑432. giadinis n.d., chochlakis d., ioannou i., kritsepi‑konstantinou m., papadopoulos e., psaroulaki a. & karatzias h. 2011. haemorrhagic diathesis in a ram with anaplasma phagocytophilum infection. j comp pathol, 144, 82‑85. giadinis n.d., chochlakis d., tselentis y., petridou e., karatzias h. & psaroulaki a. 2012. hypogalactia in dairy sheep attributed to anaplasma ovis. proceedings of 2nd veterinary congress of farm animals, thessaloniki, greece. giadinis n.d., lafi s.q., ioannidou e., papadopoulos e., terpsidis k., karanikolas g., petridou e.j., brozos c. & karatzias h. 2013. reduction of the abortion rate due to toxoplasma gondii in 3 goat herds with sulfadimidine administration. can vet j, 54, 1080‑1082. giudice e., giannetto c., torina a. & gianesella m. 2011. anaplasma phagocytophilum intragranulocytic morulae in aborting ewes: a herd case in sicily. transbound emerg dis, 58, 263‑267. granquist e.g., bardsen k., bergström k. & stuen s. 2010. variant‑ and individual dependent nature of persistent anaplasma phagocytophilum infection. acta vet scand, 52, 25. jones g.l. & davies i.h. 1995. an ovine abortion storm caused by infection with cytoecetes phagocytophila. vet rec, 136, 127. kachrimanidou m., papa a., chochlakis d., pavlidou v. & psaroulaki a. 2011. molecular evidence for anaplasma phagocytophilum in ixodes ricinus ticks from greece. vector borne zoon dis, 11, 1391‑1393. lillini e., macri g., proietti g. & scarpulla m. 2006. new findings on anaplasmosis caused by infection with references anaplasma phagocytophilum. ann n y acad sci, 1081, 360‑370. papa a., tsioka k., kontana a. papadopoulos c. & giadinis n. 2017. bacterial pathogens and endosymbionts in ticks. ticks tick borne dis, 8 (1), 31‑35. parola p., beati l., cambon m., brouqui p. & raoult d. 1998. ehrlichial dna amplified from ixodes ricinus (acari: ixodidae) in france. med entomol, 35, 180‑183. psaroulaki a., koliou m., chochlakis d., ioannou i., mazeri s. & tselentis y. 2008. anaplasma phagocytophilum infection in a child. ped infect dis j, 27, 664‑666. reppert e., galindo r.c., breshears m.a., kocan k.m., blouin e.f. & de la fuente j. 2013. demonstration of transplacental transmission of a human isolate of anaplasma phagocytophilum in an experimentally infected sheep. transbound emerg dis, 60 (suppl. 2), 93‑96. smith m.c. & sherman d.m. 2009. goat medicine. 2nd ed., wiley‑blackwell, usa. stuen s. 2007. anaplasma phagocytophilum ‑ the most widespread tick‑borne infection in animals in europe. vet res commun, 31 (suppl. 1), 79‑84. stuen s., bergström k., petrovec m., van de pol i. & schouls l.m. 2003. differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of anaplasma phagocytophilum in sheep. clin diagn lab immunol, 10, 692‑695. sumner j.w., nicholson w.l. & massung r.f. 1997. pcr amplification and comparison of nucleotide sequences from the groesl heat shock operon of ehrlichia species. j clin microbiol, 35, 2087‑2092. teglas m.b. & foley j. 2006. differences in the transmissibility of two anaplasma phagocytophilum strains by the north american tick vector species, ixodes pacificus and ixodes scapularis (acari: ixodidae). exp appl acarol, 38, 47‑58. thomas r.j., birtles r.j., radford a.d. & woldechiwet z. 2012. recurrent bacteraemia in sheep infected persistently with anaplasma phagocytophilum. j comp pathol, 147, 360‑367. woldechiwet z. 2006. anaplasma phagocytophilum in ruminants in europe. ann n y acad sci, 1078, 446‑460. 79 veterinaria italiana 2021, 57 (1), 79-82. doi: 10.12834/vetit.1077.5873.2 accepted: 03.02.2020 | available on line: 27.07.2021 1dipartimento di medicina veterinaria, università degli studi di milano, milano, italy. 2dipartimento di scienze mediche veterinarie, università di bologna, alma mater studiorum, bologna, italy. * corresponding author at: dipartimento di medicina veterinaria, università degli studi di milano, milano, italy. e-mail: sara.barbieri@unimi.it. sara barbieri1*, zita talamonti1, eleonora nannoni2, eugenio ugo luigi heinzl1, michela minero1 and elisabetta canali1 keywords animal welfare, body temperature, health status, infrared thermography, swine. summary this study aims to assess the correlation between surface temperature estimated by infrared thermography and core temperature measured with rectal thermometer in weaning and fattening pigs. a total of 108 pigs were used in this study. thermal images of the eye of each animal were recorded with a thermal imaging camera, rectal temperatures were measured using a calibrated digital thermometer. the average rectal temperature was 38.9  ±  0.4  °c (min = 37.9 °c; max = 40.1 °c) and the average eye temperature was 36.7 ± 0.1 °c (min = 34.8 °c; max = 38.8 °c). our results showed that the mean eye temperature estimated by infrared thermography was significantly correlated (r = .581, p < .01) with rectal temperature. the correlation was significant and strong for weaners (r = .739, p < .01), significant although weak for fatteners (r = .236 p < .05). thermography could be a valid method to estimate the core temperature of pigs under farm condition. use of thermography in pigs: relationship between surface and core temperature short communication temperature; based on thermal images it is possible to perform accurate temperature measurements (speakman and ward 1998). as irt is a non-contact procedure, data can be collected on animals that are difficult to reach or to approach; furthermore, the short measuring time allows the recording of data from moving animals (kastberger and stachl 2003). irt has been used in several species. the medial posterior palpebral border of the lower eyelid and the curuncula lacrimalis has been demonstrated to be the location of the eye region showing the maximum temperature (pony: johnson et  al. 2011; cattle: stewart et  al. 2008; sheep: stubsjøen et al. 2009). only a few studies have investigated the use of irt in pigs and even fewer have investigated the use of irt as a tool to identify increases in temperature (bates et al. 2014, schmidt et al. 2013, traulsen et al. 2010). the aim of the study was to assess the relationship between surface temperature estimated by irt and core temperature measured with a rectal thermometer in weaning and fattening pigs. the experimental protocol included only procedures of a common clinical examination and animals were kept in compliance with the eu council directive consumer and european union (eu) policy demand for consistent enforcement of welfare legislation in food producing animals has been increasing over the last decades. in response to this demand, the assessment of animal welfare at farm level needs to develop a science-based multidimensional approach (mason and mendl 1993). the welfare assessment aims at determining the actual status of animals, including both physical and mental state, using animal based indicators able to address areas of concern in this field (efsa 2012a). several studies report that body temperature in pigs is a valid indicator for welfare assessment (tosi et al. 2003, efsa 2012b) and fever is the earliest and one of the main clinical signs of many diseases. however, body temperature is difficult to measure under farm conditions, as the accepted methods for measuring core temperature need handling and restraining of animals (stewart et al. 2005). infrared thermography (irt) is a non-invasive technique to estimate the body temperature by detecting infrared radiation emitted by each body (mitchell 2013, speakman and ward 1998, stewart et  al. 2005). irt uses thermal radiation emitted by objects to visualize and measure their surface 80 use of infrared thermography in pigs barbieri et al. veterinaria italiana 2021, 57 (1), 79-82. doi: 10.12834/vetit.1077.5873.2 of the day. frequency distributions and pearson correlation between core and surface temperatures of the pigs were calculated. cases of animals with rectal temperature higher than a reference limit (39  °c) were selected. mean high temperatures of selected animals were compared to those of the other animals using a t test. data was normally distributed (kolmogorov-smirnov test) (ibm 2014): the average rectal temperature was 38.9  ±  0.4  °c (min  =  37.9 °c; max  =  40.1  °c) and the average eye temperature was 36.7 ± 0.1 °c (min  =  34.8 °c; max  =  38.8 °c). our results showed that the mean eye temperature estimated by irt was significantly correlated (r  =  .581, p < .01) with rectal temperature. the correlation was significant and strong for weaners (r = .739, p < .01), significant although weak for fatteners (r  =  .236 p  <  .05), showing that irt can be reliably used on pigs of different ages (figure 2). we considered the eye region in agreement with irt studies on different species, which have identified this location as the one that corresponds most to rectal temperature and that is less affected by other factors (johnson et al. 2011, stewart et al. 2008). the absence of hair around the eye allows heat dispersion that amounts to a greater emission of infrared radiation (mitchell 2013). chung and colleagues (chung et al. 2010), comparing rectal and infrared thermometry in piglets, reported a significant linear relationship for surface temperature measured on three different locations of the body (central abdomen, cranial dorsum and perianal regions), while no significant relationship was found for lower eyelid. however, under farm conditions, the measurement at body regions such as flank and back may be negatively influenced by external factors, e.g., dirtiness, contact with other pigs and with the ground. on 2008/120/ec that stipulates minimum standards for the protection of pigs. a total of 108 pigs (28 weaners of 47 day old and 80 fatteners of 232 day old) were used in this study. the experiment was carried out in the facilities of the department of veterinary medical sciences of the university of bologna (italy): pigs were kept in groups of 5 animals on a slatted floor and under controlled temperatures ranging from 20 to 27 °c, according to the age of animals. a clinical examination was performed before the measuring in order to exclude animals with clinical signs of diseases. pigs received a commercial diet, according to the consortium for parma ham production rules (consortium for parma ham 2015), and water was available ad libitum. thermal images of the eye of each animal were recorded with a thermal imaging camera (nec avio tvs500). to optimize the accuracy of the thermographic image and to reduce sources of noise, before every work session the same image of a lambert surface was taken to define the radiance emission and to nullify the effect of surface reflections on tested animals (mallick et al. 2005). only perfectly focused images were used. to determine the temperature of the eye, grayess irt analyzer 4.8 (informer technologies, inc., usa) was used and the maximum temperature (°c) within a circular area traced around the curuncula lacrimalis was measured (figure 1). this maximum value was used for subsequent analysis. rectal temperatures were measured using a calibrated digital thermometer, checked before the examination and compared to a certified mercury thermometer. in accordance with the manufacturer’s instructions, the thermometer was inserted into the anus and positioned in contact with rectal mucosa for 10 seconds, until hearing the acoustic signal. during the measurements, animals were not manually restrained. for each animal the capture of thermal image was immediately followed by the measurement of rectal temperature; temperatures were recorded at the same time figure 1. thermal image of a pig’s head showing the position of the measurement point on the eye. 38.0 36.4 34.8 33.1 31.5 29.9 28.3 26.6 25.0 °c rectal temperature (°c) > 39 °c p < .01 34.00 35.00 36.00 37.00 38.00 39.00 < 39 °c ey e te m p er at u re (° c ) figure 2. mean surface temperature estimated by infrared thermography in pigs of different ages with core temperature higher and lower than the reference limit (39 °c). 81 barbieri et al. use of infrared thermography in pigs veterinaria italiana 2021, 57 (1), 79-82. doi: 10.12834/vetit.1077.5873.2 that can be used for early disease detection. irt applied at eye level is a valid method to estimate the core temperature of pigs under farm condition; however, the results should be interpreted with caution because of the limited sample size and further research is needed. moreover, external environmental and physical conditions can negatively influence irt measurements collected in the field and these factors need to be controlled in the design of experiments in order to have a clear interpretation of temperature outcomes (church et al. 2014). irt might be a useful non-contact method to measure the core temperature of pigs under farm conditions, being valuable for a non-invasive assessment of physiological state and for monitoring pig welfare. thermal imaging cameras are still relatively expensive, but appear to be reliable under field conditions and irt provides instantaneous results since software for data analysis in real time is incorporated. therefore, such a non-contact method would save time and reduce stress on the animals. the contrary, schmidt and colleagues (schmidt et al. 2013) measured body surface temperature in sows at different body regions and concluded that, under farm conditions, the back of the ear and the eye are the most promising locations to measure body temperature in pigs. our results suggested that irt surface temperature measured at eye level is higher in animals with rectal temperature higher than the reference limit of 39 °c. other studies on adult animals (schmidt et al. 2013, traulsen et al. 2010) reported a correlation between irt body surface temperature and core temperature. a study on continuous irt measurements (schmidt et  al. 2014) showed that surface temperature increase is time-delayed compared to the increase in core temperature, proving that irt may not be an adequate early detection method. nevertheless, studies in different species validate the use of irt in assessing reaction to fear-induced stress (dai et  al. 2015, stewart et al. 2008). our study suggests that irt allows routine measurements of body surface temperatures 82 use of infrared thermography in pigs barbieri et al. veterinaria italiana 2021, 57 (1), 79-82. doi: 10.12834/vetit.1077.5873.2 bates j.l., karriker l.a., stock m.l., pertzborn k.m., balwin l.g., wulf l.w., lee c.l., wang c. & coetzee j.f. 2014. impact of transmammary-delivered meloxicam on biomarkers of pain and distress in piglets after castration and tail docking. plos one, 9 (12), e113678. doi:10.1371/journal.pone.0113678. chung t., jung w., nam e., kim j., park s. & hwang c. 2010. comparison of rectal and infrared thermometry for obtaining body temperature of gnotobiotic piglets in conventional portable germ free facility. asian-australas j anim sci, 23, 1364-1368. church j.s., hegadoren p.r., paetkau m.j., miller c.c., regev-shoshani g., schaefer a.l. & schwartzkopf-genswein k.s. 2014. influence of environmental factors on infrared eye temperature measurements in cattle. 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temperature in ponies. j equine vet sci, 31, 63-66. kastberger g. & stachl r. 2003. infrared imaging technology and biological applications. behav res methods instrum comput, 35, 429-439. mason g. & mendl m. 1993. why is there no simple way of measuring animal welfare? animal welfare, 2, 301-319. references mallick s.p, zickler t.e., kriegman d.j. & belhumeur p.n. 2005. beyond lambert: reconstructing specular surfaces using color. in ieee computer society conference on computer vision and pattern recognition (cvpr'05), san diego, ca, usa, 619-626. doi: 10.1109/cvpr.2005.88. mitchell m. 2013. thermal imaging in physiology: theoretical and practical considerations. in thermography. current status and advances in livestock animals and in veterinary medicine (f. luzi, m. mitchell, l. nanni costa & v. redaelli, eds). fondazione iniziative zooprofilattiche e zootecniche, brescia, 47-65. schmidt m., ammon c., schon p.c., manteuffel c. & hoffmann g. 2014. the suitability of infrared temperature measurements for continuous temperature monitoring in gilts. archiv tierzucht (archives animal breeding), 21, 1-12. schmidt m., lahrmann k.h. & ammon c. 2013. assessment of body temperature in sows by two infrared thermography methods at various body surface locations. j swine health prod, 21, 203-209. speakman j.r. & ward s. 1998. infrared thermography: principles and applications. zoology, 101, 224-232. stewart m., webster j.r., schaefer a.l., cook n.j. & scott s.l. 2005. infrared thermography as a non-invasive tool to study animal welfare. animal welfare, 14, 319-325. stewart m., schaefer a., haley d.b., colyn j., cook n.j., stafford k.j. & webster j.r. 2008. infrared thermography as a non-invasive method for detecting fear-related responses of cattle to handling procedures. animal welfare, 17, 387-393. stubsjøen s.m., flø a.s., moe r.o., janczak a.m., skjerve e., valle p.s. & zanella a.j. 2009. exploring non-invasive methods to assess pain in sheep. physiol behav, 98, 640-648. tosi m.v., canali e., mattiello s., ferrante v., carenzi c. & verga m. 2003. il benessere dei suini e delle bovine da latte: punti critici e valutazione in allevamento. fondazione iniziative zooprofilattiche e zootecniche, brescia. traulsen i., naunin k., müller k. & krieter j. 2010. application of infrared thermography to measure body temperature of sows. züchtungskunde, 82, 437-446. 307 introduction canine perianal gland tumours (hepatoid gland tumours, circumanal gland tumours) are common neoplasms that arise from modified sebaceous glands around the anus but are also present along the ventral midline from the perineum to the base of the skull, the dorsal and ventral tail, and the skin of the lumbar and sacral region (berrocal et  al. 1989, goldschmidt and hendrick 2000, nielsen and aftosmis 1964). these glands are often named as ‘hepatoid glands’, because the cells resemble hepatocytes. circumanal adenomas department of general and clinical pathology, faculty of veterinary medicine, trakia university, stara zagora, student’s campus, 6000 stara zagora, bulgaria. *corresponding author at: department of general and clinical pathology, faculty of veterinary medicine, trakia university, stara zagora, student’s campus, 6000 stara zagora, bulgaria. tel.: +359 42 699 565, fax: +359 42 670 624, e-mail: rsimeonov@uni-sz.bg. parole chiave canino, analisi quantitativa, morfometria nucleare, tumori perianali. riassunto sono stati esaminati campioni citologici di 36 tumori spontanei della ghiandola perianale canina (18 adenomi e 18 adenocarcinomi). le cellule neoplastiche sono state ottenute mediante biopsia per aspirazione con ago sottile, fissate immediatamente con merckofix spray® (merck kgaa, darmstadt, germania) e colorate con hemacolor® (merck kgaa, darmstadt, germania). attraverso un sistema computerizzato di analisi di immagini gli strisci citologici sono stati sottoposti ad analisi morfometriche per valutare i valori medi della superficie del nuclei (mna; µm2) e del loro perimetro (mnp; µm) nonché la lunghezza media (d media; µm), minima (d min; µm) e massima (d max) del loro diametro. i risultati hanno indicato un aumento dei valori medi dei parametri nucleari nel passare dagli adenomi perianali canini agli adenocarcinomi perianali canini. l'analisi statistica ha rilevato differenze statisticamente significative (p < 0,01) tra cellule neoplastiche benigne e maligne. l'analisi nucleare quantitativa potrebbe quindi essere utilizzata come metodo aggiuntivo per la differenziazione tra adenomi e carcinomi perianali spontanei canini. morfometria nucleare nei tumori canini della ghiandola perianale keywords canine, quantitative analysis, nuclear morphometry, perianal tumours. summary cytological samples from 36 spontaneous canine perianal gland tumours (18 adenomas and 18 adenocarcinomas) were examined. neoplastic cells were preoperatively obtained by fine‑needle aspiration biopsy, fixed immediately with merckofix spray® (merck kgaa, darmstadt, germany) and stained with hemacolor® (merck kgaa, darmstadt, germany). cytological smears were subjected to morphometric analysis by means of a digital microscope, pc station and image analysis software. the morphometric parameters evaluated in this study were mean nuclear area (mna; µm2), mean nuclear perimeter (mnp; µm), mean nuclear diameter (d mean; µm), minimal nuclear diameter (d min; µm) and maximal nuclear diameter (d max). the results indicated an increase of the mean values of the nuclear parameters from canine perianal adenomas to canine perianal adenocarcinomas. the statistical analysis revealed significant differences between benign and malignant neoplastic cells (p < 0.01). the results in this study indicate that quantitative nuclear analysis could be used as an additional method for differentiating canine spontaneous perianal adenomas from carcinomas. radostin stefanov simeonov* nuclear morphometry in 36 canine spontaneous perianal gland tumours veterinaria italiana 2019, 55 (4), 307‑310. doi: 10.12834/vetit.1031.5489.2 accepted: 07.07.2016 | available on line: 31.12.2019 308 nuclear morphometry in canine spontaneous perianal gland tumours simeonov veterinaria italiana 2019, 55 (4), 307‑310. doi: 10.12834/vetit.1031.5489.2 quantitative analysis cytological smears were subjected to morphometric analysis by means of a digital microscope1, pc station and image analysis software2. the system was previously calibrated by the built‑in micrometer ruler. nuclei were randomly selected for morphometric analysis provided that they were clearly visible and intact. at least 100 nuclei were analyzed in each case. cytologically, the hepatoid cells, characterized with abundant finely granular cytoplasm, predominated. their nuclei resembled those of normal hepatocytes appearing round with often single or multiple, prominent nucleoli. in contrast to them, the reserve cells were significantly smaller, less numerous, and had higher nuclear/ cytoplasmatic ratios and lacked features of cellular pleomorphism (figure  1). the morphometric parameters evaluated in this study were mean nuclear area (mna; µm2), mean nuclear perimeter (mnp; µm), mean nuclear diameter (d  mean; µm), minimal nuclear diameter (d min; µm) and maximal nuclear diameter (d max). the parameters were automatically calculated by the image analysis software2. statistical analysis data from the morphometric analysis were statistically analyzed by the mann‑whitney u test (statistica 6.0, statsoft, tulsa, ok, usa) at a level of significance p < 0.05. generally appear as nodular lesions affecting the perianal region (nielsen and aftosmis 1964). they represent the majority of canine perianal tumours (berrocal et  al. 1989). siberian husky, samoyed, pekingese and cocker spaniel are most likely to develop this tumour (goldschmidt and hendrick 2000). the older, intact male is at high risk (nielsen and aftosmis 1964, berrocal et  al. 1989, wilson and hayes 1979). the tumours are slowly growing but never metastasize. larger lesion commonly ulcerate, and hemorrhagic, keratinaceous material can often be extruded with local pressure (berrocal et  al. 1989, wilson and hayes 1979). up to 95 % of male dogs respond completely to castration (ross et  al. 1991). perianal adenocarcinomas occur much less frequently than its benign counterpart. they represent about 3‑21 % of all neoplasms in this region (brodey 1970). these tumours have metastatic potential and often spread to the regional lymph nodes. average age of affected dogs is 11 years. tumours occur in castrated or intact males, as well in females (ross et  al. 1991). they are generally not responsive to castration or to estrogen therapy (wilson and hayes 1979, vail et al. 1990). the rate of growth of these tumours is variable. metastases may occur via the lymphatic route to regional lymph nodes with subsequent spread to lungs and other organs (goldschmidt and hendrick 2000). pathohistological evaluation is the best mean of diagnosis in canine perianal tumours. however, there is debate about how to distinguish low grade malignant tumours from circumanal adenomas because well differentiated forms can be confused with adenomas. the aim of the present study was to determine whether the quantitative nuclear analysis could be used as an additional method for differentiating canine spontaneous perianal adenomas from carcinomas. materials and methods animals cytological samples from 36 spontaneous canine perianal gland tumours (18 adenomas and 18  adenocarcinomas) were examined. the tumours were collected following surgical removal from dogs presented to the department of surgery, faculty of veterinary medicine, trakia university. neoplastic cells were preoperatively obtained by fine‑needle aspiration biopsy, fixed immediately with merckofix spray® (merck kgaa, darmstadt, germany) and stained with hemacolor® (merck kgaa, darmstadt, germany). tumours were histopathologically confirmed according to the who international histological classification of tumours of domestic animals (goldschmidt et al. 1998). figure 1. cytological picture of a hepatoid adenocarcinoma. the reserve cells (arrows) are significantly smaller, less numerous and have a higher nuclear/cytoplasmatic ratio compared to hepatoid cells. 1 motic professional b3 digital microscope (motic, china group co ltd, hong kong, china). 2 image pro plus® analysis system (media cybernetics, silver spring, md, usa, version 4.5.0.29 for windows 98/nt/2000). 309 simeonov nuclear morphometry in canine spontaneous perianal gland tumours veterinaria italiana 2019, 55 (4), 307‑310. doi: 10.12834/vetit.1031.5489.2 discussion the most important pathohistological criteria supporting the diagnosis of perianal adenocarcinomas is invasiveness of tumour cells into surrounding tissue. increased nuclear pleomorphism, disorderly arrangement of cells and increased number of mitoses also are connected with malignancy (stannard and pulley 1978). in our previous studies (simeonov and simeonova 2008, simeonova and simeonov 2008) we investigated the prognostic value of nuclear morphometry in canine spontaneous perianal adenocarcinomas. the mean values of morphometric parameters were significantly greater in dogs with lymph node metastases compared to parameters of tumour cells from dogs which were lymph node‑negative. significant differences in mna, mnp, d mean, results the data for the investigated parameters for each of the 36 tumours examinated are presented in table i. the average numeric values of the studied parameters were significantly higher in perianal adenocarcinomas than in perianal adenomas. the results indicated an increase of the mean values of the nuclear parameters from canine perianal adenomas (mna: 81.05 ± 3.46; mnp: 33.10 ± 2.01; d  mean: 10.00 ± 0.46; d min: 8.33 ± 1.08; d max: 11.99  ±  1.67) to canine perianal adenocarcinomas (mna: 99.15  ±  19.21; mnp: 35.88  ±  3.26; d mean: 11.04 ± 1.13; d min: 9.34 ± 1.09; d max: 12.88 ± 1.48). the statistical analysis revealed significant differences (p < 0.01) between benign and malignant neoplastic cells (table ii). table i. values of the morphometric nuclear parameters in each of the examined tumours. canine hepatoid adenomas canine hepatoid adenocarcinomas cases mna(µm2) mnp (µm) d mean (µm) d min (µm) d max (µm) cases mna (µm2) mnp (µm) d mean (µm) d min (µm) d max (µm) 1 75.01 31.12 9.58 8.47 10.68 1* 139.61 42.10 13.26 11.10 14.96 2 77.43 31.07 9.75 8.90 10.46 2* 142.43 43.15 13.57 11.08 16.41 3 75.27 30.70 9.64 8.99 10.18 3* 118.32 38.73 12.02 10.31 13.49 4 79.26 31.53 9.86 8.85 11.29 4 89.34 34.88 10.43 8.74 12.66 5 84.35 32.66 10.20 9.37 11.03 5 79.54 32.06 9.85 8.79 11.48 6 83.40 32.29 10.15 9.81 10.56 6* 122.14 39.97 12.61 10.15 14.57 7 85.99 32.77 10.32 9.84 10.67 7 85.37 33.38 10.16 8.27 12.13 8 76.30 31.55 9.69 8.58 11.08 8 88.19 33.52 10.42 9.76 11.19 9 85.24 34.12 10.13 7.91 12.69 9 87.64 35.04 10.27 7.88 13.53 10 78.92 32.75 9.91 7.68 12.40 10* 102.75 38.09 11.56 8.31 14.75 11 77.28 33.33 9.50 6.82 12.99 11* 106.49 37.00 11.49 10.81 12.74 12 82.22 32.37 10.06 9.11 11.22 12 83.53 32.71 10.12 8.94 11.41 13 85.61 32.71 10.27 9.33 10.86 13 87.86 33.33 10.39 9.33 11.22 14 80.73 35.75 9.65 6.82 14.67 14* 92.93 34.95 10.69 9.17 12.77 15 79.64 32.29 9.89 8.43 11.83 15 89.48 34.12 10.46 8.85 11.93 16 85.67 38.79 11.56 6.14 16.33 16 81.32 33.14 9.92 7.61 12.06 17 83.31 34.70 9.99 7.59 12.98 17* 94.31 34.55 10.79 10.08 11.57 18 83.26 35.23 9.82 7.25 13.90 18 93.50 35.16 10.64 8.63 12.98 mna = mean nuclear area; mnp = mean nuclear perimeter; d mean = mean nuclear diameter; d min = minimum nuclear diameter; d max = maximum nuclear diameter. *metastasizing hepatoid adenocarcinomas. table ii. number of cases (n), mean (m) and standard deviation (δ m) of the measured parameters. group parameter canine hepatoid adenomas(n=18) m ± δm (range) canine hepatoid adenocarcinomas (n=18) m ± δm (range) significance p mna (μm2) 81.05 ± 3.76 (75.01-85.99) 99.15 ± 19.21 (79.54-142.43) p = 0.0004 mnp (μm) 33.10 ± 2.01 (30.70-38.79) 35.88 ± 3.26 (32.06-43.15) p = 0.004 d mean (μm) 10.00 ± 0.46 (9.50-11.56) 11.04 ± 1.13 (9.85-13.57) p = 0.001 d min (μm) 8.33 ± 1.08 (6.14-9.84) 9.34 ± 1.09 (7.61-11.10) p = 0.008 d max (μm) 11.99 ± 1.67 (10.18-16.33) 12.88 ± 1.48 (11.19-16.41) p = 0.1 310 nuclear morphometry in canine spontaneous perianal gland tumours simeonov veterinaria italiana 2019, 55 (4), 307‑310. doi: 10.12834/vetit.1031.5489.2 d max, and d min were seen between metastasizing and non‑metastasizing neoplastic formations. a statistically significant correlation was found also between survival period of dogs and each of the following variables: 1) age, 2) diameter of tumour, 3) metastases in the regional lymph nodes, 4) mna, 5) mnp, 6) d max and 7) d mean. the 87.5 % of all affected animals, that had tumour diameters > 5 cm, were already lymph node positive. a statistically significant positive correlation (p = 0.66) was found between tumour diameter and metastases to the regional lymph nodes. berrocal a., vos j., van den ingh t., molenbeek r. & van den sluijs f. 1989. canine perianal tumours. zbl vet med, 6, 739‑749. brodey r. 1970. canine and feline neoplasia. adv vet sci comp med, 14, 309‑354. goldschmidt m., dunstan r., stannard a., von tscharner c., walder e. & yager j. 1998. histological classification of tumours of the skin of domestic animals, world health organization international classification of tumors in domestic animals, second series, vol. iii, washington d.c., armed forces institute of pathology and american registry of pathology, 23‑24. goldschmidt m. & hendrick m. 2000. tumours of the skin and soft tissue. in tumors in domestic animals, 4th ed. (d. meuten, ed), iowa state press, 68‑70. nielsen s. & aftosmis j. 1964. canine perianal gland tumours. javma, 144, 127‑135. ross j., scavelli t. & matthiesen d. 1991. adenocarcinoma of the apocrine glands of the anal sac in dogs: a review of 22 cases. j am anim hosp assoc, 27, 349‑355. references stannard a. & pulley l. 1978. tumors of the skin and soft tissues. in tumors of domestic animals (j. moulton, ed), berkeley, university of california press, 16‑70. simeonov r. & simeonova g. 2008. computer‑assisted nuclear morphometry in the cytological evaluation of canine perianal adenocarcinomas. j comp pathol, 139, 226‑230. simeonova g. & simeonov r. 2008. correlation between tumour diameter and presence of metastases to the regional lymph nodes in spontaneous canine hepatoid adenocarcinomas. trakia j sci, 6 (1), 54‑57. vail d., withrow s. & schwarz p. 1990. perianal adenocarcinoma in the canine male: a retrospective study of 41 cases. j am anim hosp assoc, 26, 329‑334. wilson g. & hayes h. 1979. castration for treatment of perianal gland neoplasms in the dog. javma, 174, 1301‑1303. in this study, we found that mna, mnp, d mean and d min differed significantly among canine perianal adenomas and adenocarcinomas (p < 0.01). the mean mna, mnp, d mean, d max and d min values in malignant tumours were higher than in benign tumours. therefore, quantitative nuclear analysis could be helpful in differentiating these neoplasms in the dog. this is a good news along these lines. we are convinced that this method could be very useful for veterinary surgeons and their patients and we are confident that soon it will be carried out in various veterinary practices/ clinics on a routine basis. 223 introduction leishmania is an intracellular parasite found in mammalian macrophages and transmitted by phlebotomine sandflies of the genus phlebotomus (old world) and lutzomyia (new world). it is a heteroxenous parasite and needs 2 hosts to develop. on the vertebrate side, the principal leishmania hosts are dogs and other members of the canidae family (foxes, jackals, wolves). in italy, phlebotomus perniciosus is considered the main species responsible for the spread of the leishmania infection (bongiorno et al. 2003). canine leishmaniasis (canl) is endemic in the mediterranean basin, asia, and latin america, but is also reported in a rising number of cases from non‑endemic countries due to the travel and import of animals as pets (gramiccia & gradoni 2005, metter et  al. 2005). in italy, canl has been observed for a long time in southern, central, and insular regions (paradies et  al. 2006). recent evidence shows that canl is expanding into north‑western italy, into areas with a continental 1 department of veterinary medicine and animal production, university of naples federico ii, 80137 napoli, italy. 2 unit of vector-borne diseases and international health, mipi department, istituto superiore di sanità, rome, italy. * corresponding author at: department of veterinary medicine and animal production, university of naples federico ii, 80137 napoli, italy. gaetano.oliva@unina.it parole chiave allopurinolo, leishmaniosi canina, lesione atipica, localizzazione mucosale, miltefosina, narice. riassunto la leishmaniosi canina è una malattia zoonosica sistemica causata da un parassita intracellulare dei macrofagi, il protozoo leishmania, trasmesso dalla puntura di flebotomi vettori. nei cani l’infezione è caratterizzata dalla diffusione viscerale e cutanea del parassita; essa causa linfoadenopatia, splenomegalia, lesioni cutanee, danni renali, oculari ed articolari, determinati dalla deposizione di immunocomplessi. il parassita può essere riscontrato raramente in sedi atipiche, con coinvolgimento delle mucose. l’obiettivo del presente lavoro è descrivere un caso atipico di leishmaniosi, caratterizzato dalla presenza di una massa nasale non associata ad evidente coinvolgimento sistemico. la diagnosi definitiva è stata confermata isolando dalla lesione promastigosti con esame colturale del tessuto bioptico. il cane, trattato con un’associazione di miltefosina e allopurinolo, ha presentato piena remissione della sintomatologia a due mesi. si sottolinea l’importanza di considerare la leishmaniosi canina nella diagnosi differenziale di lesioni mucosali e simil‑tumorali. lesione atipica nasale causata da leishmania spp. in un cane keywords allopurinol, atypical lesion, canine leishmaniasis, miltefosine, mucosal localization, nostril. summary canine leishmaniasis (canl) is a systemic zoonotic disease caused by the protozoan leishmania, an intracellular macrophage parasite, transmitted by the bite of phlebotomine sandflies. in dogs, the clinical disease is mostly characterised by symptoms associated with viscerocutaneous lesions such as lymphadenopathy, splenomegaly, skin lesions, and renal and ocular disease caused by the deposition of immune complexes. the parasite may provoke mucosal lesions which cause atypical clinical signs. the aim of this study is to describe an atypical nostril mass in a dog infected by leishmania. clinical examination did not show any systemic clinical signs, while haematological, biochemical, and urinary parameters demonstrated a mild disease stage. diagnosis was confirmed through the isolation of cultured live parasites by biopsy. the dog was treated with a combination of miltefosine and allopurinol, showing full remission of clinical symptoms after 2 months. the authors outline the importance of considering canl in the differential diagnosis of mucous and tumour‑like lesions. manuela gizzarelli1*, valentina foglia manzillo1, eleonora fiorentino2, aldo scalone2 and gaetano oliva1 nostril mass caused by leishmania spp. in a dog veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1460.7898.1 accepted: 19.02.2018 | available on line: 30.09.2018 224 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx nostril mass caused by leishmania spp. gizzarelli et al. to periodic core vaccinations and anti‑parasitic treatments. two years before the dog resulted positive to an anti‑leishmania test (ifat ) with a value of 1:160. no clinical signs or urinary and biochemical alterations were present, therefore the dog was not treated for canl. clinical examination showed no relevant clinical signs. mild lymphadenomegaly was the only clinical finding. an examination of the nasal region revealed the presence of ulceration at the nasal muco‑cutaneous margin as well as a nostril mucosal hypertrophy that deformed the nasal profile (figure 1). haematological, biochemical, and urinary analysis revealed several clinicopathological alterations: mild non‑regenerative anaemia, increasing of urea, alanine aminotransferase (alt ), and total protein values; albumin/globulin ratio inversion, proteinuria and abnormal value of urinary protein‑creatinine (upc) ratio (table i, table ii). differential diagnosis included nasal tumor, systemic fungal infection, and foreign bodies. because of the particular anatomic localization, the previous history of the dog, and the presence of clinicophatological alterations such as proteinuria, canl was considered in the differential plan along with other possible granulomatous lesions. the nasal mass was first examined using brushing cytology. this revealed the moderate presence of inflammatory cells, no amastigotes of leishmania, nor fungal hyphae. the dog was submitted to anaesthesia (methadone 0.2 mg/kg, propofol 4‑6  mg/kg, isoflurane) in order to perform a tomographic examination of the nasal structures and surgical biopsy of the lesion. an anti‑leishmania specific serological test (ifat) was also performed together with popliteal lymph nodes and sternal bone marrow aspiration. a leishmania culture of bioptic tissue was carried out using a modified tobie‑evans medium. climate, far away from the recognised endemic areas along the mediterranean coast (ferroglio et al. 2005). parasite transmission occurs through the bite of an infected phlebotomine sandfly, although secondary modes of transmission (e.g. via blood transfusion and congenital transmission) have also been suggested (gaskin et  al. 2002, owens et  al. 2001). clinically, canl is characterised by chronic viscerocutaneous signs, such as lymphadenopathy, skin lesions (symmetrical alopecia, furfuraceus dermatitis, ulcers, and nodular lesions) (ciaramella et  al. 1997), keratoconjunctivitis, epistaxis, and diarrhoea (longstaffe & guy 1985). in atypical cases, the parasites can also be found in striated muscle, central nervous system, and endocrine glands or gonads, with or without functional damage (cortese et al. 1999). mucosal localizations are rarely described in dogs (aliaga et al. 2003, foglia manzillo et al. 2005, foglia manzillo et al. 2009, font et al. 1996, lamothe & poujade 2002, pinna parpaglia et  al. 2007, viegas et al. 2012). the aim of this study is to describe an atypical case of leishmania infection in a dog showing a diffuse nostril mucosal involvement, which was successfully treated with miltefosine and allopurinol. case report an english setter, 7 year old, female, was referred to the veterinary hospital of veterinary medicine faculty of naples for the appearance of unilateral mucous nasal discharge, followed by the evidence of a mass. lesions didn’t respond to antibiotic treatment (amoxicillin/clavulanic acid combination, 15 mg/kg bid x 15 days). symptoms had been present for 2 previous months. history revealed that the dog lived outdoor, in campania region, south italy. this area is considered highly endemic to canl. the dog was fed with commercial food and was submitted figure 1. nostril mass (arrow) that deformed the nasal profile. table i. haematological parameters. parameter value normal range wbc 10.400 mm3 6.000-12.000 mm3 plt 430.000 mm3 175.000-400.000 mm3 rbc 5.340.000 mm3 5.000.000-8.000.000 mm3 hgb 12.9 g/dl 16-18 g/dl hct 33.9 % 35-50% mcv 64 fl 65-75 fl mch 24.2 pg 19-24 pg mchc 38.1 g/dl 32.0-38.0 g/dl wbc= white blood cells; plt= platelets; rbc = red blood cells; hgb = haemoglobin; hct = haematocrit; mcv = mean corpuscular value; mch = mean corpuscular haemoglobin; mchc = mean corpuscular haemoglobin concentration. veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 225 gizzarelli et al. nostril mass caused by leishmania spp. were attributed to the administration of miltefosine. after 2 months, the nostril lesions were completely resolved. this was confirmed at 1‑year follow‑up (figure 3). anti‑leishmania ifat titers, evaluated 1 year after treatment, showed a reduction to 1:320. discussion canl is usually characterized by viscerocutaneous clinical signs; the mucosal localization showed in this case study is rare and mostly described in humans as a result of infection in immune depressed patients (aliaga et  al. 2003). in dogs infected by leishmania infantum, the presence of mucosal lesions is not often described (aliaga et  al. 2003, foglia manzillo et  al. 2005, foglia manzillo et  al. 2009, font et  al. 1996, lamothe & poujade 2002, pinna parpaglia et al. 2007, viegas et al. 2012), and its pathogenic origin is not fully clear. in this study, we did not identify the leishmania species involved, however we presumed the species was leishmania infantum because it is the only species actually reported in italy and the dog had no history of travel outside the country. based on these findings, our main hypotheses are similar to those considered for humans in which the mucosal lesions caused by l. infantum have been attributed to the immune suppression of the host or to the parasite tropism associated to an aberrant local tissue response after sand fly inoculation. the dog described in this study didn’t show typical ifat was found positive at 1:2560, while lymph node and bone marrow aspirates were negative to microscopic examination. after 2 weeks, the culture resulted positive for the growth of leishmania promastigotes. a histopathological examination of the nasal mucosa revealed the presence of diffuse and severe cells proliferation. these had infiltrated the connective bundles, disrupting their architecture, and reached the deeper muscle planes. cellular proliferation consisted of numerous macrophages, lymphocytes, and plasma cells (figure 2); very rare leishmania amastigotes were detected at high magnification. tomography confirmed that there was no involvement of nasal bone structures. the dog was treated with specific anti‑leishmania therapy: miltefosine at a dose of 2 mg/kg of body weight, orally, once a day for 28 days, and allopurinol at 10 mg/kg, orally, twice a day for 6 months. the dog was also treated with enalapril at a dose of 0.5 mg/kg of body weight and specific renal diet. side‑effects of the drugs were limited to 3 episodes of vomiting that table ii. biochemical and urinary parameters. parameter value normal range crea 1.06 mg/dl < 1.8 mg/dl bun 28.2 mg/dl 7-20 mg/dl glu 104 mg/dl 60-100 mg/dl alt 136 ui/l 10-47 ui/l tp 7.9 g/dl 6-7.8 g/dl a/g ratio 0.5 0.6-1.1 proteinuria 500 mg/dl upc ratio 0.9 < 0.5 crea = creatinine; bun = blood urea nitrogen; glu = glucose; alt = alanine aminotransferase; tp = total protein; a/g ratio = albumin/globulin ratio; upc ratio = urine proteine to creatinine ratio. figure 2. dog: nostril mass. cellular proliferation, due to several macrophages, lymphocytes, and plasma cells (haematoxylin-eosin 40x). figure 3. nasal region examination after treatment. nick title first author et al. 226 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx hypotheses could be considered: i) an intense local reaction at the parasite inoculation site due to possible repetitive bites during sandfly season. this hypothesis has also been considered in other studies (foglia manzillo et  al. 2005, viegas et  al. 2012); ii) a rapid progression of the first infection assessed 2 years before, with an atypical diffusion of the parasite with mucosal involvement (foglia manzillo et  al. 2005, foglia manzillo et  al. 2009, lamothe & poujade 2002, pinna parpaglia et al. 2007, viegas et al. 2012). the therapeutic efficacy of the combination of miltefosine with allopurinol, as it has been described previously in other mucosal involvement (foglia manzillo et  al. 2009), confirmed that this protocol could be considered as a first‑line approach when mucosal lesions are evident in canl. our case re‑confirmed that every atypical lesion could be caused by leishmania infection and that this type of infection should always be considered in differential diagnosis of mucosal and tumour‑like lesions. clinical symptoms associated with canl, except for a mild enlargement of the popliteal lymph nodes. however haematological, biochemical, and urinary data revealed mild anaemia, and liver and kidney involvement. during leishmania infection these findings are caused by a systemic inflammatory response and immune complexes deposition that is evidenced by the serum total protein increase and a/g ratio inversion. specific tests for leishmania showed very high antibody titres (ifat 1:2560), which allowed us to classify the clinical condition of the dog as stage ii (moderate disease) substage  b, according to the leishvet group classification (solano‑gallego et  al. 2009). in this particular case however, the visceralization of the parasite was not evident because of the negative results of direct parasitological investigations (lymph nodes and bone marrow aspirates). in addition, the good general condition of the dog seems rule out that the nostril lesion represents the result of an intense immune suppression. instead, 2 other different first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 227 aliaga l., cobo f., mediavilla j.d., bravo j., osuna a., amador j.m., martin‑sanchez j., cxorsero e. & navarro j.m. 2003. localized mucosal leishmaniasis due to leishmania infantum: clinical and microbiologic findings in 31 patients. medicine, 82,147‑158. bongiorno g., habluetzel a., khoury c. & maroli m. 2003. host reference of phlebotomine sand flies at a hypodermic focus of canine leishmaniosis in central italy. acta trop, 88,109‑116. ciaramella p., oliva g., de luna r., gradoni l., ambrosio r., cortese l., scalone a. & persechino a. 1997. a retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by leishmania infantum. vet rec, 141, 539‑543. cortese l., oliva g., restucci b., ciaramella p. & persechino a. 1999. primary hypothiroidism associated with leishmaniasis in a dog. j am anim hosp assoc, 35, 487‑492. ferroglio e., maroli m., gastaldo s., mignone w. & rossi l. 2005. canine leishmaniasis, italy. emerg infect dis, 11, 1618‑1629. foglia manzillo v., pagano a., paciello o., di muccio t., gradoni l. & oliva g. 2005. papular‑like glossitis in a dog with leishmaniosis. vet rec, 156, 213‑215. foglia manzillo v., paparcone r., cappiello s., de santo r., bianciardi p. & oliva g. 2009. resolution of tongue lesions caused by leishmania infantum in a dog treated with the association miltefosine‑allopurinol. parasit vectors, 2 (suppl 1), s6. font a., roura x., fondevila d., closa j., mascort j. & ferrer l. 1996. canine mucosal leishmaniasis. j am anim hosp assoc, 32,131‑137. gaskin a.a., schantz p., jackson j., birkenheuer a., tomlinson l., gramiccia m., levy m., steurer f., kollmar e., hegarty b.c., ahn a. & breitschwerdt e.b. 2002. references visceral leishmaniasis in a new york foxhound kennel. j vet intern med, 16, 34‑44. gramiccia m. & gradoni l. 2005. the current status of zoonotic leishmaniases and approaches to disease control. int j parasitol, 35, 1169‑1180. lamothe j. & poujade a. 2002. ulcerative glossitis in a dog with leishmaniasis. vet rec, 151, 182‑183. longstaffe j.a. &guy m.w. 1985. leishmaniasis in dogs. vet ann, 25, 358‑367. mettler m., grimm f., naucke t.j., maasjost c. & deplazes p. 2005. canine leishmaniosis in central europe: retrospective survey and serological study of imported and travelling dogs. berl munch tierarztl wochenschr, 118, 37‑44. owens s.d., oakley d.a., marryott k., hatchett w., walton r., nolan t.j., newton a., steurer f., schantz p. & giger u. 2001. transmission of visceral leishmaniasis through blood transfusions from infected english foxhounds to anemic dogs. j am vet med assoc, 219, 912‑921. paradies p., capelli g., cafarchia c., de caprariis d., sasanelli m. & otranto d. 2006. incidences of canine leishmaniasis in an endemic area of southern italy. j vet med b, 53, 295‑298. pinna parpaglia m.l., vercelli a., cocco r., zobba r. & manunta m.l. 2007. nodular lesions of the tongue in canine leishmaniosis. j vet med a, 54, 414‑417. solano‑gallego l., koutinas a., miró g., cardoso l., pennisi m.g., ferrer l., bourdeau p., oliva g. & baneth g. 2009. directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. vet parasitol, 165, 1‑18. viegas c., requicha j., albuquerque c., sargo t., machado j., dias i., pires m.a., campino l. & cardoso l. 2012. tongue nodules in canine leihmaniosis‑a case report. parasit vectors, 5, 120. 271 introduction the use of radioactive substances, sealed and unsealed, in hospitals for diagnostics and therapy produces solid, liquid, and gas forms of radioactive waste. in italy, according to the euratom directives, legislative decree 17/03/1995 n. 2301 and its subsequent changes and additions are designed to ensure high levels of protection for public health and the environment. the objectives of the present study were to investigate the radioactive waste generated by nuclear medicine hospitals departments located in abruzzo, particularly the radionuclides used in unsealed form: 131i, 99mtc, 67ga, 201tl, 123i and 111in, according to a previous study by piersanti and colleagues (piersanti et al. 1992a). the radionuclides examined have a t½ (half‑life) istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy * corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861 332456, e-mail: l.sgattone@izs.it. parole chiave contaminazione radioattiva, medicina nucleare, radionuclidi, acque di scarico, spettrometria gamma. riassunto negli ospedali i radionuclidi sono utilizzati sempre di più per la ricerca funzionale, la diagnostica per immagini e nella radioiodio terapia. il loro uso produce scorie radioattive e rischi di contaminazione ambientale. questo studio esamina 486 campioni di scorie radioattive prodotte negli ospedali abruzzesi tra il 2000 e il 2015. le misurazioni sono state effettuate con la tecnica della spettrometria gamma: rivelatore al germanio (ptg) con 8000 canali di acquisizione, gamma di potenza 59‑1836 kev, risoluzione 1 kev, efficienza complessiva 30%, tempo di misurazione 60000 s. i radionuclidi coinvolti sono i seguenti: 131i, 99mtc, 67ga, 201tl, 123i, 111in, attivi principalmente in 44 campioni. i controlli hanno permesso di certificare i livelli di concentrazione radioattiva nelle acque di scarico, pianificare la loro eliminazione e ottimizzare le procedure di gestione. valutazione dell’impatto ambientale delle acque di scarico: l’uso dei radionuclidi negli ospedali abruzzesi, 2000-2015 keywords radioactive contamination, nuclear medicine, radionuclides, waste‑water, gamma spectrometry. summary radionuclides are increasingly used in hospitals for diagnostic and therapeutic purposes, such as functional research, diagnostic imaging, and in the performance of radioiodine therapy. their use produces radioactive waste, and risks environmental contamination. the present study involves 486 samples of radioactive waste produced in hospitals in abruzzo, italy, during 2000‑2015. measurements were carried out with the gamma spectrometry technique: germanium detector (ptg) with 8000 acquisition channels, power range 59‑1836 kev, resolution 1 kev, overall efficiency 30%, measurement time 60000 seconds. the radionuclides involved were as follows: 131i, 99mtc, 67ga, 201tl, 123i, 111in, with substantial activity in 44 samples. checks allowed us to certify the levels of radioactive concentration in waste‑water, plan for their suppression, and optimise the management procedures. enrico gabriele piersanti, luigi sgattone*, luciano di stefano and giacomo migliorati environmental impact assessment of waste-water: radionuclides use in hospitals (abruzzo, italy, 2000-2015) veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1265.7053.1 accepted: 26.09.2017 | available on line: 31.12.2018 1 legislative decree 17/03/1995 n. 230. attuazione delle direttive 89/618/ euratom, 90/641/euratom, 96/29/euratom e 2006/117/euratom in materia di radiazioni ionizzanti. off j,136, 13/06/1995 (s74).. 272 veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1265.7053.1 waste‑water: radionuclides use in hospitals piersanti et al. in diagnostic activity, the main use of radionuclides is mostly linked to medium‑short‑lived radionuclides such as 99mtc (t½ 6  h) and 111in (t½ 2,8  d), which in the case of outpatient therapy involvs the 131i (t½ 8 d) and any other radionuclides. a specific waste management system is implemented in case of radionuclides administered for therapeutic purposes during hospitalisation. it includes ‘imhoff tanks’ for primary sedimentation and tanks for the collection and decay of liquid waste, in order to optimise the process for liquid radioactive waste involving the use of radionuclides with longer average life which the 131i (t1/2 8d) and higher radioactive concentrations. all organic waste from patients, washing liquids, and decontamination coming from radionuclides administered for therapeutic purposes is collected in tanks. it is crucial to evaluate the appropriate size of tank systems in order to ensure the efficiency of hospital structures that are implicated in nuclear medicine in relation to costs, number of patients to be treated and quality of services. it also depends on the concentration and quantity of radioactive substances that have been used. hospitalised patients who are under diagnostic investigations of the nuclear medicine unit, reported that liquid waste and contaminated excretions were released into the hospital sewage system. however, the controlled baths used in nuclear medicine unit lower the amount of radioactive substances released into the sewer system through liquid waste such as urination. this type of waste disposal in the environment, particularly for liquids, is supported within the exemption terms of article 154 c. 2 (legislative decree 17/03/1995 n. 230). in particular: • stakeholdes radioactive isotopes must have a t½ of less than 75 days; • concentration must be less than 1 bq/g (ref. art.  1 and annex 1 to legislative decree. 230/95) for a single radionuclide and the mixture of several radionuclides; • disposal must occur in accordance with decree. n. 22 of 5 february 1997. methods and procedures relating to disposals must be recorded and forwarded to the supervisory bodies. there is therefore a need to both verify and certify the radioactive concentration of the materials found in the tanks of liquid waste and septic tanks before leaving any waste in the environment, as well as a need to periodically review the radioactive concentration in the hospital sewer system derived from patients using services outside of controlled environments. less than 75 days and are categorised as ‘waste with other hazardous characteristics – short‑lived radionuclides’ in accordance with art. 154, legislative decree n. 230 of 17/03/1995, in which there is no reference to sealed radioactive sources. these sealed radioactive sources are used in cell sterilising processes, in calibration sources, and for the calibration of radiation therapy performance as, for example, of 60co sources. the use of these sources does not produce, as a rule, radioactive waste; and the procedures are strictly disposed of through authorised companies. unsealed radioactive substances are used in functional studies and in diagnostic imaging techniques; in liquid form are also employed for in vitro radiommunoassay investigations. radioactive substances in liquid or solid form are administered to patients during the performance of radiometabolic therapy. solid radioactive waste includes: supply tanks for individual radio‑labelled solutions, contaminated material following elution operations, administration, and investigation; contaminated test tubes, syringes, needles, blotting paper, work gloves; and any other potentially contaminated materials due to patients, workers, and environmental applications. among solid wastes potentially contaminated, there are also those originated from radiometabolic therapy. according to operating procedure rules, radioactive waste is managed in special storage tanks, by specialised companies that, in compliance with rules, are required to issue a statement for the withdrawal of materials as well as its final destination. radioactive waste in liquid form is originated by cleaning surfaces of controlled zones, such as controlled sinks, areas of radioactive substances use, zones of decontamination of patients and workers. it can further be found in the urination and excreta of patients, and in controlled baths. liquid waste resulting from radioimmunology investigations is placed in a specific container and managed in the same way as solid waste. normally, the hospital activities listed above do not constitute an appreciable source of radioactive waste in gaseous form, however it is necessary to periodically test air conditioning dilution cells and filters. the nuclear medicine hospital unit, in abruzzo region, are equipped with a radioactive waste management system that allows potentially contaminated liquids to flow into septic tanks, so‑called ‘imhoff tanks’ for primary sedimentation, and in other tanks to collect liquid radioactive waste. local and remote control checks allow to verify the management systems and monitor the levels of each tank. 273veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1265.7053.1 piersanti et al. waste‑water: radionuclides use in hospitals the following gamma‑emitting radionuclides: 241am, 113sn, 109cd, 85sr, 57co, 137cs, 139ce, 60co, 203hg, 88y, with calibration energy from 59.5 to 1,836 kev. the following software were used for the evaluation of spectra: up until 2006, range 2000 (silena international s.p.a., cernusco sul naviglio, italy), and since 2007, gammavision 32 (ortec®). tests for the detection of gamma‑emitting radionuclides were adopted in accordance with iso/ iec 17025 . check results were sent to requiring hospitals so that they could organise the unloading of liquid waste tanks and septic tanks in accordance with local regulations. in the laboratory, samples were managed in compliance with the standards for protection, safety, and working instructions on radioactive waste management, and according to the accreditation process. samples detecting an exceeding activity beyond the limits of the legislative decree 17/03/1995 n. 230, have been stored for physical decay. results between 2000‑2015, 486 waste‑water samples were analysed. the significant increase in analysis during 2009‑2015, reflects the period in which the nuclear medicine unit activity increased significantly (figure 1). table i reports the distribution of single radionuclide and findings for the distribution of activity, for each sample, of any implicated radionuclide. forty‑four samples contained values that require attention, as the sum of the concentrations of the radionuclides that were analysed was close to the critical value. we detected radioactive concentration values between 1,000 and 10,000 bq/l in 18 samples; in two samples found in 2015, values of between 10,000‑50,000 bq/l the aim of the paper is the environmental impact of waste‑water in abruzzo hospitals. materials and methods this study involved 486 waste‑water samples conferred by hospitals to istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ (izsam) during 2000‑2015, in the framework of an hospital self‑monitor on the concentration of radioactivity in the tanks, and before they were disposed in the outer sewer system. samples were received in the laboratory accompanied by a cover sheet with the following information: applicant’s name, date, amount of sample taken, type of material, mode of transport (adr code un 2910), packaging of the sample, and specifications of the radionuclides to be analysed. the removal of effluent materials was carried out by the hospitals, directly from storage tanks and storage and the sewage outlets, and before they were placed into the public sewer. the izsam lab, which was responsible for carrying out the analysis of bromatology and residues in foods for human and animal consumption, is part of the resorad network for radiation monitoring and participates in reliability programmes organised by the italian national institute for environmental protection and research (ispra) (calvarese et  al. 1997, calvarese et al. 2009, de crescenzo 2012, enea 1991, piersanti et al. 1992). this study covered concentrations of following radionuclides that were used by hospitals: 131i, 99mtc, 67ga, 201tl, 123i, 111in. one‑litre samples were placed in a marinelli beaker to proceed to the gamma spectrometry in order to determine the activity of the involved radionuclides. the shielding of measuring wells was 10 cm of lead. samples were measured for 60,000 seconds. until 2006, measurements were carried out with a germanium detector (pgt) with an overall efficiency of 25% with 4,000 acquisition channels. since 2007, high purity germanium detectors (hpge) (ortec®, south illinois, usa) with a total efficiency of 30% and 8,000 acquisition channels have been used. the detection energy range was between 59 to 1,836  kev. emission lines were identified with a resolution of 1 kev. instrument calibrations were performed with aqueous sources in metrological centres certified by the national agency for new technologies, energy and sustainable economic development (enea) until 2006, and subsequently by deutscher kalibrierdienst (dkd). to calibrate sources it was used a mixture containing 0 10 20 30 40 50 60 70 80 20 00 20 01 20 02 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 20 12 20 13 20 14 20 15 n ° sa m p le s an al ys ed year figure 1. number of waste‑water samples analysed by izsam. 274 veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1265.7053.1 waste‑water: radionuclides use in hospitals piersanti et al. carried out in two large public hospitals in granada (spain) with gamma spectrometry investigations. barquero (barquero et  al. 2008) carried out a study on the evaluation of exposure for‘worker[s] assigned to the treatment of waste water’ in a hospital that utilises radioactive substances in nuclear medicine. the results of this study demonstrate that the control of the radioactive concentrations of potentially contaminated liquid waste from hospitals is crucial. the checks that we carried out have allowed us to certify the levels of radioactive concentration in the effluent resulting from the storage tanks from the hospital and, going forward, to plan for the elimination of waste, in accordance with local regulations and limits. in several cases, significant activity was detected. this prompted new evaluations, including a methodological review of procedures in order to improve internal performance of the hospitals and environmental safety. since 2000, izsam has confirmed the following needs: • a comprehensive system for radioactive waste (liquid) management with storage tanks and decay; • a specific assessments not only limited to theoretical calculations, but based on measurements made with dedicated instrumentation and procedural protocols. radioactive waste concentration control in hospitals promotes the management of risk for workers, populations, and the ecosystem in general. for 131i were detected, and in a sample dated 2013, a value of 70,184 bq/l always for 131i was detected. discussion and conclusions sundell‑bergman and colleagues (sundell‑bergman et  al. 2008) report on the complexity of radioactive waste management in sweden. the risk of radioactivity spreading through the sewer system and the subsequent use of sludge in agricultural land has been highlighted in this study,. another study of krawczyk and colleagues (krawczyk et  al. 2013) shows as the storage of waste water in tanks to control the concentration of radionucluids prevents the dispersion of radioactivity into the environment. this conclusion descends from studies barquero r., agulla m.m. & ruiz a. 2008. liquid discharges from the use of radionuclides in medicine (diagnosis). j environ radioact, 99, 1535‑1538. calvarese s., sgattone l., zilli r. & candeloro l. 2009. la radioattività nei parchi della regione abruzzo a 20 anni da chernobyl. quaderni di veterinaria preventiva, 1, 51‑61. calvarese s., torreti l., gatti g., campana g., zitti g., piersanti e. & sgattone l. 1997. gli inquinanti ambientali nei mieli prodotti in abruzzo. rapporti istisan 97/17, roma, istituto superiore di sanità. de crescenzo s. 2012. la gestione dei rifiuti radioattivi ospedalieri. ecoscienza, 3, 66‑67. enea. direzione centrale sicurezza nucleare e protezione sanitaria. 1991. banca dati radionuclidi: manuale di riferimento e d'uso. roma, enea. references krawczyk e., pinero‑garcia f. & ferro‑garcia m.a. 2013. discharges of nuclear medicine radioisotopes in spanish hospital. j environ radioact, 116, 93‑98. piersanti e.g. , nannini d., poma c., sgattone l., macrì m.a. & di luzio s. 1992a. control on contaminated liquid wastes from a laboratory of nuclear medicine. in atti vii congresso a.i.f.b., ancona 8‑12 giugno 1992. piersanti e.g. , nannini d., poma c., sgattone l., macrì m.a. & di luzio s. 1992b. study on contamination by 137cs and 134cs of honey formulas. in atti vii congresso a.i.f.b., ancona 8‑12 giugno 1992. sundell‑bergman s., de la cruz i., villa r. & hasselblad s. 2008. a new approach to assessement and management of the impact from medical liquid radioactive waste. j environ radioact, 99, 1572‑1577. table i. number of samples analysed by izsam. activity range n. of analysed samples ≥ bq/l < bq/l activity in the range for single radionuclide for total radionuclides 131 i 99mtc 111in 67ga 123i 201tl 0 1 331 377 425 70 51 338 3 1 5 43 55 17 386 67 118 17 5 10 15 27 4 21 243 13 154 10 50 51 12 14 8 120 12 214 50 100 17 3 8 1 4 4 33 100 1,000 17 8 13 0 1 1 44 1,000 10,000 9 4 5 0 0 0 18 10,000 50,000 2 0 0 0 0 0 2 50,000 100,000 1 0 0 0 0 0 1 369 1department of veterinary medicine and animal productions, university of naples ‘federico ii’, naples, italy. 2university of salerno, department of pharmacia, via giovanni paolo ii, fisciano (sa), italy. 3azienda sanitaria locale abruzzo, l’aquila, italy. 4istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. *corresponding author at: department of veterinary medicine and animal productions, university of naples ‘federico ii’, via delpino 1, 80137 naples, italy. tel.: +39 081 2536005, e-mail: lcortese@unina.it. parole chiave citisina, ginestra odorosa, pecore, piante tossiche. riassunto nell’inverno del 2015 è stato segnalato un sospetto di malattia infettiva con prevalente interessamento del sistema nervoso centrale in un allevamento ovi-caprino sito nel comune di civitella roveto (aq). tutti gli animali hanno mostrato convulsioni tonico-cloniche seguite da paralisi muscolare associata a pupille dilatate, tremore, tachicardia e tachipnea e in alcuni casi anche diarrea. la presenza di fascine secche di ginestra odorosa spartium junceum l. con alte concentrazioni di citisina e metilcitisina e l’ingestione di tali piante da parte degli animali facevano ipotizzare un avvelenamento da tale pianta. l'esame anatomo-patologico, sierologico, batteriologico e virologico hanno escluso qualsiasi causa di malattia infettiva. l'esposizione degli animali a sostanze tossiche utilizzate in agricoltura o altre piante tossiche è stata esclusa dall'esame clinico, dai reperti anatomopatologici e dall'anamnesi ambientale. dopo la rimozione delle fascine e il cambiamento della dieta è stata osservata una remissione completa dei segni clinici in tutto il gregge. lo studio fitochimico ha mostrato che tra gli alcaloidi isolati era presente, in quantità maggiori in tutte le parti della pianta consumata dagli animali, la citosina, agonista del recettore dell'acetilcolina nicotinica. i risultati clinici e patologici associati alla remissione completa dei segni clinici dopo l'esclusione della fascina secca dalla dieta, nonché i risultati dell'analisi fitochimica hanno portato a fare una diagnosi finale di intossicazione da s. junceum l. avvelenamento da spartium junceum l. in piccoli ruminanti keywords cytisine, sheep, spartium junceum l., toxic plant. summary an outbreak of neurological disorders in a flock of 20 sheep coming from a rural farm in civitella roveto, italy, occurred in winter 2015. all the animals showed tonic-clonic convulsions followed by muscle paralysis associated with dilated pupils, tremor, tachycardia, tachypnea and diarrhea. the presence of bundles of dry broom of spartium junceum l. in the feed, eaten by the animals supported the hypothesis of plant intoxication. two animals died after worsening of clinical signs. the anatomopathological findings and the laboratory results ruled out viral or bacterial infections or accidental exposure to other toxics. phytochemical study showed the presence of large amount of cytisine, a nicotinic acetylcholine receptor agonist, in all parts of the plant eaten by the animals. clinical and pathological findings, the complete remission of clinical signs after the exclusion of dry broom from the diet, together with the results of phytochemical analyses results corroborated the hypothesis of s. junceum l. intoxication. andrea ariano1, alessandro costagliola1, massimiliano d'ambola1, 2, laura cortese1*, luigi pietrobattista3, gabriella di francesco4, stefania salucci4, valentina iovane1, orlando paciello1 and lorella severino1 spartium junceum l. poisoning in small ruminants veterinaria italiana 2019, 55 (4), 369-373. doi: 10.12834/vetit.1696.8973.2 accepted: 07.06.2019 | available on line: 31.12.2019 short communication 370 spartium junceum l. intoxication in sheep ariano et al. veterinaria italiana 2019, 55 (4), 369-373. doi: 10.12834/vetit.1696.8973.2 coprological analysis was also performed collecting feces directly from the rectum of each sheep; samples were then processed by mcmaster method. blood samples from twelve animals were collected by jugular vein for haemato-biochemical investigations. hematological parameters were performed using a semi-automatic blood cell counter (genius s, seac radom group) and included total white blood cell (wbc), neutrophils (n), lymphocytes (l), monocyte (m), eosinophil (e), basophil (b), red blood cells (rbc), hemoglobin (hb), mean corpuscular volume (mcv), hematocrit (hct ), mean corpuscular hemoglobin (mch), corpuscular hemoglobin concentration (mchc), platelet count (plt ) and mean platelet volume (mpv). regarding biochemical examination, a semi-automatic chemical chemistry analyzer (olot, spinreact) was used to analyze concentrations or activities of glucose, blood urea nitrogen (bun), creatinine, triglycerides, total cholesterol, alanine aminotransferase (alt ), aspartate aminotransferase (ast ), alkaline phosphate (alp), total bilirubin (t-bil), gamma-glutamyl transferase (gamma-gt ), amylase, serum calcium, uric acid, albumin and total serum proteins (tp). serum samples were also tested for the presence of maedi-visna antibodies by agar gel immunodiffusion test (agid). at a later stage, the flock feeding behaviour was investigated and more information about clinical history and management of the flock was obtained. the owner reported that because of restricted economic conditions of the farm, in winter the diet of the animals was extremely poor and based almost exclusively on hay; moreover, the farmer reported that for about several weeks the animals used to eat parts of bundles of dry broom subsequently identified as spartium junceum  l., stored within the same paddock of the farm. specimens of the suspect plant including stem, leaves and flowers were submitted to the laboratory at the university of salerno, department of pharmacy, for evidence of toxic compounds; in the meantime, the suspected food was removed from the farm and animals were fed only with hay and generous quantity of water. after collection, s. junceum materials (stem and bark) were analyzed. the samples, after a drying period in spartium junceum  l., commonly known as spanish broom, is a species of flowering plant in the family leguminosae (sub-family faboideae, papilionoideae) (pignatti 1982). all parts of s.  junceum  l. contain cytisine, an alkaloid reported as a nicotinic acetylcholine receptor agonist (greinwald et  al. 1990). in humans, poisoning occurs after ingestion of seed, flower or other parts of the plant. clinical signs appear within an hour of ingestion and include mild irritation of the mouth and throat, hypersalivation, diarrhea and vomit. early signs of neurotoxicity include dilated pupils, headache, delirium, mental confusion; severe cases show tremor, tonic-clonic convulsions followed by muscle paralysis, and coma. death may occur through respiratory failure because of central nervous system depression and muscle paralysis (riccardi et  al. 2006). to our knowledge no cases of poisoning from s. junceum l. have been reported in small and large ruminants. an outbreak of neurological disorders in a flock of 20  sheep coming from a rural farm in civitella roveto (aquila), italy, occurred in winter 2015 (table i). the main clinical signs observed were: dilated pupils, tachycardia, tachypnea and tremor. the animals began to stagger showing moderate to severe ataxia with unsteady posture, frequent falling pedalling and opisthotonos. some of them presented also diarrhea. the clinical signs persisted for several days and the ram and lambs were the worst affected. after few days one lamb and the ram died. because of clinical symptoms observed, the farm veterinarian suspected a possible involvement of infectious agents and notified the case to the public health department. field necropsies and laboratory analysis were carried out to ascertain the cause of death of animals and to exclude the presence of any infectious agents hazardous to both animals and humans. samples of brain, lung, kidneys and liver were collected, immersed in 10% buffered formalin (ph 7.4) and sent to diagnostic laboratory of university of naples ‘federico ii’, department of veterinary medicine and animal productions for histopathology evaluation. other organs such as intestine and stomach were in an advanced stage of putrefaction and were not eligible for histopathology. table i. clinical history of the flock after ingestion of spartium junceum l. n° of animals species age sex neurological signs duration of the illness outcome 17 sheep > 2 years female ++ 1 month complete remission of clinical signs after removal of bundles of brooms 1 sheep > 2 years male ++ 1 month death 1 sheep 3 weeks male +++ 1 month death 1 sheep 12 weeks male +++ 1 month complete remission of clinical signs after removal of bundles of brooms 371 ariano et al. spartium junceum l. intoxication in sheep veterinaria italiana 2019, 55 (4), 369-373. doi: 10.12834/vetit.1696.8973.2 membranes and poor nutritional status; there was diffuse moderate to severe bilateral congestion in pulmonary lobes with white foam fulfilling the lumen of the distal trachea, major bronchi, and intrapulmonary airways. liver was slightly enlarged and congested; petechial subdural hemorrhages were also found. no macroscopically reliable lesions were detected in other organs, including kidneys. microscopically, multifocal to coalescing areas of abundant eosinophilic cellular debris along with large numbers of macrophages and neutrophils, fibrin and edema (figure 1a) and occasional hemorrhages were observed in lungs. many alveolar septa were expanded from 2 to 4 times the normal width by similar inflammatory components. a similar exudate filled bronchial and bronchiolar lumina. the interlobular, perivascular, and subpleural interstitia were expanded up to 2 to 3 times normal by abundant fibrin deposition, occasional edema, and few neutrophils, macrophages, lymphocytes, plasma cells, and erythrocytes. there were multifocal areas of closely apposed alveoli (atelectasis) or expanded and coalescing alveolar spaces (emphysema). in the kidneys, the interstitium was mildly expanded by edema and congested blood vessels. in the brain, within the gray matter of cortex, there were multifocal areas of hemorrhages with many erytrophagocytic macrophages, severe congestion of blood vessels in cerebrum, cerebellum and mild gliosis (figure 1b). in the liver, moderate congestion of the liver parenchyma and centrilobular (zone iii) sinusoidal dilatation were evident. no other microscopically lesions were detected in other organs. phytochemical study identified five different alkaloids in dried aerial part of s.  junceum  l. at the following concentrations expressed as µg/g dry the shade, were ground into a powder, kept at 10 °c and subjected to extraction within 12  h. according to literature (barboni et  al. 1994), 2  g dried, finely pulverized samples were added with 25 ml 1n h 2 so 4 , stirred or shaked 15 min at room temperature and then filtered. this procedure was repeated twice. the combined solutions were made alkaline with nh 4 oh 20% aqueous solution and extracted three times with 30 ml chci 3 . the chci 3 extracts was evaporated to dryness under vacuum at room temperature using rotary evaporator. alkaloid extracts were analyzed by gas chromatography-mass spectrometry (gc-ms) using a fused-silica capillary column (me silicone, 20 cm x 0.2 mm, he carrier gas, column temperature 100 °c, 2 min isothermal, 10 °c min-1 to 300 °c, 5 min isothermal, split ratio 1:30; injector 250  °c) and a mass selective detector (detector temp. 280  °c, electron energy 70 ev). alkaloids were identified by comparison with ms data in the literature (ohmiya et  al. 1974, wink et  al. 1983, wink 1987, greinwald et al. 1990). all animals showed a light parasitic infestation from gastrointestinal strongyles (maximum level, 100 epg) and coccidian (maximum level, 50 opg) (reeg et  al. 2005, chandrawathani et  al. 2009). haematological and biochemical parameters showed only an increase of ast (iu/l: 109.75 ± 11.01; normal range: 32-97 iu/l) and ggt (iu/l: 53.76 ± 3.75; normal range: 0-32 iu/l) levels, suggesting a mild hepatic damage (aitken 2007). all sera tested by agid test were negative for maedi-visna virus infection. anatomo-pathological investigations performed on the ram and lamb showed abdominal distension, pale mucous figure 1. a. lamb. lung. eosinophilic amorphous material (*) fulfilling alveolar and bronchiolar spaces (severe diffuse pulmonary edema). h&e stain; 200x magnification. b. lamb. brain. moderate congestion of a blood vessel (arrow head) and mild gliosis (arrow). h&e stain; 400x magnification. 372 spartium junceum l. intoxication in sheep ariano et al. veterinaria italiana 2019, 55 (4), 369-373. doi: 10.12834/vetit.1696.8973.2 a toxic component found within the genera of the fabaceae and other plant families (wink et al. 1983). cytisine is a nicotinic acetylcholine receptor agonist and in humans has been documented to cause drowsiness, vomiting, vertigo, tachypnea, cold sweats, gastric pain, pupillary dilatation, agitation, and muscle contractions (riccardi et al. 2006). literature data reported that the alkaloid pattern of s. junceum l. is strongly dependent on the vegetative cycle of plant; cytisine and n-methycitisine are the main alkaloids present in the aerial part of the plant whereas lupanin, anagyrine and rhombifoline are present in much lower concentrations (greinwald et  al. 1990, barboni et  al. 1994). phytochemical study performed on dried aerial part of s. junceum l. revealed the presence of cytisine in relevant higher quantity compared to other alkaloids isolated in the same plant. cytisine, lupanine, and rhombifoline concentrations proved to be higher than those found in the study of barboni and colleagues (barboni et  al. 1994) but lower than those found by greinwald and colleagues (greinwald et  al. 1990). the concentrations of n-methylcitisine and anagyrine found in this study were higher and lower, respectively, than those reported in other similar studies (greinwald et al. 1990, barboni et al. 1994). the reported case demonstrates a strong causal association between clinical signs and ingestion of s. junceum l. in areas where s. junceum l. is abundant, the effect of cytisine poisoning in sheep should be considered to either prevent possible outbreaks or facilitate an early diagnosis. weight: cytisine (2,800.7 µg/g), n-methylcytisine (1,583.6 µg/g), lupanine (197.2 µg/g), anagyrine (380.7 µg/g), rhombifoline (73.5 µg/g). severe generalized weakness and clinical signs presented in animals, were consistent with well known, described nicotinic acetylcholine agonist toxicity, as exerted by some alkaloids. in small ruminants, the same clinical signs may be associated with some infectious diseases (i.e. maedi visna) or poisoning (russo et  al. 2018). anyway, laboratory examinations did not point out any viral, bacterial or prionic disease. the animal’s exposure to chemicals used in agriculture (i.e. pesticides) and other toxic substances was excluded on the basis of the environmental anamnesis. furthermore, the anatomopathological lesions described and the remission of clinical signs as a result of exclusion of s.  junceum  l. from diet, as well as the phytochemical analysis results, strongly corroborate the diagnosis of alkaloid poisoning. there is very little documentation of s.  junceum  l. intoxication in human and animals and no cases are reported in literature in small ruminants. ruminants have dietary preferences and usually exclude potentially poisonous plants. however, in some circumstances such as drought, droving or lack of forage, livestock are forced to eat, in a greater or lesser amount, toxic plants that they would otherwise avoid (simmonds et al. 2000). the toxicity seen in s.  junceum  l. is due to cytisine, an alkaloid with a bitter and unpleasant taste and 373 ariano et al. spartium junceum l. intoxication in sheep veterinaria italiana 2019, 55 (4), 369-373. doi: 10.12834/vetit.1696.8973.2 aitken i.d. 2007. appendix b clinical chemistry and reference values. in diseases of sheep. 4th edition edited by i.d. aitken obe, bvms, phd, cbiol, fbiol, dvm&s h.c. (edinburgh), blackwell publishing, p. 602. barboni l., manzi a., bellomaria b. & quinto a.m. 1994. alkaloid content in four spartium junceum populations as a defensive strategy against predators. phytochemistry, 37, 1197-1200. chandrawathani p., nurulaini r., adnan m., premalaatha b., khadijah s., jamnah o., zaini c.m., khor s.k. & zawida z. 2009. a survey of parasitic infection on small ruminant farms in kinta and hilir perak districts, perak, malaysia. trop biomed, 26 (1), 11-15. greinwald r., lurz g., witte l. & czygan f.c. 1990. a survey of alkaloids in spartium junceum l. (genisteae-fabaceae). z naturforschung c, 45 (11-12), 1085-1089. ohmiya s., otomasu h., murakoshi i. & haginiwa j. 1974. n-formylcytisine: a new alkaloid from thermopsischinensis. phytochemistry, 13, 643-644. pignatti s. 1982. flora d’italia, vol. 3. edagricole, bologna. reeg k.j., gauly m., bauer c., mertens c., erhardt g. & zahner references h. 2005. coccidial infections in housed lambs: oocyst excretion, antibody levels and genetic influences on the infection. vet parasitol, 127 (3), 209-219. riccardi a., frumento f., ghinatti m., guiddo g. & lerza r. 2006. spanish broom flower ingestion: a very unusual poisoning. euro j emerg med, 13 (5), 317-318. russo r., restucci b., vassallo a., cortese l., d’ambola m., montagnaro s., ciarcia r., florio s., de tommasi n. & severino l. 2018. toxicity of crepis lacera in grazing ruminants. bmc vet res, 14, 74. simmonds h., bourke c. & holst p. 2000. the palatability and potential toxicity of australian weeds to goats. rural industries research and development corporation. wink m., witte l., hartmann t., theuring c. & volz v. 1983. accumulation of quinolizidine alkaloids in plants and cell suspension cultures: genera lupinus, cytisus, baptisia, genista, laburnum, and sophora. planta med, 48, 253-257. wink m. 1987. quinolizidine alkaloids: biochemistry, metabolism, and function in plants and cell suspension cultures. planta med, 53, 509-514. 251 introduction fishes are considered as one of the most important sources of animal protein all over the world. because of numerous lakes, seas and a long river, egypt has a very diversified fauna of fresh and marine water fishes. vision either in wild or farmed fishes is quite different from mammalians as it has to be adapted to acquatic environment (lee 2002). the awareness of parasites that affect fish health, growth, and survival is increasing together with a developing interest for the fish farming and production. knowing fish parasites becomes important for a rapid and correct diagnosis. early diagnosis is a prerequisite for implementing preventive measures, which are the best way to reduce infection spread (abdel ghaffar et al. 2012, morsy et al. 2012). myxosporidea have a great importance in ichtyopathology. they are frequently described in freshwater, brackish and marine fishes. myxosporean parasites are the most important fish pathogens and more than 2,300 species have been reported from marine and freshwater fishes in several global areas (manrique et  al. 2015, manrique et  al. 2016). myxosporea infecting fishes are a group of parasites responsible for myxosporidiosis, a serious disease of fishes (adriano et  al. 2009, morsy 2010). myxobolus butschli, 1882, is one of the largest genus of myxosporean groups with approximately 856 species. species of myxobolus infect fishes from all over the world (eiras et  al. 2014). they can infect a diverse set of specific tissues including specifically the tegument, eyes, gills, glands, gonads, kidneys, muscle, digestive tract, and nervous system (lorna and dykova 1992). the myxobolus dermatobius (ishii, 1915) (syn. m.  dermatobia) in nile tilapia (oreochromis niloticus) causes petechial to focal haemorrhages in orbit, exophthalmia and unilateral eye opacity especially in advanced cases (abdel‑aal 2002) while m. dermatobia, isolated from tilapia zillii at giza province, causes unilateral eye opacity (mohamed et al. 2004). the ultrastructural morphology of myxosporean species has been widely studied (lorn and dykova 1992). however, in egypt, few species only have been ultrastructurally described. in this study the ultrastructural morphology of m.  dermatobius detected in nile tilapia is described. department of parasitology, faculty of veterinary medicine, zagazig university, egypt. *corresponding author at: department of parasitology, faculty of veterinary medicine, zagazig university, egypt. e‑mail: nosseur@gmail.com. keywords myxosporidae, ultrastructure, myxozoa, nile tilapia (oreochromis niloticus), transmission electron microscopy. summary a total of 1,000 cultured nile tilapia (oreochromis niloticus) were collected from different governmental and private fish farms and examined for detection of myxosporean parasites infection. the infected fishes showed slight unilateral exophthalmia with whitish cyst in the eye. numerous white cysts like plasmodia of myxobolus dermatobius were recovered from the eye of the examined fishes with low prevalence rate (1%). small intact cyst was isolated, fixed in 3% glutaraldehyde in 0.1 m sodium cacodylate (ph 7.4) and prepared for transmission electron microscopy examination. ultrathin sections myxospores of m. dermatobius revealed pair of capsulogenic cells at the apical pole of the developing myxospore. single sporoplasm containing a single nucleus and sporoplasmosomes fills nearly all the space beneath the polar capsules. the later were pyriform in shape, each one had homogenous dense core and 4 turns of polar filaments. ultrastructural characteristics of the present myxospore were described and discussed in detail. nosseur m. el‑sayed* ultrastructural morphology of the myxobolus dermatobius ishii 1915 (mixosporea: myxobolideae) microspores infecting eyes of nile tilapia (oreochromis niloticus) in egypt veterinaria italiana 2020, 56 (4), 251‑255. doi: 10.12834/vetit.1151.6322.3 accepted: 20.11.2016 | available on line: 31.12.2020 252 veterinaria italiana 2020, 56 (4), 251‑255. doi: 10.12834/vetit.1151.6322.3 ultrastructural morphology of myxobolus dermatobius microspores el‑sayed results the prevalence of infection with m. dermatobius was 1% (10 out of 1,000). it was found in the form of whitish cyst in the eye of nile tilapia. slight exophthalmia was observed in the infected eye. the myxospores were recovered from their original plasmodia found in the eye of nile tilapia (figure 1). each myxospore contains a pair of capsulogenic cells, two peripherally arranged valvogenic cells and one sporoplasm cell. capsulogenic cells are found at the apical pole of the developing spore and, together with the sporoplasm, forms a central core that is ensheathed by valvogenic cells. these cells give rise to the two shell valves surrounding each spore and the sutural ridge joining the valves. the differentiation of the capsulogenic cells starts with appearance of a club‑shaped structure ‘capsular primordium’ (figures 2 and 3). sporoplasm fills nearly all the space beneath the polar capsules. it contains a nucleus, small vesicles materials and methods a thousand of live nile tilapia were collected from different governmental and private fish farms in sharkia governorate, egypt. the collected fishes were transported to the laboratory and dissected. the different organs were examined macroscopically and microscopically for detection of any visible myxosporean cysts. several small intact cysts with minimum surrounding tissue isolated from ten positive fish samples were fixed in 3% glutaraldehyde in 0.1 m sodium cacodylate (ph 7.4) for at least 24 hours, washed in the same buffer and post‑fixed with 2% oso 4 in the same buffer. the specimens were dehydrated in series of graded ethanol, transferred to propylene oxide and, finally, embedded in araldite. ultrathin sections were stained with uranyle acetate and lead citrate and observed in a philips (208) te moperated at 80‑100 kv (vital et al. 2003). figure 1. specimen of nile tilapia (oreochromis niloticus), showing white plasmodia and exophthalmia (arrows) in the eye due to myxobolus dermatobius infection. figure 2. transmission electron microscopy of m. dermatobius premature myxospore parasite from the eye of oreochromis niloticus showing two capsulogenic cells (cc) containing capsular primordium (cp), sporoplasm (sp), valvogenic cell (vc), and suture valve (arrow). scale bar = 300 nm. figure 3. transmission electron microscopy of m. dermatobius nearly mature myxospore parasite from the eye of oreochromis niloticus showing two capsulogenic cells (cc), primordia of polar filaments (arrow head), sporoplasm (sp) with sporoplasmosomes (arrow) and glycogen body (g). scale bar = 300 nm. figure 4. transmission electron microscopy of the transverse section of m. dermatobius mature, parasite from the eye of oreochromis niloticus, showing two polar capsules (pc), sporoplasm (sp) containing sporoplasmosomes (arrows), one nucleus (n), polar filaments and small vesicles (arrow head). scale bar = 300 nm. 253veterinaria italiana 2020, 56 (4), 251‑255. doi: 10.12834/vetit.1151.6322.3 el‑sayed ultrastructural morphology of myxobolus dermatobius microspores el‑wafa (abu el‑wafa 1988) identified m. spheroidalis and m. ocularis from eye. mazen (mazen 1994) described m. heterosporus from eye and gills. abdel‑ghaffar and colleagues (abdel‑ghaffar et  al. 1995) described myxobolus sp. from the inner wall of cornea, the base of the gill arch, and roof of the mouth. hegazy (hegazy 1999) isolated m.  cornealis from the eye while, abd el‑aal (abd el‑aal 2002) and el‑mansy (el‑mansy 2005) described m. dermatobia and m. heterosporus from eye and cornea, respectively. m. dermatobia was isolated from eye of tilapia zilli at giza province (mohamed et al. 2004). most of the egyptian myxobolus species morphological descriptions have been mainly based on light microscopy and diagrammatic drawings and just few ultrastructural descriptions are available. in accordance with abd el‑aal (abd el‑aal 2002) and abdel‑ghaffar (abdel‑ghaffar et al. 2005), the ultrastructural characteristics of m.  dermatobius revealed that the spore developed from five cells. the capsulogenic cell of the present species showed capsular primordial to that described in m. stomum and myxobolus sp. by ali and colleagues (ali et  al. 2003) and abdel‑ghaffar and colleagues (abdel‑ghaffar et  al. 2005), respectively. the valvogenic cells gave rise to shell valve surrounding each spore and sutural ridge joining the valves were similar to m. dermatobia described by abd el‑aal (abd el‑aal 2002). the sporoplasm of the investigated species was composed of single mono‑nucleated cell as in m.  dermatobia described by abd el‑aal (abd el‑aal 2002) but different from bi‑nucleated sporoplasm observed in other myxobolus species (ali et  al. 2003, casal et  al. 1996, abdel‑ghaffar et  al. 1994, abdel‑ghaffar et  al. 2005). the sporoplasmosomes of the present species complied with a similar dense body found in m.  cotti reported by ei‑matbouli and colleagues (ei‑matbouli et  al. and, sometimes, exhibits dense matrices known as sporoplasmosomes. a small area of sporoplasm is occupied by a glycogen body (figure 4). two polar capsules are pyriform in shape, equal size, located side by side at the same level and occupy approximately half of the total spore length. each polar capsule has a homogenous core of medium electron‑density containing polar filaments, surrounded by an electron‑lucent layer and an outer layer of medium density (figure 5). four turns of polar filament coils are probably in each capsule. the apical portion of each mature polar capsule is plugged by a cap‑like cover (figure 6). discussion myxobolus bütschli, 1882, contains over 450 of the 1,700 species described within phylum myxospora (myxozoa). these parasites primarily infect fishes, but a small number of species have been found parasitizing amphibians and reptiles (lom and dyková 1992). the infected fishes showed slight unilateral exophthalmia with whitish cyst in the eye. similar lesion was observed in the eye of tilapia nilotica by abd el‑aal (abd el‑aal 2002) and mohamed and colleagues (mohamed et  al. 2004). numerous species of myxobolus have been reported among different african tilapia species; myxobolus agolus, m. brachysporus, m. clarii, m. cichlidarum, m.  heterosporus, m. tilapiae and m. camerounensis and m. kainjiae appeared to be common in gills, fins, eyes and teguments of oreochromis niloticus (ousman et al. 2007). in egypt, several species of myxobolus were isolated from o. niloticus. faisal and shalaby (faisal and shalaby 1987) identified m. nilei (syn. myxosoma tilapiae) from eyes, skin, gills, kidney, spleen and pancreas while, abed (abed 1987) described m.  heterosporus from eye, muscle and kidney. abu figure 5. longitudinal section through myxobolus dermatobius well developed polar capsule showing an electron-dense outer layer (arrow); a central translucent layer (lu) and inner dense core (c) with polar filament coils (pf). scale bar =500 nm. figure 6. transmission electron microscopy of longitudinal section through m. dermatobius parasite from the eye of oreochromis niloticus, showing well developed polar capsule, four turns of polar filament (pft) and apical cap (arrow). scale bar = 500 nm. 254 veterinaria italiana 2020, 56 (4), 251‑255. doi: 10.12834/vetit.1151.6322.3 ultrastructural morphology of myxobolus dermatobius microspores el‑sayed et  al. 2005), m. stomum (ali et  al. 2003), m. cotti (el‑matbouli et  al. 1990) and m. sciades (azevedo et  al. 2010). the homogenous core of each polar capsule that contains four turns of polar filaments was identical to that of m. dermatobia described by abd el‑aal (abd el‑aal 2002). the same number of polar filament coils was reported in m. hetersporus by el mansy (el mansy 2005) while different numbers of polar filament turns were mentioned in m. maculatus (14‑15), in myxobolus sp. (5), in m.  stomum (5‑6), in m. sciades (9‑10), in m. sclerii (4‑5), in m. brachysporus (6‑7) and in m. cuneus (7‑8) (casal et al. 2002, abdel‑ghaffar et al. 2005, ali et al. 2003, azevedo et  al. 2010, kaur and singh 2010, abdel‑baki et al. 2015, manrique et al. 2016). 1990); m.  dermatobia (abdel‑aal 2002), m.  stomum (ali et  al. 2003) and myxobolus  sp. (abdel‑ghaffar et  al. 2005). the glycogen body noticed in the sporoplasm is essential in the myxosporean spore as it provides the energy necessary for further developmental stages in the life cycle. it was similar to that reported in m. dermatobia (abd el‑aal 2002), myxobolus sp. (abdel‑ghaffar et al. 1994) and m. cotti (el‑matbouli et al. 1990). the fully developed polar capsule was surrounded by an electron‑lucent layer and an outer layer of medium density and covered by a cap‑like structure at its apical end. a similar finding was reported in many species of myxobolus as m. dermatobia (abd el‑aal 2002), myxobolus sp. 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freshwater fish piaractus mesopotamicus. parasitol res, 114, 2041‑2044. manrique w.g., figueiredo m.a.p., belo m.a.a., martins m.l. & azevedo c. 2016. ultrastructural description of myxobolus cuneus (myxosporea) in the skeletal muscle and kidney of tropical farmed fish piaractus mesopotamicus (characiformes: characidae). parasitol res, 115, 2505‑2510. 265 short communication veterinaria italiana 2018, 54 (4), xx‑xx. doi: 10.12834/vetit.1344.7401.3 accepted: 19.10.2017 | available on line: 31.12.2018 bluetongue virus (btv) (orbivirus; reoviridae) is an arbovirus which causes bluetongue (bt), a disease affecting wild and domestic ruminants with clinical signs being more prominent in sheep (maclachlan et  al. 2009). after inoculation into the skin of the susceptible host by the bite of some culicoides species (diptera; ceratopogonidae), which act as vectors, btv replicates in the regional draining lymph nodes from where it spreads to numerous organs, causing viraemia (darpel et  al. 2012). in the blood, btv has been demonstrated to be strictly associated with mononuclear cells and, particularly, with erythrocytes (maclachlan et al. 2009). viraemia is considered long lasting, up to 63 days, in the ruminant host (maclachlan 2004, singer et  al. 2001). based on these characteristics, detection of btv genome by real‑time reverse transcriptase pcr (real‑time rt‑pcr) and/or in vitro isolation of the virus from blood are currently the preferred diagnostic tools for confirming infection in the absence of clinical signs. viability and infectivity of virus in whole clinical blood samples are affected by storage conditions, such as temperature and time (wang et  al. 2011), virus species might, however, play an important role. establishing the duration and persistence of viability and virulence of a specific virus in blood samples will be fundamental for understanding its ability to persist under various environmental conditions. parole chiave sangue, bluetongue virus, pecora, temperatura, conservazione. riassunto in questo studio, sangue con virus bluetongue sierotipo 1 (btv‑1), prelevato nel 2006 in sardegna (italia) da una pecora con una forma clinica di bluetongue e conservato a ~ 5 °c per 10 anni è stato inoculato sperimentalmente in un ariete di razza sarda. l’inoculo ha causato nell’ariete manifestazioni cliniche tipiche della bluetongue con esito infausto. l'esame anatomo‑istopatologico ha confermato la presenza di lesioni riferibili a bluetongue, mentre la real‑time reverse transcriptase pcr ha evidenziato la presenza di btv‑1 nel sangue e in molti altri tessuti. i nostri risultati dimostrano che l'isolato btv‑1 2006 ha mantenuto nel sangue per un periodo di 10 anni a ~ 5 °c sia l’infettività che una elevata virulenza. persistenza della virulenza del sierotipo 1 del virus della bluetongue nel sangue di pecora conservato a bassa temperatura per dieci anni keywords blood, bluetongue, sheep, storage. summary this paper reports that bluetongue virus serotype 1 (btv‑1) infected blood collected during the 2006 sardinia (italy) epidemic from a ewe with clinical disease and stored at ~  5  °c for 10 years, caused bluetongue (bt)‑like clinical disease and death when inoculated into a susceptible sarda breed ram. anatomo‑histopathological examination and real‑time reverse transcriptase pcr (real‑time rt‑pcr) confirmed the presence of btv‑1 in several tissues proving that the btv‑1 2006 isolate has maintained its infectivity and virulence. giantonella puggioni1, davide pintus1, giorgio meloni1, rosario scivoli1, angela maria rocchigiani1, daniela manunta1, giovanni savini2, annalisa oggiano1 and ciriaco ligios1* 1 istituto zooprofilattico sperimentale della sardegna, via duca degli abruzzi, 07100 sassari, italy. 2 istituto zooprofilattico sperimentale dell’abruzzo e del molise, oie reference laboratory for bluetongue, campo boario, 64100 teramo, italy. * corresponding author at: istituto zooprofilattico sperimentale della sardegna, via duca degli abruzzi, 07100 sassari, italy. tel. +39 079 2892331, e‑mail: ciriaco.ligios@izs‑sardegna.it. persistence of bluetongue virus serotype 1 virulence in sheep blood refrigerated for 10 years 266 veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1344.7401.3 btv seems to be relatively resistant to lipid solvents, including ether and chloroform, but it is readily inactivated by disinfectants containing acids, alkali, sodium hypochloride and iodophors (howell and verwoerd 1971). during the btv serotype‑1 (btv‑1 it2006 ) epidemic in 2006 in sardinia (italy), samples of whole blood were routinely collected in ethylenediaminetetraacetic acid (edta) from sheep displaying clinical signs of bt. these samples were kept at 5  ±  3  °c and sent to the istituto zooprofilattico sperimentale of sardinia. a representative aliquot of these samples that tested positive to btv‑1 by nested rt‑pcr (shad et al. 1997) expecially when field samples from potentially infected animals need to be transported to remote laboratories for confirmation. virus stability should be undoubtedly taken into consideration in managing reference collections. it has been demonstrated that btv seems to be very stable in blood and tissue samples stored at 20˚c, 4˚c and ‑70˚c but not at ‑20˚c (verwoerd and erasmus 2004) and at high temperatures (howell et  al. 1967). other chemical and physiological characteristics of btv include stability in buffered lactose‑peptone solutions at freeze‑dried status (verwoerd and erasmus 2004) and instability below ph 6.5 as well as after removal of all extraneous protein (owen 1964, svehag et al. 1966). moreover, btv‑1 virulence in sheep refrigerated blood samples puggioni et al. figure 1. btv‑ serotype 1 infected ram. subcutaneous oedema, hyperemia of periorbital areas and nostrils (a); hemorrhagic and necrotic erosions in the dorsal surface of the tongue (b); sub‑intimal hemorrhage in the pulmonary artery (c); immunoreactivity for btv‑ ns2 protein of the endothelial cells of the small capillaries in the lamina propria of the tongue, diaminobenzidine chromogen and mayer hematoxylin counterstain. bar = 100 µm. 267veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1344.7401.3 erosions in the mucosa. peripheral lymph nodes were enlarged, edematous and hemorrhagic. these gross lesions were mirrored by microscopical changes characterized by severe perivasculitis/vasculatis of the small vessels with endothelial hypertrophy, oedema, vessel congestion and hemorrhages in the derma of the skin as well as in the lamina propria of the digestive mucosae. by immunohistochemical staining, btv ns2 protein was detected in the endothelial cells of the small capillaries of the tongue, under the epithelium‑lamina propria junction (figure 1d). real‑time rt‑pcr detected btv rna in several organs (figure 2c). conventional methods of long‑term preservation of viruses usually entail setting up a vial of virus suspension, prepared by specific protocols, and storage at ultra‑temperatures (‑70°c), were stored at 5 ± 3 °c to be used as positive controls in future diagnostic activities. some of samples were stored under these conditions for 10 years. in accordance with the quality control systems at our laboratory, thermometry instrumentation for monitoring the temperature was installed inside the refrigerator. in 2016, the limited experimental data available on this issue and a need to reactivate this virus for use as infectious inoculum in a larger experimental study, prompted us to determine the stability and viability of the btv‑1 it2006 isolate. the btv‑1 it2006 load in the aforementioned field blood samples was evaluated by real‑time rt‑pcr (oie manual 2014) using an ab 7900ht fast system (thermo fisher scientific). quantifications were expressed as tcid 50 /ml equivalents by using a standard curve generated from the amplification of three replicates of rnas, isolated from several (n = 5) 10‑fold dilutions of known viral infectious titer in vero cells (104.43 tcid 50 /ml btv‑1). standard curve efficiency (vaerman et  al. 2004) was 99.9% with r2 = 0.99 (p < 0.0001). rna level in blood pool resulted 103.55 tcid 50 /ml equivalents. may‑grunwald‑giemsa staining smears from these btv‑1 it2006 infected edta blood samples showed that the erythrocytes were still intact with no significant lysis. after quantification, 10 ml of this blood was mixed with a 5 ml solution of 1,000,000 u.i. of penicillin and 300,000 u.i. of streptomycin, and injected intravenously into a susceptible ram free from antibodies against btv. the ram was housed in an insect‑secure facility. clinical examination, and blood sampling were carried out daily, to evaluate the viraemia and antibody response against btv. five days post infection (p.i.), the inoculated ram displayed severe conjunctivitis, nasal discharge, hyperaemia of periorbital areas, anorexia, lameness, prostration, and subcutaneous oedema in the inter‑mandibular space (figure 1a). areas of hemorrhagic‑necrotic erosions were observed on the dorsal mucosal surface of the tongue, which exhibited an evident bluish staining (figure  1b). progressive worsening of clinical status was observed until death 10 days p.i. body temperature and viraemia monitoring are shown in figure  2a‑b. interestingly, the seroneutralization assay was not able to detect neutralizing antibodies in the collected serum samples, thus demonstrating a high virulence of this btv‑1 isolate. at necroscopy, sub‑intimal hemorrhages in the pulmonary artery (figure 1c), foci of necrosis on the papillary muscles of the left ventricle and multifocal hemorrhages in the endocardium were observed. the digestive system, particularly the rumen, showed vascular congestion, hemorrhages and puggioni et al. btv‑1 virulence in sheep refrigerated blood samples 37 37,5 38 38,5 39 39,5 40 40,5 41 0 1 2 3 4 5 6 7 8 9 r ec ta l b o d y te m p er at u re days post-infection 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 8 9 c t v al u e days post-infection 15253545 spleen testis stern skin tongue mediastinal lymph node peripheral lymph node tonsil heart lung liver kidney muscle ct values a b c figure 2. trend of rectal body temperature (a), real‑time rt‑pcr threshold cycle (ct) values for btv‑serotype 1 (btv‑1) in blood (b) and others tissues (c) of a ram intravenously inoculated with btv‑ 1 naturally infected sheep blood stored at 5 ± 3°c for 10 years. 268 btv‑1 virulence in sheep refrigerated blood samples puggioni et al. veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1344.7401.3 extended food and mouth disease virus (fmdv) activity for up to seven weeks (crick et  al. 1966), while on the other hand, viral load of mason‑pfizer monkey virus (m‑pmv), a d‑type retrovirus causing immunodeficiency syndrome and tumors in old world monkeys, significantly decreased after seven days in k 3 edta blood (qin et  al. 2014). in addition, edta reduced dengue virus (denv) liberation from bhk‑21 cells in ca++‑free culture, at contrary, no effect was observed on chikungunya virus (matsumura and yamashita 1978). it can be mention that other orbiviruses, e.g. african horse sickness virus (ahsv), are strictly associated with erythrocytes during viraemia (maclachlan and guthrie 2010) and can maintain their stability and infectivity for one year in washed erythrocytes held at ~  5  °c (house et  al. 1990). similarly, denv, also a rna arbovirus in the family flaviviridae, maintains its replication ability when stored in buffer containing platelets and erythrocytes (sutherland et al. 2016). btv is an arbovirus, hence, resistance in the environment does not seem to be of epidemiological importance. however, culicoides midges are poikilothermic and it is known that environmental temperature can influence replication and virulence of btv inside the insect (mullens et al. 1995, paweska et al. 2002). although we did not determine if there was a reduction in the virus titre over the 10‑year storage and only one sample was tested, we believe that this finding offers the opportunity to conduct retrospective studies on stored tissues and to have an easy, functional and practical way of storing btv infected samples. finally, our result stimulates interest for gaining a better understanding of the biological mechanism by which viruses preserve infectivity for extended periods in tissues and fluids collected from infected hosts. ethics statement the protocol of the slaughtering procedures, involving the ram investigated herein, was officially approved by the service for animal welfare of the izs, according to the guidelines n. i 09 044. the experiment was carried out in agreement with the prescriptions of italian national law (art. 31 decr. leg. 4 march 2014 n. 2; permission: n. 1248/2015‑pr) acknowledgments this work was supported by rc izs sa 01/13 grant from italian ministry of health. cryopreservation in liquid nitrogen and freeze‑dried techniques (gould 1999). we demonstrate that btv collected from an infected sheep does maintain infectivity and virulence in blood sample with edta for 10 years at 5  ±  3  °c. this suggests that there are alternative and easily accessible options for the long‑term storage and transport of btv. our study focused on the use of natural host bioassay as a measure of btv stability in blood samples. in this regard, the clinical, pathological and virological aspects observed correspond with those commonly reported in natural or experimental cases with highly pathogenic btv, which causes acute fatal disease (maclachlan et  al. 2008), thus excluding the possibility that other unknown infectious agents could have been preserved in the blood. among the mammalian viruses, smallpox is believed to be capable of surviving for very long periods of time (wolff and croon 1968, gould 1999). however, no other characterized examples of animal virus long‑term survival with a decade long perspective, in tissue samples or excreta kept at natural circumstances, have been reported. in vitro and in vivo experimental studies have shown the tropism of btv for a number of cell types, including monocytes/macrophages, lymphocytes, dendritic cells and microvascular endothelium (maclachlan et al. 2009). in the viremic phase of the infection, btv is engulfed into the cell membrane invagination of bovine erythrocytes, remaining infectious despite the presence of neutralizing antibodies (brewer and maclachlan 1992, parsonson and mccoll 1995). we speculate that this intimate association between btv and the erythrocyte membrane not only facilitates a prolonged viraemia and consequently the infection of biting vectors, but that it also functions as a mechanism protecting the viability of the virus, even outside of the host organism. however, our infected blood samples were stored in edta vacutainer®, and it cannot be ruled out that this anticoagulant played a role in preserving virus infectivity. indeed, edta enhanced preservation 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croon j.j. 1968. the survival of smallpox virus (variola minor) in natural circumstances. bull world health organ, 38, 492‑493. 13 veterinaria italiana 2021, 57 (1), 13-17. doi: 10.12834/vetit.1346.7413.3 accepted: 18.07.2018 | available on line: 27.07.2021 1university of prishtina “hasan prishtina”, faculty of agriculture and veterinary, kosovo. 2friedrich loeffler institute, riems, germany. 3university of agriculture, faculty of veterinary medicine, tirana, albania. *corresponding author at: university of prishtina “hasan prishtina”, faculty of agriculture and veterinary, kosovo. e‑mail: kurtesh.sherifi@uni‑pr.edu. agim rexhepi1, kerstin wernike2, kristaq berxholi3, nue marku3, jeton spahiu1 and kurtesh sherifi1* keywords schmallenberg virus, simbu serogroup, seroprevalence, kosovo, albania. summary schmallenberg virus (sbv), a novel orthobunyavirus, emerged in european domestic ruminants in 2011 causing abortions and malformations in newborns and none or mild clinical symptoms in adult animals. here, a total of 364 bovine, ovine and caprine serum samples were collected in kosovo and albania between may 2014 and august 2016 and analyzed for the presence of anti-sbv antibodies. sera were tested using an enzyme-linked immunosorbent assay (elisa), and 48 elisa-positive samples were subsequently analyzed by serum neutralization test (snt). the overall percentage of elisa positive results was 17.9%; 23.1% (53/229) was the prevalence observed in kosovo (cattle 45.5%, sheep  19.2% and goat  6.8%), while 8.9% (12/135) was that observed in albania (cattle 11.1%, sheep 0% and goat 20.0%). snt confirmed the presence of neutralizing antibodies against sbv in all samples tested. this is the first study reporting sbv circulation in domestic ruminants in kosovo and albania, with indication that this virus has been present in kosovo and albania at least since 2014 without being detected. first evidence of schmallenberg virus infection in domestic ruminants in kosovo and albania induced by sbv are arthrogryposis, torticollis, ankylosis, kyphosis, scoliosis, brachygnathia inferior, hydrocephalus, and various malformations of the central nervous system, including porencephaly and hydranencephaly (van  den  brom et  al. 2012, garigliany et al. 2012b, bilk et al. 2012, herder et al. 2012). the incidence of malformation on farms where sbv infection was confirmed varies from 2% in goat kids and 0.5%-3% in calves (veldhuis et  al. 2014, dominguez et  al. 2014) to 3%-19% in lambs (harris et  al. 2014, meloni et  al. 2017). sbv is transmitted by hematophagous biting midges (culicoides) (de regge et  al. 2012). the presence of species of culicoides of the obsoletus and pulicaris complexes are reported in kosovo (berisha et  al. 2010). direct horizontal virus transmission in sheep and cattle by contact seems highly unlikely (wernike et  al. 2014); however there is evidence for the presence of sbv in bull semen (ponsart et  al. 2014, schulz et  al. 2014). sbv infections can be detected by molecular methods or by serology (beer et  al. 2013, de regge et  al. 2013, wernike et al. 2016). the aim of this study was to investigate introduction in november 2011, a novel infectious agent named schmallenberg virus (sbv) was reported for the first time in adult dairy cows from farms located in northwest germany and the eastern region of the netherlands (hoffmann et al. 2012). sbv is a member of the genus orthobunyavirus within the family peribunyaviridae. since its emergence, circulation of sbv has been confirmed from continental europe to scandinavia and the british isles. from the initially most affected countries the following seroprevalences were reported: netherlands 72.5% in cattle (elbers et al. 2012), belgium 90.8% in cattle (garigliany et al. 2012a), in france 90% in cattle and 30% in goats (gache et  al. 2014), and in germany 61% in cattle, 24.7% in sheep and 26.4% in goats (wernike et al. 2014). clinical signs of sbv infection include reduced milk yield, inappetence, and diarrhea in adult ruminants lasting for a few days (hoffmann et al. 2012), and multiple malformations in newborns or aborted ruminants when dams are infected during a critical phase of gestation (peperkamp 2015). the main fetal malformations 14 sbv in kosovo and albania rexhepi et al. veterinaria italiana 2021, 57 (1), 13-17. doi: 10.12834/vetit.1346.7413.3 sbv antibody positive samples per municipality and animals species are given in table i. discussion sampled dairy cattle farms with reported abortions tested positive for sbv antibodies, therefore, future the exposure to sbv of cattle, sheep, and goats in kosovo and albania. materials and methods serum samples were collected during the years 2014 (march, may, july, august, september, december), 2015 (february, july, september), and in 2016 (august) and were stored at -  20  °c until use. the animals originated from 102 different ruminant farms (36 cattle, 63 sheep, and 3 goats) located in 16 different municipalities in kosovo and 6 municipalities in albania. serum samples were collected from a total of 364 animals including 154  cattle, 161 sheep and 49 goats. animals were from different breeds of cattle (simmental, holstein, busha and mixed breed), sheep (bardhoka, sharri sheep) and goat (mixed breed), and their age ranged from 1 to 10 years. abortions were reported at least in two dairy cattle farms in kosovo. all sera were tested using a commercially available enzyme-linked immunosorbent assay (id screen® schmallenberg virus competition multispecies, id.vet, montpellier, france) according to the manufacturer’s instructions. as a confirmatory test, 48 elisa-positive sera were additionally analyzed by a serum neutralization test (snt) performed as described previously (wernike et  al. 2013a). all samples were tested in triplicate and the neutralizing titers were calculated as the reciprocal of the serum dilution that still inhibited > 50% of cytopathogenic effect (nd 50 ) according to behrens and kaerber. results antibodies against sbv were detected by elisa in 17.9% (65/364) of the samples collected from domestic ruminants. since the nucleocapsid protein based elisa reacts not only with anti-sbv antibodies, but also with antibodies against closely related viruses from the simbu serogroup (cross-reactivity) (bréard et al. 2013), a subset of 48 samples that scored positive in the elisa was additionally analyzed by the more specific snt. all samples tested positive, the neutralizing titers against sbv ranged from 1/6 to 1/572. the percentage of elisa positive results in samples from kosovo was 23.1% (53 of 229 animals) and 8.9% (12 out of 135) in albania scored positive (8.9%) indicating the recent circulation of sbv in both countries. the highest percentage of seropositive results was found in cattle 36/154 (23.4%), followed by sheep 25/161 (15.5%) and then goats 4/49 (8.2%).the total number of seropositive farms was 40/102 (39.22%) and 14 of the 25 municipalities (56%) were affected, their geographical location is depicted  in  figure  1. the individual numbers of serbia montenegro north macedonia greece adriatic see 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% albania kosovo 20.0% 19.6% 22.2% 22.2% 45.0% 15.0% 10.0% 50.0% 25.0% 42.9% 34.6% 33.3% 12.3% 75.0% figure 1. geographical location of sbv in kosovo and albania. 15 rexhepi et al. sbv in kosovo and albania veterinaria italiana 2021, 57 (1), 13-17. doi: 10.12834/vetit.1346.7413.3 seropositive results has been previously observed in other affected countries as well (meroc et  al. 2013, wernike et al. 2015). it might suggest that the virus was firstly introduced into albania and kosovo in 2014 or earlier and in the following years it has circulated only to a limited extent. the reduced virus circulation in 2015 and 2016 could have led to a decline in herd seroprevalence caused by a missing infection of the young livestock. the differing percentage of seropositive results in the tested animal species, i.e. 23.4 % in cattle, 11.5% in sheep and 8.2% in goats, and their range between the municipalities are consistent with studies performed in other european countries such as the netherlands, belgium, france, germany, turkey or greece in which sbv has already circulated. ranges of 8.0% to 95% have been reported in cattle, 1.6% to 89% in sheep and 2.0% to 50.8% in goats (elbers et  al. 2012, garigliany et  al. 2012a, veldhuis et  al. 2013, azkur et  al. 2013, chaintoutis et  al. 2014, wernike et  al. 2014). this observation and the likewise wide range of percentages of seropositive results in the farms included in the present study could be related to the age of the animals, the landscape (availability of breeding sites of the insect vectors) or to the husbandry system as e.g. grazing has been identified as a risk factor for sbv infections when compared to herds in which animals are kept indoors (veldhuis et  al. 2014). the reasons for the spatio-temporal distribution of sbv seropositivity in albania and kosovo as well as the impact on animal production in these two countries should be evaluated in future studies. conclusions this is the first report on the presence of sbv in kosovo and albania. our results indicate that sbv has been circulating in domestic ruminants in kosovo and albania at least from the year 2014 without being detected. further introductions of vector-borne diseases have to be expected in these two countries, therefore strengthening of the regional surveillance and control strategies for emerging pathogens in animals as a part of global scientific efforts is required. studies should focus on the detection of the virus itself in addition to antibodies against it. however, the viremia in adult animals is very short-lived. it lasts 3 to 6 days only (hoffmann et al. 2012, wernike et  al. 2013b) making serological analysis a more appropriate tool for detecting sbv infection. in addition, not every malformed fetus suspected of sbv infection tested positive by pcr (de regge et al. 2013, van maanen et al. 2012). the overall percentage of seropositive results in the three sampled years were: 24.75% (25/101) in 2014, 17.76% (19/107) in 2015, and 13.46% (21/156) in  2016. this apparent decrease in percentage of table i. sbv seroprevalence in domestic ruminants in kosovo and albania. sbv seroprevalence: sample no. (positive samples) municipalities sample no. cattle sheep goat % drenas (kos) 9 9(2) 22.22% ferizaj (kos) 3 3(1) 33.33% gjakovë (kos) 1 1(0) 0.00% gjilan (kos) 26 2(2) 24(7) 34.62% istog (kos) 20 19(9) 1(0) 45.00% kaçanik (kos) 4 2(2) 2(1) 75.00% kamenicë (kos) 14 10(7) 4(0) 50.00% lipjan (kos) 7 7(3) 42.86% mitrovicë (kos) 9 7(1) 2(1) 22.22% pejë (kos) 2 2(0) 0.00% prishtinë (kos) 20 12(2) 8(0) 10.00% prizren (kos) 4 4(0) 0.00% rahovec (kos) 40 40(6) 15.00% shtërpc (kos) 1 1(0) 0.00% suharekë (kos) 4 4(1) 25.00% viti (kos) 65 3(2) 18(3) 44(3) 12.31% elbasan (alb) 56 49(11) 7(0) 19.64% dibër (alb) 11 11(0) 0.00% korçë (alb) 14 14(0) 0.00% lezhë (alb) 48 24(0) 24(0) 0.00% librazhd (alb) 5 5(1) 20.00% sarandë (alb) 1 1(0) 0.00% total 364 154(36) 161(25) 49(4) 17.9% 16 sbv in kosovo and albania rexhepi et al. veterinaria italiana 2021, 57 (1), 13-17. doi: 10.12834/vetit.1346.7413.3 azkur a.k., albayrak h., risvanli a., pestil z., ozan e., yılmaz o., tonbak s., cavunt a., kad h., macun 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conraths f., zanella g., granzow h., gache k., schirrmeier h., valas s., staubach c., marianneau p., kraatz f., höreth-böntgen d., reimann i., zientara s. & beer m. 2014. schmallenberg virus two years of experiences. prev vet med, 116, 423-434. wernike k., holsteg m., sasserath m. & beer m. 2015. schmallenberg virus antibody development and decline in a naturally infected dairy cattle herd in germany, 2011-2014. vet microbiol, 181, 294-297. high within-herd seroprevalence and malformations in calves, and its impact on productivity. vet microbiol, 168, 281-293. wernike k., beer m. & hoffmann b. 2016. schmallenberg virus infection diagnosis: results of a german proficiency trial. transbound emerg dis, 64 (5), 1405-1410. wernike k., eschbaumer m., schirrmeier h., blohm u., breithaupt a., hoffmann b. & beer m. 2013a. oral exposure, reinfection and cellular immunity to schmallenberg virus in cattle. vet microbiol, 165, 155-159. wernike k., hoffmann b., bréard e., bøtner a., ponsart c., zientara s., lohse l., pozzi n., viarouge c., sarradin p., 279 diagnostica generale e benessere animale, istituto zooprofilattico sperimentale dell'umbria e delle marche ‘togo rosati’, via g. salvemini 1, 06126 perugia, italy * corresponding author at: diagnostica generale e benessere animale, istituto zooprofilattico sperimentale dell'umbria e delle marche ‘togo rosati’, via g. salvemini 1, 06126 perugia, italy. e-mail: c.pesca@izsum.it. parole chiave ovine herpesvirus 2, bovini, pecora, italia, febbre catarrale maligna. riassunto viene descritto un caso di febbre catarrale maligna (fcm) in un vitello di razza chianina di 4  mesi presso un allevamento semi‑intensivo in italia centrale. nella stessa stalla era stabulato un gregge ovino. al momento dell’insorgenza della sintomatologia all’esame clinico il vitello mostrava depressione del sensorio, diarrea, dimagrimento, febbre, scolo nasale, epifora, pallore delle mucose apparenti, rafforzamento del murmure vescicolare della porzione apicale dei polmoni. nel settembre 2016, dopo 10 giorni dall’insorgenza della malattia, l’animale veniva a morte. la diagnosi è stata effettuata in vivo nel sangue rilevando il dna di ovine herpesvirus di tipo 2 attraverso un saggio di real-time pcr. alla necroscopia, i reperti post mortem erano tipici di fcm e i test istologici e molecolari hanno confermato, infatti, la presenza del virus. si presume che la fonte dell'infezione sia da rintracciare nel gregge di pecore con cui i bovini condividevano la stalla. in italia, così come in europa, sono disponibili pochi dati sull'epidemiologia e sulla ri‑emergenza della malattia negli allevamenti bovini. l’intento della seguente indagine è quello di arricchire la bibliografia già presente di segnalazioni riferibili a questa patologia e di sensibilizzare i colleghi veterinari ad includere la fcm in diagnosi differenziale con altre patologie. febbre catarrale maligna in un bovino dell’italia centrale keywords ovine herpesvirus 2, cattle, sheep, italy. summary a case of malignant catarrhal fever (mcf) occurred in a 4‑month‑old calf housed in a semi‑intensive herd in central italy is described. the herd was in strict cohabitation with a group of domestic sheep. the calf displayed clinical signs that resembled the acute form of mcf and, after a few days of antibiotic and anti inflammatory therapy, died in september  2016. the diagnosis was confirmed in vivo in blood by detection of ovine herpesvirus type 2 dna through real‑time pcr. at necropsy, the gross post‑mortem findings were typical of mcf and the histological and molecular assays confirmed the presence of the virus. the sheep flock was suspected to be the source of the infection. in italy, as well as in europe, there is little data regarding the epidemiology and the recurrence of the disease in herds of cattle, due to the lack of an active surveillance plan and to a major consideration of mcf between differential diagnosis. cristina pesca*, marco gobbi, claudio palombi, claudio forte, silvia pavone, marica stazi, michela pela, deborah cruciani and nicoletta d'avino bovine malignant catarrhal fever: case reporting in central italy veterinaria italiana 2019, 55 (3), 279‑283. doi: 10.12834/vetit.1708.9037.4 accepted: 16.09.2019 | available on line: 30.09.2019 short communication herpesvirus 1 (cphv‑2), ibex malignant catarrhal fever virus, alcelaphine herpesvirus  2 like virus and a virus responsible for mcf in deer (modesto et  al. 2015). the most prevalent virus in europe is ovhv‑2 which is associated with outbreaks of the disease in domestic cattle. sporadic cases are usually observed in european cattle breeds (bos taurus), because they malignant catarrhal fever (mcf) is a systemic viral disease of domestic and wild ruminants such as deer, bison and buffalo, and occasionally pigs. the disease is spread worldwide and it is caused by six viruses belonging to the subfamily gammaherpesvirinae, genus macavirus including alcelaphine herpesvirus 1 (aihv‑1), ovine herpesvirus 2 (ovhv‑2), caprine 280 viral diarrhea virus antibodies (prima check® pi‑3 ab, agrolabo spa scarmagno ‑ italy; svanovir® bcv ab, boeringer ingelheim svanova, uppsala, se; prima check® brsv, agrolabo spa, scarmagno ‑ italy; ab idexx® bvdv p80 ab, idexx laboratories, westbrook, me), whereas nucleic acids purified from the blood sample were tested by real‑time pcr for the detection of ovhv‑2 (traul et  al. 2004). pcrs performed on the nasal swab resulted constantly negative. the blood sample was also analyzed also for the determination of total protein concentration and biochemical values. analysis of the blood sample revealed no significant alteration regarding total protein concentration and biochemical values but revealed the presence of ovhv‑2 dna by real‑time pcr (ct 27.84). samples from different tissues were collected for molecular analysis. in details, spleen was analyzed by end point rt‑pcr and real‑time pcr for detection of bovine viral diarrhoea virus and ovhv‑2, respectively. real‑time pcr on nucleic acids purified from spleen resulted positive for ovhv‑2. our genetic study was based on the orf50 characterization (russell et al. 2014). viral dna was extracted from blood sample previously identified as ovhv2‑positive. the purified amplicons were sequenced on both strands. nucleotide sequences were aligned using clustal w algorithm. manual editing was performed using bioedit software, version 7.0. the phylogeny was estimated using the neighbour‑joining algorithm (nj). the strain described in this study (36409/um/2016; genbank accession number lr697102) shared a 99.80 % identity to strain ku499856.1/itcal allele, previously identified in two water buffalo farms in southern italy (amoroso et  al. 2017). the products of the polymerase chain reaction of the expected size (444 nt) was obtained after amplification with the primer pair designed for the orf50 region. the topology of the tree indicated that the isolate clustered into known genotypes (figure 1). the post mortem examination showed severe bilateral keratoconjunctivitis. grossly, enlarged lymph nodes and mucosal erosive and ulcerative lesions on the gastrointestinal tract were observed (figure 2). renal congestion and necrohemorrhagic cystitis were also detected. lastly, lung showed mild oedema and congestion of the anteroventral lobes. bacteriological examination was carried out on visceral tissue samples by culturing on blood agar, macconkey agar and mannitol salt agar but the samples did not showed any significant pathogenic bacteria. tissue samples from the spleen, omasum, abomasum, larinynx, liver, kidney, urinary bladder, brain, eye globes, mesenteric lymph nodes, prescapolar lymph are a relatively resistant species. bali cattle, bison, and some but not all cervid species (eg., white‑tailed deer, pere david’s deer) instead are highly susceptible to the virus (wiyono et  al. 1994, berezowski et  al. 2005). mcf outbreaks in herds typically have low morbidity and high mortality rates, and generally affect younger individuals (8‑24  months). a feature of mcf, with regards to cattle, is that many outbreaks are sporadic and affect a single or only a few individuals in the herd. however, occasionally there are more serious outbreaks that can affect up to 40% of the herd. the disease has been reported worldwide, including north and south america (rech et al. 2005), europe (yus et  al. 1999), the middle east (abu elzein et  al. 2003), asia (dabak and bulut 2003), africa (rossiter 1981) and new zealand (wilson 2002). in italy, few cases of mcf have been described over the past 20 years; in 2003 decaro and colleagues (decaro et  al. 2003) reported the first two cases of mcf in the country that were confirmed by pcr assays; in 2015 a case of mcf was also described in a captive pudu (pudu puda) by modesto and colleagues (modesto et  al. 2015) and, more recently, in 2017 a mcf outbreak was reported in two water buffalo farms in southern italy (amoroso et  al. 2017). moreover, other cases have been described in cattle and in bison increasing the attention to mcf (campolo et  al. 2008, grattarola et  al. 2011). however, even if sporadic, the prevalence of the disease seems to be underestimated in cattle. this is probably due to the lack of reports and, in some cases, of accurate diagnosis. to the best of the authors’ knowledge, there are no reports of mcf in central italy. the study describes a case of mcf in a 4‑month‑old chianina calf. the case occurred in august 2016. the calf belonged to a semi intensive herd of 15 chianina and mixed‑breed individuals, living in close proximity with a group of domestic sheep. clinical symptoms started in august 2016 when the calf showed hyperthermia (41 °c), anorexia, reluctance to stand, depression, ocular and nasal discharge, lacrimation, generalized lymph nodes enlargement, diarrhoea, bilateral keratoconjunctivitis. auscultation of the lungs assessed an increased pulmonary murmur of the apical lobes of the lungs. the animal died ten days after the onset of the symptoms. serum sample and nasal swab were collected intra vitam for laboratory testing. the nasal swab was analyzed by end point pcr and real‑time pcr for histophilus somni, mycoplasma spp., bovine respiratory syncytial virus, bovine coronavirus, bovine viral diarrhea virus, bovine parainfluenza type 3 virus (moustacas et  al. 2013, lierz et al. 2007, vilcek et al. 1994, decaro et al. 2008, oie 2018, thonur et al. 2012). the serum sample was analyzed by elisa for bovine parainfluenza type 3 virus, bovine coronavirus, bovine respiratory syncytial virus, bovine herpesvirus type 1and bovine bovine mcf in central italy pesca et al. veterinaria italiana 2019, 55 (3), 279‑283. doi: 10.12834/vetit.1708.9037.4 281 pesca et al. bovine mcf in central italy veterinaria italiana 2019, 55 (3), 279‑283. doi: 10.12834/vetit.1708.9037.4 were described in a holstein calf and in a cow in southern italy by decaro and colleagues (decaro et  al. 2003) and diagnosed by pcr test and immunofluorescence. an interesting case was reported in 2006 in a mediterranean water buffalo farm in which calves presented the “head and eye” nodes and mediastinal lymph nodes were fixed in 10% neutral buffered formalin and embedded in paraffin. paraffin‑wax embedded tissue samples were submitted for histological examination via hematoxylin‑eosin (he). microscopical examination revealed systemic mild to marked necrotizing lymphocytic vasculitis. the gastrointestinal tract, brain, and eye globes were most affected. in particular, severe necrotic and hemorrhagic transmural omasitis with lymphocytic endarteritis and necrotizing vasculitis was observed. in the central nervous system, a marked nonsuppurative meningoencephalitis, with disseminated perivascular cuffing and small areas of malacia were detected especially in the brainstem. histological eye lesions consisted in perivascular and intramural lymphocytic infiltrates (perivasculitis and vasculitis) in the periocular skeletal muscle, sclera, and in the connective tissues of the cornea associated with haemorrhages, oedema, neovascularization and keratinocytes necrosis. overall, the histological findings suggested a viral infection that was afterconfirmed by molecular tests. the clinical manifestations and the post mortem findings of the affected calf were strongly suggestive of mcf. the diagnosis was then confirmed by the detection of ovhv‑2 dna in the blood and in the spleen by real‑time pcr and by histological examination. although ovhv‑2 has been found worldwide, data regarding the epidemiology of the disease in italy is limited; the first two cases 0.001 85 62 hg813089.1 allele orf50*0301 bos taurus united kingdom 2014 hg813088.1 allele orf50*0201 bos taurus united kingdom 2014 hg813087.1 allele orf50*0103 bos taurus united kingdom 2014 hg813085.1 allele orf50*0101 bos taurus united kingdom 2014 hg813092.1 allele orf50*0601 bos taurus united kingdom 2014 hg813091.1 allele orf50*0501 bos taurus united kingdom 2014 hg8130900.1 allele orf50*0401 bos taurus italy 2016 ku499855.1 orf50*itcamp allele water bu�alo italy 2016 kj454355.1 isolate 55 bos taurus italy 2016 kj454350.1 isolate 6653 bos taurus italy 2016 kj454361.1 isolate 78009 bos taurus italy 2016 kj454358.1 isolate 288 bos taurus italy 2016 kj454359.1 isolate 408 bos taurus italy 2016 kj454356.1 isolate 127 bos taurus italy 2016 kj454353.1 83090 bos taurus italy 2016 hg813086.1 allele orf50*0102 bos taurus united kingdom 2014 ku499856.1 orf50*itcal allele water bu�alo italy 2016 lr697102 36409/um/2016 bos taurus italy 2016 figure 1. phylogenetic tree based on orf50 sequences. phylogenetic analysis was performed on an alignment of 420 nucleotides with mega7 using the nj method. distances were computed using the kimura two-parameter model. bootstrap values higher than 60% are shown for 10,000 replicate data sets. the tree describes the relationship between selected orf50 sequences retrieved from the genbank database and the italian novel nucleotide sequence (36409/um/2016; genbank accession number lr697102) labelled in bold. bar number of substitutions per site. figure 2. chianina calf showing mcf lesions: (a) corneal opacity, (b) mucosa of the small intestinal tract with congestion and ulcerative lesions. 282 bovine mcf in central italy pesca et al. veterinaria italiana 2019, 55 (3), 279‑283. doi: 10.12834/vetit.1708.9037.4 of caustic materials or some toxic plants. clinical signs can be elusive if not related to risk factors such as evidence of contact with a carrier species (sheep, goats, or wildebeest) as suspected in this case and as described in literature (decaro et  al. 2003, campolo et al. 2008, martucciello et al. 2006). in this occasion, unfortunately, the sheep flock could not be adequately investigated to identify the potential reservoirs of the virus. anyway, the present study provides new information regarding the circulation of ovhv‑2 in central italy. malignant catarrhal fever is an important and underestimated disease with many unclear questions concerning transmission, prevalence and pathogenesis. the authors believe that the inclusion of this pathology in routine differential diagnosis protocols could help to fill these gaps. importantly, some susceptible species, including cattle and bison, may be latently infected. recrudescence of latent infections is possible and must be considered for cases with unknown history of contact with carriers. in this regard, it could be important to consider the establishment of an active surveillance plan for mcf in european herds. in addition due to the lack of a valid immunization strategy and the rapid course of the disease, is of fundamental importance to rapidly report field outbreaks in order to create a baseline data for more extensive epidemiological investigations. form and died within 8 days (martucciello et al. 2006). later, in 2008, an adult american bison, housed in a zoo in southern italy, displayed clinical signs of mcf that was confirmed by molecular and serological assays (campolo et  al. 2008). other two cases, one in a cattle farm in northern italy and the other in a captive pudu (pudu puda), have been registered until 2015 (grattarola et  al. 2011, modesto et  al. 2015). the latest outbreak of mcf was reported in southern italy in two water buffalo farms (amoroso et  al. 2017). in the majority of the cases described above, a contact between the affected animals and carriers like sheep was documented. diagnosis of mcf is first based on clinical signs and gross pathological examination even if these can be extremely variable, thus causing difficulties during diagnosis. histological examination can be considered a useful diagnostic tool and it can be performed on a variety of tissues. however, detection of viral dna is rapidly becoming the method of choice for confirmation of clinical cases. as presented in this case report, the inclusion of mcf in differential diagnosis can represent the first step toward the final diagnosis (oie 2018). primary differential diseases include bovine viral diarrhoea/ mucosal disease, infectious bovine rhinotracheitis, bluetongue, epizootic haemorrhagic disease, foot and mouth disease, vesicular stomatitis, or ingestion 283 pesca et al. bovine mcf in central italy veterinaria italiana 2019, 55 (3), 279‑283. doi: 10.12834/vetit.1708.9037.4 abu elzein e.m.e., housawi f.m.t., gameel a.a., al afaleq a.i. & el bashir a.m. 2003. sheep‑associated malignant catarrhal fever involving 3‑5‑week‑old calves in saudi arabia. j vet med b infect dis vet public health, 50, 53‑59. amoroso m.g., galiero g. & fusco g. 2017. genetic characterization of ovine herpesvirus 2 strains involved in water buffaloes malignant catarrhal fever outbreaks in southern italy. vet microbiol, 199, 31‑35. berezowski j.a., appleyard g.d., crawford t.b., haigh j., li h., middleton d.m., o'connor b.p., west k. & woodbury m. 2005. an outbreak of sheep‑associated malignant catarrhal fever in bison (bison bison) after exposure to sheep at a public auction sale. j vet diagn invest, 17, 55‑58. campolo m., lucente m.s., mari v., elia g., tinelli a., laricchiuta, p. caramelli m., nava d., buonavoglia c. & decaro n. 2008. malignant catarrhal fever in a captive american bison (bison bison) in italy. j vet diagn invest, 20 (6), 843‑846. dabak m. & bulut h. 2003. outbreak of malignant catarrhal fever in cattle in turkey. vet rec, 152, 240‑241. decaro n., tinelli a., pratelli a., martella v., tempesta m. & buonavoglia c. 2003. first two confirmed cases of malignant catarrhal fever in italy. new microbiol, 26 (4), 339‑344. decaro n., elia g., campolo m., desario c., mari v., radogna a., colaianni m.l., cirone f., tempesta m. & buonavoglia c. 2008. detection of bovine coronavirus using a taqman‑based real‑time rt‑pcr assay. j virol methods, 151 (2), 167‑171. grattarola c., iulini b., zoppi s., varello k., dondo a., rumello g., baglivo t., rondoletti m., savini g., amorisco f. & casalone c. 2011. investigation on three outbreaks of malignant catarrhal fever in cattle in piedmont. large anim rev, 17 (2), 49‑55 lierz m., hagen n., harcourt‑brown n., hernandez‑divers s.j., lüschow d. & hafez h.m. 2007. prevalence of mycoplasmas in eggs from birds of prey using culture and a genus‑specific mycoplasma polymerase chain reaction. avian pathol, 36 (2), 145‑150. martucciello a., marianelli c., capuano m., astarita s., alfano d. & galiero g. 2006. an outbreak of malignant catarrhal fever in mediterranean water buffalo (bubalus bubalis). large anim rev, 12 (5), 21‑24. modesto p., grattarola c., biolatti c., varello k., casalone, c., mandola m.l., caruso c., dondo a., goria m., rocca f., decaro n., leonardi c., iulini b. & acutis p.l. 2015. first report of malignant catarrhal fever in a captive pudu (pudu puda). res vet sci, 99, 212‑214. moustacas v.s., silva t.m.a., costa l.f., xavier m.n., references carvalho c.a. jr, costa e.a., paixão t.a. & santos r.l. 2013. species‑specific multiplex pcr for the diagnosis of brucella ovis, actinobacillus seminis, and histophilus somni infection in rams. bmc vet res, 9, 51. rech r.r., schild a.l., driemeier d., garmatz s.l., oliveira f., riet‑correa f. & barros c.s.l. 2005. malignant catarrhal fever in cattle in rio grande do sul, brazil: epidemiology, clinical signs and pathology. pesqui vet brasil, 25, 97‑105. rossiter p.b. 1981. antibodies to malignant catarrhal fever virus in sheep sera. j comp pathol, 91, 303‑311. russell g.c., scholes s.f., twomey d.f., courtenay a.e., grant d.m., lamond b., norris d., willoughby k., haig d.m. & stewart j.p. 2014. analysis of the genetic diversity of ovine herpesvirus 2 in samples from livestock with malignant catarrhal fever. vet microbiol, 172 (1‑2), 63‑71. thonur l., maley m., gilray j., crook t., laming e., turnbull d., nath m. & willoughby k. 2012. one‑step multiplex real time rt‑pcr for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3. bmc vet res, 28 (8), 37. traul d.l., taus n.s., lindsay oaks j., o'toole d., rurangirwa f.r., baszler t.v. & li h. 2007. validation of nonested and real‑time pcr for diagnosis of sheep associated malignant catarrhal fever in clinical samples. j vet diagn invest, 19 (4), 405‑408. vilcek s., elvander m., ballagi‑pordany a. & belak s. 1994. development of nested‑pcr assay for detection of bovine respiratory syncytial virus in clinical samples. j clin microb, 32 (9), 2225‑2231. yus e., guitian j., diaz a. & sanjuan m.l. 1999. outbreak of malignant catarrhal fever in cattle in spain. vet rec, 145, 466‑467. wilson p.r. 2002. advances in health and welfare of farmed deer in new zealand. new zeal vet j, 50, 105‑109. wiyono a., baxter s.i.f., saepulloh m., damayanti r., daniels p. & reid h.w. 1994. pcr detection of ovine herpesvirus‑2 dna in indonesian ruminants ‑ normal sheep and clinical cases of malignant catarrhal fever. vet microbiol, 42, 45‑52. world organisation for animal health (oie). 2018. malignant catarrhal fever. in manual of diagnostic tests and vaccines for terrestrial animals. chapter 2.4.14, 1‑13. paris, oie. world organisation for animal health (oie). 2018. bovine viral diarrhea. in manual of diagnostic tests and vaccines for terrestrial animals. chapter 3.4.7, 1073‑1096. paris, oie. 257 introduction camels are bred in some provinces of turkey for tourism and social and cultural purposes. currently, there are approximately 2000 camels living in turkey (turkstat 2017, www.turkstat.gov.tr). aydin province is the area with the largest camel population approximately 1,500 animals. traditionally, camels have been brought to the aydin province from several regions of turkey including mediterranean, central anatolia, and south and north regions for camel wrestling or tourism purposes. majority of camels are near other ruminants, and thus, frequently in contact with sheep, goats, and cattle in husbandry. there are four major ruminant viruses that can potentially infect camels: bovine enterovirus‑1 (bev‑1), bovine herpesvirus type‑1 (bhv‑1), bovine viral diarrhea virus (bvdv), and bovine parainfluenza‑3 (pi‑3). bovine enterovirus‑1 is a member of the genus enterovirus within the family picornaviridae, a group of small, non‑enveloped, positive strand rna viruses (hyypia et  al. 1997). although it usually causes subclinical infection with mild symptoms, rare fatal cases with low morbidity have also been reported (blas‑machado et  al. 2007). clinical symptoms associated with the infection include digestive and respiratory system disorders and abortion (grooms 2004). the virus has a wide range of host spectrum including cattle, water buffaloes, sheep, and goats (gur et al. 2008). bovine herpesvirus‑1 classified in subfamily alphaherpesvirinae, family herpesviridae, is the cause 1aydın adnan menderes university, faculty of veterinary medicine, department of virology, 09016 efeler-aydin, turkey. 2afyon kocatepe university, faculty of veterinary medicine, department of virology, ans campus, 03200 afyonkarahisar, turkey. 3department of virology, faculty of veterinary medicine, university of selcuk, 42075 konya, turkey. *corresponding author at: adnan menderes university, faculty of veterinary medicine, department of virology, 09016 isikli-aydin, turkey. tel.: +90 256 247 07 00 / 6203, e-mail: nuralerol@adu.edu.tr. keywords camels, bovine enterovirus‑1, bovine herpesvirus‑1, bovine viral diarrhea virus, parainfluenza‑3 virus, antibody, prevalence. summary camels (camelus dromedarius) are bred in western turkey, particularly in the province of aydin, for touristic, social and cultural purposes. bovine enterovirus‑1 (bev‑1), bovine herpesvirus type‑1 (bhv‑1), bovine viral diarrhea virus (bvdv ), and parainfluenza‑3 (pi‑3) virus infections are significant causes of health and/or economic concerns in several animal species. these agents have not been investigated in the camel population in turkey. the objective of this study was to serologically investigate the presence and infection rates of these viruses in camels in aydin province, western turkey. ninety‑two serum samples were taken from clinically healthy camels that were kept in private farms or brought to the local slaughterhouses. serum neutralization test was performed to assess the presence and the titers of specific antibodies against bev‑1, bhv‑1, bvdv, and pi‑3 virus in camel sera. of the 92 camels tested, 30 (32.61%), 2 (2.17%), 54 (58.7%), and 20 (21.74%) were seropositive for bev‑1, bhv‑1, bvdv, and pi‑3, respectively. these results suggest that, except for bhv‑1, these viral infections are common among camels in western turkey. to our knowledge, this the first comprehensive, large‑scale study investigating these viral infections in camels in turkey. nural erol1*, sibel gür2, b. taylan koç1 and sibel yavru3 a serological investigation of bovine enterovirus-1, bovine herpesvirus-1, bovine viral diarrhea virus, and parainfluenza-3 infections in camels in western turkey veterinaria italiana 2020, 56 (4), 257‑262. doi: 10.12834/vetit.1730.9136.2 accepted: 08.04.2019 | available on line: 31.12.2020 258 ruminant viruses in camels from western turkey erol et al. veterinaria italiana 2020, 56 (4), 257‑262. doi: 10.12834/vetit.1730.9136.2 local farms in the aydin province for breeding. all camels were apparently healthy at the time of blood collection. the animals that were brought to the clinics were suffering from long teeth or lameness that required surgical intervention and did not show any signs of systemic disease. individual features of animals were recorded according to owners’ and/or veterinarians’ declaration. one of the most important evidence was that camels brought to clinics had been raised with ruminants (cattle, sheep, and goats). the blood samples were collected in serum separator tubes. serum was obtained by centrifugation and stored at ‑  20  °c until testing. the camels brought to the clinic and the camels from local farms were housed with cattle. no housing history was instead available on animals brought to slaughterhouses. therefore, it was not known whether they have had any contact  with cattle. cell culture madin darby bovine kidney (mdbk) cells were used for virus propagation, titration, and serum neutralisation test (snt). dulbecco’s modified eagle medium (dmem) (biochrom‑ germany) containing 10% inactivated fetal calf serum (biochrom, germany) was used as the cell culture medium. viruses in this study, bev serotype 1, colorado strain of bhv‑1, cytopathic nadl strain of bvdv, and sf‑4 strain of pi‑3 were used as test viruses. for each virus suspension 50% tissue culture infection dose (tcid 50 ) was calculated using spearman and kaerber method (ramarkrishnan 2016). serum neutralization test serum samples were examined for the presence of antibodies against bev‑1, bhv‑1, bvdv and pi‑3 virus using standard snt according to the procedure by frey and liess (frey and liess 1971). in total, 368 serum neutralization tests were performed to investigate the four viruses in the 92  samples. microneutralization test was used after serum samples were diluted 1:1 with medium to test for bhv‑1, bev‑1, bvdv and 1:5 to test for pi‑3. fifty microliters of the diluted samples were added in duplicates into tissue culture microplates with the same volume of virus suspension containing 100 tcid 50 virus. after an hour of incubation at 37 °c in 5% co 2 , 50 μl cell suspension (300,000 cells/ml) was added and the plates were further incubated for 72 hours. test results were evaluated based on the cytopathic effects on cells observed under an inverted microscope. of infectious bovine rhinotracheitis characterized by reduced milk yield, abortions, and respiratory symptoms such as conjunctivitis and nasal mucopurulent discharge in cattle (muylkens et  al. 2007). it was isolated in different countries from aborted fetuses and lungs of camels with pneumonia (ismail 2017). bovine viral diarrhea virus is a member of the pestivirus genus, a group of serologically closely related single‑stranded rna viruses classified in the family flaviviridae. bvdv infections are common world‑wide. in cattle infection can cause diarrhea, mucosal disease, hemorrhagic lesions in respiratory and digestive system and reproductive disorders such as abortion, teratogenesis, embryonic resorption, fetal mummification, stillbirth, and congenital infections (baker 1995, van amstel and kennedy 2010, moening and becher 2018). bovine parainfluenza‑3 virus recently renamed as bovine respirovirus 3 is a respirovirus classified in the family paramyxoviridae (intisar et  al. 2010b, adams et al. 2017). it is the cause of acute respiratory tract disease and enzootic bronchopneumonia in calves (oros et al. 1997). in adults, pi‑3 substantially does not trigger a direct respiratory disease but, suppressing the respiratory system, leads to opportunistic infection agents infect these animals (intisar et  al. 2010b, saeed et al. 2015). these four listed above viral infections are significant causes of health and/or economic concerns in ruminants, especially cattle. data on the prevalence of these infections in camels in turkey are very limited because of low numbers and minor economic value compared to the other ruminants. as of february 2018, entering the key words ‘bovine enterovirus’, ‘bovine herpesvirus’, ‘bovine viral diarrhea’, or ‘parainfluenza’ along with ‘camel’ and ‘turkey’ into the pubmed database (http://www/ pubmed.gov) produced only the following three publications. in these few studies which involved few animals, no bev‑1, bvdv or bhv‑1 antibodies were detected in camels (gur et  al. 2008, albayrak et al. 2010, yesilbağ et al. 2011). the objective of this study was to investigate the seroprevalence of these infections in dromedary camels (camelus dromedarius) in western turkey. materials and methods samples ninety‑two samples were taken from camels that were brought from various regions of turkey to incirliova municipal slaughterhouse for slaughter, to the faculty of veterinary medicine clinics at the adnan menderes university for treatment, or to 259 erol et al. ruminant viruses in camels from western turkey veterinaria italiana 2020, 56 (4), 257‑262. doi: 10.12834/vetit.1730.9136.2 estimate potential association between recorded data and study outcomes. all tests were performed at the 0.05 level of significance. results the serological results are presented in table  i. the seropositive rates were 32.61%, 2.17%, 58.7%, and 21.74% for bev‑1, bhv‑1, bvdv, and pi‑3, respectively. the effect of sample source on the seropositive rates were presented in table ii. multi‑infection status were summarised in pie‑chart (figure 1). antibody titers in camels were generally low (figure 2). the highest titer (1/80) was detected in two samples against bev‑1. the highest titer against bvd and pi‑3 was 1/40. the sn 50 value of two bhv‑1 positive samples was 1/2. discussion to our knowledge, this is the first comprehensive study on seropositive rates of bev‑1, bhv‑1, bvdv, and pi‑3 in camels in turkey. due to their high prevalence and their capacity to cause economic losses, these viruses represent a major threat to livestock in turkey (erol et al. 2007, gur et al. 2008, determination of sn 50 value all positive sera were serially diluted in a two‑fold series. this assay is different from first serum neutralization assay. first serum neutraliztion assay was performed to investigate sera positivity in which samples has been diluted 1:1 or 1:5. second neutralization assay were performed to determine titration of antibody in positive‑sera which has provided to detect sn 50 value. the snt was used to determine 50% serum neutralisation (sn 50 ) antibody titer values. statistical analysis chi‑squared statistical test was used in order to table i. seropositive rates in camels from western turkey. virus n strain used positive samples seropositivity % bev-1 92 serotype 1 30 32.61 bhv-1 92 colorado 2 2.17 bvdv 92 cytopathic nadl 54 58.7 pi-3 92 sf-4 20 21.74 n = number of tests performed; bev-1 = bovine enterovirus-1; bhv-1 = bovine herpesvirus-1; bvdv = bovine viral diarrhea virus; pi-3 = parainfluenza-3. table ii. effect on sample source on seropositive rates. animal source animals tested animals tested seropositive bvd (%) bhv-1 (%) bev-1 (%) pi-3 (%) slaughterhouse 65 34 (52.3%) 1 (1.5%) 15 (23.1%) 7 (10.8%) p > 0.05 clinics 13 9 (69.2%) 1 (7.7%) 8 (61.5%) 6 (46.2%) p < 0.05 local farms 14 11 (78.6%) 0 (0%) 7 (50%) 7 (50%) p > 0.05 total 92 54 (58.7%) 2 (2.17%) 30 (32.61%) 20 (21.74%) snt = serum neutralization test; bev-1 = bovine enterovirus-1; bhv-1 = bovine herpesvirus-1; bvdv = bovine viral diarrhea virus; pi-3 = parainfluenza-3. 12 dual 17% 17 triple 25% 1 quaternary 1% 39 single 57% figure 1. multi-infection status of wetern turkey camels according to neutralising antibodies against bovine enterovirus-1 (bev-1), bovine herpesvirus type-1 (bhv-1) bovine viral diarrhea virus (bvdv), and bovine parainfluenza-3 (pi-3). 30 25 20 15 10 5 0 1/5 1/10 1/20 antibody titers se ro p o si ve s am p le s 1/40 1/80 bvd bev-1 pi-3 figure 2. bovine enterovirus-1 (bev-1), bovine viral diarrhea virus (bvdv), and bovine parainfluenza-3 (pi-3) neutralising titers in camels of western turkey. 260 ruminant viruses in camels from western turkey erol et al. veterinaria italiana 2020, 56 (4), 257‑262. doi: 10.12834/vetit.1730.9136.2 in our study, 21.74% of the camels were exposed pi‑3 virus. pi‑3 infection rates in camels can be very different in different countries. it was 82.2% in sudan (intisar et al. 2010b) but 5.6% in united arab emirates (afzal et al. 1994). ayelet and colleagues (ayelet et al. 2013) found no pi‑3 seropositive camels in ethipoia. in this study, the source from where samples were taken had a significant impact on seropositive rates. camels brought to the clinics for treatment had higher infection rates than those brought to slaugtherhouse and local farms (table ii, p < 0.05). this is probably due to the fact that in the clinics animals were kept together with other ruminants, especially cattle. camels housed near or along with other ruminants may have been infected with viruses from other ruminants. this should be considered when drawing plans to control these infections. in summary, our results showed that bev‑1, bvdv, and pi‑3 infections are common in camels in turkey. these infections are also common in cattle causing significant economic losses. the camels with high seropositive rates were those kept together with other ruminants, especially cattle. this finding might suggest a possible transmission between camels and other ruminants. therefore, camels may play a critical role in the transmission cycle of these viruses under field conditions and should be considered in future potential prevention program against these infections. acknowledgements the authors would like to thank to the staff of the incirliova municipal slaughterhouse, and adnan menderes university faculty of veterinary medicine clinics for their help and cooperation. ethical statements this study was approved by the animal ethical committee of the adnan menderes university (approval no: b.30.2.adu.0.06.00.00/124‑h ek/2009/018). tan et al. 2006 a, b). although the viruses can infect camels, data on prevalence of these viruses in camels are very limited in the literature probably because camel populations have little economic impact in turkey. therefore, it is not known whether camels might play a role in the epidemiology and spread of these viruses in rumiinants in turkey. in this study, 30 of the 92 (32.6%) samples collected from camels were positive for bev‑1. although new camel enteroviruses have been recently described (woo et  al. 2015), data on susceptibility of camels to this infection are very limited. there is only one study by gur and colleagues (gur et  al. 2008) that didn't detect antibodies against bev‑1 in 18 camels in turkey. our study showed that 32.6% of the camels were seropositive to this infection. the reason for this discrepancy could be due to the difference in sample size between the two studies [i.e., 18 in the study by gur and colleagues (gur et al. 2008) vs 92 in our study]. the low seropositive rate to bhv‑1 found in this survey is similar to those reported in some other studies. yesilbağ and colleagues (yesilbağ et al. 2011) didn't detect bhv‑1 antibodies in two camels in turkey. no antibodies against bhv‑1 were detected in 111 sera collected from camels in algeria (saidi et  al. 2018). however, 76.9% seropositivity was reported in sudan (intisar et al. 2009), suggesting that bhv‑1 infection rates can vary greatly in different countries. in our study, 58.7% of the camels were seropositive to bvdv. this is a common finding world‑wide. high bvdv seroprevalence in cattle in different regions of turkey include 41.4% (yesilbag et  al. 2008), 58.2% (gur et  al. 2011), and 70.8% (aslan et  al. 2015). tan and colleagues (tan et  al. 2006a) detected antibodies against bvdv in 86% and bvdv antigen in 12.85% of cattle in aydin province; 4.9% of the animals were persistently viremic. high seropositive rates in camels have been reported in several countries including sudan (84.6%, intisar et  al. 2010a) and egypt (52.5%, zaghawa et  al. 1998). in a recent comprehensive review, wernery (wernery 2012) suggested that bvdv infection in camels is common and that camels may play a role in epizootiology of the bvdv. 261 erol et al. ruminant viruses in camels from western turkey veterinaria italiana 2020, 56 (4), 257‑262. doi: 10.12834/vetit.1730.9136.2 adams m.j., lefkowitz e.j., king a.m., harrach b., harrison r.l., knowles n.j., kropinski a.m., krupovic m., kuhn j.h., mushegian a.r., nibert m., sabanadzovic s., sanfaçon h., siddell 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italiana 2021, 57 (3), 175-180. doi: 10.12834/vetit.1763.9298.3 accepted: 09.09.2019 | available on line: 31.12.2021 1institut des sciences vétérinaires, université de blida 1, algeria. 2ecole superieure des sciences de l'aliment et des industries alimentaires, algeria. *corresponding author at: institut des sciences vétérinaires, université de blida 1, algeria. e‑mail: s_lafri@hotmail.fr. ismail lafri1* and idir bitam2 keywords algeria, leishmania, phlebotomine, phlebovirus, sand flies. summary being the only established vectors of the protozoan parasites of the genus leishmania, sand flies have become very important in all countries where leishmaniasis exists. it is caused by a variety of species, each one having specific mammalian reservoir hosts and vectors. leishmania and sand fly classification has always been a controversial matter, and the increasing number of sand fly species described complicates the task. until recently, sand flies distributed in the old world were known as vectors for few phleboviruses including two known species (sandfly fever naples virus, and salehebad virus), and tentative species such as sand fly fever sicilian virus and corfou virus. these infections are emerging in the mediterranean region and will likely spread in forthcoming decades, posing a complex threat to public health. here, we reviewed the current literature on phlebotomine sand flies fauna and epidemiology of sand fly-borne infections in algeria. phlebotomine sand flies and associated pathogens in algeria: update and comprehensive overview contributions provided information but now some reports contain data that need to be updated. with the growing importance of phlebotomine-borne diseases, entomological and epidemiological studies with the inventory of phlebotomine sand flies fauna are the first step to prevent infections and the risk of transmission. whereas, it is necessary to map the risk areas in relation to the phlebotomine population vector distribution and their pathogens. we propose an updated view of events that have played important roles in the geographical distribution of sand flies (table i), in relation to both the leishmania and phleboviruses pathogens associated and circulated in algeria (table ii). the information gathered in this review was mainly obtained from the online literature using the pub med, google search engines and a personal research of data. a comprehensive literature search using the terms (phlebotomine, sand flies, leishmania, phlebovirus and algeria) was conducted. updated inventory of phlebotomine sand fly fauna in algeria the name ‘sand fly’ can be misleading, as it wrongly introduction phlebotomine sand flies (diptera: psychodidae: phlebotominae) are blood-feeding insects of great significance for physicians and veterinarians. indeed, they are vectors of numerous pathogens to humans and animals, including protozoa, bacteria and viruses (dantas-torres et  al. 2012, maroli et  al. 2013). for instance, species of the genus phlebotomus are vectors of phleboviruses (e.g., sand fly fever naples virus, and sand fly fever sicilian virus) causing the sand fly fever, which is a transient febrile illness that is mainly prevalent in the mediterranean region (maroli et  al. 2013). most importantly, phlebotomine sand flies are the biological vectors of leishmania parasites which still cause disfiguring lesions and claim the lives of thousands of dogs and humans each year in more than 98 endemic countries (alvar et  al. 2012). in algeria, 25 phlebotomine sand flies species and 3 synonyms were inventoried up to 2018 (table i), including two genera and seven subgenera (belazzoug 1991, berdjane-brouk et  al. 2011, benallal et  al. 2013). some of them are known to be leishmania parasite vectors (boubidi et  al. 2011) and phleboviruses vectors (alkan et  al. 2015) (table ii). knowledge about phlebotomines in algeria is currently incomplete. some scientific 176 veterinaria italiana 2021, 57 (3), 175-180. doi: 10.12834/vetit.1763.9298.3 xxxxxxxxxxxxxxxx lafri & bitam external structures (male genitalia) and descriptions of internal structures such as the spermathecae, cibarium, and the pharynx were employed (perfil’ev 1968). a distinction must therefore be made for the vectors of the leishmaniases and other diseases of public health concern, which are correctly termed ‘phlebotomine sand flies’ (killick-kendrick 1999). to date, over 800 species have been estimated to exist in different regions of the world. approximately 464 species are found in the new world and 375 in the old world (galati 2003, seccombe et al. 1993). suggests to laypeople that they may be at risk of vector-borne disease while on holiday on the beach. actually, the english denomination refers to the pale (sandy) color of this small insect. sand flies belong to the order diptera, suborder nematocera, family psychodidae, and subfamily phlebotominae. phlebotomine sand flies are principally present in the warm zones of asia, africa, australia, southern europe and the americas (killick-kendrick 1999). sand flies were identified according to two distinct methods (akhoundi et al. 2016). the analysis of certain table i. phlebotomine sand flies species of algeria: current known distribution ‑ december 2018. phlebotomine sand flies species geographical distribition phlebotomus phlebotomus (phlebotomus) papatasi (scopoli, 1786) northern steppe fringe (highland) phlebotomus (phlebotomus) bergeroti (parrot, 1934) djanet, in amguel, tamanrassat phlebotomus (paraphlebotomus) sergenti (parrot, 1917) south, tell, saharian steppe phlebotomus (paraphlebotomus) alexandri (sinton, 1928) pre‑saharian steppes, atlas phlebotomus (paraphlebotomus) chabaudi (croset, abonnenc et rioux, 1970), synonym of p. riouxi pre‑saharian steppes (arid climatic), ghardaia, aures phlebotomus (paraphlebotomus) kazeruni (theodor et mesghali, 1964) hoggar phlebotomus (larroussius) ariasi (tonnoir, 1921) tell phlebotomus (larroussius) chadlii (rioux, juminer et gibily 1966) tell phlebotomus (larroussius) perniciosus (newstead, 1911) all bioclimatic stage phlebotomus (larroussius) longicuspis (nitzulescu, 1911) tell, highlands, saharian steppe, central sahara phlebotomus (larroussius) langeroni (nitzulescu, 1930) tell phlebotomus (larroussius) perfiliewi (parrot, 1930) tell phlebotomus (transphlebotomus) mascittii (grassi, 1908) kabylie phlebotomus hirtus (parrot et de jolinière, 1945) hoggar sergentomyia sergentomyia (sergentomyia) minuta parroti (adler et theodor, 1927) all bioclimatic stage sergentomyia (sergentomyia) fallax (parrot, 1921) pre‑saharian steppes, atlas sergentomyia (sergentomyia) antennata (newstead, 1912), synonym of s. cincta pre‑saharian steppes, central sahara sergentomyia (sergentomyia) schwetzi (adler, theodor et parrot, 1929) tamanrassat sergetomyia (parrotomyia) eremitis (parrot et de jolinière, 1945), synonym of s. africana asiatica in amguel, tamanrassat sergetomyia (parrotomyia) lewisi (parrot, 1948), synonym of s. palestinensis biskra, djanet, iherir, tamanrassat sergentomyia (grassomyia) dreyfussi (parrot, 1933) biskra sergentomyia (sintonius) clydei (sinton, 1928) northern steppe fringe, central sahara (hoggar, tassili) sergentomyia (sintonius) christophersi (sinton, 1927) northern steppe fringe, central sahara (hoggar, tassili) sergentomyia (sintonius) hirta (parrot et de jolinière, 1945) central sahara sergentomyia (sintonius) tiberiadis (adler, theodor et louric, 1930) djanet table ii. associated pathogens detected in phlebotomine sand flies of algeria up to december 2018. phlebotomine sand flies species pathogens detected phlebotomus (phlebotomus) papatasi (scopoli, 1786) leishmania major / novel phlebovirus related to sandfly fever sicilian virus phlebotomus (paraphlebotomus) sergenti (parrot, 1917) leishmania killicki phlebotomus (larroussius) ariasi (tonnoir, 1921) sandfly fever sicilian virus phlebotomus (larroussius) perniciosus (newstead, 1911) leishmania infantum / toscana virus phlebotomus (larroussius) longicuspis (nitzulescu, 1911) leishmania infantum / novel phlebovirus related to sandfly fever naples virus / toscana virus phlebotomus (larroussius) perfiliewi (parrot, 1930) leishmania infantum / toscana virus first author et al. nick title 177veterinaria italiana 2021, 57 (3), 175-180. doi: 10.12834/vetit.1763.9298.3 from the genus leishmania (trypanosomatida: trypanosomatidae). leishmaniases are endemic in large areas of the tropics, subtropics, and the mediterranean basin, where there are a total of 350 million people at risk and 12 million cases of infection (moreno and alvar 2002, alvar et  al. 2012) with few exceptions; phlebotomine sand flies are the unique haematophagous insects proven to transmit leishmaniases through the bite of infected female that have previously fed on an infected mammal. after afghanistan, algeria is the second largest focus of cutaneous leishmaniasis (cl) in the world. in algeria, the first report dates back to the time of sergent who succeeded to produce an experimental lesion of cl to a volunteer by filing on a scarification dermal seven specimens of p.  papatasi captured in biskra (sergent et al. 1921); it was the first evidence of the role vector of leishmaniasis played by a sand fly. after that, parrot and colleagues, recorded the infection of four females of p.  perniciosus out of 53 induced to feed on a dog with vl in algiers (parrot et al. 1930). in 1931, parrot and colleagues, observed the spontaneous infection of p.  perniciosus by promastigotes of l.  infantum in 14 of 58 females gorged from dog having leishmaniosis (parrot et  al. 1931); this experience continued for several consecutive years (parrot et al. 1941). it was the first evidence of l.  infantum in algeria. in 1941, parrot observed a natural infection in 16.5% of p. longicuspis females gorged from dogs affected by leishmaniosis (parrot et  al. 1941). this fact suggested to consider p.  longicuspis as possible vector of vl, associated to p.  perniciosus. a very interesting investigation done in kabylia by izri and colleagues lead to isolate l.  infantum mon-1 from p.  perniciosus (izri et  al. 1990), to confirm its role vector of vl in algeria. after this work, the same team isolated l.  major in p.  papatasi in biskra (izri et  al. 1992); this supports classical observations of sergent, that p.  papatasi is the main vector in this focus in 1921 (sergent et  al. 1921). during these epidemiological series in concern to phlebotomine-associated pathogens of algeria, this team realized another record and proved that p.  perfiliewi was found naturally infected with dermotropic l. infantum at tenes (izri and bellazoug 1993). in the last decade, some researcher have adopted molecular tools to control phlebotomine sand flies in algeria. in 2011, a team of pasteur institute of algeria suggested that cl caused by l.  killicki could be a zoonotic disease, with p. sergenti sand flies acting as hosts and vectors and gundi rodents as reservoirs in ghardaia, from the south (boubidi et al. 2011). going back in time, parrot's hypothesis raised in 1941 (parrot et  al. 1941) concerning the role of p.  longicuspis in the transmission of l.  infantum was strongly supported by the detection of l.  infantum dna in p.  longicuspis from vl endemic focus in kabylia (berdjane-brouk et al. 2012). in algeria, phlebotomine sand flies were reported for the first time in 1912 (foley and leduc 1912). sand flies have been the subject of very important work carried out in pasteur institute of algeria, under the direction of parrot and the sergent brothers, with description of several new species (phlebotomus sergenti in 1917, sergentomyia fallax in 1921, s. dreyfussi in 1933 and p. bergeroti in 1934) in 1980 and 1981 respectively, phlebotomine sand flies population of tassili n'ajjer and hoggar from the southern part of algeria was described (belazzoug et  al. 1980, belazzoug et  al. 1981). after that, the same team reported for the first time the presence of s.  minuta (belazzoug et  al. 1982). later in literature, after an epidemiological survey on leishmaniases conducted in algeria between 1972 and 1976, dedet and colleagues reported the result of their entomological investigations with special reference to taxonomy, distribution, ecology and pathogenic role of the 15 species found. the check-list of sand flies was then actualized to 21 species and a key was provided to aid identification of algerian sand flies (dedet et  al. 1984). in 1991, belazzoug established a new status of algerian sand flies fauna with 22  species recorded (belazzoug 1991). some studies in the northeast of the country have been realized in concern of ecological status of phlebotomine sand flies (kabbout et  al. 2016), and morphological distinction between two sympatric species: p.  perniciosus and p.  longicuspis (berchi et al. 2007). in 2011, p. mascittii was reported for the first time in algeria during an entomological study conducted in endemic visceral leishmaniasis (vl) focus from the north part of the country (kabylia) (berdjane-brouk et  al. 2011). another entomological survey carried out in tamanrasset enabled us to identify a new location for p. kazeruni in algeria (benallal et al. 2013), which lead to enlarge phlebotomine fauna into 25  species. recently, lafri and colleagues established the application of maldi-tof ms for monitoring and identification of field caught sand flies (lafri et  al. 2016). more recently, an entomological study provided for the first time the presence of atypical form of p. perniciosus in algeria (benallal et al. 2017). phlebotomine sand flies pathogens reported in algeria leishmaniases among the over 800 phlebotomine sand fly species estimated to exist, only 98 species of phlebotomus and lutzomyia genera are currently proven or suspected vectors of human leishmaniases (maroli et  al. 2013). leishmaniases are vector-borne diseases caused by obligate protozoan parasites 178 veterinaria italiana 2021, 57 (3), 175-180. doi: 10.12834/vetit.1763.9298.3 xxxxxxxxxxxxxxxx lafri & bitam is limited to the work of relatively few groups of entomologists experienced in phlebotomine research. if this trend continues, aspects of sand fly behaviour that might be relevant to target control may remain unknown or neglected. leishmaniases and other tropical infectious diseases, are generally regarded as neglected diseases because of the lack of effective, affordable and easy-to-use drug treatments. otherwise, as most affected patients live in developing countries, the pharmaceutical industry has ignored these diseases. understanding these evolutionary relationships between phlebotomine and associated pathogens is of epidemiological importance for the future prediction of leishmania transmission patterns in the first instance and other phlebotomine-associated pathogens. we have expanded the understanding and updated the phlebotomine sand flies repertoire and associated pathogens reported in algeria. this review will help researcher, human and veterinary clinicians to enlarge the spectrum of pathogens to be considered in differential diagnoses. further work is needed to map phlebotomine-associated pathogens distribution in relation to environmental and climatic characteristics. educational health programmes seem to have been neglected, when they have been implemented, they have been poorly evaluated in many countries. this fact should be taken into consideration by all public health actors institutions in algeria. acknowledgments we would like to acknowledge all our contributors in the field. viral diseases phlebotomine sand flies are involved in the transmission of several viral agents, among which the most important are grouped into the phlebovirus genus (family bunyaviridae), which includes the sand fly fever sicilian virus and toscana virus, and the vesiculovirus genus (family rhabdoviridae), which encompasses vesicular stomatitis, chandipura and isfahan viruses (maroli et  al. 2013). the risk for infection with sand fly-transmitted phleboviruses has been shown to pertain to very large areas of the old world in association with the presence of sand fly vectors (tesh et al. 1976). in algeria, phleboviruses have also been highlighted and detected in humans (izri et  al. 2008), dogs (tahir et  al. 2016), and sand flies (alkan et al. 2015). a molecular evidence for the presence of a phlebovirus closely related to sand fly fever sicilian virus was detected in a p. ariasi (izri et al. 2008). after that, another investigation conducted by the same team in the north of algeria indicated that a viral sequence from p. papatasi was closely related to, but distinct from, a sequence obtained from p. ariasi (izri et al. 2008), and that two viral sequences from p. longicuspis were genetically distantly related to sequences corresponding to virus members of the sand fly fever naples virus species, although 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175-180. doi: 10.12834/vetit.1763.9298.3 xxxxxxxxxxxxxxxx lafri & bitam catalogue of old world phlebotomine sandflies (diptera: psychodidae). occas pap syst entomol, 8, 1-57. sergent e.d., serget e.t., parrot l., donatien a. & beguet m. 1921. transmission du clou de biskra par le phlébotome (phlebotomus papatasi). crc acad sci paris, 173, 1030. tahir d., alwassouf s., loudahi a., davoust b. & charrel r.n. 2016. seroprevalence of toscana virus in dogs from kabylia (algeria). clin microbiol infect, 22 (3), e16-17. tesh r.b., saidi s., gajdamovic s.j., rodhain f. & vesenjak hirjan j. 1976. serological studies on the epidemiology of sandfly fever in the old world. bulletin who, 54, 663-674. nouveiles sur le développement du parasite de la leishmaniose viscérale du chien chez un phléhotome (phlebotomus perniciosus). arch inst pasteur algérie, 9, 438-441. parrot l., donatien a. & plantureux e. 1941. sur l’infection naturelle des phlébotomes par la leishmaniose générale de i ‘homme et du chien en aigérie. arch inst pasteur algérie, 19, 209-217. perfil’ev p.p.  1968. phlebotomidae. translation of perfil’ev, 1966, diptera: family phlebotomidae fauna. sssr, 93, 1-382. seccombe a.k., ready p.d. & huddleston l.m.a. 1993. 261 1central veterinary research laboratory, united arab emirates. 2european union reference laboratory for equine diseases, anses, laboratory for animal health, bacterial zoonosis unit, maisons-alfort, france. 3department of microbiology, li ka shing faculty of medicine, the university of hong kong, hong kong. *corresponding author at: central veterinary research laboratory, po box 597, dubai, united arab emirates tel.: +971 4 337 5165, e-mail: cvrl@cvrl.ae. parole chiave ematologia, inoculo, melioidosi, sierologia, sottocutaneo, burkholderia pseudomallei. riassunto in questo studio stati infettati sei cavalli con un ceppo di burkholderia pseudomallei isolato da un dromedario infetto deceduto nell'emirato di dubai. il batterio è stato inoculato per via sottocutanea in tre cavalli e somministrato per via orale negli altri tre. quattro cavalli, da cui è stato isolato nuovamente l'agente patogeno, sono risultati sierologicamente positivi ai test oie per la morva. solo uno dei cavalli infettati per via sottocutanea ha avuto un'alterazione febbrile della durata di 3 giorni. i loro valori di globuli bianchi e di neutrofili erano considerevolmente elevati. lo studio ha confermato che nei cavalli il test sierologico per la morva non è sufficiente a differenziare la diagnosi di morva da quella di melioidosi. positività alle prove sierologiche ufficiali per morva in cavalli infettati con burkholderia pseudomallei keywords haematology, inoculum, melioidosis, serology, subcutaneous, burkholderia pseudomallei. summary six horses were challenged experimentally with a strain of burkholderia pseudomallei isolated from a fatal case of the infection in a dromedary camel years earlier in the emirate of dubai. three horses were inoculated subcutaneously and in 3 the bacterium was administered by the oral route. four of the horses became serologically positive based on reactions to one or more of the oie described tests for glanders. b. pseudomallei was re-isolated from the 4  serological positive horses. only one of the subcutaneously infected horses, developed fever for 3 days. the white blood cell values and the neutrophil counts were also elevated. the study confirmed that existing serological test for diagnosing glanders cannot differentiate between glanders and melioidosis in horses. ulrich wernery1* marina rodriguez caveney¹, renate wernery¹, rekha raghavan¹, karine laroucau², ginu syriac¹, shruti miriam thomas¹, jeeba john¹, marina joseph¹, shantymol jose¹, sunitha joseph¹ and patrick woo3 evaluation of serological responses in horses challenged with burkholderia pseudomallei using current diagnostic tests for glanders veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 accepted: 19.04.2019 | available on line: 30.09.2019 caseous lesions in infected organ. b.  pseudomallei, is a motile, gram-negative, facultative anaerobic bacillus which grows readily on routine diagnostic media, but preferably on ashdown’s medium on which it develops a distinctive colonial morphology (markey et al. 2013). b. pseudomallei is usually found in soil and water and may affect a wide range of animal species (lefèvre 2010). in terms of global distribution, melioidosis is predominantly associated introduction melioidosis is an infectious disease which resembles glanders. it is caused by burkholderia (b.) pseudomallei a gram-negative bacillus which share over 99% genetic homology with b. mallei, the etiologic agent of glanders (rainbow et  al. 2002). melioidosis is a disease common to both, animals and people. it is associated with the development of suppurative or 262 glanders serological test in b. pseudomallei infected horses wernery et al. veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 horse boxes in a desert area. two experienced equine grooms were responsible for the horses which were fed timothy hay ad libitum and 2  kg of grain mixture twice a day in the morning and afternoon. all horses had access to fresh water via automatic drinkers in their boxes. the front and rear of the barn were kept closed by big doors which as a precautionary method, were covered with an insect proof-netting outside each door. the entire barn was air-conditioned with no open windows. grooms and researchers entered through a separate side door, which led to a separate room. furthermore, the entrance to this room was secured by an insect proof netting and a mat containing disinfectant. this room was used by 2 grooms and 3 researchers to change into disposable gowns and rubber boots; each person put on three layers of gloves one of which (middle one) had shoulder protection (veterinary gloves, henry schein, usa). besides wearing a particulate respirator n95 mask (3m-8210, mexico) and goggles, all 5 persons also wore an anti-fog, protection-face shield (vwr, usa). inoculum the source of the strain of b.  pseudomallei used for this study was a dromedary camel that died of the infection in 1997 (wernery et  al. 1997). the strain was grown on ashdown’s medium. the inoculum had a concentration of 6.6 x 107 cfu/ml. three of the horses were injected with 2  ml of the inoculum subcutaneously on the left side of the neck (figure  1). the remaining 3 horses were fed a piece of bread that had been injected with 5  ml of the same inoculum. samples blood was collected for serological tests, which are discussed in results section and temperature was monitored daily. blood was withdrawn from the jugular vein until horses were euthanased (please see with tropical and subtropical areas of thailand, vietnam and india, but also with north australia. for many years, it was considered a tropical disease of warm and humid countries (limmathutotsakul et al. 2015). since 1970 however, melioidosis has also been reported in countries with a temperate climate such as parts of africa, southern australia, latin america and europe. on occasion, b.  pseudomallei has been introduced into new environments in which it may cause sporadic disease as was the case with regard to the outbreak in the zoological gardens in paris in 1975 (nouvel et  al. 1976) and in brazil in 1982 (galimand et  al. 1982). shipment of contaminated soil, water and infected animals (ryan et al. 2018) could potentially give rise to melioidosis outbreaks anywhere, including countries in which glanders occurs. on this basis, melioidosis could be considered a re-emerging disease. while serological tests such as the complement fixation, indirect haemagglutination, elisas and other assays are effective herd surveillance tools, they do not differentiate between melioidosis and glanders infected horses. false-positive reactions for glanders may occur in areas where melioidosis is endemic since available serological tests may detect antibodies that cross-react with those of b.  mallei. in the case of melioidosis, no serological tests have yet been validated for use in veterinary medicine (oie 2018). an experimental study of b. pseudomallei infection in horses was undertaken in dubai, united arab emirates, with a triple aims, the first of which was to investigate the clinical signs and pathological lesions of melioidosis in this species. the second objective was to raise positive equine sera to b.  pseudomallei for the purpose of developing serological tests for the diagnosis of the disease in horses and thirdly, to investigate if the sera of infected horses cross-react in currently available diagnostic tests for glanders. the results of the third objective is reported herein. material and methods design study the experiment lasted 2 months. the duration of the study was based on the development of clinical signs and health deterioration, which are detailed in the ‘outcome of the study’ section. horses six retired horses each above 25 years of age and of different gender, all with a degree of intercurrent bone and ligament diseases were selected for this experiment. they were kept isolated in individual figure 1. subcutaneous infection of horse 2 with burkholderia pseudomallei resistance. 263 wernery et al. glanders serological test in b. pseudomallei infected horses veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 subcutaneously and one (3) challenged by the oral route, became infected with b. pseudomallei. horse 1: 30 years of age, had pre-existing chronic arthritis and after subcutaneous infection, it developed fever within 4 days, followed by weight loss, nasal and ocular discharge. abscessation at the site of injection developed on day 7 post infection (pi). horse was euthanased on day 13 pi. horse 2: 31 years of age. the severity of ‘rocked back stance’ which was primarily due to previous chronic ailments, increased. after subcutaneous injection, there was severe swelling at the injection site. mucopurulent discharge from both nostrils and a swelling at the right side of the brisket were observed. the horse was euthanised on day 31 pi. horse 3: 26 years of age. severe swelling of the left chest area followed by weight loss and severe lameness of both front legs was observed after oral infection. the horse was euthanased on day 37 pi. horse 4: 28 years of age, it developed slight fever on day 14 pi. following subcutaneous injection. abscessation at the site of injection was also observed from day 4 pi. and from day 17 pi. the left retropharyngeal lymph node swollened at the size of a golf ball. the animal lost appetite and weight. it was euthanased on day 44 pi. horse 5: 23 years of age, with one eye only. it was severely lame for many years after a fracture of the left tibia. the animal developed mild nasal discharge from day 7 to 13 pi and lost weight. it was euthanased on day 51 pi. horse 6: 28 years of age, it suffered from severe chronic suspensory ligament injury on the right front leg and was emaciated. after oral infection, it developed mild nasal discharge from day 6 to day 12 pi. the horse was euthanased on day 58 pi. outcome of the trial in the results section). the blood was allowed to clot and was transported in cool boxes to cvrl where it was centrifuged and serum was frozen and stored at - 20 °c until tested. additionally, edta blood was also regularly withdrawn and blood parameters (rbc, wbc, pcv, haemoglobulin, neutrophils and lymphocytes) were assessed by using the automatic haematology analyser sysmex xt 2000i (kobe, japan). the reference values for the parameters tested were adapted from the ‘diagnostic cytology and haematology of the horse’ (cowell and tyler 2002). all horses were regularly monitored twice a day, in the morning and evening, rectal temperatures were recorded and also the haematological values were examined during each visit. sera were tested using the following serological tests for the diagnosis of glanders. complement fixation test (cft) using either two antigens; a commercial antigen from c.c.pro (oberdorla, germany) or dubai-7 (scholz et  al. 2006) which was produced according to the oie manual of diagnostic tests and vaccines for terrestrial animals (oie 2018); two enzyme-linked immuno-sorbent assays (elisa), an in-house elisa using dubai-7 antigen and a commercial elisa from idvet, montpellier, france (laroucau et al. 2014). the fifth serological test used was an immunoblot assay (ib) (elschner et  al. 2011). the different serological tests used in this study were carried out in accordance with the detailed methodology described in the manual of diagnostic tests and vaccines for terrestrial animals of the oie in 2018, chapter 2.5.11. results outcome of the trial out of the six trial horses, three (1, 2, and 4) injected table i. summary of clinical signs and pathological lesions observed in 6 horses infected with b. pseudomallei. horse id age in years route of infection clinical signs loss of weight abscess at injection site euthanazed days p.i pathology 1 30 sub cutaneous fever (40 °c), nasal and ocular discharge yes yes, opened 13 pulmonary and renal abscesses; acute hemorrhagic cystitis. no lesions in nasal septum/ conchae 2 31 sub cutaneous no fever, mucopurulent nasal discharge no yes, swelling not opened 31 pulmonary hemorrhages; subacute pyogranulative cystitis. no lesions in nasal septum/ conchae 3 26 oral no fever, lameness of both front legs swelling at chest, no nasal or oral discharge yes swelling left chest 37 granulative cystitis. no pulmonary and renal lesions. no lesions in nasal septum/conchae. 4 28 sub cutaneous slight fever (38.6 °c), weight loss, stopped laming, no ocular or nasal discharge yes yes, opened 44 granulative cystitis. no pulmonary and renal lesions. no lesions in nasal septum/conchae. 5 20 oral none no no 51 no pathological lesions. 6 29 oral none no no 58 no pathological lesions. 264 glanders serological test in b. pseudomallei infected horses wernery et al. veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 haematological profile of horse 1 which also had fever (38.8  °c-39.9  °c) for 4 days (from day 3 to day 8 pi). the white blood cell count (wbc) increased from 12 to 38 106/l and the neutrophil count from 73% to 89%. haematological values did not change considerably in the remaining infected horses (figure 2). serology blood samples were analyzed for antibodies to b.  mallei using 5 serological tests, 2 cfts, 2 indirect elisas and one ib. with the exception of the ib test, serological results are summarized in figure 3. interpretation of the ib test is based on a qualitative threshold, indicated by a protein band (colorimetric method), not by a numerical value. other clinical signs clear to mucopurulent bilateral nasal and eye discharge was observed in horse 1, less copious discharges were also seen in horses 2 and 3. these clinical signs persisted until days 13 pi, 31 pi and 37  pi, respectively. no clinical signs were observed in horses 5 and 6 and they were euthanased within two months. horses subcutaneously infected displayed multiple pyogranulomatous nodules in lungs and kidneys, but did not exhibit conchal ulcers as seen in glanders horses. blood parameters infection with b.  pseudomallei influenced the 65 32 4 1 range high range low65 32 4 1 range high range low 65 32 4 1 range high range low65 32 4 1 range high range low 5 6 7 8 9 10 11 12 13 0 10 20 30 40 50 60 1 0 6 /µ l days post infection a. red blood cells 10 12 14 16 18 20 0 10 20 30 40 50 60 g /d l days post infection b. haemoglobulin 0.3 0.4 0.5 0.6 0 10 20 30 40 50 60 l/ l days post infection c. packed cell volume 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 1 0 6 /µ l days post infection 65 32 4 1 range high range low65 32 4 1 range high range low days post infection days post infection d. white blood cells 25 35 45 55 65 75 85 95 0 10 20 30 40 50 60 % e. neutrophils 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 % f. lymphocytes figure 2. haematological results of 6 horses before and after infection with b. pseudomallei. 265 wernery et al. glanders serological test in b. pseudomallei infected horses veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 the ib and the in-house elisa remained positive for horses 1 and 2 until they were euthanased on days 13 and 31 pi, respectively. on the contrary, horse  2 was negative when tested by the idvet elisa. this discrepancy may be explained by the fact that the recently isolated dubai-7 strain of b.  mallei is antigenically closer to b.  pseudomallei whereas the antigen used in the idvet elisa was an atcc strain of the bacterium. of the 6 horses challenged with b. pseudomallei, this commercial elisa gave positive reactions only on samples collected from horse  1 which developed severe disease. the animal had showed an increasing cft titer which continued to increase until the end of the study. from horse  1 and horse  2 b.  pseudomallei was re-isolated. horse 3, which was orally infected, became cft, elisa using dubai-7 strain of b.  mallei and ib positive from day  21  pi. its cft titre remained low and the idvet elisa negative. the animal was euthanased on day 37 pi as in the case of horse 2. also from this animal b. pseudomallei was re-isolated. horse 4, which was infected subcutaneously, did not produce cft and idvet elisa antibodies but reacted with the dubai-7 strain of b. mallei elisa and ib assay from day 21 pi until day 44 pi. as for horse 1, 2, and 3, b.  pseudomallei was also re-isolated from horse  4. although due to the exiguous sample size any conclusion should be interpreted with caution, all subcutaneously infected and one orally infected horses (horse 3) developed antibodies, detected by cft and / or elisa. horses 5 and 6 which were challenged by oral route remained seronegative throughout the study period. horses 1 and 2 which were infected by the subcutaneous route developed a detectable serological response in both, elisa and ib tests, starting from day 7 pi. three days later horse 1 was positive to both cfts, while horse 2 from day 10 pi to ccpro and from day 35 to the dubai-7 cft. discussion the six horses used for this experimental study of b. pseudomallei infection were above 25 years of age and all suffered since many years from ligament and bone diseases. in the 10 years preceding the study, they had been kept in a horse retirement enclosure and were to have been euthanased shortly because of age and clinical ailments. however, permission was granted to include them in the b.  pseudomallei challenge study before euthanasia (see animal welfare issue). as it is evident from figure 3, three subcutaneously (horses 1, 2 and 4) and 1 orally infected horses (horse 3) developed antibodies. cfts, cut-o� 654 321 cut-o� 654 321 cut-o� 654 321 cut-o� 654 321 a. idvet elisa assay b. in-house elisa c. complement fixation test ccpro d. complement fixation test dubai-7 0 10 20 30 40 50 60 70 80 0 7 14 21 28 35 42 49 56 % o f p o si ti vi ty days post infection 0 0.2 0.4 0.6 0.8 1 1.2 0 7 14 21 28 35 42 49 56 o p ti ca l d en si ty days post infection 0 50 100 0 7 14 21 28 35 42 49 56 63 1/ n days post infection 0 50 100 150 200 250 300 350 0 7 14 21 28 35 42 49 56 63 1/ n days post infection figure 3. serological results of 6 horses infected with b. pseudomallei using 4 serological tests for diagnosing glanders. 266 glanders serological test in b. pseudomallei infected horses wernery et al. veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 which it has not been investigated yet. it is hoped to use the sera obtained from this study as the basis for further studies that hopefully will lead to a serological test for the diagnosis of melioidosis in equids. statement of animal rights when reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. acknowledgement the authors herewith express their gratitude to h. h. sheikh mohammed bin rashid al maktoum, ruler of dubai and vice president of the united arab emirates as well as saeed al tayer, chairman of dubai racing club and dr. ali ridha, director general of the central veterinary research laboratory. we thank the following persons who helped us with this project: brenda cooke, heather copland, zulfiqar ali kiani, tahir zaman, grooms: shyam singh and karan singh. according to these findings, the idvet elisa appears to be more specific than the other serological tests used in this study. horses 5 and 6, which were both orally challenged, did not seroconvert until the entire length of the trial. b.  pseudomallei was not re-isolated from these horses. horses which produced antibodies after infection also showed lesions in some organs. the two serologically negative horses, instead, did not have any observable lesions. this study confirmed that the serological diagnosis of melioidosis is difficult and that glanders and melioidosis cannot be differentiated based on current diagnostic tests for glanders. although specific assays have been developed to detect antibodies to b.  pseudomallei, most of them use poorly characterized antigens and are not standardized (wiersinga et  al. 2018). a protein microarray that contains 20 recombinants and purified b.  pseudomallei proteins, provides a standardized test for detection of antibodies in humans (kohler et al. 2016). a similar approach may have the potential to improve the serodiagnosis of this infection in other species including equids in 267 wernery et al. glanders serological test in b. pseudomallei infected horses veterinaria italiana 2019, 55 (3), 261-267. doi: 10.12834/vetit.1701.9026.2 cowell r.l. & tyler r.d. 2002. diagnostic cytology and haematology of the horse. 2nd ed. mosby, st louis, p. 201. elschner m.c., scholz h.c., melzer f., saqib m., marten p., rassbach a., dietzsch m., schmoock g., de assis santana v.l., de souza m.m., wernery r., wernery u. & neubauer h. 2011. use of a western blot technique for the serodiagnosis of glanders. bmc vet res, 7, 4. galimand m. & dodin a. 1982. le point sur la mélioidose dans le monde. bull soc path exot, 75, 375-383. kohler c., dunachie s.j., müller e., kohler a., jenjaroen k., teparrukkul p., baier v., ehricht r. & steinmetz i. 2016. rapid and sensitive multiplex detection of burkholderia pseudomallei specific antibodies in melioidosis patients based on a protein microarray approach. plos negl trop dis, 10(7): e0004847. laroucau k., bertin c., roche m., donnet f., colaneri falcão m.d., madani n., zientara s., pourquier p., santana v.l., saqib m. & comtet l. 2014. a new elisa assay for glanders diagnosis. iii pan-american congress of zoonoses, la plata, 4-6 june 2014. lefèvre p.-c., blancou j., chermette r. & uilenberg g. 2010. infectious and parasitic diseases of livestock. paris, lavoisier, 1189-1196. limmathurotsakul d., golding n., dance d.a.b., messina j.p., pigott d.m., moyes c.l., rolim d.b., bertherat e., day n.p.j., peacock s.j. & hay s.i. 2016. predicted global distribution of burkholderia pseudomallei and burden of melioidosis. nat microbiol, pii: 15008.2015. markey b., leonard f., archambault m., cullinane a. & maguire d. 2013. clinical veterinary microbiology. 2nd ed. mosby, elsevier, 275-284. references nouvel j., dodin a., demontory m.c. & chove m.a.1976. la mélioidose à la ménagerie du jardin des plantes de paris. bull acad vet, 49, 223-230. rainbow l., hart c.a. & winstanley c. 2002. distribution of type iii secretion gene clusters in burkholderia pseudomallei, b. thailandensis and b. mallei. j med microbiol, 51, 374-384. ryan c.w., bishop k., blaney d.d., britton s.j., cantone f., egan c., elrod m.g., frye c.w., maxted a.m. & perkins g. 2018. public health response to an imported case of canine melioidosis. zoonoses public health, 65 (4), 420-424. scholz h.c., joseph m., tomaso h., al dahouk s., witte a., kinne j., hagen r.m., wernery r., wernery u. & neubauer h. 2006. detection of the reemerging agent burkholderia mallei in a recent outbreak of glanders in the united arab emirates by a newly developed flip based polymerase chain reaction assay. diagn microbiol infect dis, 54, 241-247. wernery r., kinne j., hayden-evans j. & ul haq a. 1997. melioidosis in a seven-year-old camel, a new disease in the united arab emirates (uae). j camel pract, 4 (2), 141-143. wiersinga w.j., virk h.s., torres a.g., currie b.j., peacock s.j., dance d.a.b. & limmathurotsakul d. 2018. melioidosis. nat rev dis primers, 4, 17107. world organisation for animal health (oie). 2018. manual of diagnostic tests and vaccines for terrestrial animals. chapter 2.5.11 glanders and mmelioidosis. paris, oie. 201 veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 accepted: 17.06.2019 | available on line: 31.12.2021 1kazakh scientific research veterinary institute, kazakhstan. 2diavak-abn scientific and production center, kazakhstan. 3kazakh national agrarian university, kazakhstan. *corresponding author at: kazakh national agrarian university, kazakhstan. e-mail: m.umitzhanov@mail.ru. mynbay umitzhanov3*, asiya k. musaeva1, abdikalyk a. abishov2, toktar m. zhamansarin3, urzhan zh. omarbekova3, sholpan zh. turyspayeva3 and sarsenbek t. siyabekov3 keywords parenchymal organs, lymphohistiocytic proliferative, pneumonia, bronchospasm, echinococcosis. summary the authors have conducted experiments to study the pathoanatomical and histological pattern of organs and tissues of adult sheep affected by unsymmetric dimethylhydrazine (udmh). this highly toxic fuel was spilled on the territory of the karsakpay and ulytau districts of karaganda region, kazakhstan, because of the fall of the rocket ‘proton-m’ after an unsuccessful launch from the baikonur cosmodrome in 2007. in the experiment, the study group was consisted of 7 adult sheep that grazed in the area of possible intoxication with rocket fuel udmh. the main objects of the study were histological preparations obtained from “fixed structures” live and dead cells and tissues. as the structures have a flat contrast and are poorly detected in the ordinary light microscope, the specially processed preparations were used. after preparing, the authors studied organs and tissues using a microscope, which allowed to reveal in detail the level of damage caused by intoxication and to establish the negative effect of udmh on the internal organs. the group of sheep showed a high index of macroscopic signs of interstitial pneumonia (85.7 ± 14.3%), and histologically quite high index was granulomatous inflammation of liver (71.4 ± 18.4%). kidneys also showed a high level of abnormalities. examination of organs and tissues of adult sheep grazed in an area with possible intoxication with rocket fuel, kazakhstan alcohols, ethers. the molecular weight is 60.08. udmh is a highly dangerous compound with strong irritant properties. its vapors irritate the mucous membranes of the eyes and respiratory tract. the effect of udmh at a concentration of 400 mg/m3 causes a person to be fatal poisoning. the strong odor of udmh is felt at its concentration in the air above 5.0 mg/m3 in the first minutes of contact. subsequently, olfactory adaptation may occur (schmidt 2011, ushakova et al. 2004). the accumulation, transformation and migration of any toxicant generally depends on its chemical nature and possible variants of transformations in biological objects, as a result of which secondary and even tertiary pollutants of the environment can be formed, which are even more dangerous than the raw material itself. according to the literature data, udmh, a derivative of hydrazine, is a sufficiently reactive substance. in chemical introduction in peacetime, the damage by the rocket fuel components generally occurs in emergencies, violation of safety rules during fueling, as well as in the destruction of storage (petrenko 2006, nauryzbaev et al. 2006). the most common type of liquid fuels is two-component rocket fuels, consisting of oxidizing agent and combustible agent. oxidizing agents are nitric tetraoxide and nitric acid, and combustible agent is unsymmetrical dimethylhydrazine (udmh or heptyl). this composition is self-igniting upon contact of the components with each other, which simplifies the engine start system and reduces the risk of explosion in the combustion chamber (fedorov 2005). udmh is a colorless, transparent, highly volatile liquid with a strong unpleasant smell. the substance is hygroscopic, readily soluble in water, hydrocarbons, 202 veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 possible intoxication with rocket fuel in kazakhstan umitzhanov et al. be carried out in transmitted light when light passes through a thin transparent histological preparation, or in reflected light, when, for example, a thick or opaque object is examined. similarly, electron microscopy can be a transmission microscopy, when an electron beam passes through the studied ultrathin slice, or raster microscopy or scanning microscopy, when the electron beam is reflected from the surface of the studied object. the authors studied the tissues and organs of adult sheep, which allowed to reveal in detail the level of damage caused by intoxication (back and thomas 1963). results and discussion echinococcosis of the liver and lungs in the pathoanatomical examination of organs and tissues in seven adult sheep, macroscopic changes were detected in four animals (no. 4235, no. 3223, no. 06931, no. 5470), which were presented as granulomatous inflammations, mainly in the liver, less often in the lungs (figure 1). one ewe no. 4235 had visible macroscopic changes in liver and lungs at the vesicular stage of echinococcosis. they were characterized by multiple polymorphic foci of greyish-greenish or greyish-bluish color with a size of 3 х 5 cm mainly with surface-subcapsular localization. fibrotic folds with costal pleura were observed in places of their localization in the lungs. among the echinococcal vesicles there were single, but denser (calcified) small gray-white areas, consisting of two or three paired foci from 1 to 2 cm. the liver in the areas of absence of granulomas was slightly indurated, gray-brown or light-brown in color; the gallbladder contained a small amount of bile (30 ml). in one sheep, a disorder of the coordination of nature, this substance is a strong reducing agent, the oxidation of which causes the release of more substances that have a negative effect on the biota (clements and stoye 2014). udmh is readily soluble in water, alcohols, amines and mixed with petroleum products and many organic solvents. udmh forms mixtures with water with significant heat release (tulupov et al. 1991). materials and methods the authors conducted experiments to study the pathoanatomical and histological pattern of organs and tissues of adult sheep affected by unsymmetric dimethylhydrazine (udmh, heptyl). this highly toxic fuel was spilled on the territory of the karsakpay okrug of ulytau district of karaganda region as a result of the fall of the rocket ‘proton-m’ after an unsuccessful launch from the baikonur cosmodrome in 2007 (zhubatov et  al. 2006). the experiment was conducted in 2018. four rams and 3 ewes (animal age 5 years), which grazed in the zone of possible intoxication with udmh rocket fuel, were studied. the main stages of cytological and histological analysis were the choice of the object of the study, its preparation for microscopic examination, the use of microscopic methods of investigation, and the qualitative and quantitative analysis of images (nurtazin et  al. 2006). the objects of the study were live and dead (fixed) cells and tissues, as well as their images obtained as a result of light and electron microscopy. the main objective of the study were histological preparations obtained from fixed structures. samples can be an imprint (for example, from spleen, thymus, liver), a film from the tissue (for example, connective tissue or peritoneum, pleura) and a thin section (bratkov et  al. 1987). the authors selected tissue and organ sites for the experiment (liver, lungs, thyroid gland). histological preparations can be studied without special treatment (winter et  al. 2015). but due to the fact that the structures have a flat contrast, they are poorly detected in the ordinary light microscope, and the use of special microscopes (phase-contrast, etc.) is required. therefore, specially processed preparations, i.e. fixed, embedded in a solid medium and stained are used more often. the process of making the histological preparation included the following main steps: sampling and material fixing, compacting, section cutting, staining or contrasting the sections (angell et  al. 2015, angell et al. 2018). after preparing, the authors studied organs and tissues using a microscope. microscopy allows to examine an object at the cellular level to determine the level of exposure to organs. microscopy can be light and electron microscopy. light microscopy can figure 1. internal organs of the ewe no. 4235: multiple large‑focal echinococcosis of the liver and lungs in a vesicular form in the calcification stage. 203veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 umitzhanov et al. possible intoxication with rocket fuel in kazakhstan of islets (ferreira et al. 2016). following the necrotic mass there was a layer with a perifocal, rich cell composition of polynuclears, histiocytes and a small number of giant cells of pirogov-langhans (figure 2). the third layer was a capsule of dense connective tissue, consisting of fibrocyte-fibroblastic cells from groups of modified, multinucleated hepatocytes. the fourth layer (densely adjacent to the third layer) was consisted of homogeneous cellular structures, but also characterized by heterogeneity, multinuclear cells-hepatocytes with a symplastic and dense layer growth. the layer of pericapsular hepatocytes is very different from the comparatively distant, described hepatocytes. giant multinucleated liver cells are formed from aggregated cells 3 x 4, round, sometimes oval in shape and contain 3, 4 or 5 nuclei with condensed basophilic-eosinophilic cytoplasm. a large and variant number of described macroscopic granulomas indicate a development in the liver of more significant processes with a tendency to form connective tissue and cirrhosis (figure 1). the histostructure of the section from more distant from granulomas site of animal liver no. 06931 characterizes the cytolysis of hepatocytes with significant disturbance of the hemodynamics of the microcirculatory bloodstream, a significant extension of the sinusoids, the deposition of the fibrillar structure of fibrin, the discomplexation and atrophy of the hepatic tubules with the proliferation of the stellate endothelial cells and endothelium. in histopreparations from more distant site of the liver in animal no. 5470, small granulomas with a size of 15 x 25 µm could be seen intralubularly. in the perivascular vessels and ducts of extralobular zones, small granulomas with lymphohystocyte cells and fibroblasts were found. there were also lymphoid infiltration and erythrocyte stasis, dystrophic-necrobiotic changes in hepatocytes in the swelling state in some lobules. the other adult animals (no. 5550, no. 5056, no. 3223, no.  4235) movements in the form of a blind-staggers (a true turn sickness) was observed. however, at the autopsy, 5 larvae of oestrus ovis in the frontal sinus area were found, the so-called and known veterinary disease – sheep oestrosis. the integrity of the anterior part of the cerebral cavity was broken; polycystosis was found on the right side of the brain, in three areas; the brain was atrophied, gray-dirty in color; gray and white substances were indurated, thinned and pale (petersen et  al. 2018). the nasal cavity in the area of the ethmoid bone was dirty-green in color, with ichorous smell. the stud ram no. 06931 had changes that differed somewhat from the previous cases in that among the gray-yellow indurated foci there were gray, relatively large, tallow-like, considerably protruding above the liver surface, multiple growths 3 x 4 cm in size with soft consistency. in addition, along with the above described pathology of granulomatous inflammation, in two areas of the liver of stud ram no. 06931 there were small-focal, round-oval, exploded immature granulomas with infiltrative growth, wherein an occupied area was about 10 х 5 cm. the stud ram had ruffled hair, fatness below the average, frank thinning, pallor of the eye sclera and change in vesicular breathing before the slaughter. against the background of more frank granulomatous inflammations, perifocal encapsulation, proliferation of connective tissue, slight indurations, as well as flabbiness and congestion were observed in the liver of animals. also, slight changes in the lung tissue were detected. in this case, a slight corrugation of the capsule, pink-reddish color of the tissue and monotonicity, despite the bleeding by decapitation, were detected. liver granulomatosis the most significant granulomatous inflammation was found in the liver of two adult sheep. the stud ram no. 06931 had small multiple microscopic visual intralobular or extralobular granulomas. histologically granulomas were characterized by a rounded shape, of different size with necrosis or without necrosis in the center. some of them contained lymphohystocytes, fibroblasts and fibrocytes with a tendency to form connective tissue. generally, such granulomas did not have foreign bodies, fragments or detritus of parasitic origin and cholicosis in the center. the granulomas were larger, partially visualized without a microscope, and had a more complex, layered structure and consisted of 4 layers, i.e. with necrosis in the center and the distinctiveness of cellular proliferates in the direction from the center to the periphery. in the center of the granuloma there was a cell necrotic mass with an admixture of groups of hepatocytes in the form figure 2. the liver of stud ram no. 06931: granuloma with necrosis in the center, a giant cell such as pirogov‑langhans cell, hypertrophic cirrhosis. 204 veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 possible intoxication with rocket fuel in kazakhstan umitzhanov et al. subacute glomerulonephritis many animals (no. 5056, no. 5470, no. 4235) had an extracapillary serous edema expressed in varying degrees; less often they had mild plasmorrhage with expansion of the extracapillary space, compression and atrophy, necrosis and necrobiosis of the glomerulus with eccentric localization. in addition, quite often the studied adult sheep had intracapillary hyperemia, palmateness, expansion of the interstitium, plasmorrhage, less often they had edema; some of the sheep had granular dystrophy and excretory insufficiency of tubules (figure 4). the dimensions of the glomerular capsule were 150  x  90  µm, and the dimensions of the glomeruli were 65 x 120 µm. morphological criteria were collectively classified as subacute glomerulonephritis. as a rule, in the vast majority of cases, we observed a hypoexcretory function with tubulopathy processes with a total delay of eosinophilic, homogeneous and small (from 0 to 1.5 x 2 µm) substance in the center in the lumens, with simultaneous rejection of the apical part of the nephrocyte of the straight and convoluted tubules of the kidney. disorders of the normal structure of the thyroid gland endocrine glands, including the thyroid gland, are actively involved in the period of adaptation of the organism to adverse environmental influences. in functional terms, the thyroid gland performs regulation of metabolic, thermoregulatory, reparative and other processes, which under a certain pathology undergo a change in their coloidal secretory structural-morphological unit. in five sheep out of seven, the thyroid gland functionally and morphologically had definite changes, in particular in the secretory-colloidal sector and its cell proliferative components. in general, the gland had follicles of round-oval had less significant changes in the liver. they were characterized by the presence of small single granulomas with activation of stellate cells with slight expansion of sinusoids and maintenance of the normal structure; macroscopic findings were echinococcosis and oestrosis of sheep with a lesion of the nervous system and a slight exacerbation of vascular disorders. interstitial pneumonia as a result of scanning the sections of the lung parenchyma of the lungs of sheep no. 069131 and no. 5056 with light-optical microscope, we saw the development of focal-spread interstitial pneumonia (figure 3). in this case, the alveolar septa were unevenly thickened (650 x 200 µm) and accompanied by a frank congestion of the capillaries, diapedesis hemorrhage in the perifocal zones. in sheep no. 5056 we observed activation and proliferation of alveolar epithelium with conversion into macrophages. respiratory epithelium is not straightened, more often is dentiform. a mild peribronchial fibrosis, was noted bronchospasm and the formation of small outgrowths of up to 100 µm in the inside of the lumen of the bronchi and bronchioles of the respiratory epithelium. in the perifocal sites, we could see the thinning and breaking of the septum, and the increase in the size of the alveoli with the activation of regional processes of emphysema. in some places they reached a size of up to 200  x  100  µm. these processes were somewhat less significant in the ram no. 5056, but there was a frank vascular disorder, provoking old foci of interstitial pneumonia. changes in foci of interstitial pneumonia were found in ewes no. 06980, no. 3232, no. 4235. the results of histological changes indicate the development of interstitial pneumonia processes with periods of exacerbation against a background of mild vascular disorders of hemodynamics in the microcirculatory bloodstream of the lungs. figure 3. lung of the ram no. 5056: small‑focal interstitial pneumonia. figure 4. kidney of the ewes no. 4235: acute glomerulonephritis. 205veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 umitzhanov et al. possible intoxication with rocket fuel in kazakhstan background of the proliferates of the interfollicular zone (figure 6). thus, interpreted morphological criteria indicate the manifestation of hyposecretion with a disorder of the normal structure of the thyroid gland, which occurs as a result of residual exudative-proliferative and age-related changes in the basic functional structures of the parenchyma. as is known from literature sources (simonyan and khisamutdinov 1995, ioffe et  al. 1979, zrelov and seregin 1975), the adrenal gland, participating in the endocrine system of the animal organism, actively participates in the restoration of disturbed homeostasis, weakened by various disorders and damages of various body systems. the histostructure of the adrenal glands was the same in most adult sheep. adjacent to the adrenal gland extraorganic tissue and connective tissue capsule was loosened, disorganized and had a serous edema in some areas. quite often, the most significant shape and varied size. despite the age and sex characteristics of animals, large forms and microfolliccles, expressed in interstitium, were dominant. the stud rams no.  06931, no. 5470 and ewes no. 3223 and no. 06980 can be classified as such animals. most animals had a moderate, and the animal no. 066931 (figure 5) had a more significant interstitial edema of the septum with a disturbance in the normal structure. despite the dominance of large follicles, we observed many generating microfollicles. lymphohistiocytic proliferates were in the interfollicular zones. we observed activation of cell flattening from low cubic epithelium, a marked decrease in secretion properties of thyroid cells, indicating the detachment of the parietal part of the colloids or the absence of colloids and diffusely-spotty vacuolization of colloid-unfilled follicles. some animals had mild violation of the integrity of the follicle, desquamation and proliferation of the epithelium in the intra-follicular fluid against the figure 5. thyroid gland of the ram no. 06931: interstitial serous edema, total hypofunction of the gland. figure 6. thyroid gland of the ewe no. 3223: gland dysfunction, small‑follicular hypoxecretion table i. statistical data of sectional and histological studies of the organopathology of adult sheep. pathology of organs and tissues small ruminants (adult sheep) number of examined number of affected percentages m± m± liver echinococcosis a) vesicular stage; 7 2 28.6 18.4 b) calcified and uncalcified small foci. 7 2 28.6 18.4 echinococcosis of the lungs a) vesicular stage 7 1 14.3 14.3 liver granulomatosis a) multiple granulomas with necrosis, giant cells such as pirogov-langhans cells and foreign bodies; 7 1 14.3 14.3 b) single granulomas without necrosis; 7 5 71.4 18.4 dystrophy and necrobiosis of hepatocytes 7 6 85.7 14.3 expansion of sinusoids, deposition of fibrillar structures in sinusoids. 7 2 28.6 18.4 interstitial pneumonia 7 6 85.7 14.3 subacute glomerulonephritis 7 5 71.4 18.4 disorders of the normal structure of the thyroid gland 7 4 57.1 20.2 206 veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 possible intoxication with rocket fuel in kazakhstan umitzhanov et al. signs of interstitial pneumonia (85.7 ± 14.3%) was established, and histologically, granulomatous inflammation of the liver had a rather high index (71.4 ± 18.4%). granulomatous inflammation was accompanied by dystrophic-necrobiotic processes (85.7 ± 14.3%) of hepatocytes. the percentage ratio of multiple granulomas in the liver with necrosis and fibrosis and the presence of giant pirogov-langhans cells in them was within 14.3%. in addition, the authors noted the significant expansion of sinusoids of hepatocytes in adult sheep, followed by the deposition of fibrillar structure (28.6 ± 18.4%), such as pre-stage of granulomatosis and single microgranules. among organ pathology, the frequency of histological changes in the kidneys was also highwhich was accompanied by extra(71.4 ± 18.4%) and intracapillary (85.7 ± 14.3%) exudative process in glomeruli and the development of dystrophy and necrobiosis of tubular nephrocytes (71.4 ±  18.4%), granulomas and necrosis (71.4 ± 18.4%) and a decrease in the excretory function of the kidney (71.4 ± 18.4%). at the same time, it should be noted that the morphofunctional differences of the thyroid gland were decreased (57.1 ± 20.2%). changes were observed in zona fasiculata of the animals, which is due to a local-total disorders of hemodynamics. at the same time, they were characterized by large-rounded, sometimes oblong, focal degenerative-destructive changes, swelling of adrenocorticocytes, a decrease in dark cells, and increase in light cells. discomplexation with the phenomenon of vagueness of the contours of cells, their dystrophy and necrobiosis were quite distinct. in addition, we observed a significant loosening, expansion of capillaries, plasmorrhagia and enlightenment of zona fasiculata. statistical data of sectional and histological studies of the organopathology of adult sheep in the zone of possible udmh intoxication after the emergency fall of the rocket ‘proton-m’ are presented in table i. conclusions the authors conducted sectional and histological studies of the detection frequency of organopathology in adult sheep in the area of possible intoxication with unsymmetric dimethylhydrazine after the fall of the rocket ‘proton-m’. a high percentage of the development of macroscopic 207veterinaria italiana 2021, 57 (3), 201-207. doi: 10.12834/vetit.1680.8924.3 umitzhanov et al. possible intoxication with rocket fuel in kazakhstan angell j.w., grove-white d.h. & duncan j.s. 2018. sheep and farm level factors associated with footrot: a longitudinal repeated cross-sectional study of sheep on six farms. vet rec, 182 (10), 293. back k.g. & thomas a.a. 1963. pharmacology and toxicology of 1, 1-dimethylhydrazine (udmh). am ind hyg ass j, 24 (1), 23-27. bratkov a.a., seregin e.p. & gorenkov a.f . khimmotologiya raketnykh i reaktivnykh topliv [chemmotology of rocket and jet fuels]. moscow, khimiya, 1987. fedorov l.a. 2005. study on persistent organic pollutants. green world, 1‑2, 22-23. ferreira m.g., duarte c.g., oliveira m.s. & castro k.l.p. 2016. toxicity of crude and detoxified tityus serrulatus venom in anti-venom-producing sheep. j vet sci, 17 (4), 467-477. ioffe b.v., kuznetsov m.a. & 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implementation of programs to assess the impact of launches rockets from the cosmodrome “baikonur” on the environment and human health. scientific and practical conference, 27-29 june, almaty, karaganda. zrelov v.n. & seregin e.p. 1975. liquid propellant. moscow, chemistry. 219 introduction porcine circovirus type 2 (pcv2) is one of the smallest, non‑enveloped, single‑stranded dna virus belonging to the circovirus genus of the circoviridae family (meehan et  al. 1998). several studies have confirmed the association of the pcv2 infection with disease syndromes collectively named porcine circovirus diseases (pcvd). the predominant clinical signs of pcvd are wasting and retarded growth among grow‑finish pigs (harding et  al. 1998, cino‑ozuna et  al. 2011). among pcvd porcine circovirus systemic disease (pcv2‑sd) (previously named post‑weaning multi‑systemic wasting syndrome) is one of the most devastating and economically damaging diseases to the swine industry (segalés 2012). in the european union, the cost of pcv2‑sd was assessed to be between 1 department of swine diseases, national veterinary research institute, 24-100 pulawy, poland. 2 department of preclinical sciences and infectious diseases, faculty of veterinary medicine and animal science, poznań university of life sciences, 60-637, poznań, poland. * corresponding author at: department of swine diseases, national veterinary research institute, 24-100 pulawy, poland. tel.: +48 81 889 34 01, e-mail: ewelina.czyzewska@piwet.pulawy.pl. parole chiave porcine circovirus di tipo 2, maialini da latte, vaccinazione, condizioni di campo, efficacia vaccinale. riassunto l'obiettivo di questo studio è stato determinare, in condizioni di campo, l'effetto della vaccinazione pcv2 sui livelli di viremia, il numero di suini viremico‑positivi e i parametri di produzione. questo studio ha interessato 140 allevamenti di maiali da ingrasso. la vaccinazione è stata attuata in 82 delle 140 mandrie. sono stati raccolti campioni di sangue da tutte gli allevamenti e, per ciascun allevamentoa, è stato fornito un questionario relativo ai parametri di produzione. i risultati dimostrano che la vaccinazione dei maialini ha impedito lo sviluppo della viremia nel 23,2% degli allevamenti. nel siero e nel numero di suini positivi sono stati inoltre osservati diminuzioni significative dei livelli di dna di pcv2. sorprendentemente, non è stata osservata nelle mandrie vaccinate l'influenza significativa della vaccinazione sui parametri di produzione. effetto della vaccinazione contro il porcine circovirus 2 (pcv2) in condizioni di campo keywords porcine circovirus type 2, piglet, vaccination, field conditions, vaccine efficacy. summary the objective of this study was to determine the effect of the porcine circovirus type 2 (pcv2) vaccination on the levels of viremia, the number of viremic‑positive pigs, and production performance [i.e. nursery mortality, post‑weaning mortality, and average daily weight gain (adwg)] under field conditions. there were 140 farrow‑to‑finish pig herds involved in this study. the vaccination of piglets was implemented in 82 of the 140 herds. in each herd blood samples were collected from sows and pigs in different age category. in addition, a questionnaire regarding the production performance was provided for each herd. results demonstrate that the vaccination of piglets prevented the development of viremia in 23.2% of herds. significant decreases in the levels of pcv2 dna in serum and in the number of viremic pigs were also noted. these results indicate that the vaccination of piglets against pcv2 is a useful tool in controlling the pcv2 infection in herds with a high risk of a wide range of viral and bacterial agents, poor management strategies, and a low level of biosecurity practices. ewelina czyżewska‑dors1*, arkadiusz dors1, małgorzata pomorska‑mól1,2, katarzyna podgórska1 and zygmunt pejsak1 efficacy of the porcine circovirus 2 (pcv2) vaccination under field conditions veterinaria italiana 2018, 54 (3), 219‑224. doi: 10.12834/vetit.1009.5377.3 accepted: 20.05.2017 | available on line: 30.09.2018 220 veterinaria italiana 2018, 54 (3), 219‑224. doi: 10.12834/vetit.1009.5377.3 pcv2 to piglets at the age of 3‑4 weeks as a part of their regular farming practices. in 61.4% and 8.6% of herds, vaccinations against enzootic pneumonia and porcine pleuropneumonia were administered to piglets and/or growers, respectively. immunization programmes for sows included vaccination against porcine parvovirus, erysipelothrix rhusiopathiae, colibacillosis, and porcine reproductive respiratory syndrome virus (prrsv) in 92.3%, 90.6%, 54.1%, and 9.3%, respectively. in none of the examined herds sows were vaccinated against pcv2 and swine influenza virus (siv). the herds were affected by several endemic diseases. specific antibodies (avoiding vaccination interferences) against mycoplasma hyopneumoniae, actinobacillus pleuropneumoniae, siv, pcv2, and prrsv were detected in 85.2%, 96.1%, 87.9%, 100%, and 37.8% of the investigated herds respectively. low levels of biosecurity practice, inadequate sanitation, and poor management strategies were observed in all of the examined farms. five established biosecurity measures – a fence around the farm, disinfection mats within the herd, the use of boots and clothes provided by the farm, a changing room with showers, and quarantine for purchased pigs – were only implemented in a very low percentage of investigated farms. above mentioned biosecurity measures were applied in 4.9% of farms vaccinated piglets against pcv2 and in 3.5% of farms non‑vaccinated piglets against pcv2. the adoption of an all in – all out (aiao) pig flow at all production stages (i.e. farrowing, nursery, and finishing) was respected in 22% and 10% of farms with piglets vaccinated and non‑vaccinated against pcv2, respectively. however, significant differences were not observed (data not shown). serum samples in total, 1,160 and 760 serum samples were collected from vaccinated and non‑vaccinated pigs, respectively. blood samples were taken randomly, from sows and pigs that were 4‑5, 6‑7, 8‑9, 10‑11, 12‑13, 14‑15, 16‑17, 18‑19, 20‑21, and 22‑24 weeks old. in a particular farm, the number of different age groups selected to collect the serum samples depended on the herd size and the types of batch farrowing system (1‑, 2‑, 3‑, 4‑ week farrowing batch interval) operated in the farm. six pigs in each age group were always sampled. production performance data regarding production performance, including nursery mortality, overall post‑weaning mortality, and adwg were gathered using a questionnaire at each farm. to minimise confusion and maximise €  562 million and €  900 million per year. the need for the introduction of effective control strategies is therefore essential (segalés and domingo 2002, tucker 2006, alarcon et al. 2013). one of the most effective pcvd control strategies is the administration of pcv2 vaccines in piglets/ weaners and/or sows, before farrowing. the vaccination of sows has been shown to reduce the prevalence of pcv2 viremia and improve the performance of their offspring (pejsak et  al. 2010, seo et  al. 2014). vaccines are mainly administered to newborn piglets in herds with a high risk of pcv2 infection. several studies have found that the vaccination of piglets against pcv2 significantly reduces the viral load in their blood and thus the prevalence of positive animals, which in turn is associated with a reduction of clinical signs and improved production performance (fort et al. 2008, horlen et al. 2008, kixmoller et al. 2008, martelli et al. 2011, hemman et al. 2012, heissenberger et al. 2013, seo et al. 2014). previous studies investigating the efficacy of the pcv2 vaccination under field conditions were either performed within single farms with relatively healthy animals, good management practices, and high levels of biosecurity (cline et  al. 2008, fachinger et  al. 2008, jacela et  al. 2011) or with no data regarding management, biosecurity, and environmental conditions (lyoo et al. 2011, martelli et al. 2011, heissenberger et al. 2013). little is known about the efficacy of the pcv2 vaccination with regards to levels of viremia and production performance on farms with low health status, poor management strategies, and inadequate biosecurity, sanitation, and environmental conditions. the aim of this study was to address this gap by assessing the efficacy of the pcv2 vaccination on farms with poor management strategies, low levels of biosecurity practices, and inadequate sanitation. selected production performance (i.e. nursery mortality, post‑weaning mortality, and adwg), the level of pcv2 dna in serum, and the proportion of viremic pigs were evaluated. materials and methods herd characteristics the study design was discussed and approved by the polish institutional ethics committee (allowance number: 37/2014). the study was conducted in 140 conventional, farrow‑to‑finish pig farms, with between 22 and 2,000 (200.67  ±  336.39) sows per farm. eighty‑two (58.6%) of the 140 farms administer vaccines against efficacy of the porcine circovirus 2 vaccination czyżewska‑dors et al. 221veterinaria italiana 2018, 54 (3), 219‑224. doi: 10.12834/vetit.1009.5377.3 all different age categories) as compared to the non‑vaccinated group (p < 0.05). these results indicate lower viral loads (i.e. the level of virus in the blood) in pigs vaccinated against pcv2, compared to their non‑vaccinated counterparts (figure 1). association of vaccination with the number of positive herds and the proportion of viremic pigs pcv2 dna was detected in all of the non‑vaccinated herdsand in 68 (82.9%) out of 82 vaccinated herds. the total number of positive pigs was significantly reduced in vaccinated herds, compared to their non‑vaccinated counterparts (p <  0.05). the same trends were observed in all different age categories (figure 2). the lowest prevalence of pcv2 dna was observed in pigs aged 4‑5 weeks or 6‑7 weeks, from vaccinated and non‑vaccinated herds, respectively. the highest prevalence of the pcv2 virus was observed in pigs from 14 to 21 weeks of age, in the case of both vaccinated and non‑vaccinated herds. association of vaccination with production performance none of the evaluated production parameters were significantly affected in vaccinated herds compared to non‑vaccinated herds (p > 0.05) (table i). discussion in recent years, a wide range of commercial pcv2 vaccines, designed to diminish the negative impact of pcvd in pigs have become available. previous experimental and field reports have proved that pcv2 vaccines are capable of decreasing the prevalence of pcv2 dna and viral load in serum and improving production parameters (fachinger et  al. 2008, fort the accuracy of responses, questions were written in clear and intelligible language. aside from questions regarding production parameters, the questionnaire included queries about biosecurity measures. polymerase chain reaction (pcr) the individual sera were pooled 3:1 within each age category and tested by real‑time pcr according to the method described by opriessnig and colleagues (opriessnig et  al. 2003). nucleic acids were isolated with a commercially available kit (magna pure lc total nucleic acid isolation kit, roche) according to the manufacturer's recommendations. results of real‑time pcr were expressed as the ct (threshold cycle) value. statistical analysis samples with ct values equal to or lower than 39 were considered ‘positive’. a herd was classified as positive for pcv2 dna if at least 1 serum sample taken from the herd had a positive pcr result. differences in ct among vaccinated and non‑vaccinated pigs were determined by a mann‑whitney u test. differences in the prevalence of positive herds, as well as the proportion of viremic‑positive pigs in vaccinated and non‑vaccinated herds, were determined by a chi‑squared test (statistical significance at p < 0.05). a mann‑whitney u test was applied in order to compare production performance. results association of vaccination with the levels of pcv2 dna mean ct values in serum samples from vaccinated pigs were significantly higher (in total and in czyżewska‑dors et al. efficacy of the porcine circovirus 2 vaccination 0 5 10 15 20 25 30 35 40 45 50 total sow 4-5 6-7 8-9 10-11 12-13 14-15 16-17 18-19 20-21 22-24 m ea n c t va lu e age category in weeks * farms vaccine piglets against pcv2 farms non vaccine piglets against pcv2 figure 1. mean ct (± sd) values in pigs sera (in total and in different age categories) when tested by pcv2 real time pcr. * p < 0.05 between bars. 0 20 40 60 80 100 % v ir em ic p ig s total sow 4-5 6-7 8-9 10-11 12-13 14-15 16-17 18-19 20-21 22-24 age category in weeks farms vaccine piglets against pcv2 farms non vaccine piglets against pcv2 * figure 2. proportion of pcv2 viremic pigs (% ± ci) (in total and in different age categories). * p<0.05 between bars. 222 efficacy of the porcine circovirus 2 vaccination czyżewska‑dors et al. veterinaria italiana 2018, 54 (3), 219‑224. doi: 10.12834/vetit.1009.5377.3 vaccinated and non‑vaccinated herds, is at this age. it has previously been shown that blood is a suitable specimen for pcv2 detection using pcr (shibata et al. 2003, grau‑roma et al. 2008). numerous studies have reported that the vaccination of pigs against pcv2 can improve adwg and decrease mortality rates (cline et  al. 2008, fachinger et  al. 2008, horlen et  al. 2008, lyoo et  al. 2011, martelli et  al. 2011). this study also compared production performance between vaccinated and non‑vaccinated herds. adwg, nursery mortality, and overall, post‑weaning mortality, were slightly better in herds practicing pcv2 vaccination in 3‑week‑old piglets, although without statistical significance (p  >  0.05). discrepancies between the results obtained previously and the results of this study could be associated with differences in study design (i.e. field experimental studies versus a field cross‑sectional study). possible reasons for the lack of significance between vaccinated and non‑vaccinated herds with regards to production performance may be a result of varied health status, management strategy, biosecurity and sanitation practices, environmental conditions, antibiotics usage, the breed of reared pigs, and the geographical location of the evaluated farms. in general, management strategies and biosecurity practices were poor in the farms included in this study. another potential reason for vaccine failure with regard to production performance were factors such as the lack of use of the recommended dosage of vaccine, administering vaccines to sick or immune‑compromised pigs, non‑compliance with proper vaccine schemes, and/or vaccinated pigs registering the presence of interfering, maternally‑derived antibodies (mda). in addition, the antigenic difference between the vaccine strain and field strain, possible strain mutations under field conditions, the adjuvant type, and the amount of pcv2 antigen in the vaccine could all have had an influence on the results reported here (lefebvre et al. 2008, opriessnig et  al. 2009, guo et  al. 2010, lyoo et  al. 2011, prpić et  al. 2014). nevertheless, further studies are required in order to explain the obtained findings. this relates especially to the analysis of the identification of possible risk factors affecting production performance in commercial pig farms under field conditions and how these risk factors are relate to pcv2 vaccination efficacy. in summary, on the basis of the obtained results, we can conclude that under field conditions, the vaccination of piglets with a commercial vaccine reduces the viral load in blood and proportion of pcv2‑viremic pcv2‑positive pigs. moreover, vaccination protected the animals against early infection, as shown by the delayed onset of viremia in vaccinated piglets. although the pcv2 vaccination et al., 2008, horlen et al. 2008, fort et al. 2009, pejsak et  al. 2010, martelli et  al. 2011, heissenberger et  al. 2013). however, previous studies were conducted under laboratory or field‑experimental conditions, mainly in herds with a reasonably good health status, effective management, and high level of farm biosecurity strategies. under the conditions of this study, pcv2 vaccination in piglets aged 3 weeks prevents the infection in 23.2% of vaccinated herds. by contrast, the pcv2 infection was detected in 100% of non‑vaccinated herds. in addition, significantly lower amounts of pcv2 dna were detected in sera, and a lower proportion of viremic pigs were observed in vaccinated herds. this trend has been observed in vaccinated pigs of all different age groups, including sows that were not vaccinated against pcv2. these results suggest that the implementation of the pcv2 vaccination substantially reduces the viral burden in the housing facilities. decreasing the prevalence of pcv2 in the sow sector is important because it reduces the risk of foetal infection (pensaert et  al. 2004, sarli et  al. 2012). similarly, findings from us showed a decrease of pcv2 infectious pressure in the american pig population 5 years after the implementation of a widespread pcv2 vaccination programme (shen et al. 2012). in the vaccinated herds, only 1.3% of 4‑5 week‑old piglets were pcv2 positive, while in non‑vaccinated herds, 41.7% (p < 0.05) of pigs of the same age were pcv2 positive. these results suggest that the vaccination of piglets significantly delayed the development of pcv2 viremia. this finding is in agreement with previous studies, demonstrating the effectiveness of pcv2 vaccines in controlling viremia in piglets (fort et  al. 2008, seo et  al. 2014). moreover, the postponed onset of viremia in piglets also diminished the risk of pcv2‑sd developing (rose et al. 2003, lopez‑soria et al. 2005). in both vaccinated and non‑vaccinated herds, the highest percentage of pcv2 positive pigs was observed among pigs from 14 to 21 weeks of age. this finding indicates that the best time to detect pcv2 dna in blood, under field conditions, in both table i. production parameters from herds vaccinated and non‑vaccinated against pcv2. production parameters vaccination against pcv2 p value yes no mean sd mean sd nursery mortality (%) 2.8 1.5 3.2 1.5 0.184 overall postweaning mortality (%) 5.0 3.0 5.3 3.0 0.408 average daily weight gain (g) 642.8 43.5 628.1 47.9 0.273 223 czyżewska‑dors et al. efficacy of the porcine circovirus 2 vaccination veterinaria italiana 2018, 54 (3), 219‑224. doi: 10.12834/vetit.1009.5377.3 acknowledgements this work was supported by a grant from the national science centre, no. n n308 571740. the authors are grateful to the polish veterinary practitioners and farmers who contributed to this study. we also thank karolina kus for her excellent technical assistance. was only administered to piglets, it also decreased the 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biopathology and biotechnology, university of palermo, 90134 palermo, italy. 2istituto zooprofilattico sperimentale della sicilia ‘a. mirri’, via g. marinuzzi 3, 90129 palermo, italy. * corresponding author at: istituto zooprofilattico sperimentale della sicilia ‘a. mirri’, via g. marinuzzi 3, 90129 palermo, italy. tel.: +39 091 6565236, e-mail: sara.villari@izssicilia.it. parole chiave brucella abortus, bovini, cellule ifn-γ+t, yersinia enterocolitica o:9. riassunto uno dei principali limiti nella diagnosi delle brucellosi animali è la cross-reattività che si verifica tra gli antigeni di superficie di brucella e yersinia. con l’intento di cercare un metodo in grado di discriminare tra le infezioni causate dai due patogeni, nel presente lavoro è stata messa a confronto l’espansione di linfociti t in grado di produrre interferon gamma (ifn-γ+), ottenuti da cellule mononucleate di sangue periferico (pbmc) isolate da bovini con infezione da brucella abortus, con quella indotta in animali sperimentalmente immunizzati con yersinia enterocolitica o:9. i linfociti sono stati quindi analizzati mediante citofluorimetria dopo che le pbmc erano state riesposte in vitro ad antigeni di yersinia o brucella. i risultati ottenuti hanno evidenziato una differenza statisticamente significativa nell’espansione di linfociti t cd4+ e cd8+ / ifn-γ+ nei casi in cui le pbmc di animali immunizzati con yersinia erano state esposte in vitro agli antigeni di y. enterocolitica o:9, che invece non si verificava nel caso di esposizione ad antigeni di brucella. questo metodo, pertanto, potrebbe risultare di interesse per la conferma di casi sierologici con diagnosi dubbia e/o per l’esclusione di potenziali cross-reattività. analisi dell’interferon gamma prodotto nelle cellule durante le infezioni da yersinia enterocolitica o:9 e brucella abortus nei bovini keywords brucella abortus, cattle, ifn-γ+t cells, yersinia enterocolitica o:9. summary one of the major constraints in the diagnosis of animal brucellosis is the cross-reactivity that occurs between brucella and yersinia surface antigens. with the aim to find a method to distinguish brucella from yersinia infection, the expansion of interferon gamma producing (ifn-γ+) t cell subsets obtained from peripheral blood mononuclear cells (pbmc) isolated from cattle either infected by brucella abortus or experimentally immunized with yersinia enterocolitica o:9 were compared. the lymphocytes were analyzed by flow cytometry after pbmc were in vitro re-exposed to yersinia or brucella antigens. the results highlighted a statistically significant difference in the expansion of the cd4+ and cd8+ ifn-γ+ t cells occurring when pbmc of animals immunized with yersinia are in vitro exposed to y. enterocolitica o:9 antigen but not to brucella antigen. this method could thus be suggested in those cases where results obtained by serodiagnosis need to be further clarified. analysis of interferon-gamma producing cells during infections by yersinia enterocolitica o:9 and brucella abortus in cattle veterinaria italiana 2019, 55 (2), 149-155. doi: 10.12834/vetit.1374.7538.2 accepted: 31.08.2018 | available on line: 30.06.2019 annalisa agnone1, marco pio la manna1, gesualdo vesco2, valeria gargano2, giusi macaluso2, francesco dieli1, guido sireci1 and sara villari2* (pappas et  al. 2006, skendros et  al. 2011), and the mediterranean basin is an acknowledged endemic region of human brucellosis (seimenis et  al. 2006). the infection is transmitted to humans mostly by ruminants, through direct contact with infected materials or indirectly by ingestion of animal introduction brucellosis is an infectious disease mainly affecting cattle, swine, goats, sheep and dogs caused by members of the genus brucella. brucellosis is the most common zoonotic infection worldwide 150 ifn-γ+ cells to distinguish brucella and yersinia infections agnone et al. veterinaria italiana 2019, 55 (2), 149-155. doi: 10.12834/vetit.1374.7538.2 detected by enzyme linked immunosorbent assay (elisa) (weynants et  al. 1995). it has also been demonstrated that ifn-γ secreted by peripheral blood in response to a commercial brucella antigen,a was not functional to distinguish between infection caused by brucella or yersinia enterocolitica o:9, representing the most important source of false positive reactions in the serological diagnosis of brucellosis in animals and humans (kittelberger et al. 1997). the role of cell-mediated immunity in brucella infection has been analyzed in murine models (skyberg et al. 2011, weynants et al. 1998). protective immunity requires activated antigen-presenting cells (mainly macrophages and dendritic cells) as well as cd4+, cd8+ and γδ+t lymphocytes activation. in particular, cd4+ifn-γ+ t cell subset was described as essential for the clearance of brucella infection in murine model activating killing mechanisms in macrophages as well as other type of cells that are reservoir of replicating bacteria (skendros and boura 2013). brucella may thus survive in host macrophages and dendritic cells evading adaptive immune mechanisms (carvalho neta et  al. 2010, kittelberger et al. 1997). to our knowledge, the ifn-γproducing lymphoid subsets following initial in  vivo and then in  vitro re-exposure to brucella and yersinia antigens in cattle have not been identified yet. it was reported that cd4+ ifn-γ+ lymphocytes play a key role in containing brucella infection in cattle (skendros and boura 2013). the novelty of our approach is represented by the effort to detect the ifn-γ+t cell subsets during natural brucella infection and experimental yersinia immunization after in  vitro re-exposure with specific antigens. this approach was designed to evaluate the potential diagnostic use of these parameters to discriminate between the two infections in cattle. materials and methods animal trial a total number of 15 female cattle were enrolled for the study. blood samples were collected weekly by venipuncture in heparinized tubes and tubes with no anticoagulant from the groups of cattle. sera were obtained after centrifugation and stored at - 20°c until used for diagnostic purposes. in group 1, the animals were immunized with inactivated yersinia enterocolitica o:9 (charolaise breed, 6-8  months old). group 2 (holstein breed, 2-5 years old) consisted of cattle naturally infected with b. abortus. group 3 (charolaise breed, 1-3 years old) was the control group, including seronegative products (milk and derivatives) or by inhalation (olsen and palmer 2014). infected cows can abort, once only after the exposure to the pathogen, usually at about 7 months of pregnancy; otherwise, newborn calves are very weak and usually die shortly after birth. brucella colonization of the udder results in a severe drop in milk production, which may also present swelling and inflamed knees (hygromas) (olsen and palmer 2014), while bulls with brucellosis usually become sterile (carvalho neta et al. 2010). brucella is able to survive in immune cells and persist in tissues of the reticuloendothelial system (de jong et  al. 2010), using several strategies of immune evasion. one of these includes the production of cyclic glucan molecules that impede the lipid raft-mediated vacuole maturation to interfere with the membrane transport systems (skendros and boura 2013). moreover, lps from brucella abortus bears a non canonical lipid a active only at very high concentration, therefore even if the lps is recognized by tlr4 receptors of phagocytes it does not induce early activation of macrophages and consequent production of pro-inflammatory cytokines (barquero-calvo et  al. 2007). brucellosis diagnosis in livestock is mainly based on serological methods; the italian national eradication program in ruminants, based on test-and-slaughter policy, prescribes to perform the official diagnosis by two serological tests, rose bengal test (rbt ) and complement fixation test (cft ) (o.m. 24-06-2015). unfortunately, they both lack of specificity due to the use of the whole lipopolysaccharide fraction of brucella cell envelope as antigen (corbel 1985, nielsen et  al. 2004). in fact, the most relevant constraint of the serological diagnosis consists in the frequently observed cross-reactions after the animal exposure to other microorganisms which all share common features with brucella polysaccharide ‘o’ chain, e.g. yersinia enterocolitica o:9, salmonella enterica serotype typhimurium and escherichia coli o157 (muñoz et  al. 2005, nielsen et al. 2004). despite the presence of a wide body of literature showing efforts to discover antigens that can be useful in discriminating between brucella and other gram negative bacteria infections (denoel et  al. 1997, guzman et  al. 2012, ko et  al. 2012), the lack of brucella specific antigen still highlights the need to develop new diagnostic methods, in order to improve the efficacy of eradication strategies and to avoid un-necessary animal sacrifices. the reaction of cattle immune system has been investigated in terms of delayed type hypersensitivity, pbmc proliferative response and interferon gamma (ifn-γ) secretion by whole lymphocyte fraction in presence of brucella antigens, 151 agnone et al. ifn-γ+ cells to distinguish brucella and yersinia infections veterinaria italiana 2019, 55 (2), 149-155. doi: 10.12834/vetit.1374.7538.2 commercial reagentf and then incubated with phicoeritrina (pe) labelled  anti ifn-γ mab (clone cc302, mouse anti cow igg1)e for 30 minutes at 4 °c, washed three times and resuspended in 500  µl of phosphate buffer solution. ifn‑γ measurement and data analysis stained pbmc were acquired by flow cytometry analysis using a cytometerg and a commercial software.g each acquisition was performed collecting 10,000 events of lymphocytes region gated using forward scatter (fsc) and side scatter (ssc) parameters. acquired cells were analyzed in order to detect the subset of ifn-γ producing cells; the percentage of ifn-γ+ cells detected in cultures with rpmi only (negative control) was subtracted from the same measurement observed in cells cultured with antigens. statistical analysis of data was performed by mann whitney test, for any significant difference among the average percentages of a three times-repeated sample for each experimental group. in order to evaluate a parameter able to discriminate between animals immunized with y.  enterocolitica and naturally infected with b.  abortus, the ratio between the percentages of ifn-γ+ cells in response to brucella and yersinia antigens was also analyzed. the confidence value was fixed at 0.05 unless otherwise specified. results ifn‑γ+ cells expansion in response to yersinia results showed that in animals immunized with y. enterocolitica an expansion of ifn-γ+ lymphocytes was detected following the re-exposure to ye  o:9 (2.36% in immunized versus 0.36% in uninfected controls) but not to brucella antigen (0.21%) (p < 0.05) (figure  1a). there was a statistically significant difference in the expansion of cd4+ ifn-γ+t cells when pbmc from animals immunized with yersinia were exposed to ye o:9, but not to brucella antigen (1.26% vs 0.14%) (figure  1b). the percentage of cd8+ ifn-γ+t cells was 0.93% in yersinia-immunized animals versus 0.08% in seronegative controls. taking into account that γδt-lymphocytes are represented in a consistent amount in cattle in an age-dependent manner (guzman et  al. 2012) and are relevant in murine as well as in bovine immune response to brucella (skyberg et  al. 2011), the role of γδ+ ifn-γ+ t cells was also analyzed (figure  1d). results show that these cells display a minor contribution to ifn-γ production in tested animals. the comparison of γδ+ ifn-γ+ t cells in vitro expansion was statistically different when pbmc of animals obtained from farms officially free of brucellosis. animal trial was accomplished following current european legislation (directive 2010/63/ue and following commission implementing decisions) and the corresponding italian law (d. leg. 26, march 4th, 2014). the whole procedure was conducted according to the regulations of the italian health ministry (decreto ministeriale no. 101/2006-a). all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. as for group 1, five cattle were immunized with 100 ml of heat inactivated (65  °c for 1.5 minutes) y.  enterocolitica o:9 (1012 colony forming units – cfu – per os). a 10 ml dose containing 1012  cfu with aluminium hydroxide as adjuvant was also administered subcutaneously, in the neck region, weekly for two months. antibodies against y.  enterocolitica o:9 were measured weekly for each animal by a specific cft procedure, using a commercial antigen,b as recommended by the manufacturer. sera showing at least 75% of fixation at 1:10 dilution were considered positive. furthermore, the same serum samples from each animal were tested by rbt and cft specific for brucellosis before the experimental procedure, resulting all negative. as for group 2, we collected samples from naturally infected cattle resulted positive to the official serological tests. sera showing agglutination and/or at least 50% of fixation at 1:4 (20 iu/ml) dilution were diagnosed as positive, as prescribed by the national legislation (o.m. 24-06-2015). they were also tested for y.  enterocolitica o:9 isolation from faeces and resulted all negative. pbmc cultivation pbmc were isolated from heparinized blood samples of each experimental group as described elsewhere (la manna et al. 2011). then, 5 x 105 pbmc from each sample were cultured for 48 h at 37 °c-5% co 2 with two different stimuli: yeo:9b (10 μg/ml) and b.  abortusa (10 μg/ml), in a final volume of 0.2  ml/ well of complete rpmic in 96 well u-bottomed microplatesd. the concentration of stimuli added to the wells was chosen following a dose-response curve assay for the best antigen-specific expansion of t cell subsets (data not shown). after 48 h of culture, pbmc were collected and stained with fluorescein isothiocyanate (fitc) labelled monoclonal antibodies (mab) -anti cd8 (clone cc63, mouse anti cow igg2a),e -anti cd4 (clone 44.38, mouse anti cow igg2a)e and -wc1 (clone 19.19,  mouse anti sheep – cross reactive with cowigg1a)e. after 30  minutes of incubation at 4  °c, cells were washed three times, fixed and permeabilized using a specific 152 ifn-γ+ cells to distinguish brucella and yersinia infections agnone et al. veterinaria italiana 2019, 55 (2), 149-155. doi: 10.12834/vetit.1374.7538.2 yersinia treated animals were exposed to yersinia and brucella antigen (p < 0.05). ifn‑γ+ cells expansion in response to brucella the results obtained in cattle naturally infected by brucella (figure 2) showed a statistically significant increase of ifn-γ+ lymphocytes when the cells were stimulated both by brucella and yersinia antigens in comparison with the control group (p  <  0.05) (figure 2a). data showed that in the same animals ifn-γ+ lymphocytes are similarly represented when in  vitro exposed to yersinia (5.99%) or brucella (5.70%). the analysis of t-cell subsets showed that cd4+ t cells produce a similar percentage of ifn-γ+ cells in response to both antigens, thus confirming cross-reactivity in cell-mediated immunity (figure  2b). brucella abortus-infected samples showed a higher percentage of ifn-γ+ t lymphocytes compared to controls, with statistically significant differences. ifn-γ+ pbmc and cd4+ ifn-γ+ in brucella abortus-infected samples were also higher than in yersinia-infected animals, maybe due to natural infections with other cross-reactive gram-negative bacteria (kittelberger et al. 1997). statistical analysis in details, cd4+ ifn-γ+ t-cell subset expanded similarly in response to yersinia (2.96% for cattle positive for brucella abortus and in vitro stimulated with yersinia a. ifn-γ+ t lymphocites * * ag yersinia immunized (yersinia) seronegative controls ag brucella 0 1 2 3 in vitro stimuli % o f p o si ti ve c el ls b. ifn-γ+ cd4+ t lymphocites c. ifn-γ+ cd8+ t lymphocites d. ifn-γ+ γδ t lymphocites * * * * * * ag yersinia immunized (yersinia) seronegative controls ag brucella 0.0 0.5 1.0 1.5 in vitro stimuli % o f p o si ti ve c el ls ag yersinia immunized (yersinia) seronegative controls ag brucella 0.0 0.5 1.0 1.5 in vitro stimuli % o f p o si ti ve c el ls ag yersinia immunized (yersinia) seronegative controls ag brucella 0.0 0.5 1.0 1.5 in vitro stimuli % o f p o si ti ve c el ls figure 1. difference of ifn-γ production between cells stimulated with the two antigens in y. enterocolitica immunized animals. a. in yersinia‑immunized animals the antigen‑specific stimulation resulted in a statistically significant difference of ifn‑γ+ t‑lymphocytes with respect to the seronegative controls in all the cellular subpopulation analyzed. in these animals, also a higher percentage of ifn‑γ+ t‑lymphocytes was detected, when compared with the same cells stimulated with brucella antigen. b. detection of cd4+ ifn‑ γ+ t‑lymphocytes. c. detection of cd8+ ifn‑γ+ t‑lymphocytes confirmed a similar contribute of these two subsets to total ifn‑γ+ t‑lymphocytes in the two experimental groups, with a slightly higher cd4+ t cells detection. d. detection of γδ+ ifn‑γ+ t cells amounted to 0.1% in response to yeo:9 and to 0.04% in seronegative animals. *p < 0.05. 153 agnone et al. ifn-γ+ cells to distinguish brucella and yersinia infections veterinaria italiana 2019, 55 (2), 149-155. doi: 10.12834/vetit.1374.7538.2 significant (p < 0.05). following the same approach, we also compared the ratios: between cd4+ ifn-γ+ t cells and cd8+ ifn-γ+ t cells within the same groups. when the percentages of cd4+ifn-γ+ t cells are analyzed (p  <  0.05), the difference was statistically significant, whereas it was not for cd8+ ifn-γ+ t cells. conclusions previous data related to antigen-specific ifn-γ production measured by elisa in y.  enterocolitica experimentally infected cattle re-exposed to brucellergenea are available (kittelberger et  al. 1997), but in the present work, for the first time, the production of antigen-specific cd4+cd8+and γδifn-γ+ t lymphocytes were assessed by flow cytometry in animals naturally infected with brucella antigen) and brucella (2.77% in vitro stimulated with brucella antigen). we did not observe any statistically significant difference when cd8+ cells were analyzed in response to the two antigens (figure  2c). data regarding the analysis of γδt cells revealed that the difference of ifn-γ production was not statistically significant between animals positive for brucella and in  vitro stimulated with both antigens, while a statistically significant difference was detected when pbmc from infected and seronegative cattle were exposed to yersinia (figure 2d). the mann-whitney test has been performed to study the difference in the ratio of ifn-γ+ lymphocytes stimulated with yersinia over those stimulated with brucella antigen, between the animals immunized with yersinia versus those infected with brucella. our results showed that this difference is statistically a. ifn-γ+ t lymphocites * ag yersinia immunized (brucella) seronegative controls ag brucella 0 2 6 8 in vitro stimuli % o f p o si ti ve c el ls b. ifn-γ+ cd4+ t lymphocites c. ifn-γ+ cd8+ t lymphocites d. ifn-γ+ γδ t lymphocites * * * 4 ag yersinia immunized (brucella) seronegative controls ag brucella 0 1 2 4 in vitro stimuli % o f p o si ti ve c el ls 3 ag yersinia immunized (brucella) seronegative controls ag brucella 0.0 0.5 2.0 2.5 in vitro stimuli % o f p o si ti ve c el ls 1.0 1.5 ag yersinia immunized (brucella) seronegative controls ag brucella 0.0 0.5 1.0 1.5 in vitro stimuli % o f p o si ti ve c el ls figure 2. difference of ifn-γ production between cells stimulated with the two antigens in brucella infected animals. a. in cattle naturally infected by brucella ifn‑γ+ t‑cells detection is similar when cells are in vitro exposed to yersinia or brucella antigens. b. cd4+ t cells produce ifn‑γ when exposed to both antigens. statistical significances were assessed and percentages of ifn‑γ+ and cd4+ ifn‑γ+ t cells were compared to seronegative controls. c. no statistically significant expansion of cd8+ ifn‑γ+ t cellswas observed in response to both the antigens. d. no statistically significant expansion of γδ+ ifn‑γ+ t cellswas observed in response to both the antigens. *p < 0.05. 154 ifn-γ+ cells to distinguish brucella and yersinia infections agnone et al. veterinaria italiana 2019, 55 (2), 149-155. doi: 10.12834/vetit.1374.7538.2 to assess the efficacy of this method also with animals naturally infected with yersinia. acknowledgements we are grateful to dr. paolo li donni (palermo university) for statistical analysis. sources and manufacturers a. brucellergene, ocb®zoetis, usa. b. yersinia enterocolitica o:9 yop® institutvirion/ serion gmbh, würzburg ,germany. c. gibco media of fisher scientific uk ltd, loughborough, uk. d. nunc a/s, roskilde, denmark. e. abdserotec, kidlington, uk. f. fix & perm® cell fixation and cell permeabilization kit – thermofisher scientific, waltham, massachusetts, usa. g. facscan and cell quest pro, becton dickinson, new jersey, usa. funding this study was funded by a grant of the italian ministry of health “ricerca corrente izs si 10/09”. the funding body was not involved in the study design and analysis. and experimentally immunized with yersinia after in  vitro re-exposure to the specific antigens. our data show that in animals immunized with yersinia there is an antigen-specific expansion of cd4+ and cd8+ t lymphocytes without any appreciable cross-reactivity, at least with brucella. in cattle naturally infected with cross-reactivity between the in vitro yersinia and brucella stimulated cells has been observed and cd4+ t lymphocytes were the main producer of ifn-γ. the increase of cd4+ ifn-γ+ t cell subset was expected, since its crucial role has been already demonstrated in a murine model for the control of brucella infection (baldwin and parent 2002, vitry et al. 2012). the γδ t cells do not play a key role in response to both antigens either in vivo or in vitro, being a minor ifn-γ+ producing t cell subset when compared to cd4+ and cd8+ lymphocytes, as also reported by previous results (vitry et al. 2012). the work carried out during this study allowed the detection, for the first time in cattle, of lymphocyte populations, i.e. cd4+ and cd8+ifn-γ+t cells, that expand differently when pbmc of animals immunized with yersinia are in  vitro exposed to yersinia or to brucella. even if this scenario was not described viceversa, the analysis of the expansion of ifn-γ+ cells in response to brucella and y.  enterocolitica could be useful to sort out between yersinia and brucella infection in cattle in case of doubtful results with the official serological methods. anyway, further studies will be necessary 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dispensable function of cd8+ t cell, b cell, th2, and th17 responses in the control of brucella melitensis infection in mice. infect immun, 80, 4271-4280. weynants v., walravens k., didembourg c., flanagan p., godfroid j., & letesson j.j. 1998. quantitative assessment by flow cytometry of t-lymphocytes producing antigen-specific gamma-interferon in brucella immune cattle. vet immunol immunopathol, 66, 309-320. weynants v., godfroid j., limbourg b., saegerman c. & letesson j.j. 1995. specific bovine brucellosis diagnosis based on in vitro antigen-specific gamma interferon production. j clin microbiol, 3, 706-712. 247 1area diagnostica sierologica, istituto zooprofilattico sperimentale della sicilia “a. mirri”, palermo, italy. 2area sorveglianza epidemiologica, istituto zooprofilattico sperimentale della sicilia “a. mirri”, palermo, italy. 3area territoriale palermo, istituto zooprofilattico sperimentale della sicilia “a. mirri”, palermo, italy. *corresponding author at: area diagnostica sierologica, istituto zooprofilattico sperimentale della sicilia “a. mirri”, palermo, italy. tel.: +39 091 65 65 230, e-mail: giuseppina.chiarenza@izssicilia.it. parole chiave bovino, sieroprevalenza, coxiella burnetii. riassunto la febbre q è una zoonosi diffusa causata da coxiella burnetii, un batterio intracellulare obbligato capace di infettare diversi ospiti. lo scopo di questo studio è stato quello di stimare la prevalenza di c. burnetii nei bovini presenti nelle aziende agricole siciliane, al fine di adottare misure preventive utili a ridurre la prevalenza della malattia nel territorio regionale, visti anche i potenziali rischi zoonotici. sono stati esaminati mediante test elisa nr. 4661 campioni di siero, provenienti da bovini appartenenti a 198 aziende siciliane; di questi, nr. 246 sono risultati positivi. la sieroprevalenza a livello aziendale è stata 38.8% (77/198) (95% ci), mentre a livello animale è risultata 5,28% (246/4661) (95% ci). diffusione di coxiella burnetii nei bovini da latte in sicilia keywords bovine, coxiella burnetii, seroprevalence. summary q fever is a widespread zoonotic disease caused by coxiella burnetii, an obligate intracellular bacterium with a wide range of hosts. the aim of this study was to estimate the seroprevalence of c. burnetii infection in cattle in sicilian farms. a total of 4,661 serum samples, from cattle belonging to 198 sicilian farms, were examined by elisa test and 246 resulted positive. the average seroprevalence at the farm level was 38.8% (77/198) (95% ci), while at the animal level it was 5.28% (246/4,661) (95% ci). the present study highlights the need for continuous monitoring of c. burnetii spread as it represents a serious risk for human health. paola galluzzo1, sara villari1, francesco geraci2, carmela sciacca1, francesca grippi1, vittoria currò3 and giuseppina chiarenza1* seroprevalence of coxiella burnetii in dairy cattle from sicily veterinaria italiana 2019, 55 (3), 247-252. doi: 10.12834/vetit.1391.7602.2 accepted: 31.08.2018 | available on line: 30.09.2019 vaginal mucous and feces post parturition (roest et  al. 2012). the main route of human exposure to c. burnetii is the inhalation of contaminated aerosols from excreta, especially birth products (maurine and raoult 1999). the role of raw milk and unpasteurized dairy products in the transmission of q fever to humans is debated but, until now, not proven for either acute infection or clinical disease (capuano et al. 2012, eldin et al. 2013, gale et al. 2015, oie 2015). moreover, low levels of c.  burnetii were detected in sewage water (schets et al. 2013). c. burnetii is well equipped to resist to drought (kazar 2005), and when infected animal excreta dry and turn to dust, the bacterium spreads to the environment. c. burnetii is extremely infectious; also a low dose can cause contamination (madariaga et al. 2003). introduction coxiella burnetii is an intracellular zoonotic bacterium able to cause q fever in humans as well as several animal species: sheep, goats and cattle are the primary animal reservoirs. moreover, ticks and rodents also are natural reservoirs of c. burnetii (oie 2015). q fever is a recognized occupational infection in workers having regular contact with ruminants or their products, such as farmers, veterinarians, laboratory technicians, slaughterhouses and cheese factories personnel, all categories at higher risk of infection (schimmer et al. 2014). infected animals shed large numbers of organisms in their placenta, birth fluids and milk (agerholm 2013). c.  burnetii can also be excreted through 248 detection of coxiella burnetii in sicily galluzzo et al. veterinaria italiana 2019, 55 (3), 247-252. doi: 10.12834/vetit.1391.7602.2 among the other studies, a survey carried out throughout the campania region has shown a q fever seroprevalence of 11.8% within sheep, 6.3% within goats, 14% in cattle and 7% in dogs (capuano et al. 2001). a seroprevalence around 8% was found in cattle from an apennines area of the emilia-romagna region (martini et  al. 1994). data showed a high occurrence of c.  burnetii in dairy cattle in the pavia province (38%), in cremona province (80%) and in lodi province (78%) (vicari et  al. 2013). in northern italy, 44.9% of cattle that experienced abortion were seropositive for c.  burnetii (cabassi et  al. 2006). in a serological survey in the province of bologna, 0.87% of dogs were found to have antibodies to c. burnetii and 35% of dog owners were also found seropositive (baldelli et al. 1992). the seroprevalence for c. burnetii in dogs was 31.5% in sicily (torina et al. 2006) and 7% in southern italy (capuano et al. 2001). more recently, the prevalence of c. burnetii in cattle and sheep raw milk farms was determined in central italy, showing a higher value for cattle (50%) than sheep (21%) farms (guidi et al. 2017). knowing q fever prevalence in animals is necessary to prevent the human disease. in fact, the identification and removal of any head of cattle with intrauterine infection would prevent the shedding of large amounts of bacteria into the environment via placenta and birth fluids (after both abortion or normal delivery), thereby lowering the risk of spread of c. burnetii to animals and humans (sánchez et al. 2006, rousset et al.2009, roest et al. 2012). concerning the human disease, seasonal agricultural workers were recently tested in sicily and coxiella antibodies were found in the 21.4% of serum samples from women and in the 25.0% of serum samples from men (verso et al. 2016). the highest prevalence of antibodies was demonstrated in trapani (45.0%), higher than that observed in agrigento (22.7%) and palermo (17.7%). none of the sampled individuals reported in the anamnesis spreading of c. burnetii from contaminated farms to the environment may e.g. occur with soil, animal skin, wool or fur, non-pasteurized milk and wastewater. in fact, c.  burnetii survives in the environment for months to years due to its resistance to heat, pressure and chemical stress (kazar 2005), and the most likely route of dispersion of the bacterium is through air with aerosols and dust particles (astobiza et al. 2011, raoult et al. 2005). abortion is an important symptom of infection for dairy goats and sheep, while in cattle this is rarely observed and shedding of c. burnetii is of lower level (rodolakis et  al. 2007, hansen et  al. 2011). infected cows shed the bacterium in feces, milk and birth products (guatteo et al. 2012). the pathogen can be excreted for up to 13 months in cow’s milk (kargar et al. 2013). since the clinical symptoms are often generic and the infection could be asymptomatic, in most instances, the diagnosis of q fever relies upon serology. among the various techniques useful for animal serological diagnosis, the most common are the indirect immunofluorescence assay (ifa), the enzyme-linked immunosorbent assay (elisa) and the complement fixation test (cft). currently, no ifa commercial kit is available for ruminants; therefore, elisa is the preferred choice for seroepidemiological surveys, also due to practical reasons (easier and faster to perform than cft) (natale et al. 2012). in italy, q fever surveys concerning seroprevalence in animals are very scarce, as reports have been mainly focused on reproductive disorders and, particularly, on abortion as the major clinical problems (parisi et  al. 2006, natale et  al. 2009). to our knowledge, the only extensive investigation conducted to date was carried out in sardinia among flocks, revealing a seroprevalence of 38% and 47% on sheep and goat farms, respectively; furthermore, c. burnetii was also found by pcr in 10% and 6% of ovine and caprine fetuses (masala et al. 2004). table i. n. of tested herds according to winepi software (http://www.winepi.net/), and positive farms distribution for each province. province n. tot of farms per province (and distribution as %) n. of farms to test according to winepi n. of examined farms n. of farms with at least 1 positive sample % of positive farms for each province agrigento 489 (5) 9 12 9 75 caltanissetta 281 (3) 6 6 3 50 catania 650 (6) 11 11 3 27 enna 1,251 (12) 23 27 11 41 messina 2,311 (22) 41 41 11 27 palermo 2,503 (24) 45 46 13 28 ragusa 1,724 (16) 30 31 20 64.5 siracusa 985 (8) 17 18 7 39 trapani 378 (4) 8 6 0 0 total 10572 190 198 77 249 galluzzo et al. detection of coxiella burnetii in sicily veterinaria italiana 2019, 55 (3), 247-252. doi: 10.12834/vetit.1391.7602.2 was composed of 375,840 cattle belonging to 10,572 farms. study design blood samples were collected in 2014 and 2015. all specimens examined in the study were randomly selected among those routinely conferred to the istituto zooprofilattico sperimentale della sicilia (izssi) for the brucellosis national eradication program. . this program establishes to test twice per year all the animals older than 12 months present in each cattle herd within the regional territory. as the average number of animals present in sicilian herds, according to the italian national livestock registration database (www.vetinfo.sanita.it) is around 50, only farms within this size were included in the present study. the total number of cattle herds (n = 198) to be sampled was selected considering an expected prevalence of 50%, with 5% precision at the 95% confidence level (as no other epidemiological data were available), according to winepi software (http://www.winepi.net/) (see tables i-iv for all sampling details and for the descriptive of cattle farms in sicily).a significant number of animals per herd was then selected by random sampling, based on farms’ size and according to winepi software. serological tests a total of 4,661 blood samples were collected from cattle belonging to 198 sicilian farms (tables i-iv). risk factors like working in stables, or being in direct contact with animals; only one male worker in the province of agrigento reported the occurrence of a tick bite in the past. this study compared data originating from human samples with those coming from animals raised in the same areas: cattle (14.6% in agrigento, 1.9% in palermo and no positive animal in trapani) and sheep (17.4% in agrigento, 15.1% in palermo and 17.6% in trapani). materials and methods study area sicily is an island located in the mediterranean basin and it is divided into nine provinces: palermo (pa), agrigento (ag), enna (en), caltanissetta (cl), catania (ct), messina (me), ragusa (rg), siracusa (sr) and trapani (tp). the region is strongly devoted to animal productions and according to the italian national livestock registration database (www.vetinfo. sanita.it) in 2013 the regional cattle population table ii. number of serum samples examined in all sicilian provinces and distribution of positive samples for each province. province n. of examined samples n. of positive samples (%) agrigento 133 23/133 (17.3) caltanissetta 164 4/164 (2.4) catania 186 8/186 (4.3) enna 775 29/775 (3.7) messina 650 13/650 (2) palermo 1,260 28/1,260 (2.2) ragusa 1,011 114/1,011 (11.3) siracusa 389 27/389 (6.9) trapani 93 0/93 (0) total 4,661 246/4,661 table iii. number of animals to test in each farm based on its size according to winepi software(http://www.winepi.net/). farm size ≤ 30 ≤ 40 ≤ 50 n. of animals to test ≤ 28 ≤ 37 ≤ 45 table iv. distribution of the average farms’ size within each sicilian province according to the italian national livestock registration database (www. vetinfo.sanita.it). province minimum 1st quartile median 3rd quartile maximum agrigento 1 3 10 23,25 266 caltanissetta 1 13 33 41,2 250 catania 1 8 23 61 231 enna 1 9 25 47,7 264 messina 1 8 20 39 425 palermo 1 5 14 31 664 ragusa 1 11 24,5 53 619 siracusa 1 12 31 66 781 trapani 1 1 2 5 14 250 detection of coxiella burnetii in sicily galluzzo et al. veterinaria italiana 2019, 55 (3), 247-252. doi: 10.12834/vetit.1391.7602.2 distributed in space; get information about the areas identified as at higher disease prevalence. results seroprevalence and spatial distribution of c. burnetii seropositive herds the seroprevalence at the farm level was 38.8% (77/198) (95% ci), while at the animal level it was 5.28% (246/4,661) (95% ci). only nine samples resulted as ‘doubtful’; they were all retested by elisa confirmed either positive (2/9) or negative (7/9). the serological results obtained in each province by elisa are shown in tables i and ii. epidemiologic analysis the territory of chiaramonte gulfi (rg), in particular with 41 positive samples out of 51 animals controlled in just one herd, was identified as the one with the highest prevalence of antibodies. moreover, 5 farms in cammarata (ag) fell into the i secondary cluster, 1  farm in regalbuto (en) fell in the ii secondary group, 9  herds in ragusa fell in the iii secondary group and 9 farms near ferla, carlentini, melilli and canicattini bagni (all in sr province) fell in the iv secondary cluster (figure 1). discussion and conclusions the present study shows that c.  burnetii is widespread in sicily. the provinces of agrigento and ragusa showed the most intense serological prevalence, having the two highest rates of positive farms and animals (75% and 17.3% for agrigento, 64.5% and 11.3% for ragusa, respectively), and thus representing the areas where control measures should be particularly accurate. furthermore, the territory of chiaramonte gulfi (rg), with 41 positive samples out of 51 tested in one herd, and cammarata (ag), with 5 farms in the i secondary cluster, showed the highest c.  burnetii serological prevalence. the high seroprevalence in chiaramonte gulfi involved a dairy farm with intensive management system, may suggest that animals in intensive breeding are at greater risk to contract the disease than those raised in extensive systems, as previosly reported (paul et  al. 2012). this is probably due to an indirect transmission from contamination with the barn environment, as cows in intensive management breeding usually spend more time inside the barns, thus being more exposed to the bacterium (paul et  al. 2012). furthermore, dairy herds have a greater risk to develop the infection blood samples were taken from the coccygeal vein into a 10 ml vacuum tube, stored in a refrigerated bag and conferred to izssi. sera were then removed by centrifugation and stored at -20  °c until tested by elisa. antibodies to c.  burnetii were detected by a commercial elisa test (id screen® q fever indirect multi-species, idvet, grabels, france) according to the manufacturer’s instructions. as recommended by the manufacturer, any sample was considered positive if the od percent was over 50. if the od percent was between 40 and 50, the result was considered as doubtful, while any sample with an od percentage under 40 was considered as negative. any farm with at least one positive result was considered as positive. epidemiologic analysis epidemiologic analysis was carried out using two different softwares: mapinfo (version 8.5) and sat-scan (version 9.0). mapinfo was used to analyze the spatial position of each farm identified by geographic coordinates (latitude and longitude), expressed in decimal degrees. sat-scan software was used in order to: check for the existence of statistically significant clusters of disease; verify if the disease was randomly figure 1. spatial distribution of c. burnetii seropositive herds in sicily. each line represents the borders of the municipality involved in the cluster, with a color-scale descending intensity (from dark brown for the most likely cluster to light pink for the iv secondary cluster). each dot represents the location of the farm(s) involved in the cluster. furthermore, the bigger dots correspond to the farms with the higher seroprevalence (most likely cluster (80.4%), i secondary cluster (48%), ii secondary cluster (44.4%), iii secondary cluster (36%) and iv secondary cluster (34%); the smaller dots represent each positive farm within 10 km radius from the clusters. most likely cluster i secondary cluster ii secondary cluster iii secondary cluster iv secondary cluster n s ew 0 50 100 km 251 galluzzo et al. detection of coxiella burnetii in sicily veterinaria italiana 2019, 55 (3), 247-252. doi: 10.12834/vetit.1391.7602.2 in light of the significant presence of specific c.  burnetii antibodies, it appears quite essential to deepen the knowledge on local epidemiological situations for q fever. a high seroprevalence in dairy cattle should lead to take preventive measures, including a control strategy to reduce the disease circulation. acknowledgements this work was supported by grant no. izs si 05/12 rc from the italian health ministry. the authors thank dr. gesualdo vesco for his contribution to the study design and his support during the study. than beef or mixed-breeding herds, in accordance with other studies (paul et al. 2014, mccaughey et al. 2010), maybe as beef cattle are maintained for a 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prevalence of coxiella burnetii antibodies in portuguese dairy cattle herds. trop anim health prod, 47 (1), 227-230. raoult d., marrie t.j. & mege j.l. 2005. natural history and pathophysiology of q fever. lancet infect dis, 5 (4), 219-226. rodolakis a., berri m., hechard c., caudron c., souriau a., bodier c.c., blanchard b., camuset p., devillechaise 27 parole chiave antibiotico-resistenza, colibacillosi, escherichia coli patogeno aviario (apec), geni di virulenza, pcr. riassunto la colibacillosi è la malattia batterica che si riscontra più frequentemente nelle specie aviari e i farmaci antimicrobici sono l'arma più utilizzata per ridurre sia l'incidenza che la mortalità ad essa legate. l'uso indiscriminato di antibiotici può, tuttavia, portare al fallimento della terapia e a ingenti perdite economiche da parte dell’allevatore. questo studio ha l’obiettivo di determinare i tassi di resistenza agli antibiotici da parte di ceppi di escherichia coli, valutare la possibile correlazione tra l'isolamento di e. coli e le tipologie di allevamento prese in considerazione e rilevare la presenza di e. coli patogeni aviari (apec) tra i vari e. coli isolati. mediante l’impiego di 19 agenti antimicrobici, sono stati sottoposti al test di suscettibilità antimicrobica 51 ceppi di e. coli; gli stessi ceppi sono stati analizzati per valutare la presenza di otto geni di virulenza mediante metodica pcr. la resistenza è stata riscontrata più frequentemente verso ampicillina e acido nalidixico, mentre gli isolati di e. coli hanno mostrato una minore resistenza nei confronti delle cefalosporine. complessivamente, il 40% degli isolati ha mostrato resistenza ad almeno tre o più agenti antimicrobici. sedici di 51 isolati sono stati definiti ceppi apec poiché in essi sono stati rilevati almeno cinque degli otto geni di virulenza ricercati. mentre i geni di virulenza iucd, cvi / cva, irp2 e iss sono stati rilevati in tutti i 16 ceppi apec, tsh, vat, papc e asta rispettivamente da 11, 7, 5 e 3 ceppi apec. i nostri risultati dimostrano quanto sia importante approfondire la diffusione del fenomeno dell’antibiotico resistenza e indagare la distribuzione di ceppi apec in italia, anche al fine di valutare un’eventuale correlazione tra i due fenomeni, con l’obiettivo di fornire uno strumento preventivo utile. inoltre, è stato dimostrato come la tipologia di allevamento adottata può influenzare i tassi di antibiotico resistenza. resistenza antibiotica e geni di virulenza in e. coli patogeni aviari (apec) isolati in varie tipologie di allevamento keywords antibiotic resistance, avian pathogenic escherichia coli (apec), colibacillosis, pcr, virulence gene. summary colibacillosis is the most frequent bacterial disease in avian species and antimicrobials are the main weapon to reduce incidence and mortality associated to it. however, indiscriminate use of antibiotics may lead to therapy failure and economic losses for the breeder. the aims of this study were to, determine the antibiotic resistance of escherichia coli isolates, evaluate the correlation between e. coli isolation and systems of breeding included in this study, and identify the avian pathogenic e.coli (apec) amongst the e. coli strains isolated. a total of 51 e. coli strains were subjected to antimicrobial susceptibility test and they were screened for the presence of virulence genes through pcr. resistance was most frequently detected against ampicillin and nalidixic acid meanwhile e. coli isolates showed less resistance to the cephalosporins. overall, 40% of the isolates showed resistance to at least three or more antimicrobials and 16/51 isolates were defined apec strains. the virulence genes iucd, cvi/ cva, irp2 and iss were detected from all 16 apec strains. the virulence genes tsh, vat, papc, and asta were detected from 11, 7, 5 and 3 apec strains, respectively. results demonstrated the importance of studies on apec and antibiotic resistance genes in italy, and it was shown that the systems of breeding might influence the antibiotic resistance. veterinaria italiana 2019, 55 (1), 27-33. doi: 10.12834/vetit.1617.8701.1 accepted: 19.12.2018 | available on line: 31.03.2019 istituto zooprofilattico sperimentale dell'umbria e delle marche ‘togo rosati’, via g. salvemini 1, 06126 perugia, italy #these authors contributed equally to this work. *corresponding author at: istituto zooprofilattico sperimentale dell'umbria e delle marche ‘togo rosati’ via g.salvemini 1, 06126 perugia, italy. e‑mail: elisa.sgariglia86@gmail.com. elisa sgariglia*#, nicholas aconiti mandolini#, maira napoleoni, laura medici, roberta fraticelli, michela conquista, paola gianfelici, monica staffolani, stefano fisichella, marinella capuccella, marta sargenti and gianni perugini antibiotic resistance pattern and virulence genes in avian pathogenic escherichia coli (apec) from different breeding systems 28 veterinaria italiana 2019, 55 (1), 27-33. doi: 10.12834/vetit.1617.8701.1 antibiotic resistance pattern and virulence genes in apec sgariglia et al. introduction escherichia coli (e.  coli) is considered a commensal microorganism in people and animals and it is part of normal intestinal microflora in birds (aarestrup et  al. 2008, de carli et  al. 2015). some strains might be pathogenic and cause colibacillosis, an extraintestinal disease characterised by pericarditis, air sacculitis, perihepatitis, peritonitis. colibacillosis is responsible for high economic losses in chicken industry (matthijs et al. 2009, matter et al. 2011, de carli et  al. 2015). it is the most frequent bacterial disease in avian species and e. coli is considered the first cause of death in poultry sector, even if usually it plays a secondary role during infection (lutful kabir 2010). colibacillosis is caused by avian pathogenic e.  coli (apec) (matin et al. 2017) and its pathogenic ability may be localized or systemic. the apec pathogenic ability is facilitated by broad range of virulence factors which are coded by virulence-associated genes (de carli et al. 2015). according to molecular criteria, apec is defined by presence of at least five virulence genes. according to molecular criteria, five genes carried by plasmids were considered as being the most significantly associated with highly pathogenic apec strains (de carli et al. 2015). escherichia coli strains are also considered good indicators of antimicrobial resistance because they are part of the physiological microbiota both in man and animals, and they are also present in the environment (aarestrup et al. 2008). antibiotic resistance represents a serious problem to global public health, resulting in a significant impact on animal health and food safety (aarestrup 2004). the misuse of antimicrobial agents could lead to selection and diffusion of resistant microorganisms with related increase of antibiotic resistance rate (spellberg 2014). furthermore, the problem of multi-drug resistance (mdr) can be transmitted and disseminated between animal and human pathogens, leading to treatment problems both animal and human diseases (collignon et al. 2005). poultry industries consume wide range of antibiotics, because only few regulations are controlling their use (hvistendahl 2012). in poultry industries, antibiotics have been used in chicken broilers as growth promoter and disease preventive measures (bhandari et  al. 2004, osti et  al. 2017, shrestha et al. 2017). the main goals of this study were (1) to determine the rates of antibiotic resistance of e.  coli isolated from several avian species, (2) to evaluate possible correlations between e.  coli isolation and the types of breeding and (3) to detect the presence of apec among e. coli isolates. table i. origin of escherichia coli strains isolated. progressive number type of breeding species and/ or production class type of samples 1 industrial breeding chicken intestinal swab 2 industrial breeding chicken liver 3 industrial breeding chicken liver 4 industrial breeding chicken intestinal swab 5 industrial breeding chicken liver 6 industrial breeding chicken intestinal swab 7 industrial breeding chicken lung 8 industrial breeding chicken femoral swab 9 industrial breeding chicken spleen 10 industrial breeding chicken liver 11 industrial breeding chicken intestinal swab 12 industrial breeding chicken femoral swab 13 industrial breeding chicken vertebral swab 14 industrial breeding chicken vertebral swab 15 industrial breeding chicken pool organs 16 industrial breeding chicken yolk sac 17 industrial breeding hen liver 18 industrial breeding hen liver 19 industrial breeding goose intestinal swab 20 industrial breeding goose heart 21 industrial breeding goose heart 22 chick dealer chicken liver 23 chick dealer chicken intestinal swab 24 chick dealer chicken liver 25 chick dealer chicken intestinal swab 26 chick dealer chicken liver 27 chick dealer chicken intestinal swab 28 chick dealer capon intestinal swab 29 chick dealer hen intestinal swab 30 chick dealer duck intestinal swab 31 chick dealer guinea fowl intestinal swab 32 chick dealer guinea fowl liver 33 chick dealer goose lung 34 chick dealer goose intestinal swab 35 chick dealer goose lung 36 chick dealer goose intestinal swab 37 rural breeding chicken intestinal swab 38 rural breeding chicken liver 39 rural breeding chicken intestinal swab 40 rural breeding chicken liver 41 rural breeding chicken intestinal swab 42 rural breeding hen intestinal swab 43 rural breeding hen lung 44 rural breeding hen heart 45 rural breeding hen liver 46 rural breeding hen spleen 47 rural breeding hen intestinal swab 48 rural breeding capon liver 49 rural breeding capon intestinal swab 50 rural breeding pigeon liver 51 rural breeding turkey intestinal swab 29veterinaria italiana 2019, 55 (1), 27-33. doi: 10.12834/vetit.1617.8701.1 sgariglia et al. antibiotic resistance pattern and virulence genes in apec and 1 minute at 72°c. the amplicons were analyzed by 2% agarose gel electrophoresis (euroclone®) prepared in 1x tbe buffer (biorad®). all the pcr products were stained with appropriate intercalating dye and the bands were visualized and photographed under uv light. the amplified product was considered to contain virulence gene if it produced band of the expected size. the amplicon size of the toxin genes of apec is described on the manufacturer’s instructions. data related to resistance and virulence genes rates were analyzed by the fisher exact test. a value of p < 0.05 was considered significant. results the e.  coli resistant strains found in this study are displayed in table ii. the resistance most frequently observed was against ampicillin and nalidixic acid (23/51, 45%) followed by tetracycline (22/51, 43%), sulphonamide (21/51, 41%), flumequine (17/51, 33%). escherichia coli isolates exhibited lower resistance to cefotaxime and cefepime (0%) followed by cefoxitin and ceftazidime (1/51, 2%). overall, 41% (21/51) of the isolates showed resistance to at least three or more antimicrobial agents with a significantly (p  >  0.05) higher number of isolates from animal of industrial breeding (13/21) than isolates obtained from animals of rural breeding (2/21). no significant differences (p > 0.05) were observed between the number of resistent isolates obtained from samples collected from animals of dealer and those from industrial and rural breeding. an e.  coli strain isolated from liver of an animal of industrial breeding was resistant to 11 antimicrobial molecules, including all antimicrobial categories. the rates of resistance according to the type of breeding were illustrated in figures 1, 2 and 3. of the 51 e.  coli isolates, 16 (31%) were found to be apec strains because they contain at least five virulence genes. out of 16 apec strains, 7 were isolated from samples collected from animals of industrial breeding (6 chicken and 1 hen), 5  from samples collected from animals of dealer (3 chicken and 2 geese), and 4 from samples collected from animals of rural breeding (2 geese, 1 capon and 1 hen). seven virulence genes were present in 2 apec strains, 6 in six strains and 5 in 8 strains. the virulence genes iucd, cvi/cva, irp2 and iss were detected in all 16 apec strains. the virulence genes tsh, vat, papc, and asta were detected in 11, 7, 5 and 3 apec strains, respectively. the virulence-associated genes iucd, iss, irp2, and cvi/cva were found in both apec and non-apec materials and methods fifty-one e.  coli strains from chickens (n  =  27), hens (n  =  9), geese (n  =  7), capons (n  =  3), guinea fowls (n = 2), duck (n = 1), pigeon (n = 1), and turkey (n = 1) originated from chick dealer (n = 15) , rural (n = 15) and industrial (n = 21) breeding were included in the present study. these isolates were from different types of samples (table i). e.  coli identification was performed with standard microbiological techniques which include studies of colony morphology, gram staining, and biochemical tests (api biomerieux®). the e.  coli isolates were tested for antibiotic susceptibility against 19 antimicrobial agents using disk agar diffusion method according to the clinical laboratory standard institute (clsi) and eucast guidelines, based on available clinical breakpoints. the antibiotics used in this study, belonged to 7 categories of antimicrobial agents, and included ampicillin (amp 10  µg), amoxicillin/clavulanic acid (amc 30  µg), cefotaxime (ctx 30  µg), cefepime (fep 30  µg), cefoxitin (fox 30  µg), ciprofloxacin (cip 5  µg), cefazolin (kz 30  µg), ceftazidime (caz 30 µg), chloramphenicol (c 30 µg), enrofloxacin (enr 5  µg), gentamicin (cn 10  µg), kanamicin (k 30  µg), meropenem (mem 10 µg), nalidixic acid (na 30 µg), streptomycin (s 10  µg), compound sulphonamides (s3 300  µg), tetracycline (te 30  µg), trimethoprim/ sulfamethoxazole (sxt 25  µg) and flumequine (ub 30  µg). these antibiotics were selected based on world health organization and regional pharmacovigilance center recommendations. furthermore, these antibiotics were a global representation of all antimicrobial classes. subsequently, e.  coli isolates were investigated for the presence of eight virulence genes with kylt® apec anicon kit. these genes are enteroaggregative toxin (asta), increased serum survival protein (iss), iron-repressible protein (irp2), p fimbrie (papc), aerobactin (iucd), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin v plasmid operon genes (cvi/ cva) (exers et al. 2005). for dna extraction the colony was immersed in 500  µl of dna extraction-mix ii and it was resuspended carefully. consequently, the sample vortexed, incubated for 10-15 minutes at 100  °c  ±  3  °c, vortexed again and centrifuged at 10,000-12,000 g for 5 minutes. the pcr was performed in 20 µl volume containing 18 µl master-mix (10 µl 2x pcr-mix, 2 µl 10x loading dye, 6  µl primer-mix) and 2  µl dna template. the cycling conditions were as follows: 94  °c for 3 minutes, 35 cycles of 30 sec at 94 °c, 30 sec at 57 °c, 30 veterinaria italiana 2019, 55 (1), 27-33. doi: 10.12834/vetit.1617.8701.1 antibiotic resistance pattern and virulence genes in apec sgariglia et al. strains but their detection rates were significantly (p < 0.05) higher in apec isolates. conversely, the virulence associated gene  papc was found in the apec isolates only. discussion colibacillosis caused by apec results in huge economic losses in poultry industry throughout the world (roy et al. 2006, barnes et al. 2008, dziva et al. 2008). antimicrobials are the main weapon to fight both the incidence and the mortality associated with colibacillosis (harisberger et  al. 2011). antibiotics were also used as feed additives to improve weight gain (bower et  al. 1999). however, indiscriminate 0 2 4 6 8 10 12 c h ic ke n animal species n u m b er c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n h en h en g o o se g o o se g o o se antimicrobial resistance antimicrobial category resistance figure 1. number of antimicrobials and antimicrobial categories in animals of industrial breeding. table ii. number of resistant, intermediate and susceptible strains according to the clinical laboratory standards institute (clsi) and the european committee on antimicrobial susceptibility testing (eucast) guidelines. antimicrobial antimicrobial acronym concentration (µg) resistant (%) intermediate (%) susceptible (%) ampicillin amp 10 45 0 55 amoxicillin/clavulanic acid amc 30 12 0 88 cefotaxime ctx 30 0 0 100 cefepime fep 30 0 0 100 cefoxitin fox 30 2 0 98 ciprofloxacin cip 5 18 6 76 cefazolin kz 30 16 18 66 ceftazidime caz 30 2 2 96 chloramphenicol c 30 22 0 78 enrofloxacin enr 5 18 22 60 gentamicin cn 10 6 6 88 kanamicin k 30 6 8 86 meropenem mem 10 0 0 100 nalidixic acid na 30 45 4 51 streptomycin s 10 24 12 64 compound sulphonamides s3 300 41 0 59 tetracycline te 30 43 4 53 trimethoprim/sulphametoxazole sxt 25 25 0 75 flumequine ub 30 33 12 55 0 2 4 6 8 10 c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n c ap o n h en d u ck g u in ea fo w l g u in ea fo w l g o o se g o o se g o o se g o o se animal species n u m b er antimicrobial resistance antimicrobial category resistance figure 2. number of antimicrobials and antimicrobial categories in animals of chick dealer. 0 1 2 3 4 5 6 c h ic ke n c h ic ke n c h ic ke n c h ic ke n c h ic ke n h en h en h en h en h en h en c ap o n c ap o n pi g eo n tu rk ey animal species n u m b er antimicrobial resistance antimicrobial category resistance figure 3. number of antimicrobials and antimicrobial categories in animals of rural breeding. 31veterinaria italiana 2019, 55 (1), 27-33. doi: 10.12834/vetit.1617.8701.1 sgariglia et al. antibiotic resistance pattern and virulence genes in apec iucd, both related to iron acquisition system, tend to be present on the same strain; in this study all the apec strains contained either iron acquisition systems. although at lower frequency, the asta gene was also detected in both the apec and non-apec isolates. in contrast, the virulence gene papc was detected in apec strains only. this finding confirms what reported in other similar studies and supports the hypothesis that it is an important virulent gene. the vat and tsh genes were distribuited in both the apec and non-apec strains. in this study, the frequency of virulence genes iucd, cvi/cva, irp2 and iss observed in apec strains was higher than that found by subedi and colleagues (subedi et  al. 2018) which reported frequency of irp2 and cvi/cva of 73.3% and 57.8%, respectively. in other studies the frequency of irp2 observed (59.3% and 67%) was also much lower (sadeghi bonjar et  al. 2017, kwon et  al. 2008). furthemore, kwon and colleagues reported 16% frequency of cvi/cva virulence gene (kwon et  al. 2008). the frequency of tsh virulence gene (69%) was comparable with literature data (subedi et al. 2018). lower frequencies were found for vat, papc and asta virulence genes (44%, 31% and 19%, respectively). four apec strains from rural breeding showed no resistance (2 strains) or only resistance to nalidixic acid (other 2 strains). however, it should be highlighted that 3/7 isolates from industrial breeding resulted susceptible to all drugs. out of 9 resistant apec strains, 6 showed multidrug resistance to at least three or more antimicrobials agents (4 from industrial breeding and 2 from dealer). although four strains from rural breeding were identified as apec, they did not show high level resistance. conversely, the strains from industrial breeding showed multidrug resistance suggesting a possible excessive use of antibiotics in poultry industries (subedi et al. 2018). these results demonstrate the importance of studies on apec, antibiotic resistance rates in our country and its correlation with poultry breeding, aiming to acquire preventive measures to minimize losses due to apec and multidrug-resistance that plays an important role and have high significance to public health. acknowledgements the authors would like to thank valentina stefanetti for her valuable technical assistance and andrea felici for his help in statistical analysis. use of antibiotics has provided selective pressure for the emergence of drug resistance strains which may lead to therapy failure and potential economic losses for breeders (scioli et al. 1983, quednau et al. 1998, bower et al. 1999, oosterik et al. 2014). of the nineteen antibiotics tested in this study, only two showed 100% efficacy against all e. coli strains. the highest rates of resistance (45%) were found with ampicillin and nalidixic acid. other studies have also shown similar resistance but with higher rates (yang et  al. 2004, li et  al. 2010, shrestha et  al. 2011, yassin et  al. 2017, matin et  al. 2017, shrestha et  al. 2017, bakhshi et  al. 2017, subedi et  al. 2018). similarly, for quinolones such as ciprofloxacin and enrofloxacin which in this study showed moderate resistance (18%) in other studies resistance rates higher than 50% were reported (yassin et  al. 2017, subedi et al. 2018). the resistance rates of the e. coli strains included in this study to ceftazidime and cefoxitin were very low. in contrast with a previous study of younis and colleagues (younis et  al. 2017), that found 95.8%, 90.4% and 76.7% resistance rates against cefepime, cefoxitin and cefotaxime, respectively, all the e.  coli isolates included in this study were susceptible to cefotaxine and cefepime.. tetracycline resistance in our isolates was quite high (43%) which is consistent with what found by other authors (vandemaele et  al. 2002, smet et  al. 2008, salehi et al. 2010, persoons et al. 2012, younis et al. 2017). the high levels of resistance to sulfonamides (41%) revealed in this study, was not unexpected. indeed sulfonamides were widely and continuously used for a long time and resistance was already described before 1950 (yassin et al. 2017). the resistance to aminoglycosides was quite low in our study (gentamicin and kanamicin 6%, streptomycin 24%), that is in disagreement with other authors which reported higher levels of resistance (yassin et al. 2017, yousin et al. 2017). in this study, the frequency of eight virulence genes and their correlation with phenotypic antibiotic resistance were evaluated. e. coli strains are defined apec when at least five virulence genes are detected. the virulence genes iucd, iss, irp2, and cvi/cva were found in both, apec and non-apec strains; the presence of these genes in both groups of strains might indicate that they are not associated with virulence. however, iss gene has been described as a virulence gene of recognised importance in e. coli of chickens (ellis et  al. 1988). furthermore, irp2 and 32 veterinaria italiana 2019, 55 (1), 27-33. doi: 10.12834/vetit.1617.8701.1 antibiotic resistance 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production, istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. 2laboratory animal and vaccine production, istituto zooprofilattico sperimentale della lombardia e dell’emilia romagna ‘b. ubertini’, via bianchi 9, 25124 brescia, italy. 3viral vaccines and diagnostic devices production, istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. *corresponding author at: department of bacterial vaccines and diagnostic devices production, istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861 332492, e-mail: f.profeta@izs.it. parole chiave vaccini stabulogeni veterinari, prova di “tossicità anormale” (att), mtt test, metodi alternativi. riassunto la produzione dei vaccini stabulogeni e degli autovaccini ad uso veterinario in italia è disciplinata dal decreto ministeriale n. 287 del 17 marzo 1994, recante indicazioni relative alla “produzione, l'impiego ed il controllo dei medicinali veterinari immunologici inattivati, aventi caratteristiche di vaccini stabulogeni ed autovaccini”, e rientra tra le attività principali svolte dagli istituti zooprofilattici sperimentali. la normativa prevede che, prima del rilascio, ciascun lotto di vaccino sia sottoposto alla prova di “tossicità anormale” (att abnormal toxicity test) su animali da laboratorio. il decreto legislativo n. 26 del 4 marzo 2014, “sulla protezione degli animali utilizzati a fini scientifici”, recepimento della direttiva europea 2010/63/ue, ribadisce l'importanza di identificare e validare metodi alternativi in accordo con i principi delle 3r di riduzione, affinamento e sostituzione (replacement, reduction, refinement) delle prove biologiche sugli animali da esperimento. a tal fine, 49 lotti di vaccini stabulogeni di origine batterica, precedentemente sottoposti, con esito positivo, alla prova di tossicità anormale, sono stati testati con il test di citotossicità cellulare mtt (methyl tetrazolium test) utilizzando la linea cellulare continua l929. tutti i vaccini saggiati alla diluizione 1:128 non hanno manifestato azione citotossica, alla diluizione 1:32 il 68% dei vaccini risultava idoneo, valutazione della sicurezza di vaccini stabulogeni veterinari attraverso il methyl tetrazolium test (mtt) keywords veterinary autogenous vaccines, att mtt assays, alternative methods. summary in italy, veterinary autogenous vaccines manufacturing is regulated by the legislative decree of the ministry of health, march 17th, 1994, n. 287. the production is performed by the network of the ‘istituti zooprofilattici sperimentali’ (izss), public health institutes scattered all over the italian territory. the aim of this research was to evaluate the feasibility of an in  vitro method to test the abnormal toxicity of autogenous bacterial vaccines as an alternative to animal models routinely employed. for this purpose, the istituto zooprofilattico sperimentale dell’abruzzo e del molise (izsam) in partnership with the istituto zooprofilattico sperimentale della lombardia e dell’emilia romagna (izsler), evaluated the toxicity of 49 batches of autogenous bacterial vaccines, previously shown to be safe in guinea pigs and mice, on animal model, by means of the methyl tetrazolium (mtt) assay. all vaccines showed cytotoxic effects when tested 1:2 diluted and undiluted; overall, all vaccines lost toxicity at 1:128 dilution. as expected, these findings suggest a different susceptibility of this assay compared to the laboratory animal model. on the other hand, these results do not clarify which components of the vaccines are responsible for the cytotoxic effect. overall, more experiments are warranted in order to standardize the mtt assay which could be coupled with the trials in laboratory animals. francesca profeta1*, osvaldo matteucci1, gianluca orsini1, luigina sonsini1, guerino lombardi2, sara capista3, daniela antonucci3, gaetano federico ronchi3 and mauro di ventura3 evaluation of veterinary autogenous vaccines safety by mtt in-vitro cytotoxicity assay veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 accepted: 23.04.2019 | available on line: 31.12.2019 300 veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 abnormal toxicity evaluation of autogenous bacterial vaccines by mtt profeta et al. correct design of a scientific project4. this task will be achieved by defining the most suitable animal model to be employed and by reducing their number with increased animal welfare (combrisson 2014). the three r’s principles expressed in 1959 by russell and burch, represent the main ethical guideline addressing the eu directive 63/2010. it foresees a reduction in the number of animals (reduction), their substitution with effective in vitro and in silico models or with animals with the lowest capacity to experience pain (replacement) and an improvement of welfare conditions (refinement)4 (fabre 2009). while in many fields it hasn’t been possible yet to replace animal models, in some other, as toxicology (mainly acute topic toxicity, such as skin irritation), several in  vitro tests were validated3 (faller and bracher 2002, oecd 439 2015, ohtake et al. 2018). within these alternative tests, the 3-(4,5-dimethyl-2-t hiazoly)-2,5-diphenyl-2 -h-tetrazolium bromide-mtt assay is included. mtt assay has been widely employed in many fields, ranging from regenerative medicine, dermatology and orthodontics, to immunology and toxicology (thoneman et al. 2002, di francesco et  al. 2005, malkoc et  al. 2010, patnaik and padhy 2018, qi et al. 2018). this test recognizes live cells for their capability to reduce tetrazolium mtt salt, a yellowish reagent, to an intracellular, purple product, named formazan; on the contrary, dead cells lack the ability to process any substrate, therefore they still not induce any color change and wells remain yellow. the amount of live/dead cells can be quantified by a spectrophotometer that measures the amount of light that passes through the purple/yellow solution. introduction autogenous vaccines veterinary vaccines are an important tool for controlling animals infectious diseases with a tremendous impact on antimicrobial resistance. their use has been indeed promoted in the last years (thibault 2004, monath 2013, oie 2018) rather than treat animals with antibiotics, into a ‘one health’ vision (kaplan et al. 2009, evans and leighton 2014). the current legislation regarding autogenous vaccines, decree of the italian ministry of health, 17 march 1994, n. 2871, establishes that these products can be released after a safety control, named ‘abnormal toxicity test’ (att), that has to be carried out in laboratory mice and guinea pigs (ep 01/2008:20609) by injecting subcutaneously a specified volume of final product, and waiting the following 7 days for any adverse reaction to occur2. anyway, side effects in these animals were never reported by the istituti zooprofilattici sperimentali (izss), the legal italian publich health authorities which are in charge for this task. for this and for other reasons, such as the low specificity of the test (e.g. strain-related differences, or the fact that stressful conditions can produce different results), the reliability of in  vivo methods is questionable3 (kumar et al. 2018). on september 2010, a new european union directive for the protection of vertebrate animals used for experimental purposes, directive of 22 september 2010, n. 63 of the european parliament and of the council, increased the protection of experimental animals and posed specific rules regarding the 1 ministry of health. ministerial decree of 17 march 1994, n. 287. regolamento recante norme sulla produzione, l'impiego ed il controllo dei medicinali veterinari immunologici inattivati, aventi caratteristiche di vaccini stabulogeni ed autovaccini. gu, 111, 14.05.1994. 2 coordination group for mutual recognition and decentralized procedures veterinary medicine (cmdv) & heads of medicines agencies (hma). 2017. recommendations for the manufacture, control and use of inactivated autogenous veterinary vaccines within the eea. ema/cmdv/452656/2016. rec-002-01. london, 20 march 2017. http://www.hma.eu/fileadmin/dateien/veterinary_medicines/cmdv_website/procedural_guidance/miscellaneous/ recommendations_ manufacture_control_use_inact_autogenous_vaccines.pdf. 3 tellner p. & european federation of pharmaceutical industries and associations (efpia). 2017. deletion of test for abnormal toxicity from european pharmacopoeia. european federation of pharmaceutical industries and associations (efpia). version: final, 30/06/2017 https://www.efpia.eu/ media/219814/deletion-of-test-for-abnormal-toxicityfrom-european-pharmacopoeia.pdf. 4 european parliament and council union (eu). 2010. directive 2010/63/eu, 22 september 2010 on the protection of animals used for scientific purposes. off j, l 276/33, 20.10.2010. mentre alle diluizioni 1:2 e tal quale c'è stata una riduzione di vitalità cellulare > 30% in tutti i vaccini. dai dati ottenuti si evidenzia una diversa suscettibilità del test mtt rispetto al saggio di tossicità anormale su animali. non è stato finora possibile comprendere quali costituenti siano effettivamente responsabili della citotossicità dei vaccini, inoltre è emersa la necessità di produrre controlli positivi affinché la prova possa essere standardizzata. dai risultati ottenuti si evince che, se ulteriormente saggiato e standardizzato, il test mtt potrebbe costituire un metodo alternativo e complementare al tradizionale saggio in vivo attualmente in uso. 301veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 profeta et al. abnormal toxicity evaluation of autogenous bacterial vaccines by mtt l929 cells were grown in 75 cm2 cell culture flasks with filtered cap (thermo fisher scientific, waltham, massachusetts, usa) and were cultured in mem (modified eagle’s medium sigma aldrich, saint louis, missouri, usa) without phenol red, supplemented with 10% fetal bovine serum (bfs) (sigma aldrich, saint louis, missouri, usa), nahco 3 2.2 g/l and glutammine 200 mm (sigma aldrich, saint louis, missouri, usa). cells were incubated at 37  °c at 5% co 2 and were daily observed under inverted microscope (20x-40x); at 90-100% of confluence, cells were trypsinized, centrifuged (300  g x 10 min at 4  °c) and diluted in mem medium with phenol red (sigma aldrich, saint louis, missouri, usa) supplemented with 10% bfs, to obtain a final concentration of 3 x 105 cells/ml. cell suspension was transferred onto 96-well flat bottomed cell culture plates (bd biosciences, massachutes, usa) (100 µl of cell suspension/well) excluding the peripherals wells of the plate filled with 100 µl of mem without phenol red. plates were incubated for 24 hours at 37 °c at 5% co 2 . at the end of incubation, plates were observed under inverted microscope (magnification 20x-40x) to verify that a minimum of 70% of growth, necessary to perform the mtt assay, has been reached. thirty-five out of forty-nine vaccines were tested starting from the original and undiluted material up to dilution  1:32. a positive control, consisting of sterile saline solution containing 0.5% phenol, was also enrolled for the study. the remaining 14/49 vaccines were tested with the same procedure but up to 1:256 dilution. for these samples, a different positive control, consisting of a sterile saline solution containing sodium ethylmercurithiosalicylate 0.005%, was used (table i). to perform the mtt test, the supernatant of the cell culture plates, at 70% of confluence, was discharged. samples included by columns 3,4,5 and rows b-g, contained 100  µl of undiluted and two-fold diluted vaccines, in triplicate. mem without phenol red was employed as diluent. wells included by columns the present study aimed to evaluate the effectiveness of the mtt assay in assessing the toxicity of bacterial autogenous vaccines. materials and methods autogenous vaccines manufacturing a total number of 49 autogenous vaccines, produced by izsam, during the years 2013-2018, were tested by mtt. vaccines were produced following an official request using, as starting material, microbial agents isolated during outbreaks in domestic animals (figure 1). the isolates were propagated into specifics growth medium and subsequently, after purity and identity tests, pure liquid cultures were amplified in the proper amount of growth medium, depending on the number of requested doses. after growth, bacterial cultures were harvested and inactivated by dilution in 0.4% sterile saline solution containing formaldehyde (37%), followed by incubation at 37  °c for 24 hours. for clostridium spp., the inactivation was achieved by adding 0,6% sterile saline solution containing formaldehyde (37%), followed by incubation for 21 days at 37 °c. once bacterial inactivation was confirmed, the product was diluted in saline solution containing sodium ethylmercurithiosalicylate 0.005% as preservative, to reach a final concentration ranging between 2.2 x 109 to 3 x 109 cfu/ml. finally, 10% of aluminum hydroxide was added as adjuvant. before the delivery, final products were tested to establish the residual concentration of free-formaldehyde, that should not exceed 20 ppm (ph. eur. 01/2008:20418), and were also submitted to evaluate the abnormal toxicity in laboratory animals, in accordance to md n. 287/1994. three mice were inoculated subcutaneously with 0.5 ml of each vaccine and observed for 7 days (21  days for clostridium spp. vaccines) for any adverse reaction. for clostridium spp. autogenous vaccines, 3 guinea pigs were also injected subcutaneously of vaccine, following the same protocol provided for mice. vaccines were rejected when a local or a systemic reaction was observed (e.g. increase in body temperature, variation in feed and water intake) within 7 days (ph. eur. 04/2013:50209). mtt test l929 cell line, provided by izsler (cod. bs cl 56), deriving from mouse fibroblast cells located in the adipose tissue (nordin et al. 1991, badole et al. 2013), were employed for the mtt assay. clostridium spp. 12% salmonella spp. 16%pasteurella spp. 6% escherichia spp. 43% pseudomonas spp. 2% staphylococcus spp. 21% figure 1. bacterial autogenous vaccines tested by mtt assay. 302 veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 abnormal toxicity evaluation of autogenous bacterial vaccines by mtt profeta et al. mean od samples = mean optical density for each sample, tested in triplicate, columns 3, 4, 5, rows b-g; mean od blank = mean optical density of the blanks, for each sample tested in duplicate in columns 2-11, row b-g. the results for each one of the 49 mtt assays were considered valid if mean od values for blank and pc were ≥ 0.2 and < 0.2, respectively (table ii). a minimum of 70% cell viability was considered as cut-off (final dilution fd70). statistical analysis the percentages of non-cytotoxic vaccines for each dilution were calculated. in order to associate an uncertainty with this probability, the 95% confidence intervals, according to a bayesian approach, were calculated by means of the beta distribution. results vaccines were not toxic in laboratory animals the att test performed with the 49 autogenous vaccines did not elicit adverse reaction in laboratory animals. 2 and 11 and rows b-g were used as blank by adding 100 µl of mem without phenol red. wells belonging to columns 6 and 7 were employed as negative control (nc) by adding 100 µl of mem without phenol red but containing 10% bfs. finally, wells belonging to columns 8, 9 and 10, rows b-g, served as positive control (pc). plates were then incubated for 24 hours at 37 °c, 5% co 2 . after 24 hours, plates were evaluated to detect cytotoxic effect; then, the medium was discharged and plates washed once with 100 µl of pbs. subsequently, 50 µl of mtt 1x (50 mg/50ml) reagent (merck millipore, italy) was added to each well. plates were incubated at 37 °c for 2 hours. after incubation, 100 µl of isopropyl alcohol 100% (sigma aldrich, italy) was added to each well. after a gentle shaking of the plates for 30 minutes at room temperature, the optical density (od) was measured at a 550 nm wavelength on a spectrophotometric benchmark microplate reader and results were processed by microplate manager 5.2 (biorad, italy). for each dilution the mean cell viability (cv%) was calculated as follows: mean cell viability (cv%) = [mean od (samples)/ mean od blank] x100 where: table i. organization of the 96 wells cells culture plates for each vaccine. a total of 35 vaccines were tested up to the 1:32 dilution (a); the remaining 14 vaccines were tested up to the 1:256 dilution (b). a 1 2 3 4 5 6 7 8 9 10 11 12 a mem mem mem mem mem mem mem mem mem mem mem mem b mem blank sample u sample u sample u nc nc pc pc pc blank mem c mem blank sample 1:2 sample 1:2 sample 1:2 nc nc pc pc pc blank mem d mem blank sample 1:4 sample 1:4 sample 1:4 nc nc pc pc pc blank mem e mem blank sample 1:8 sample 1:8 sample 1:8 nc nc pc pc pc blank mem f mem blank sample 1:16 sample 1:16 sample 1:16 nc nc pc pc pc blank mem g mem blank sample 1:32 sample 1:32 sample 1:32 nc nc pc pc pc blank mem h mem mem mem mem mem mem mem mem mem mem mem mem b 1 2 3 4 5 6 7 8 9 10 11 12 a mem mem mem mem mem mem mem mem mem mem mem mem b mem blank sample 1:64 sample 1:64 sample 1:64 nc nc pc pc pc blank mem c mem blank sample 1:128 sample 1:128 sample 1:128 nc nc pc pc pc blank mem d mem blank sample 1:256 sample 1:256 sample 1:256 nc nc pc pc pc blank mem e mem mem mem mem mem mem mem mem mem mem mem mem f mem mem mem mem mem mem mem mem mem mem mem mem g mem mem mem mem mem mem mem mem mem mem mem mem h mem mem mem mem mem mem mem mem mem mem mem mem u = undiluted; nc = negative control; pc = positive control; mem = modified eagle’s medium. 303veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 profeta et al. abnormal toxicity evaluation of autogenous bacterial vaccines by mtt cytotoxic effect is due to one or more vaccines components. when employed as positive control the preservative itself damaged l929 cells in all pc wells, suggesting that vaccine’s cytotoxic effect may be related mainly to it. our data show that mtt has a different susceptibility with respect to the in vivo att. in our settings, in order to find the dilution at which cytotoxicity is completely lost, 14  vaccines, that showed df70  ≥  1:32, were re-tested at higher dilutions, employing the protocol previously described. all 14 vaccines showed values ranging from 1:32 to 1:128 (table  iii); therefore, with all due caution, 1:128 dilution could represent the dilution limit (df70) from which, if cytotoxic effects are absent, the safety of autogenous vaccines can be ensured. moreover, in order to apply and validate the assay, vaccines which were previously rejected by att, so toxic for live animals, need to be tested by mtt. in this way, we could easily define the df70 for safe and unsafe vaccines with relevant consequences in terms of reliability of the assay and reduction of the use of live animals. reasonably we do not most of vaccines lost toxicity at 1:32 dilution all vaccines under investigation were cytotoxic when tested undiluted and 1:2 diluted, as they reduced more than 70% of l929 cell viability; cytotoxicity progressively decreased up to 1:128 dilution (table iii). interestingly, 32 out of 49 vaccines lost their citotoxicity at 1:32 dilution (p  value: 87.76%; lower confidence limit: 75.6; higher confidence limit: 94.18) (table iv). discussion the mtt assay (fotakis and timbrell 2006), is a cell viability test able to evaluate the in vitro cytotoxic effect of several compounds after interaction with a cell monolayer (mosmann 1983, lin et al. 2015). in our study the mtt assay was employed to detect vaccines cytotoxicity; low dilutions induce cytotoxicity which decreases in a concentration-dependent manner. nevertheless, the assay lacks of the ability to recognize if the table ii. spectrophotometric reading (wavelength: 550 nm) for autogenous vaccine n. 35. the mean od values for blanks and pc were 0.58, and 0.04, respectively; therefore mtt assay for vaccine n. 35 was considered valid. blank 35 35 35 nc nc pc pc pc blank undiluted 0.515 0.045 0.049 0.047 0.673 0.749 0.041 0.043 0.057 0.546 1:2 0.595 0.046 0.045 0.045 0.748 0.716 0.046 0.047 0.049 0.62 1:4 0.532 0.043 0.049 0.047 0.641 0.629 0.048 0.05 0.051 0.675 1:8 0.663 0.228 0.242 0.241 0.825 0.802 0.051 0.05 0.066 0.593 1:16 0.693 0.662 0.645 0.6 0.879 0.879 0.045 0.043 0.088 0.771 1:32 0.503 0.475 0.48 0.634 0.724 0.641 0.049 0.05 0.059 0.582 mean value 0.54 0.04 0.05 0.05 0.62 nc = negative control; pc = positive control. table iii. numbers in bold indicate how many vaccines showed (per pathogen) a reduction of cytotoxicity for at least the 70% of l929 cells; the corresponding dilution represents the dilution limit for each vaccine (final dilution-fd70). cv values ≥ 70% dilutions s. abortus ovis s. abortus equi clostridium spp. e. coli pseudomonas spp. staphylococcus spp. pasteurella spp. total undiluted 0 1:2 0 1:4 1 1 1:8 1 1 1:16 1 3 2 1 2 9 1:32 1 4 18 1 8 32 1:64 2 1 3 1:128 1 2 3 1:256 total 4 4 6 21 1 10 3 49 304 veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 abnormal toxicity evaluation of autogenous bacterial vaccines by mtt profeta et al. in conclusion, further investigations are required to standardize att for autogenous vaccines in order to promote, if solid and robust data are obtained, amendments to the current legislation. grant support this study was supported by the italian ministry of health, in the framework of the research project ‘sviluppo di metodiche alternative all’utilizzo di animali nelle attività diagnostiche e di controllo dei prodotti biologici negli istituti zooprofilattici sperimentali (iizzss)’ (grant number: ricerca corrente 2016 msaabs0116. scientific coordinator: dr. guerino lombardi, izsler). acknowledgments the authors are grateful to dr. romolo salini (izsam), for his assistance in the statistical analysis. support the single use of this assay. mtt can, indeed, efficiently couple att as a front-line screening tool for the mandatory in vivo trials of the most promising vaccines (schwanig et al. 1997, kumar et al. 2018). table iv. p values, corresponding to the cumulative number of vaccines that lost cytotoxicity at a specific dilution/ total of examined vaccines. for each p value, confidence limits for beta distribution (95%) are also reported. dilution loss of cytotoxicity examined p l.c.l. (95%) u.c.l. (95%) 1:4 1 49 2.04% 0.49% 10.65% 1:8 2 49 4.08% 1.25% 13.71% 1:16 11 49 22% 13.06% 35.96% 1:32 43 49 87.76% 75.69% 94.18% 1:64 46 49 93.88% 83.45% 97.78% 1:128 49 49 100.00% 94.18% 100.00% 305veterinaria italiana 2019, 55 (4), 299-305. doi: 10.12834/vetit.1778.9390.2 profeta et al. abnormal toxicity evaluation of autogenous bacterial vaccines by mtt badole g p, madhukar m. m., meshram g.k., bahadure r. n., tawani s., tawani g. & badole s.g. 2013. a comparative evaluation of cytotoxicity of root canal sealers: an in vitro study. 2013. restorative dentistry endodontics, 38 (4), 204-209. ciuchini f., fischetti r. & piccininno g. 1986. manuale per il controllo di qualità di sieri, vaccini e prodotti diagnostici di origine batterica, per uso veterinario. roma, istituto superiore di sanità (rapporti istisan 86/13). combrisson h. 2014. the directive 2010/63/ue: the explicit and the implicit. bull acad vet france, 167 (2), 137-142. di francesco c.e., leone a., lombari v., luciani m. & 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fibroblasts, primary and immortalized bovine dental papilla-derived cell lines to dental resin components. dental materials, 18 (4), 318-323. world organisation for animal health (oie). 2018. principles of veterinary vaccine production. chapter 1.1.8. in manual of diagnostic tests and vaccines for terrestrial animals. 7th ed. oie, paris. 343 short communication veterinaria italiana 2018, 54 (4), 343‑348. doi: 10.12834/vetit.1502.8097.1 accepted: 28.01.2018 | available on line: 31.12.2018 bluetongue (bt) is a sub‑acute to acute culicoides‑borne viral disease of ruminants in temperate and tropical areas throughout the world (toussaint et  al. 2006, mellor et  al. 2008). the world organization for animal health (oie) listed bt as an economically important emerging disease, especially in sheep. according to savini and colleagues (savini et  al. 2017), the causative orbivirus, bluetongue virus (btv), exists as 28 serotypes (hofmann et  al. 2008, maan et al. 2011, zientara et al. 2014), the last parole chiave bluetongue, cammello, marocco, rischio. riassunto tra il 2010 e il 2013 è stata condotta un’indagine sierologica per valutare se i cammelli (camelus dromedaries) possano essere utilizzati come animali sentinella per rilevare la circolazione del virus della bluetongue (btv). campioni di siero di 537 cammelli provenienti da varie località del marocco sono stati quindi testati mediante elisa competitiva e nel 41.8 % degli animali sono stati rilevati anticorpi nei confronti di btv. mediante il saggio di sieroneutralizzazione, effettuato verso btv‑1, ‑4, ‑6, ‑8, ‑11, ‑14 e ‑16, è stato possibile rilevare la presenza di anticorpi verso tutti i sierotipi testati ad eccezione del btv‑11. questo studio, dunque, non solo conferma la circolazione in marocco dei sierotipi btv‑1, ‑4, ‑8, ma prova, per la prima volta nel paese, la circolazione di btv‑6, ‑14, e ‑16. si può quindi concludere che i cammelli potrebbero essere utilizzati come animali sentinella per rilevare l’eventuale circolazione e possibili nuove incursioni di btv in marocco. il valore dei cammelli come sentinelle per il virus della bluetongue in marocco keywords bluetongue, camels, morocco, risk. summary a serosurvey was conducted to determine the value of camels (camelus dromedaries) as sentinel animals for the detection of bluetongue virus (btv) in morocco. between 2010 and 2013, camels from various localities in morocco were randomly tested for antibodies against btv serotypes‑1, ‑4, ‑6, ‑8, ‑11, ‑14, and ‑16. antibodies against 1 or more serotypes were detected in 41.8% of 537 camels tested with a competitive enzyme‑linked immunosorbent assay (elisa) diagnostic test. of the 7 tested serotypes, only btv‑11 antibodies were not detected with serum neutralisation assays. this study not only confirms the epidemiological presence of btv‑1, ‑4, and ‑8 in morocco, but also presents the first evidence of btv‑6, ‑14, and ‑16 in the country. as such, we conclude that camels would be ideal sentinel animals to determine the potential risk of btv in morocco. kamar drif1*, gert venter2,3, mehdi el harrak4, ouafaa fassi fihri1, chafiqa loutfi6, nadia touil5 and bachir harif6 1 agronomy and veterinary institute (iav) hassan ii, al irfane, rabat, morocco. 2 epidemiology, parasites and vectors, agriculture research council‑ onderstepoort veterinary research, onderstepoort, south africa. 3 department of veterinary tropical diseases, faculty of veterinary science,university of pretoria, onderstepoort, south africa. 4 clinvet, benslimane, morocco. 5 equipe de recherche en virologie moléculaire et onc‑biologie, faculté de médecine et de pharmacie, université mohamed v, av. mohamed belarbi el alaoui, rabat, morocco. 6 biopharma, société de production biologique et pharmaceutique vétérinaire, rabat, morocco. * corresponding author at: agronomy and veterinary institute (iav) hassan ii, al irfane, rabat, morocco. tel.: +212 6 62761668, e‑mail: drif.kamar@gmail.com. the value of camels as sentinels for bluetongue virus in morocco 344 veterinaria italiana 2018, 54 (4), 343‑348. doi: 10.12834/vetit.1502.8097.1 year, thereby providing a possible mechanism for viral overwintering in morocco (baylis et  al. 1997). other species encountered by baylis and colleagues (baylis et  al. 1997) include culicoides circumscriptus kieffer, c.  newsteadi, culicoides puncticollis (becker), culicoides obsoletus meigen, and culicoides pulicaris  (l.). while c.  imicola and c. obsoletus are considered proven vectors of btv (purse et al. 2015), the detection of btv in field collected c. newsteadi in the 2012 to 2014 bt epidemics in italy implicated this species as a potential vector (goffredo et al. 2015). bluetongue virus infects all known species of ruminants, but severe disease is usually restricted to certain breeds of sheep, especially european breeds, e.g., dorset horn and some species of deer, e.g., white‑tailed deer (odocoileus virginianus) (taylor 1986, barnard 1997). experimental infection has shown that although camels do not display clinical signs of bt, they can seroconvert and develop btv specific neutralising antibodies (batten et  al. 2011). when infected with the virus, camels developed viremia from 7 days post infection, albeit at lower levels than in experimentally infected sheep and cattle (batten et al. 2011). the isolation of viable virus from the blood of experimentally infected camels suggests that they might act as reservoir hosts, and as such play a role in the epidemiology of bt. according to the food and agriculture organization (fao) of the united nations, the number of camels in morocco increased from 52,000 in 2010 to 59,000 in 2017. in 2017, about 19,863,000 sheep were estimated to be at risk of btv (faostat 2017). in the same year, the number of cows, which are also considered as a reservoir host for btv, was estimated at 3,364,000 (fao stat2017)1. this preliminary study focuses on the extent to which camels in the south of morocco were exposed to the btv serotypes present in and around the mediterranean basin. information on the role of camels in the epidemiology of btv, and the extent to which they can be used to monitor circulating serotypes and therefore help determine the risk of this disease occurring in morocco will contribute to the implementation of timeously and effective control programmes. from may 2010 to february 2013, sera were randomly collected from 538 camels at abattoirs and from animals intercepted by veterinary services at border posts in southern and southwestern morocco (table i). intercepted camels came from neighboring countries and crossed the border illegally. when seized, the animals were kept in quarantine until tested for a number of infectious and contagious of which was detected in 2014 in china (sun et  al. 2016). further potential novel btv serotypes have recently been described. amongst them we cite: btvxitl2015 detected in sardinian goats in 2015 (savini et  al. 2017), that which was detected in a sheep pox vaccine preparation in israel (bumbarov et al. 2016) and another strain, for which only partial genome sequences exists, which was isolated from an alpaca in south africa (wright 2014). the spread of at least 5 serotypes of btv – btv‑1, btv‑2, btv‑4, btv‑9, and btv‑16 – throughout mediterranean europe since 1998 suggests changes in the abiotic and biotic factors that first defined orbivirus distribution, and highlights the importance of understanding these underlying mechanisms (purse et  al. 2015). the timeously detection of circulating serotypes in morocco will contribute to the implementation of effective control programmes. in morocco, bt (btv‑10) was identified for the first time in 1956 in the southern area of laarach and west of arbua (placidi 1957). in september 2004, after an epidemiological silence of almost 50 years, 1876 sheep exhibited subclinical disease involving btv‑4 in morocco (oie 2004). a serological survey conducted in camels (camelus dromedaries) in 2003 suggested that btv had actually been subclinically present in camels in morocco before 2004 (touil et  al. 2012). in august 2006, btv‑1 spread into algeria and reached tunisia and sardinia during late 2006. in september 2006, it re‑emerged in morocco, causing widespread and severe clinical disease in sheep (oie 2006). the disease intensified in 2007 with outbreaks spreading to different areas of morocco (oie 2007). although it is generally accepted that btv is almost exclusively transmitted by certain species of culicoides‑biting midges (diptera: ceratopogonidae) (purse et  al. 2015), vector‑free transmission has recently been demonstrated. transmission can either be vertical, from dam to fetus (rasmussen et  al. 2013, van der sluijs et  al. 2013, savini et  al. 2014), or horizontal, via direct contact (batten et  al. 2013, batten et al. 2014). vector surveys conducted from april 2009 to march 2010 at 14 sites in morocco indicated that the 2 most abundant livestock associated with culicoides species were culicoides imicola kieffer (94.2% to 95.9%) and culicoides newsteadi austen (2.2% to 2.7%) (lhor et  al. 2015). earlier surveys conducted between 1989 and 1991 indicated that c. imicola was widely distributed in morocco (baylis et  al. 1997). indeed, c.  imicola was absent at only 1 (near settat) of 22 sites sampled across most of morocco. culicoides were most abundant in the low‑lying northwestern areas (between tangier and rabat) and at marrakech, where at least one adult c. imicola per night was collected with a light trap over one camel as btv sentinels drif et al. 1 faostat. 2017. http://www.fao.org/faostat/fr/#data/qa accessed on 20 december 2018. 345veterinaria italiana 2018, 54 (4), 343‑348. doi: 10.12834/vetit.1502.8097.1 serotyped for antibodies against btv serotypes that had previously encountered in the mediterranean region i.e., btv‑1, btv‑4, btv‑6, btv‑8, btv‑11, btv‑14, and btv‑16 (toussaint et  al. 2006). neutralisation titers were determined by serum neutralisation testing (snt) according to reed and muench (reed and muench 1938) as the reciprocal of the highest dilution of serum that resulted in a 50% neutralisation endpoint titration. the snt samples were heat inactivated at 56°c for 30 minutes. neutralising antibody titers were determined by the micro‑method of snt in 96‑well plates according to the oie manual of diagnostic tests and vaccines for terrestrial animals (oie 2009). back‑titrations of each virus were included to confirm the 100 tcid 50 dose. the number of camels tested annually, which ranged from 87 in 2010 to 273 in 2012, as well as the proportion of antibodies that were detected at the various collection sites, are shown in table  i. the presence of antibodies against one or more serotypes of btv in 225 of 538 camels tested (41.8%), indicates that btv is highly prevalent in camels in morocco (table i). this result is consistent with those obtained by touil and colleagues et  al. (touil et  al. 2012), who reported a 42% seroprevalence in camels in guelmim, morocco, in 2003. the proportion of positive samples ranged from 13.3% in 2013 to as high as 60.4% in 2012. the highest number (68.3%) of camels showing high antibody titers against btv was registered at errachidia in march 2012 (table i). as sampling was not conducted at the same time of the year, direct comparison of prevalence between the various sites is not possible. overall, the highest seroprevalence rate (66.6%) was obtained in camels bled in march 2012. relatively high seroprevalence rates (53.6%) were also obtained in may 2010 and july 2012 (table i). this period coincides with the end of the rainfall season and a gradual increase in temperature. it also coincides with the potential abundance of culicoides diseases, including bt. while quarantined, all contact with local animals was prohibited. the facilities were, however, not insect‑proof. in morocco, the results of the entomological investigations showed a great abundance of culicoides populations, particularly c.  imicola and c. newsteadi, for two periods: spring (april‑june) for both species, and autumn (october‑november) for c.  imicola (lhor 2017). although camels bled at abattoirs usually represent camels from the immediate vicinity, their origin and age are not always known. blood was collected from the jugular vein of adult animals (older than 5 years) using 10‑ml vacutainer tubes without anticoagulant. after collection, the blood was left to clot overnight at 4°c. the following morning, after centrifugation at 2,500 rpm, the serum was transferred to 2.5‑ml eppendorf tubes and stored at ‑20°c. btv antibodies were detected using a commercial competitive elisa (celisa) kit from vmdr, inc. (pullman, wa, usa) following the manufactures instructions. the celisa micro‑plates were read using a biokit spectrophotometer (bio tektm elx 808‑fisher scientific‑usa) with an interference filter 620‑650 nm. sera displaying a mean optical density (od) of < 50% of the mean of the negative controls obtained from the reference sera bank of biopharma, were considered positive. a total of 225 celisa‑positive serum samples were drif et al. camel as btv sentinels table i. number of camels bled yearly at various abattoirs and border posts in southern morocco to determine btv seroprevalence and the number of sera that tested positive for the presence antibodies against btv. year month sampling locality no of sera collected no of celisa pos sera (%) 2010 may intercepted at zagora/ ouerzazate 35 22 (62.9) oct imported from mauritania (dakhla) 19 3 (15.8) nov intercepted at ouerzazate 33 4 (12.1) total 87 29 (33.3) 2011 aug intercepted at errachidia 10 0 (0) aug abattoir laâyoune province 48 15 (31.3) total 58 15 (25.9) 2012 march intercepted at errachidia 41 28 (68.3) march abattoir laâyoune province 97 63 (65.0) july abattoir guelmim‑es semara 79 48 (60.8) july abattoir laâyoune province 56 26 (46.4) total 273 165 (60.4) 2013 feb laâyoune province 120 16 (13.3) total 120 16 (13.3) grand total 538 225 (41.8) table ii. viral neutralisation results of 225 celisa positive camel sera collected between 2010 and 2013 in morocco and tested against seven btv serotypes. year no of pos camels btv serotype 1 (%) 4 (%) 6 (%) 8 (%) 11 (%) 14 (%) 16 (%) total 2010 29 8 (27.6) 2 (6.9) 0 6 (20.7) 0 7 (24.1) 6 (20.7) 29 2011 15 2 (13.3) 0 1 (6.7) 5 (33.3) 0 3 (20.0) 4 (26.7) 15 2012 165 108 (24.7) 43 (9.7) 10 (2.2) 113 (25.4) 0 71 (16.0) 100 (22.5) 445 2013 16 7 (28.0) 7 (28.0) 1 (4.0) 3 (12.0) 0 2 (8.0) 5 (20.0) 25 total 225 125 (24.3) 52 (10.1) 12 (2.3) 127 (24.7) 0 83 (16.1) 115 (22.4) 514 346 camel as btv sentinels drif et al. veterinaria italiana 2018, 54 (4), 343‑348. doi: 10.12834/vetit.1502.8097.1 detected in a further 15 camels. although the ages of the camels were unknown, it can be expected that the number of circulating serotypes will increase as the camel get older (figure 1). the serotypes btv‑4 and btv‑1 had been previously detected in morocco (oie 2004, oie 2006). between 2009 and 2012, btv‑8 was detected for the first time in sheep and cattle and appears now to be endemic (drif et  al. 2014). the present study presents the first evidence of btv‑6, btv‑14, and btv‑16. although only antibodies were detected in this survey, the relative abundance and widespread occurrence of seropositive camels indicates that the virus must have been circulating in morocco during the past 5 years. it has previously been demonstrated that btv‑1 can persist asymptomatically for up to 2 months (i.e. low levels of viral rna was found in the blood) in artificially infected camels (batten et  al. 2012). in addition, infected camels were found a year before the onset of the bt epidemic in morocco; results of a serological survey of 1,392 camel sera showing 42% positive sera in guelmim in 2003 clearly indicate that btv was circulating in camels before the first notified outbreak in sheep in 2004 (touil et al. 2012). in a study conducted in 2011 in neighboring algeria, btv seroprevalence was 14% in sheep, 21% in goats, and 29% in cattle (madani et  al. 2011). in the same study, the seroprevalence was 21% in camels. in 2016, lorusso and colleagues (lorusso et  al. 2016) reported the circulation of btv‑26 in neighbouring mauritania, with btv‑26 antibodies present in 108 of 159 camels that were tested. since btv‑26 was not included in this study, it remains to be determined if this serotype is present in morocco. touil and colleagues (touil et  al. 2012) indicate a relatively high abundance and geographical distribution of camels in morocco in addition to the sub‑clinical infection of btv in camels. the fact that the camels move frequently across morocco increases their potential to transport btv over long distances. due to an apparently low viremia, the potential reservoir contribution of camels to btv epidemiology may be low, but a steady rise in population, along with the sub‑clinical nature of their infections could cause the species to contribute to the short‑term persistence of btv. the clandestine movement of animals across borders could be one potential entry route for other serotypes of btv into southern morocco. the fact that at least seven different btv serotypes – btv‑1, btv‑2, btv‑4, btv‑8, btv‑9, btv‑14, and btv‑16 – have invaded the west mediterranean basin since 1999 (youssef et  al. 2017) emphasises the need for a reliable early warning sentinel system in morocco. the present study not only confirms the presence of btv at epidemic levels in morocco, but in morocco (baylis et  al. 1997, baylis and rawlings 1998). the lowest percentages were recorded in february (13.3%) and november (12.1%) (table  i). although the samples were not all taken in the same year and/or location, our findings seem to indicate significant differences in seasonal frequencies of btv‑positive animals. results of the serum‑neutralisation test obtained with samples taken between 2010 and 2013 indicated that antibodies to at least 5 serotypes, btv‑1, btv‑6, btv‑8, btv‑14, and btv‑16, were present in camels. the most abundant serotypes were btv‑8 (24.7%) and btv‑1 (24.3%). representing 22.4% of all tested sera, btv‑16 serotype was found in 115 of 225 camels (51.1%) that were tested (table  ii). although the 5 serotypes were identified every year, their seroprevalence varies from year to year. we did not research the co‑circulation of antibodies to more than 1 btv serotype in the 44 camels tested in 2010 and 2011. during 2012 and 2013, 55 sera (40 of 2012 and 15 of 2013) were tested for co‑infections with 10 serotypes (1, 2, 4, 6, 8, 9, 11, 14, 16 and 24). co‑infections were detected in 18 of 40 of the sera tested in 2012 and 4 of 15 camels tested in 2013; up to 4 serotypes (btv‑1, btv‑8, btv‑14 and btv‑16) could be detected in a single sample. co‑circulation of btv‑1 and btv‑8 was detected in 7 of the camels tested in 2012. the co‑circulation of 2 to 3 serotypes, in no particular frequency, were relative frequency of serum (%) with co-infection collected from slaughterhouse (unknown origin, el-aaiún) feb 2013 (n = 15) relative frequency of serum (%) with co-infection collected from slaughterhouse (local breeders, el-aaiún) march-july 2012 (n = 40) number of serum with co-infection 30 25 20 15 10 5 0 bt -1 , b t8 bt -2 , b t8 bt -1 , b t6, b t8, b t9 bt -1 , b t4, b t8 bt -1 , b t6, b t8 bt -1 , b t8, b t14 , b t16 , b t24 bt -2 , b t16 , b t24 bt -4 , b t6, b t16 bt -4 , b t8, b t16 bt -4 , b t6, b t8, b t16 bt -6 , b t8, b t9, b t16 bt -6 , b t8 bt -8 , b t14 bt -8 , b t16 bt -4 , b t16 figure 1. relative frequency of bluetongue virus co-infection detected in camel serum samples collected between 2012 and 2013 in morocco. bt = bluetongue virus serotype. 347 also supports previous studies (madani et  al. 2011, touil et al. 2012, youssef et al. 2017) that demonstrate that camels will be reliable sentinels to monitor the abundance and occurrence of btv. drif et al. camel as btv sentinels veterinaria italiana 2018, 54 (4), 343‑348. doi: 10.12834/vetit.1502.8097.1 barnard b.j.h. 1997. antibodies against some viruses of domestic animals in southern african wild animals. onderstepoort j vet res, 64, 95‑110. batten c., darpel k., henstock m., fay p., veronesi e., gubbins s., graves s., frost l. & oura c. 2014. evidence for transmission of bluetongue virus serotype 26 through direct contact. plos one, 9 (5), e96049. batten c.a., harif b., henstock m.r., ghizlane s., edwards l., loutfi c., oura c.a.l. & el harrak m. 2011. experimental infection of camels with bluetongue virus. res vet sci, 90 (3), 533‑535. batten c.a., henstock m.r., bin‑tarif a., steedman h.m., waddington s., edwards l. & oura 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huang l.p., xu q.y., wang h.x., xue x.m., lu p., li w.j., liu w., bu z.g. & wu d.l. 2016. emergence of a novel bluetongue virus serotype, china 2014. transbound emerg dis, 63, 585‑589. taylor w.p. 1986. the epidemiology of bluetongue. rev sci tech off int epiz, 5, 351‑356. touil n., cherkaoui z., lmrabih z.c., loutfi c., harif b. & el harrak m. 2012. emerging viral diseases in dromedary camels in the southern morocco. transbound emerg dis, 59 (2), 177‑182. toussaint j.f., vandenbussche f., mast j., de meester l., goris n., van dessel w., vandenbosche e., kerkhofs p., de clercq k., zientara s., sailleau c., czaplicki g., depoorter g. & dochy j.m. 2006. bluetongue in northern europe. vet rec, 159 (10), 327. van der sluijs m.t.w., schroer‑joosten d.p.h., fid‑fourkour a., vrijenhoek m.p., debyser i., moulin v., moormann r.j.m. & de smit a.j. 2013. transplacental transmission 404 not found 319 department of biochemistry, faculty of veterinary medicine, university of life sciences in lublin, poland *corresponding author at: department of biochemistry, faculty of veterinary medicine,university of life sciences, 20-033 lublin, akademicka 12, poland. tel.: +4881 445 6608, fax: +4881 445 6608, e-mail: marta.kankofer@up.lublin.pl. parole chiave status antiossidativo, stress ossidativo, saliva, vacche, vitelli. riassunto scopo di questo studio è stato valutare i livelli di antiossidanti e ossidanti presenti nel plasma e nella saliva di bovine da latte con diversi profili ormonali per determinarne lo stato fisiologico. sono state condotte analisi spettrofotometriche e spettroflurimetriche su sangue e saliva di vitelle clinicamente sane (n = 18), vacche sessualmente mature non gravide (n  =  19) e vacche in gravidanza (n = 15). il livello più basso (p <0,05) di capacità antiossidante totale (tac) è stato rilevato nel plasma del gruppo di vacche sessualmente mature, non gravide (2,375  ±  0,500  μmol/g). è stata evidenziata una correlazione negativa statisticamente significativa (tau b = 0,248, p <  0,05) tra i valori antiossidanti totali rilevati nel plasma e nella saliva. i valori medi più alti dei gruppi tiolici sono stati trovati sia nel plasma (0,007  ±  0,0015  mmol/g) che nella saliva (0,276 ± 0,116 mmol/g, p < 0,05) delle bovine sessualmente mature non gravide mentre la concentrazione di formilchinurenina ha raggiunto livelli più alti (p < 0,05) nella saliva e nel plasma delle bovine gravide (11,535 ± 3,785 e 0,133 ± 0,0237 μg/mg). tra il contenuto di residui di tirosina nel plasma e nella saliva delle vacche è stata invece riscontrata una correlazione positiva (tau b = 0,255, p < 0,05). in conclusione, per quanto riguarda i livelli di antiossidanti e ossidanti presenti, la saliva rispecchia parzialmente quanto contenuto nel plasma ma mostra differenze legate all'età che possono essere utilizzate nella determinazione dello stato fisiologico delle bovine. antiossidanti e ossidanti presenti nel plasma e nella saliva di bovine con diversi profili ormonali keywords antioxidant status, oxidative stress, saliva, cows, calves. summary the aim of this study was to evaluate changes in the antioxidant status and oxidative stress parameters in plasma and saliva in order to investigate the physiological conditions of dairy cows. blood and saliva were collected from clinically healthy female calves (n = 18), sexually mature, non-pregnant cows (n = 19), and pregnant dairy cows (n = 15). spectrophotometric and spectroflurimetric analyses were carried out in the body fluids of these animals. the level of total antioxidant capacity (tac) in plasma reached the lowest (p  <  0.05) value in the group of sexually mature, non-pregnant cows (2.375  ±  0.500 µmol/g). a significant negative correlation (tau b = -  0.248, p  <  0.05) was found between tac values detected in plasma and saliva of examined animals. the highest (p < 0.05) mean values of thiol groups were detected in both plasma (0.007 ± 0.0015 mmol/g) and saliva (0.276 ± 0,116 mmol/g) of mature, non-pregnant cows. conversely, the highest (p < 0.05) levels of formylokinurenine concentration were detected in saliva (11.535 ± 3.785 µg/mg) and plasma (0.133 ± 0.0237 µg/ mg) of pregnant dairy cows. a significant positive correlation (tau b = 0.255, p < 0.05) was also found between the bityrosine content detected in plasma and saliva of the examined cows. in conclusion, although with regards to antioxidant/oxidative parameters saliva reflects the content of plasma only in part, however it shows age-related differences that can be used in the description of the physiological status of cows. witold puzio, lukasz chrobak, marcin rutkowski, monika franczyk and marta kankofer* antioxidative and oxidative profiles in plasma and saliva of cows in different ages and hormonal status veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 accepted: 14.01.2017 | available on line: 31.12.2019 320 veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 antioxidative and oxidantive profiles in saliva and plasma of bovines puzio et al. the use of saliva as a diagnostic material has garnered growing interest from scientists. veterinarians are also interested in this procedure because saliva collection is a non-invasive procedure (lamy and mau 2012). muthukumar and colleagues (muthukumar et  al. 2014) suggested that estrus in buffaloes is indicated by the presence of beta enolase and tlr 4 in saliva. the aim of our study was to compare the levels of total antioxidant capacity (tac) and the intensity of protein peroxidation in bovine plasma and saliva collected from sexually immature, sexually mature, non-pregnant, and pregnant cows. the results could provide a biochemical salivary profile in physiological conditions, which may be of use in future diagnoses. materials and methods plasma and saliva were collected during routine veterinary procedures in accordance with the principles of antiseptics from clinically healthy female calves (aged between 1 and 5-6 months; n  =  18), sexually mature, non-pregnant cows (aged 8 months-10 years; n = 19, secretory phase of cycle), and pregnant polish black and white dairy cows (aged 4-8 years; n = 15, 3-5 months pregnant). blood samples were collected via puncture of the jugular vein into tubes with anticoagulant and centrifuged. plasma samples were portioned and stored at - 20 °c until they were used for analysis. saliva was collected at a similar time of day with sponges that were mounted in the space between the teeth and cheeks. materials were then centrifuged, portioned, and frozen in -  200  °c until they were tested. feeding regimes were adjusted according to physiological requirements, and were similar between animals within the examined groups. total antioxidant capacity the total antioxidant capacity (tac) was measured according to the method described by benzie and strain (benzie and strain 1996), based on the ferric reducing ability of plasma (frap), with some modifications. changes in absorbance were directly related to the ‘total’ reducing capacity of the electron donating antioxidants that were present in the examined plasma and saliva samples. the working reagent contained 300 mmol/l acetate buffer (ph 3.6), 10 mmol/l 2,4,6-tri-pyridyl-striazine (tptz, sigma, poznań, poland) solution in 40 mmol/l hcl, and 20 mmol/l fecl 3 x 6h 2 o solution in h 2 odest, mixed to the ratio of 10:1:1. the working reagent was prepared immediately before use. the working introduction reactive oxygen species (ros) are constantly produced in the body and are involved in the regulation of certain physiological processes, including cell signalling as secondary messengers that activate transcription factors, and inducing gene expression (bartosz 2003). the balance between the formation and neutralisation of ros is modulated by the body’s antioxidant systems. disturbances in this balance can be the result of the excessive production of ros or because of the reduced efficiency of antioxidants. this, in turn, leads to oxidative stress and consequently to various pathological conditions (bansal and kaushal 2014), including, among others, coronary disease, atherosclerosis, acute myocardial infarction, and rheumatoid arthritis (čolak 2008). ros can also regulate several of the processes that take place in cells affecting the activity of participating substances. in the female reproductive system, ros acts as essential to signalling molecules involved in, among others, ovarian steroid synthesis, ovulation, corpus luteum formation, luteolysis, and maintenance of pregnancy (rizzo et al. 2012). proteins are extremely sensitive to the action of free radicals. unlike the lipid peroxidation process, the oxidative modification of proteins is faster and linear (with regards to time and concentration). it is therefore a more sensitive indicator of the action of ros on the cellular components than lipid peroxidation (čolak 2008). ros may act on the primary, secondary, and tertiary structures of proteins, and likewise, with amino acids. the amino acid that most frequently undergoes oxidation is sulfamino acid cysteine, by the presence of -sh group in its structure (as a result of the oxidation of this group, disulfide bonds are created) and aromatic amino acids (tryptophan, tyrosine, phenylalanine) and the effects of their modification are formylkynurenine, kynurenine (from tryptophan), and bis-tyrosyl bridges (from phenylalanine and tyrosine). any alterations in the protein structure results in the modifications of protein molecules, which leads to changes in the functions and activities of enzymes, and changes in the binding capacity of receptors or other biologically active substances. these changes are reflected in disturbances in metabolic pathways and clinical symptoms are expressed, e.g. retained placenta in cows (kankofer 2001). the metabolism of proteins is also hormonally regulated by sex steroids. metabolism may vary depending on the hormonal status and sexual maturity (ayres et  al. 1998, massafra et  al. 2000, pajović and saicić 2008). sex steroids also control the synthesis and activity of antioxidant enzymes (pajović and saicić 2008). 321veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 puzio et al. antioxidative and oxidantive profiles in saliva and plasma of bovines ml in 0.1 mol/ h₂so₄) at excitation (350 nm) and emission wavelength (445  nm). the results were expressed as µg/mg protein. protein content the protein content of the plasma samples was measured according to the biuret method, using commercial assay kits (cormay, łomianki, poland) based on the method described by gornal and colleagues (gornal et al. 1949). the protein content of the saliva samples was measured according to the bradford method using commercial reagents (bradford reagent, sigma, poznań, poland). statistical analysis the biochemical results of duplicates were statistically analysed and compared using spss software (spss inc., chicago, il). a nonparametric mann-whitney u test was used to determine differences among the biochemical variables of all 3 groups. a kendall's tau-b correlation test was used to determine the association between plasma and saliva oxidative stress parameters. the p-value < 0.05 was considered to be statistically significant. results analysis of the tac in plasma (figure  1a) showed that, among the 3  groups, the highest values were observed in the group of pregnant cows (2.906  ±  0.361 µmol/g). in the group of sexually immature cows, the value was lower (2.872  ±  0.688  µmol/g). the lowest value (p  <  0.05) was in the group of sexually mature, non-pregnant cows (2.375 ± 0.500 µmol/g). tac values in saliva (figure 1b) were significantly higher as in plasma of all the examined groups of animals when recalculated in relation to the protein content of respective biological fluids. the highest average tac values in saliva were found in the group of sexually mature, non-pregnant cows (1,154.11  ±  387 µmol/g). in the other groups values reached 957.77 ± 313 µmol/g (in the group of sexually immature cows) and 917.14  ±  256 µmol/g (in the group of pregnant cows). tac results in saliva did not reveal significant differences between the groups. a significant negative correlation (tau b = -  0.248, p  <  0.05) was found between the tac values detected in plasma and saliva of the examined cows. the increase of the tac values in plasma was accompanied by a decrease of values in saliva. the mean values of sh groups in plasma (figure 2a) were similar in immature (0.0065  ±  0.0016  mmol/g reagent (2250 µl) was mixed with 25 µl of sample and absorbance was measured at 593 nm. the absorbance of the working reagent alone served as control. after exactly 10 minutes of incubation at room temperature, the absorbance was measured again. the difference in absorbance at 0 and at 10 minutes was compared with a standard curve prepared with 10 different dilutions of fe (ii) between 0 and 1000 µmol/l. the results were expressed as µmol/g protein. the content of sulfhydryl groups the concentrations of sulfhydryl residue (sh) in plasma and saliva were measured by spectrophotometry, as detailed by rice-evans and colleagues (rice-evans et  al. 1991). a volume of 300  µl 10% (w/v) sodium dodecyl sulphate (sds, sigma, poznań, poland) in 10 mmol/l sodium phosphate buffer (ph 8.0) was added to 300 µl of sample and mixed precisely. a 2.4 ml of 10 mmol/l sodium phosphate buffer (ph 8.0) was then added. then 300 µl 20 mg of 5,5-dithiobis2-nitro benzoate (sigma, poznań, poland) in 50  ml of buffer (dtnb) was added and the aborbance was measured at 412 nm. the control sample contained 300 µl of the same buffer, instead of dtnb. all samples were incubated for 1 hour at 37  °c. after incubation, the absorbance was measured again at 412 nm. the difference in absorbance before and after incubation (after subtracting the respective absorbance of the control) referred to the content of sh groups. the content was calculated using a standard curve prepared with different dilutions of glutathione (gsh, sigma, poznań, poland) ranging from 0 to 1 mmol/l in 10 mmol/l sodium phosphate buffer (ph 8.0) and expressed in mmol/g protein. the content of formylokinurenine formylokinurenine was determined by a spectroflurimetric method described by rice-evans and colleagues (rice-evans et  al. 1991). the diluted plasma and saliva samples were excited by light at 360 nm and emission was measured at a 454  nm wavelength. the spectrofluorimeter (jasco, tokyo, japan) was standardised, as described above (rice-evans et  al. 1991). the results were expressed as µg/mg protein. the content of bityrosine bridges bityrosine bridges were determined by a spectroflurimetric method according to rice-evans and colleagues (rice-evans et  al. 1991). the diluted plasma and saliva samples were excited by light at 325 nm and emission was measured at a wavelength of 410 mm. the spectrofluorimeter was standardised to 100 deflections with chinine sulphate (0.1 µg/ 322 veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 nick title puzio et al. significantly (p  <  0.05) lower values were detected in pregnant animals (0.163 ±  0.072 mmol/g) and in sexually immature cows (0 134 ± 0.094 mmol/g). the last 2 groups did not differ significantly. the concentrations of formylokinurenine in the plasma (figure 3a) of immature (0.0805 ± 0.0274 μg/mg protein) and mature, non-pregnant cows (0.0926± 0.0303 μg/mg protein) were similar. protein), mature non-pregnant (0.007 ± 0.0015 mmol/g protein) and pregnant cows (0.0065 ± 0.0020 mmol/g protein). the u mann-whitney test did not show any significant differences. the concentration of sh groups in saliva (figure 2b) were significantly higher than that detected in plasma. the highest values were found in mature, non-pregnant cows (0.276  ±  0.116 mmol/g). a b 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 si smnp smp ta c [µ m o l/ g p ro te in ] a b ac 0 200 400 600 800 1000 1200 1400 1600 si smnp smp ta c [µ m o l/ g p ro te in ] figure 1. total antioxidant capacity (tac) values in plasma (a) and in saliva (b) of sexually immature as well as sexually mature non pregnant and pregnant cows. si = sexually immature; smnp = sexually mature, non-pregnant; smp = sexually mature, pregnant; a, b different letters denote significant differences at p < 0.05. a b 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 si smnp smp sh [m m o l/ g p ro te in ] 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 si smnp smp sh [m m o l/ g p ro te in ] a b ac figure 2. sulfhydryl (sh) group content in plasma (a) and in saliva (b) of sexually immature as well as sexually mature non pregnant and pregnant cows. si = sexually immature; smnp = sexually mature, non-pregnant; smp = sexually mature, pregnant; a, b, c different letters denote significant differences at p < 0.05. 323veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 puzio et al. nick title the concentrations of bityrosine in plasma (figure  4a) differed significantly in all examined groups. the lowest values were detected in immature cows (0.249  ±  0.114 µg/mg protein). values increased to 0.314 ± 0.122 µg/mg in mature, non-pregnant animals and to 0.506  ±  0.084 µg/ mg in pregnant cows. the highest values of bityrosine was found in the saliva of pregnant significantly (p < 0.05) higher results were detected in pregnant cows (0.133 ± 0.0237 μg/mg protein). the results were also significantly higher in saliva (figure 3b) for this group, but showed a similar tendency in immature (8.038 ± 5.344 μg/mg protein) and mature, non-pregnant cows (9.387 ± 6.401 μg/ mg protein). in pregnant cows, values reached the highest level of (11.535 ± 3.785 μg/mg protein). a b 0 0.04 0.08 0.12 0.16 0.2 0.24 si smnp smp fo rm yl o ki n u re n in e [µ g /m g p ro te in ] a a b 0 2 4 6 8 10 12 14 16 si smnp smp fo rm yl o ki n u re n in e [µ g /m g p ro te in ] a ab b figure 3. formylokinurenine content in plasma (a) and in saliva (b) of sexually immature as well as sexually mature non pregnant and pregnant cows. si = sexually immature; smnp = sexually mature, non-pregnant; smp = sexually mature, pregnant; a, b different letters denote significant differences at p < 0.05. a b 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 si smnp smp b it yr o si n e [µ g /m g p ro te in ] c a b 0 50 100 150 200 250 300 350 si smnp smp b it yr o si n e [µ g /m g p ro te in ] ab a b figure 4. bityrosine content in plasma (a) and in saliva (b) of sexually immature as well as sexually mature non pregnant and pregnant cows. si = sexually immature; smnp = sexually mature, non-pregnant; smp = sexually mature, pregnant; a, b, c different letters denote significant differences at p < 0.05. 324 veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 nick title puzio et al. of antioxidant enzymes is thought to be influenced by other sex steroid hormones (massafra et  al. 2000, pajović and saicić 2008), and is reinforced in particular by the effect of age. both saliva and plasma are rich in antioxidants. uric acid (primary salivary antioxidant), albumin, ascorbic acid, glutathione, and antioxidant enzymes are present in saliva (miricescu et al. 2011). in this study the results of the tac determinations showed significant differences between pregnant and non-pregnant animals both in plasma and saliva. these differences may indicate a possible antioxidant role of oestrogen, which increases during pregnancy. the difference between the group of sexually immature cows and sexually mature, non-pregnant cows could be related to changes in hormonal profiles. interestingly, the tac of saliva was higher than the tac of plasma, and the lowest levels of the tac in saliva were found in the group of sexually mature, pregnant cows (in contrast to plasma levels). it should be noted that our results were re-calculated per protein content. reyes and colleagues (reyes et  al. 2006) demonstrated that, in vitro, 17β-oestradiol and oestriol, in addition to its hormonal action, play a role in materno-foetal auto-protection against free radicals. however, in  vivo studies are necessary to confirm these results. in the same study, reyes and colleagues claimed that this protection is necessary because pregnancy is a condition that leads to oxidative stress. in other studies, it was found that during the last trimester of pregnancy, human placenta contains enough oestrogen (12.5 µmol/kg wet tissue) to protect cell membranes from being damaged by lipid peroxidation in mild oxidative conditions (diczfalusky and mancuso 1969). the oxidative modification of proteins has a great impact on a variety of cellular functions involving protein receptors, signal transduction mechanisms, transport systems, and enzymes. it may affect the majority of amino acids, leading most often to irreversible alterations based on the introduction of carbonyl or hydroxyl groups to amino acid chains. this may, in turn, result in the fragmentation of the peptide chain, as well as the modification of the amino acid chains, synthesis of interand intra-molecular bonds, and the aggregation of molecules (skrzydlewska and farbiszewski 1995, petropoulos and friguet 2006). the sh groups, which are present in cysteine and the majority of peptides and proteins, are susceptible to ros attack and may react with a wide range of ros and electrophilic compounds. the alterations in this parameter can easily reflect the intensity of protein peroxidative damage as well as the status of proteins possibly damaged by ros. cows (225.643  ±  121.542  μg/mg protein) (figure 4b). concentrations decreased to 135.609 ± 88.644 µg/mg in immature cows, and to 73.179  ±  39.496 µg/mg in mature, non-pregnant cows. values in pregnant and non-pregnant animals were significantly different (p < 0.05). a significant positive correlation (tau b  =  0.255, p  <  0.05) was detected between bityrosine content in plasma and saliva of examined cows. discussion and conclusions the study compared the antioxidant status, represented by the tac, and oxidative status, represented by the content of protein peroxidative metabolites and sh groups, in plasma and saliva of cows. in this study the animals were sampled according to their different sexual maturity, physiological,and hormonal statuses. results showed the influence of hormonal status, including sex steroids, on the antioxidant/oxidative status of the examined animals. saliva can be defined as a ‘reflection of the body’ (lima et  al. 2010) because its composition contains materials of both local (including salivary glands) and systemic (from blood) origin. this peculiarity makes it a potential source of information about physiological, as well as biological materials, which could be applied in the diagnosis of diseases, including animal diseases. it is worth noting that saliva is easy to obtain and does not cause any stress in animals. because human saliva proteins have antimicrobial activity and 30% are of blood origin (schulz et al. 2013), saliva composition is dependent on plasma composition. together with the somatic development of cows, changes in the concentrations of hormones, especially in sexual hormones like oestrogen, appear at the time of sexual maturation. changes in hormonal profile are also detected during the course of pregnancy, where the levels of both progesterone and oestrogen fluctuate with significant increase in the third trimester of pregnancy. as these profiles are well defined, this study did not cover the determinations of sex hormones, but only referred to known alterations that appear during sexual maturation and pregnancy. unlike all other natural steroids, oestrogens – especially 17β-estradiol (mooradian 1993) – have a phenolic structure in their molecules (sugioka et al. 1987) that determines their antioxidant ability. one of the mechanisms lying upon their antioxidant activity is probably a stimulating effect on cellular antioxidant enzymes (bednarek-tupikowska 2002) as well as scavenging action against free radicals (ayres et  al. 1998, pfohl et  al. 2002). the regulation 325veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 puzio et al. nick title to gonadotropin therapy (onuma, hahn, and foote 1970). differences in protein profiles expressed in the endometrium between sexually immature and mature cows have been demonstrated (giergiel et al. 2016). this is why appropriate defences against ros may assure sex-steroid-dependent physiological processes. faulkner and colleagues (faulkner et  al. 2013) confirmed that particular protein profile is associated with progesterone as well as the stage of cycle. apart from the negative effects of ros on reproductive performance, ros is associated with several advantageous reactions: sperm capacitation, the regulation of luteal function, the regulation of prostaglandin, and ovarian sex steroid synthesis. in conclusion, with regards to antioxidant/oxidative parameters, saliva reflects the content of plasma only in part, due to the local metabolism of salivary gland tissues. however, it reveals age-related differences that can be used in the description of the physiological status of cows. further studies are necessary to define the usefulness of saliva as a biological material in routine laboratory tests. this study demonstrated the relatively stable concentrations of sh groups in plasma that are probably protected from peroxidative damage by multifunctional enzymatic and non-enzymatic antioxidant systems. saliva samples more profoundly reflected the differences in the hormonal status of the examined animals. these differences require further elucidation. the highest concentrations of protein peroxidation products found in both plasma and saliva of pregnant animals might confirm the possibility of oxidative stress during pregnancy. the increased values of tac detected in the plasma of this group were probably a consequence of it. there is evidence that pregnancy is accompanied by an increase in tissue oxygen needs (öztürk et  al. 2010). it determines the increase in ros synthesis and turnover. it might also be related to the oxidation of sh groups and alterations in other examined parameters of protein peroxidation. sexual maturation is related to an increase in the size and activity of ovaries as well as their susceptibility to gonadotropins. with the increase of oestrogen secretion, ovaries of 2-month-old calves can react 326 veterinaria italiana 2019, 55 (4), 319-326. doi: 10.12834/vetit.1178.6557.2 nick title puzio et al. ayres s., abplanalp w., liu j.h. & subbiah m.t. 1998. mechanisms involved in the protective effect of estradiol-17beta on lipid peroxidation and dna damage. am j physiol, 274 (pt l): e1002-8. bansal m. & kaushal n. 2014. introduction to oxidative stress mechanism and their 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doświadczalnej, 49, 747-766. sugioka k., shimosegawa k. & nakano m. 1987. estrogens as natural antioxidants of membrane phospholipid peroxidation. febs letters, 210, 37-39. 77 introduction meat and meat products represent an important source of proteins for the human diet (font‑i‑furnols and guerrero 2014). recently, due to changes in animal production, product processing, consumer needs and people awareness of meat safety, the authenticity of meat and meat products has been at the forefront of attention of consumers, manufacturers, and regulators (mousavi et  al. 2015, danezis et  al. 2016). in particular, according to the european union (eu) regulations, these products must respect the requirements of food hygiene regulations and must be labeled with detailed information about their ingredients (european regulations1). the authenticity of meat and meat products should be assessed by checking the ingredients declared in the label and excluding the presence of unauthorized tissues and cells (ballin 2010). moreover, the monitoring of animal species present in these products is fundamental to prevent adulteration and to protect consumers in terms of economic, health and religious aspects (ballin 2010, kane and hellberg 2015, mousavi et al. 2015). since different types of meat have a different quality and price (e.g. usually red raw meat such as beef and sheep is more expensive than the other meat), the fraudulent addition or replacement of valuable species by less valuable ones may occur. for example, several authors reported the practice 1dipartimento di scienze veterinarie, università degli studi di torino, torino, italy. 2istituto zooprofilattico sperimentale del piemonte liguria e valle d'aosta, torino, italy. *corresponding author at: dipartimento di scienze veterinarie, università degli studi di torino, l.go braccini 2, 10095 grugliasco (to). tel:. +39 011 670 90 32, e‑mail: tiziana.cannizzo@unito.it. keywords bovine, dna microarray, histology, meat products, minced meat. summary adequate testing and adulterant detection of food products are required to assure its safety and avoid fraudulent activities. adulteration/substitution of costlier meat with a cheaper or inferior meat is one of the most common fraudulence in meat industry. aim of this study was to check the correct labelling of meat and ready to cook bovine meat products, combining the dna microarray approach to identify the animal species with the histological examination, to check the composition and safety of meat. one hundred and one samples of bovine minced meat (group 1) and ready to cook meat products (group 2) were collected from supermarkets in turin, italy. dna microarray revealed that 25.7% of samples were positive for species not declared on the label, swine being the most common. histology showed the presence of cartilage, bone and glandular tissue. a higher presence of bacteria and inflammatory cells was detected in group 1. bacterial cells associated to inflammatory cells were detected with a higher score in group 2. sarcocystis spp. were present in 83.3% samples of group 1 and 49.1% of group 2. this study confirmed that the mislabelling of meat products is not uncommon. the combination of dna microarrays and histology can increase the monitoring capacity in bovine meat industry. hamidreza sohrabi1, francesca tiziana cannizzo1*, paola pregel1, frine eleonora scaglione1, chiara beltramo2, pier luigi acutis2, alessandra dalmasso1 and bartolomeo biolatti1 tissue and species identification in minced meat and meat products from italian commercial markets by dna microarray and histological approach veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 accepted: 11.10.2019 | available on line: 31.12.2020 1 european commission (ec). 2011. regulation (eu) no 1169/2011 of the european parliament and of the council of 25 october 2011 on the provision of food information to consumers, amending regulations (ec) no 1924/2006 and (ec) no 1925/2006 of the european parliament and of the council, and repealing commission directive 87/250/eec, council directive 90/496/eec, commission directive 1999/10/ec, directive 2000/13/ec of the european parliament and of the council, commission directives 2002/67/ec and 2008/5/ec and commission regulation (ec) no 608/2004. oj, l 304, 22/11/2011, 18‑63. 78 veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 tissue and species identification in minced meat sohrabi et al. (group 2, n = 53) were collected from different local supermarkets (referred as a, b, c, d, e and f) in turin, italy. in both experimental groups the samples were collected from retail distributors a, b, c, and d, while the market e & f were designed for exclusive sampling for group 1 and group 2, respectively. samples characteristics were recorded, including product type, ingredients, and date of sampling. each sample was divided into two aliquots: the first aliquot was stored in sterile tubes at ‑  20  °c to prevent dna degradation for species identification by dna microarray analysis; the second aliquot was fixed in 10% buffered formalin and processed for tissue identification by histological methods. dna microarrays meat samples were thawed and put in a sterile petri dish, then they were manually mixed by using sterile scalpel and nipper. dna was extracted using a nucleo spin food kit (macherey‑nagel, düren, germany), following the manufacturer’s instructions. dna was extracted from 1 g of tissue (5 aliquots of 200 mg each, in order to have good sample representation). dna was stored at ‑ 20 °c until further use. pcr assays were performed using a 2x “all‑in‑one” master mix (roche, basel, switzerland) in a final volume of 25 μl: each reaction contained 1x master mix, 1.5 μl of the meat pcr biotinilated primers supplied with the meat lcd array kit (chipron, berlin, germany) and 5 μl of sample’s dna. amplifications were performed in a geneamp pcr system 9700 thermal cycler (applied biosystems, foster city, ca, usa) according to the following protocol: 10 min at 95  °c to activate the hot‑start taq polymerase; then 40 cycles with a denaturation step at 94  °c for 30 seconds, an annealing step at 57 °c for 45 seconds, an elongation step at 72 °c for 45 seconds, and a final step at 72 °c for 2 minutes. dna for each sample was analyzed using the meat 1.6 lcd array kit. each array presents 8 chips, with 14 species‑specific capture probes fixed to each chip, spotted as duplicates. the detectable species with this kit are: cattle (bos taurus), buffalo (bubalus bubalis), pork (sus scrofa), sheep (ovis aries), goat (capra hircus), horse (equus caballus), donkey (equus asinus), rabbit (oryctolagus cuniculus), hare (lepus europaeus), chicken (gallus gallus), turkey (meleagris gallopavo), goose (ansa albifrons), mallard duck (anas platyrhyncos) and muscovy duck (cairina moschata). the pcr products were identified on the lcd array following the manufacturer's instructions (chipron). biotinilated amplicons were linked to a streptavidin‑peroxidase conjugate after hybridization at 35  °c to the probes on the array. a peroxidase substrate was added to highlight spots with amplicon‑probe hybridization. a pf3650u of mixing beef with cheaper meats such as chicken, horse and pork (ballin 2010; parchami nejad et  al. 2014) or with plant proteins such as soybean or grain derivatives (flores‑munguia et al. 2000). moreover, mislabelling or improper labelling may not cite allergens that can be matter of concern for food‑allergic people (pascoal et  al. 2004, de la cruz et al. 2017), as well as the risk of cross‑contamination with pork meat that raises religious problems. alongside the possible presence of not declared species, some authors also reported undesirable organs from slaughtered animals, including viscera, hyaline cartilage, udder, skin, spleen, fat, bone and central nervous tissue, replacing the meat (botka‑petrak et  al. 2011, latorre et  al. 2015, tafvizi and hashemzadegan 2016). apart from the adulteration issues, some animal tissues such as central nervous tissue can be vectors of infectious agents transmissible to humans (herde et al. 2005). currently, molecular techniques are the methods of choice for species identification in meat products. dna analysis has some advantages, such as the high thermal stability of dna and the specificity of the genetic code (ballin 2010, yosef et  al. 2016). several authors developed methods for species identification in meat products based on end point pcr (calvo et  al. 2002), real time pcr (martín et  al. 2009), and sequencing analysis (iijima et  al. 2006). meanwhile dna microarray approach is one of the fastest‑growing technologies, based on the classical pcr followed by a lcd array hybridisation. previous studies already reported efficient and reliable meat species identification by dna microarray technique (iwobi et al. 2011, yosef et al. 2014, beltramo et al. 2017). formerly, many researchers reported histological methods as a simple, economic and efficient approach for the identification of unauthorized tissues, herbal content, different microorganisms and parasites in meat products (prayson et al. 2008, botka‑petrak et al. 2011, sadeghinezhad et al. 2015, hafeez et al. 2016). aim of this study was to check the correct labelling of meat and ready to cook bovine meat products, combining the dna microarray approach to identify the animal species with the histological examination, to check the composition and safety of meat. materials and methods sample collection in this study, 101 samples of bovine minced meat (without other ingredients reported in label) (group 1, n = 48) and ready to cook bovine meat products 79veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 sohrabi et al. tissue and species identification in minced meat statistical analysis the number of observations for each parameter was recorded from each section and the mean score of five slides was obtained for each sample, and the two groups (group 1 and 2) were compared. scores obtained were reported as the mean ± standard deviation. the normality of distributions was evaluated by kolmogorov and smirnov test. mann‑whitney test was applied to compare the two groups. regarding the presence of animal tissues and of parasites, each sample was considered positive if the investigated items were present at least in 1 slide (gibson‑corley et al. 2013). fisher’s exact test was applied to determine the possible association of the presence of bacteria and inflammatory cells, parasites and different tissues in the two groups, as well as the species contamination. statistical analysis was performed using graph pad prism (graph pad software, la jolla, ca, usa). p‑values smaller than 0.05 were considered statistically significant. lcd‑array scanner (pacific image electronics, torrance, ca, usa) was used to visualize the dark precipitate. the default detection cut‑off threshold was the 1,700‑pixel value (pv). histological screening test for histological screening, five different aliquots were randomly obtained from each sample. each aliquot was fixed in 10% neutral buffered formalin and paraffin embedded. paraffin blocks were cut into 4 μm‑sections and stained with haematoxylin and eosin (h&e) by means of a leica st5010 auto‑strainer xl machine (leica biosystem, wetzlar, germany). in total, 505 sections from 101 meat products were prepared and observed under a light microscope (u‑mdob3, olympus optical co. ltd, tokio, japan) to detect the presence or absence of different tissues, and to identify the presence of microorganisms, parasites, inflammatory cells and other ingredients. table i. results of dna microarray analysis for group 1 (minced meat, n = 13) and group 2 (meat products, n = 13) sample id group product label market id substituted species 4 1 choicest minced of adult bovine c beef, pork, chicken and turkey 5 1 ground from adult bovine ragu c beef, pork, chicken and turkey 6 1 ground chosen from adult bovine b beef, pork and turkey 7 1 ground chosen from adult bovine b beef and pork 11 1 adult bovine hamburger (without other ingredients) d beef and pork 16 1 adult bovine hamburger (without other ingredients) b beef and pork 17 1 adult bovine hamburger (without other ingredients) b beef and pork 22 1 thigh minced of adult bovine e beef, chicken and turkey 23 1 choicest minced of adult bovine c beef, pork, chicken and turkey 24 1 minced of adult bovine c beef, pork, chicken and turkey 30 1 minced of adult bovine c beef and pork 32 1 choicest minced of adult bovine c beef and pork 44 1 adult bovine hamburger (without other ingredients) b beef, pork and chicken 3p 2 hamburger a beef and pork 5p 2 adult bovine hamburger b beef and pork 15p 2 pizzaiola hamburger a beef and pork 16p 2 hamburger a beef and pork 17p 2 hamburger a beef and pork 23p 2 the american 100% black angus meat f beef and pork 24p 2 the american 100% black angus meat f beef and pork 29p 2 cheeseburger c beef and pork 33p 2 cheeseburger c beef and pork 39p 2 calf hamburger with olive b beef and turkey 41p 2 calf hamburger with olive b beef, turkey and chicken 44p 2 minced for sauce c beef and pork 53p 2 the piedmont hamburger f beef and chicken 80 veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 tissue and species identification in minced meat sohrabi et al. no significant association was revealed between the species contamination results and the examined groups. histology screening results light microscopy showed skeletal muscle tissue characterized by muscular fibers surrounded by nuclei as well as peripheral nerves, vascular tissue and adipose tissue in samples of both minced meat and meat products (figure 1a). fifteen (31.3%) samples out of 48 in minced meat, and 13 (24.6%) out of 53 in meat products, showed the presence of hyaline cartilage fragments in at least 1 out five aliquots (table ii), characterized by the presence of chondrocytes in a collagenous extracellular matrix. forty (83.3%) samples in group 1 and 38 (71.7%) in group 2 revealed pieces of bone tissues in at least one out the 5 aliquots analysed, with the osteocytes embedded in mineralized bone matrix, whereas 5 (10.4%) samples in group 1 and 4 (7.6%) in group 2 presented glandular tissue (figure 1b) in at least 1 out of the 5 aliquots analysed, recognisable by the characteristic epithelial structure. the number and distribution of the positive slides in the two groups results dna microarray results dna microarray analysis showed that 26 out of 101 samples (25.7%) were incorrectly labelled, containing different animal species besides the declared ones (table i). in group 1, the minced meat samples showed contamination in 13 out of 48 samples (27.1%). in 6 cases the contamination was due to the presence of a single species (swine), whereas in 7 cases the contamination was due to multiple species. totally, contaminations were found in 4 out of the 5 supermarkets investigated (table i). in group 2, the samples of meat products analysed were from 5 different manufacturers. incorrect labelling was found in 13 samples out of 53 (24.5%), showing the presence of different species aside from bovine. in 10 cases the contamination was due to swine, whereas in the other cases the contaminant species were chicken and turkey. the non‑compliant samples were found in all the 5 supermarkets taken in account (table i). figure 1. a. histological image of adult bovine minced meat. cross and longitudinal‑sections of muscular fibres, with peripheral nuclei (h&e). b. glandular tissue found in an adult bovine hamburger along with striated muscle fibres (h&e). c. adult bovine hamburger, bacterial contamination associated with inflammatory cells infiltrating the skeletal muscle (h&e). d. bovine minced meat, longitudinal and cross sections of sarcocystis spp. localized in the muscle fibres (h&e). 81veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 sohrabi et al. tissue and species identification in minced meat presence of different types of inflammatory cells. in group 1, expected to be without ingredients other than meat, 6 (12.5%) samples out of 48 showed plant cells. a significantly higher presence of bacteria (p < 0.01) and inflammatory cells (p  <  0.01) was detected (figure 3) in meat products compared to minced meat samples, when comparing the mean score calculated for the 5 replicates of each sample. bacteria associated to inflammatory cells were detected with a significantly higher mean score in group 2 (p  <  0.05). moreover, 1 (2.1%) sample of minced meat and 7 (13.2%) samples from meat products showed the simultaneous presence of concerning cartilage, bones and glandular tissue is reported in the figure 2 (a, b, c). no statistically significant associations were found between the presence of the analysed tissues and the group. central nervous tissue and skin tissue were never found in the examined samples. in addition to the identification of tissues other than meat, histology revealed the presence of inflammatory cells and bacteria (figure 1c, table  ii) in 46 (95.8%) samples out of 48 in group 1 and in 50 (94.3%) out of 53 in group 2. the bacteria were represented by cocci‑shaped bacteria, and rod‑shaped bacteria. twenty‑five (52.1%) samples in group 1 and 40 (75.5%) in group 2 showed the table ii. tissues other than meat as well as bacteria and inflammatory cells found in minced meat and meat products revealed by histological examination. groups cartilage bones glands bacteria inflammatory cells minced meat 15 (31.3%) 40 (83.3%) 5 (10.4%) 46 (95.8%) 25 (52.1%) meat products 13 (24.6%) 38 (71.7%) 4 (7.6%) 50 (94.3%) 40 (75.5%) a b c d n u m b er o f s am p le s group 1 group 2 60 40 20 0 n u m b er o f s am p le s group 1 group 2 80 60 20 0 40 n u m b er o f s am p le s group 1 group 2 80 60 20 0 40 n u m b er o f s am p le s group 1 group 2 60 40 20 0 5 positive slides 4 positive slides 3 positive slides 2 positive slides 1 positive slide negative 5 positive slides 4 positive slides 3 positive slides 2 positive slides 1 positive slide negative 5 positive slides 4 positive slides 3 positive slides 2 positive slides 1 positive slide negative 5 positive slides 4 positive slides 3 positive slides 2 positive slides 1 positive slide negative figure 2. number and distribution of the positive slides concerning cartilage (a), bones (b), glandular tissue (c), and parasites (d) in the two examined groups. slides were considered positive when at least one finding of investigated items (cartilage, bones, glandular tissue or parasites) was observed. 82 veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 tissue and species identification in minced meat sohrabi et al. furthermore, the prevalence of sarcocystis spp. was easily assessed by histology, suggesting considerable concerns in this regard. our study reveals a high substitution rate and insufficient or improper labelling in minced meat and meat products sold on the different chain supermarket in italy without a significant association with the groups. overall trends indicate that cheaper species can be mixed with more expensive species for economic purposes although unintentional cross contamination in the processing procedures may occur. the rate of mislabeling in the present study was 25.7% which is slightly higher than a recent study conducted in u.s. which reports the 21% of mislabeling rate for ground meats (kane and hellberg 2015). another study carried out in istanbul reported a 53.4% of samples of meat and meat products incorrectly labelled (özpinar et al. 2013). the reasons for the presence of dna of undeclared species could be either the deliberate introduction of meat from other species in order to commit a fraud or the consequence of a cross‑contamination in the production chain. dna microarray method used in the present study is essentially qualitative but the manufacturer declares that the signal intensity, given by the dna probe hybridization, is somehow proportional to the amount of dna in the sample. the majority of non‑compliant samples showed a weak signal, probably explainable with unintentional cross‑contamination, but 7 samples had a very strong signal: all of them were minced meat and five of them were prepared directly at selling points. this finding shows that a particular attention should be paid by controllers to minced meat prepared in small production sites belonging to shops and supermarkets: in these sites there could be a higher risk of contamination either intentional, to get rid of leftover meat, or unintentional, due to very poor cleaning procedures, giving rise to heavy contamination loads. the histological technique based on light bacteria and inflammatory cells in at least 1 out 5  slides, without revealing a significant association between the findings and the groups. examination of histological sections revealed the presence of at least one parasite in one slide per sample (figure 1d), which were identified as sarcocystis, in 40 (83.3%) out of 48 samples of group 1 and 26 (49.1%) out of 53 samples of group  2, revealing a statistically significant association (p  <  0.01) of the finding with the groups, with the highest incidence in the group 1. the number and distribution of the positive slides in the two groups is reported in figure 2d. inflammation was not detected along with parasites in the positive samples. discussion due to the rising awareness of the public health and lifestyle improvement, consumers pay more attention to quality and safety of meat. on the other hand, high importance of a clear and trustworthy identity of the species in meat products has become important due to economic, safety and religious issues. analytical methods are applicable based on different types of fraud, but it seems that there is not a perfect analytical tool able to provide an answer for all the existing problems. each single technique has its own characteristics and individual limitations, particularly in minced and homogenised meat, where animal tissues may occasionally be mixed with various ingredients. recently, to strengthen analytical methods, some multivariate techniques have been suggested to be more effective to determine meat authenticity as reviewed by vlachos and colleagues (vlachos et al. 2016). in this survey the histological analysis allowed to detect specific tissues, sometimes unwanted, as well as to identify various microorganisms, inflammation and other ingredients in different meat products. a b m ea n s co re group 1 group 2 150 100 50 10 5 0 m ea n s co re s group 1 group 2 4 3 1 0 2 figure 3. a. bacterial cells detected in the two groups (mean scores), p < 0.01. b. inflammatory cells detected in the two groups (mean scores), p < 0.01. 83veterinaria italiana 2020, 56 (2), 77‑85. doi: 10.12834/vetit.1669.8871.3 sohrabi et al. tissue and species identification in minced meat 2012), in our investigation particular attention was devoted to detect the presence of central nervous tissue, though all slides were negative. similarly, ghisleni and colleagues (ghisleni et  al. 2010) and prayson and colleagues (prayson et al. 2008) did not report any positive samples for bse risk material from tortellini meat filling. a recent study carried out in piedmont (italy) (meistro et  al. 2015), examining six histological sections for each sample, revealed the presence of sarcocystis spp. in 16 (64%) out of 25 samples of bovine minced meat, which indicates a lower frequency compared with the first group of our study (83.3%). conversely, the frequency was higher compared to the second group (49%). previous studies carried out in italy showed a prevalence of sarcocystosis in cattle above 80% (domenis et  al. 2011) and 91% (chiesa et  al. 2013). in some european countries, there is a trend to raw or undercooked bovine minced meat consumption. in italy and particularly in piedmont region, this type of consumption is common (meistro et  al. 2015). therefore, it is highly recommended to cook fresh beef products before consumption or, alternatively, to freeze them for at least 3‑5 days, in order to destroy sarcocystis cysts (roberts et al. 2005). conclusions within the last decades, a wide range of analytical methods have been employed to reveal the composition of meat and meat products as well as other aspects related to meat authenticity. seeking out the development of molecular methods for food authentication, still it seems that the use of traditional methods along with innovative techniques may provide useful advantages to achieve a more comprehensive outcome of the food quality control. in conclusion, the combination of dna microarrays and histology will increase the monitoring capacity of bovine meat food process. microscopy has been extremely useful to determine the content of specific animal tissues like skeletal muscle, blood vessels, peripheral nerve, connective and fat, or components not mentioned in the label such as bone, cartilage, glands and vegetables in different meat products (prayson et  al. 2008, ghisleni et  al. 2010, sentandreu and sentandreu 2014, sadeghinezhad et al. 2015, hafeez et al. 2016, malakauskiene et  al. 2016). in the present study, histology revealed high frequency of unauthorized tissues in various commercial meat products sold in the italian supermarkets. a similar survey was carried out by ghisleni and colleagues (ghisleni et  al. 2010) who investigated tortellini meat‑filling coming from four italian commercial brands by light microscopy in combination with image analysis, confirming that histology and image analysis are reliable tools in order to identify various animal tissues in a processed meat product. in another study, microscopic examination was used in order to estimate the meat content in american hotdogs; outcomes revealed that the amount of skeletal muscle in most of brands was less than 10% of the cross‑sectional surface area while bone and cartilage were present in all samples (prayson et  al. 2008). although, due to the meat processing procedures, the presence of bone and cartilage fragments is not completely unexpected, according to prayson and colleagues (prayson et al. 2008) there is a general correlation between cost of the products and proportion of meat and bone. bacteria were a frequent finding in 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pathol, 12 (6), 406‑409. roberts t.a., cordier j.l., gram l., tompkin r.b., pitt j.i. & gorris l.g.m. 2005. meat and meat products, in micro‑organisms in foods. 6. ed., springer, boston, ma, 36‑39. sadeghinezhad j., hajimohammadi b., izadi f., yarmahmoudi f. & latorre r. 2015. evaluation of the morphologic method for the detection of animal and herbal content in minced meat. czech j. food sci,  (6), 564‑569. sentandreu m.á. & sentandreu e. 2014. authenticity 1 parole chiave dige, endometrio, mucche, proteome, vitelli. riassunto il profilo proteico di ciascun tessuto dipende dall'espressione di geni che sono regolati anche da steroidi sessuali. confrontare i profili delle femmine sessualmente mature e immature dovrebbe quindi definire i marcatori delle variazioni legate all'età. scopo di questo studio è confrontare il pattern di proteine nell'endometrio di giovenche e vitelli pre‑puberali utilizzando l'elettroforesi differenziale su gel (dige) e identificare la presenza e la quantità di proteine simili o significativamente differenti. in mattatoio sono stati raccolti campioni endometriali da giovenche di età compresa tra 14 e 27 mesi (n = 6, sessualmente mature) e vitelle tra 0,5 e 2 mesi (n = 6, sessualmente immaturi). gli animali, tutti di razza holstein‑friesian, erano sani. i campioni sono stati sottoposti a colorazione fluorescente e elettroforesi 2d. il 73% degli oltre 900 prelievi era simile sia nell'endometrio delle giovenche che in quello dei vitelli. i punti selezionati sono stati identificati. l'angiopoietina‑2 e la dinamina‑like protein, invece, coinvolte rispettivamente nell'angiogenesi e nel rimodellamento della membrana cellulare, sono state rilevate solo nelle giovenche. la aldoso reduttasi e la fosfolipasi, importante per il metabolismo delle prostaglandine, erano presenti in quantità diverse in entrambe le fonti. i risultati dovrebbero aiutare a comprendere meglio il meccanismo d'azione degli ormoni steroidei e cercare i marcatori dello stato dell'endometrio bovino. la colorazione fluorescente, inoltre, si è rivelata uno strumento utile nel confronto tra 2 campioni esaminati. comparazione tra profili proteici endometriali di giovenche e di vitelle di razza holstein-friesian keywords calves, cows, dige, endometrium, proteome. summary the protein profile of each tissue depends on the expression of genes that are regulated, among others, by sex steroids. comparing the profiles of sexually immature and mature females should therefore define markers of age‑related changes. the aim of this study is to compare the pattern of proteins in the endometrium of heifers and pre‑pubertal female calves by using difference in gel electrophoresis (dige), and to identify the presence and amount of similar or significantly different proteins. endometrial samples were collected in slaughterhouse from heifers aged between 14‑27 months (n = 6; sexually mature) and calves between 0.5‑2 months (n = 6; sexually immature). all animals were healthy, holstein‑friesian bred animals. samples were subjected to fluorescent staining and 2d electrophoresis. out of more than 900 spots detected in the endometrium of heifers, 73% were similar to calves. selected spots were identified. angiopoietin‑2 and dynamin‑like protein were detected only in heifers. these proteins are involved in angiogenesis and cell membrane remodelling, respectively. aldose reductase, and phospholipase, which are important for prostaglandin metabolism, were present in different amounts in both sources. these results help to further understand the mechanism of steroid hormone action and look for markers of bovine endometrium status. moreover, fluorescent staining appeared to be an useful tool when comparing 2 samples from different sources. veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 accepted: 04.11.2016 | available on line: 30.09.2018 department of biochemistry, faculty of veterinary medicine, university of life science in lublin, 20-033 lublin, akademicka 12, poland. *corresponding author at: department of biochemistry, faculty of veterinary medicine, university of life sciences, 20-033 lublin, akademicka 12, poland. tel.:+4881 445 6608, fax: +4881 445 6608, e-mail: marta.kankofer@up.lublin.pl. marta giergiel, jacek wawrzykowski and marta kankofer* comparison between endometrial protein profile in holstein-friesian heifers and female prepubertal calves 2 veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 protein profile in the endometrium of heifers and female pre‑pubertal calves giergiel et al. due to, for example, the post‑transcriptional modification of protein or catalytic capacity of enzymes (fu et al. 2009). two‑dimensional electrophoresis is a useful tool to determine protein molecules in a particular biological sample. each run, however, may differ slightly. difference in gel electrophoresis (dige) overcomes this disadvantage by allowing 2 samples to be labelled with fluorescent dye whilst also allowing for their separation in the same run. this way of comparison between 2 samples can reveal the details of which proteins are characteristic for one or another or both biological sources. this technique is used in human medicine to search for markers of diseases (gharbi et  al. 2002, tian et  al. 2008) but could also be used to search for markers of physiological status in animals. the aim of the present investigation is to describe the endometrial pattern of proteins and to compare the presence as well as the amount of selected proteins between heifers (sexually mature) and female calves (sexually immature) by using fluorescent staining. materials and methods an experimental protocol was approved by the appropriate ethics committee appointed at the university of life sciences in lublin (decision no 60/2011). the uterus samples were sourced from a slaughter‑house. in particular, uteri were collected from heifers in their luteal phase, aged between 14‑27 months (n = 6), and pre‑pubertal female calves, aged between 2 weeks‑2 months (n = 6). the classification of luteal phase was based on gross detection of a corpus luteum, which was mature in all the heifers included in the experiment (ireland et al. 1980). all animals were healthy, neither inseminated nor pregnant, and of holstein‑friesian breed. tissues were collected immediately after the animals were slaughtered, washed in 0.9 % nacl, portioned, and frozen. uteri were separated manually into myometrium and endometrium, samples were taken consistently from the same region of uterus. the endometrium was used for further determinations. tissues were homogenised in phosphate buffer (0.05  mol/dm3, ph 7.2), centrifuged at 4  ºc for 20  minutes at 6000 x g. supernatant was used for protein precipitation, which was performed with ready prep 2‑d clean‑up kit (bio‑rad, warszawa, poland) according to the manufacturer’s instructions. dige labelling fifty micrograms of resolubilised protein preparations were labelled with 333 pmol of cydye introduction uterine endometrium is a very dynamic tissue. its appearance and function in bovine species is determined by the phase of oestrus cycle, which is regulated, among other compounds, by the concentration of the sex steroid hormones: estrogens and progesterone (spencer et  al. 2004). during pregnancy, the endometrium is responsible for creating the interplay between the mother and developing fetus. bauersachs and colleagues (bauersachs et  al. 2005) examined the profile of gene expression in bovine endometrium during the late oestrus (day 0, low progesterone) and dioestrus phases (day 12, high progesterone) of the cycle using a combination of subtracted cdna libraries and cdna array hybridisation. different functional groups of proteins dominated during oestrus (day 0, low progesterone), while in dioestrus (day 12, high progesterone), the genes of several groups of enzymes and proteins increased. forde and colleagues (forde et al. 2011) focused on the early (d7) and late luteal (d13) phases in their study, which describes the occurrence of significant differences in endometrial gene expression in the bovine endometrium. faulkner and colleagues (faulkner et  al. 2013) described the impact of the phase of cycle and progesterone plasma level on the protein profile of the bovine endometrium. the activity of the endometrium is reflected by concert action of different proteins that are synthesised as the response to stimuli coming from hormones and their receptors. the endometrium undergoes temporal remodelling during the cycle, but also develops during pregnancy. the profile of endometrial proteins is responsible for the appropriate function of the endometrium. cytological changes related to the sequence of sex steroid activity are well described, but proteins that are directly involved in morphological changes have not yet been fully established. the available literature about protein profile in the bovine endometrium is focused on the differences between pregnant and non‑pregnant animals and the proteins that are characteristic for pregnancy (berendt et  al. 2005, ledgard et  al. 2009, forde et  al. 2014). however, there is still a lack of information about the protein profile in non‑pregnant animals and the associated changes in this profile during ageing or sexual maturation. it is well‑known that the influence of sex steroids differentiates immature from mature organisms, which is furthermore reflected in their different protein profiles. protein profile dependent on sex steroids was not elucidated yet. analysis of mrna often does not reflect the real amount/biological activity of certain proteins 3veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 giergiel et al. protein profile in the endometrium of heifers and female pre‑pubertal calves temperature, the solution was replaced by 100 µl of 50 mm iodoacetamide in 50 mm of nh 4 hco 3 buffer. the gel pieces were incubated in this solution in the dark for 45 minutes at room temperature. the gel pieces were then washed 3 times with 100 ml of a 100 mm nh 4 hco 3 buffer for 5 minutes at room temperature, dehydrated with 100 µl acn, and dried in the centrivap (labconco, local seller a.g.a analitical, warsaw, poland) for 15 minutes. the enzymatic digestion of the proteins was carried out on ice by a stepwise addition of 10 µl of 12.5 ng/ ml trypsin (trypsin gold, mass spectrometry grade, promega, madison, usa) in a 50 mm nh 4 hco 3 buffer, until they were totally rehydrated. finally, 30  µl  50  mm nh 4 hco 3 buffer was added to keep the gel pieces covered during digestion at 37  °c overnight. after digestion, the supernatant was collected and the peptides were extracted 3 times with 50 µl 70% acn with 1.5% trifluoroaceticacid (tfa) by sonification for 15 minutes at room temperature in an ultrasonic water bath (ultron u‑507, ultron, dywity, poland). the supernatant was collected and dried in the centrivap (labconco, local seller a.g.a analitical, warsaw, poland) for 45 minutes at 40 °c. peptide pellet was allowed to re‑swell in 10  µl of 0.1% tfa and purified with µc18 ziptip (eppendorf, poznań, poland) according to the manufacturer’s instructions. the 1  µl of cleaning peptide mixture was picked to a pre‑spotted hcca‑pac (with 3,5‑dimethoxy‑4‑hydroxycinnamic acid) frame (bruker, poznań, poland) and allowed to dry at room temperature. mass spectra were acquired with an ultraflex iii maldi tof/tof spectrometer (bruker, poznań, poland). acquisition was performed in positive ion reflector mode with a 25‑kv acceleration voltage. external calibrations were performed using the peptide calibration standard (bruker, poznań, poland). flex analysis 3.0 software bruker‑daltonics was used for the selection of the monoisotopic peptide masses. the identification of peptides and proteins from mass spectrometry data was achieved by mascot algorithm interrogating, the uniprotkb database was restricted to the ‘other mammalia’ taxonomy. search parameters were set as follows: enzyme – trypsin; modification obligatory – carboamoinodmethylacion cysteine; possible modification – oxidation of methionine; error of 50 ppm. statistical analysis the spots were selected for further examination and identification based on the statistical analysis of gels [anova, wilcoxon (1945) and kolmogorov (1933) tests]. the selection was based on the presence of spots and their intensity, which reflects the amount dige fluor minimal dyes (ge, warsaw, poland). pre‑electrophoretic labelling was performed according to the manufacturer’s instructions. samples from cows were stained with cy 3 and samples from calves with cy 5. in order to avoid non‑specific labelling, dye swap was made. the internal standard was created as a pool from equal amounts of all samples in particular experiment and was stained with cy 2. electrophoresis isoelectric focusing was performed by loading 10 µg proteins by in‑gel rehydration in a volume of 250 µl; denaturating the 2d buffer (8 m urea, 4 % chaps, 70  mm dtt, 0.5% ampholyte ph 4‑7) onto 11  cm ipg‑ready strip linear ph 4‑7 nl (bio‑rad, warsaw, poland) and focused for 30 kvh, using a protean® ief system (bio‑rad, warsaw, poland). the same amount of protein from both examined sources was loaded. before loading ipg strips onto sds‑polyacrylamide gels, they were incubated for 15 minutes in equilibration buffer (50  mm tris‑hcl, ph  8.8, 6  m urea, 30% glycerol, 2% sds) containing 1% dtt, and then for another 15 minutes in equilibration buffer containing 2.5% iodoacetamide. we proceeded with the sds‑polyacrylamide gels (20 x 20 cm, 1.5 mm, t = 11%, c = 2.6%) in accordance to laemmli (laemmli 1970). the second dimension was performed using a protean ii xi (bio‑rad, warsaw, poland) according to the manufacturer’s instructions. the gels with separated labelled proteins were scanned using the typhoon® 9410 imager (ge warsaw, poland) and the protein patterns were displayed with iq tools software. all sample gel images were processed by the image master platinum dige 7.0 software (ge, warsaw, poland) in order to co‑detect and differentially quantify the protein spots. in order to extract manually spots of interest, gels were stained using protein silver nitrate, according to mass spectrometry compatible protocol (shevchenko et al. 1996). selected spots of interest were excised from the gels, chopped into pieces, and transferred into 0.5 ml tubes. the gel pieces were washed 3 times with 100 µl of 100 mm nh 4 hco 3 buffer (ph 8.5) (sigma, poznań, poland) for 5 minutes. the gel pieces were subsequently dehydrated by adding 100 µl acetonitrile (acn) and dried in centrivap (labconco, local seller a.g.a analitical, warsaw, poland) (room temperature, 15 minutes). they were then allowed to re‑swell in 100 µl of 10 mm dtt in 50 mm nh 4 hco 3 of buffer in order to perform reduction (56 °c, 60 minutes). after cooling to room 4 veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 protein profile in the endometrium of heifers and female pre‑pubertal calves giergiel et al. spots 4 and 7 were identified as similar proteins, however they do differ in molecular weight. this may suggest that one of them is simply a fragment. further analysis of the spots is planned. discussion in this study we performed a preliminary examination of protein profiles from bovine endometrium and conducted a comparison between heifers (sexually mature) and female calves (sexually immature). to our knowledge this is the first time such an experiment has been performed. the 2‑d dige analysis allowed for the detailed detection of spots that belonged to either 1 of 2 compared samples, or both examined sources. the elucidation of protein profiles in this particular tissue should help further understanding about the relationship between their biological role and the mechanism of action of sex steroids. moreover, any differences related to age and the same status of prepuberty‑puberty may help to establish possible markers of alterations. the gene profiles of uteri in humans (borthwick et  al. 2003), mice (tan et  al. 2003), rhesus monkeys (ace and okulicz 2004), and bovines (bauersachs et al. 2008) were determined and described. steroid hormones, using genomic way of action, are responsible for the expression of genes leading to the synthesis of the particular proteins necessary for of protein. statistical analyses allowed us to select spots that were most different and similar between heifers and pre‑pubertal female calves. p‑value was defined as < 0.05. results the statistical analysis of data is presented in figure 1 (scatter plot). gel analysis detected 947 spots in the endometrium of heifers. out of this number, 73% of spots (689) were similar to the endometrium of pre‑pubertal female calves. the endometrium of calves showed 816 spots. out of this number, 34% of spots were specific to calves, while 538 spots (66%) were similar to the endometrium of heifers. the protein profile of bovine endometrium from heifers and pre‑pubertal female calves is shown in figure 2. proteins were selected for further analysis based on the statistical analysis of gels (anova test, wilcoxon and kolmogorov). statistical tests allowed us to select the most different and the most similar spots between heifers and pre‑pubertal female calves. eight spots were selected for preliminary identification (figure 1). spots 1, 6, 7, and 8 were detected only in heifers while 2, 3, 4, and 5 were present in both sources. spot number 5 showed a similar intensity of staining (similar amount of protein) both in heifers and pre‑pubertal female calves, while spots 2, 3, and 4 showed a higher intensity of staining (higher amount of protein) in heifers than in pre‑pubertal female calves. the results of the identification of proteins (shown in figure 1) are presented in table i. figure 1. scater plot of spots from endometrium of heifers and pre‑pubertal female calves. ca lv es cows 0 1 3 5 7 9 11 2 4 6 8 10 12 0 2 4 6 8 10 12 1 3 5 7 9 11 figure 2. 2‑d dige image of gel with endometrial proteins from heifers and pre‑pubertal female calves. identified proteins are marked with numbers. ph 4 7 1 2 3 4 5 6 7 8 5veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 giergiel et al. protein profile in the endometrium of heifers and female pre‑pubertal calves to bin et  al. (2012), oestrogens increased relevant changes in ang‑2 concentration in uteri from mice, while progesterone inhibited it. the actions of steroid hormones were compared with the action of its antagonists ici 182,780 and ru486, respectively. on the contrary, hirchenhain and colleagues (hirchenhain et al. 2003) did not observe significant differences in the concentration of ang‑2 during the human menstrual cycle. this could suggest that ang‑2 might not be regulated by progesterone. krikun and colleagues (krikun et  al. 2004) also did not notice major changes in the amount of ang‑2 after estradiol or medroxyprogesterone acetate treatment in cultured human endometrial endothelial cells. genes for ang‑2 like angptl2 and endothelial tyrosine kinase (tek, angiopoietin receptor tie‑2) were expressed during oestrus in bovine endometrium (bauersachs et al.2008). another protein that was identified in examined samples is dynamin‑1‑like, which was present only in heifers. the protein belongs to a superfamily of large guanosine triphosphatase gtpase proteins. the main function of this particular protein is membrane remodelling in a variety of cellular membranes. it is also associated with receptor‑mediated endocytosis at the plasma membrane and is involved in the process of fission of mitochondria. the lack of dynamin‑like protein, which is essential to the remodelling process described above, may lead to a defect in division (bleazard et  al. 1999, sesaki and jensen 1999). for more details about the dynamin‑like protein, see williams and kim’s (williams and kim 2014) review. among the identified proteins that were present in both groups of animals, and which also showed a similar intensity of staining (similar amount of protein), there was aldose reductase (ec 1.1.1.21). the aldose reductase enzyme plays an important role in the endometrium because there is a strong a cellular response to receptor‑hormone interaction. their biological activity may differ depending on age, sex, and the phase of cycle. bauersachs and colleagues (bauersachs et  al. 2005) suggested that proteins representing ‘cytoskeleton’, ‘cell motility’, ‘cell adhesion’, ‘extracellular matrix structural proteins’, ‘ecm remodelling’, and ‘proliferation’ dominate during oestrus, while in dioestrus, genes of several groups of enzymes and proteins connected with ‘transport’ are increased. berendt and colleagues (berendt et  al. 2005) observed higher amount of proteins such as rho gdp dissociation inhibitor beta; 20 alpha‑hydroxysteroid dehydrogenase; soluble nadp1‑dependent isocitrate dehydrogenase 1; and acyl‑coa‑binding protein in pregnant cows than in non‑pregnant animals. these proteins are involved in redox reactions and cell signalling. in the present study we identified angiopoietin‑2 (ang‑2) in endometrium samples from heifers, which was absent in the endometria from female pre‑pubertal calves. angiopoietin‑2 belongs to a family of vascular growth factors and plays a role in embryonic and postnatal angiogenesis. it influences angiogenesis through the activation of endothelial cell‑specific receptor tyrosine kinase tie‑2. ang‑2, which is antagonist of ang‑1, takes part in regulation of vascular re‑modelling together with vascular endothelial growth factor (vegf). when vegf is present, ang‑2 stimulates the proliferation and migration of endothelial cells, but while vegf is absent, ang‑2 causes endothelial cell apoptosis and vessel regression (holash et  al. 1999). ang‑2 is very important in appropriate female reproduction related to uterine vascular permeability and angiogenesis, and its presence has been confirmed in the ovaries, uteri, and placentas of mice (matsumoto et al. 2002) as well as in human endometrium (hirchenhain et al. 2003), human placenta during normal pregnancy (schiessl et al. 2009), and in uterine natural killer cells in the late secretory phase (li et al. 2001). according table i. the list of identified proteins from endometrium of heifers and prepubertal female calves selected based on similarities and significant differences in intensity of staining. spot id* protein name uniprot score anova wilcoxon kolmogorov 1 angiopoietin-2 angp2_pig 41 0.001 0 1 2 phospholipase ddhd1 ddhd1_bovin 36 0.489 2 0.5 3 putative rna exonuclease nef-sp rexon_bovin 54 0.001 0 1 4 dehydrogenase/reductase sdr family member 4 dhrs4_bovin 39 0.025 0 1 5 aldose reductase aldr_bovin 59 0.982 1 0.5 6 cytochrome p450 3a6 cp3a6_rabit 38 0.001 0 1 7 dehydrogenase/reductase sdr family member 4 dhrs4_bovin 39 0.013 0 1 8 dynamin-1-like protein dnm1l_bovin 60 0.016 0 1 * see figure 2. 6 veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 protein profile in the endometrium of heifers and female pre‑pubertal calves giergiel et al. its activity. this is released from phospholipids by phospholipase. this enzyme was identified in both examined sources of tissues, but a higher intensity of staining (higher amount of protein) was detected in heifers than in pre‑pubertal female calves. putative rna exonuclease, which cleaves phosphate bonds in rna, is necessary for appropriate cellular metabolism and in the present study was detected in higher amounts in heifers than in pre‑pubertal female calves. dehydrogenase/reductase sdr family member 4 known as nadph‑dependent retinol dehydrogenase/reductase (1.1.1.184) reduces all‑trans‑retinal and 9‑cis retinal. it is well known that vitamin a is crucial for the proper functioning of epithelia not only in the reproductive tract. the proteins mentioned above were identified in the present study and were known earlier. moreover, their biological activity was described in different tissues and processes that were not limited to the reproductive tract. these proteins have not yet been considered with respect to age and hormone related alterations in concentrations in bovine endometrium. conclusions in this preliminary study we used dige and partial identification to compare proteins that were absent or present in different amounts based on sex steroid influence in conditions of current experiment (i.e. the age of used animals and used tissues) in the endometrium of heifers and pre‑pubertal female calves. obtained results demonstrate that differences in protein profile can be expected depending on pre‑pubertal or pubertal status. acknowledgements authors are grateful to mr tomasz banach for his technical support with maldi analysis. grant support the study was funded by statutory activity of university of life sciences in lublin (wkb‑ds‑4). connection between akr1b5 – recently renamed as bos taurus – akr1b1 (gene id 317748), and prostaglandin f2α (pgf2α) production, and because it is involved in alternative pathway of pgf2α synthesis (madore et al. 2003). it is moreover associated with additional enzymatic activity of 20α‑hsd and glucose metabolism activities. the new activity has been confirmed in bovine (madore et  al. 2003) and human endometrium (bresson et al. 2011). it is interesting that human and bovine akr1b1 belong to the akr1b family and share 86% identity or homology. the same protein was identified in the peri‑implantation uterine luminal fluid that was taken from pregnant sheep. according to rita and colleagues (rita et  al. 1998), the protein was synthesised by the trophoblast and detected even before the formation of the placenta. bresson and colleagues (bresson et al. 2011) studied the presence of both akr1b1 and akr1c3 at the mrna and protein levels in human endometrium during the menstrual cycle. they observed that both mrnas were present throughout the cycle without significant variation. the akr1c3 protein immunostaining was constant during the cycle in epithelial cells, but completely absent in the stromal compartment. this has also been confirmed in other studies (pelletier et  al. 1999). at the same time, the akr1b1 protein was present in luminal and glandular epithelial cells as well as in stromal cells of the endometrium. a higher amount of akr1b1 protein was observed in the early proliferative and mid‑late secretory phases compared with other phases of the menstrual cycle. the results were surprising taking into consideration that human endometrial stromal cells produce high concentrations of pgf2α (chapdelaine et al. 2006, kang et al. 2006). this leads to the hypothesis that the corresponding human enzyme akr1b1 could possess pgfs activity in the human endometrium as similar to akr1b5 in bovine endometrium (madore et al. 2003). the human akr1b1 can metabolise pgh2 and form pgf2 with a high efficiency. according to kabututu and colleagues et al. (kabututu et al. 2009), akr1b1 is 20 times more efficacious than akr1c3 in producing pgf2 from its precursor pgh2. aldose reductase may require arachidonic acid for 7veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1002.5292.1 giergiel et al. protein profile in the endometrium of heifers and female pre‑pubertal calves ace c.i. & okulicz w.c. 2004. microarray profiling of progesterone‑regulated endometrial genes during the rhesus monkey secretory phase. reprod biol endocrinol, 2, 54‑58. bauersachs s., ulbrich s.e., gross k., schmidt s.e., meyer h.h., einspanier r., wenigerkind h., vermehren m., blum h., sinowatz f. & wolf e. 2005. gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes. j mol endocrinol, 34, 889‑908. bauersachs s., mitko k., ulbrich s.e., blum h. & wolf e. 2008. transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of 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jensen r.e. 1999. division versus fusion: dnm1p and fzo1p antagonistically regulate mitochondrial shape. j cell biol, 147, 699‑706. 404 not found 63 parole chiave percezione del rischio, pratiche preventive, professionisti della salute animale, salute pubblica, nigeria, zoonosi. riassunto questo studio valuta la conoscenza delle zoonosi, la percezione del rischio e l’uso di pratiche preventive in coloro che si occupano di salute degli animali in nigeria. è stata condotta un’indagine trasversale su 582 partecipanti; mediante statistiche descrittive e modelli di regressione logistica multivariata sono stati analizzati i dati raccolti dai 529 questionari pervenuti. la percentuale dei veterinari (92,0%)che conosce le zoonosi è risultata essere significativamente (p < 0.001) più alta di quella dei para-veterinari (32,4%). la maggior parte dei veterinari (76,7%) e il 46.2% dei para-veterinari considerano elevato il rischio di contrarre infezioni zoonotiche durante le necroscopie o la raccolta di tessuti. ugualmente, una percentuale più alta (p < 0.001) di veterinari (54%) rispetto ai paraveterinari (25,0%) giudica lavare le mani sul posto di lavoro prima dei pasti una soluzione efficace per ridurre il rischio. coloro che sono impegnati in attività con i grossi animali adottano misure protettive inadeguate (0,35; 95% ic: 0,16; 0,77). questi risultati possono contribuire a fornire informazioni sulla riduzione del rischio a supporto di misure per la prevenzione delle zoonosi in nigeria. conoscenza, percezione del rischio e pratiche per prevenire le infezioni zoonotiche tra coloro che si occupano di sanità animale in nigeria keywords animal health professionals, preventive practices, public health, risk perception, zoonosis, nigeria. summary this study was aimed to assess zoonotic disease knowledge, risk perceptions, and preventive practices of animal health professionals in nigeria. cross-sectional questionnaire survey was conducted on 582 participants and 529 responded. collected data were analyzed by descriptive statistics and multivariate logistic regression models. the proportion of veterinarians (92.0%) which knowledge about zoonosis was much higher (p  <  0.001) than that of para-veterinarians (32.4%). in contrast to para-veterinarians (46.2%), the majority of veterinarians (76.7%) perceived high risk of zoonotic infections during necropsy/ tissue collections. similarly, a much higher (p < 0.001) proportion of veterinarians (54.0%) considered hand washing before eating at work as effective way of risk mitigation, compared to para-veterinarians (25.0%). professionals in large animal practice were less likely (or 0.35; 95% ci: 0.16, 0.77) not to be engaged in satisfactory protective measures. these results constitute public health contributions to the risk mitigation information that may support measures for zoonosis prevention in nigeria. veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 accepted: 17.07.2017 | available on line: 31.03.2019 public health and epidemiology unit, niger state veterinary hospital, minna *corresponding author at: public health and epidemiology unit, niger state veterinary hospital, minna, niger. e‑mail: nmabida62@gmail.com. nma bida alhaji*, ismail ayoade odetokun and abdullahi abubakar erena animal health professionals’ knowledge, risk perceptions and preventive practices towards zoonotic infections in nigeria: any challenging gap? 64 veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 risk perceptions and preventive practices in nigeria nma bida alhaji et al. veterinarians that contracted zoonotic diseases at work have been reported to be as high as 60-65% in two studies conducted in the uk and south africa (constable and harrington 1982, gummow 2003). while the literature is rich in documentation of particular circumstances elsewhere on protective practices against zoonotic pathogens, they tend to be restricted to disease-specific interventions (sultana et  al. 2009, max et  al. 2011), high-risk occupational settings (rabinowitz et al. 2013, odo et al. 2015), and responses to zoonotic outbreaks (ferguson et  al. 2006, somrongthong et al. 2012). results of previous studies on the subject matter in nigeria have only highlighted the risk of exposures of farm workers and pet owners to zoonotic infections (adesokan et  al. 2013, awosanya and akande 2015). to-date, information in scientific literature on the levels of zoonotic infections risk perceptions and mitigation practices by animal health professionals in nigeria have received surprisingly very little attention. understanding the infectious disease protective behaviours in animal health workers is important to inform effective first-line public health and preventive practices programs on zoonoses. therefore, this study was aimed to assess knowledge and risk perceptions about zoonotic infections, and preventive practices of a subset of animal health professionals that work in the field in nigeria. we hypothesize that demographic and professional specialization characteristics of animal health professionals cannot influence protective practices against zoonotic infections. we anticipate that our preliminary findings will help to identify challenging gap against zoonotic infections and assist the professionals and public health authorities to enhance strategies of zoonoses control and prevention programs in nigeria. materials and methods study area and population the study was conducted in the north-central zone of nigeria. the zone comprised of six states and abuja, out of the 36 states in nigeria. each state has its own veterinary services directorate. the zone also accommodates three veterinary teaching hospitals at the universities of abuja, ilorin and makurdi as well as the only national veterinary research institute in nigeria, located at vom in plateau state (figure 1). the target population was animal health professionals practicing in the study area within the survey period. it is made up of veterinarians (with doctor of veterinary medicine degree as basic professional qualification) and para-veterinarians (with ordinary national diploma and/or higher introduction zoonoses are infectious diseases transmitted from vertebrate animals to humans (who/fao/oie 2004). zoonoses have become more prominent in recent years with outbreaks of some emerging infectious diseases, such as severe acute respiratory syndrome, highly pathogenic avian influenza, and ebola virus disease, leading to human deaths across many countries (lau et  al. 2010, oladokun et  al. 2012, fasina et  al. 2014). however, some are listed as endemic zoonoses and include brucellosis, rabies, human african trypanosomiasis, bovine tuberculosis, cysticercosis, echinococcosis and anthrax, (who 2006). an emerging zoonosis is “a pathogen that is newly recognized or evolved, or has occurred previously but shows an increase in incidence or expansion in geographical, host or vector range”1. endemic zoonoses, often neglected, are found throughout the developing world where conditions for their maintenance and spread exist, and may occasionally give rise to epidemics. they are associated with people living in close proximity to their animals, affecting not only the health of people in the poorest communities but also causing morbidity and mortality of their livestock (maudlin et  al. 2009). it has been estimated that around 60% of the infectious organisms pathogenic to humans are zoonotic and that 75% of the all emerging infectious diseases are zoonotic (cleaveland et al. 2001, taylor et al. 2001, woolhouse and gowtage-sequeria 2005). animal health professionals are broadly trained to help prevent transmission of zoonoses; to promote public health through recognition and treatment of diseases in companion and food animals; and to educate clients about diseases that may be transmitted from animals to humans (glickman 1992, wohl and nusbaum 2007). because these professionals are often the first to have contacts with potentially infected animals during clinical investigations, they are at risk of contracting zoonotic infections and may serve as a bridge for disease entry into the human population (wright et al. 2008, dowd et al. 2013). some preventive practices, such as use of sanitizers and protective clothing, have been used by veterinarians in nigeria to mitigate risks of infections, but their effectiveness depends on the risk perceptions and appropriate applications during veterinary procedures. for many years, veterinarians and associate staff have been recognized as being at high risks of many zoonotic infections at work, possibly as a result of the nature of exposures such as bites and scratches of animals. the proportions of 1 www.who.int/zoonoses/emerging_zoonoses/en. 65veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 nma bida alhaji et al. risk perceptions and preventive practices in nigeria questionnaire design, pretesting and implementation the survey instrument, designed specifically for this study, was a 2-page structured paper based questionnaire developed in english, and contained mostly close-ended questions to ease data processing, minimize variation, and improve precision of responses (thrusfield 2009). the questionnaire focused on various sub-themes like the respondents’ demographic information and professional practice specialization; knowledge about zoonoses; risk perceptions on zoonoses at work; practices used for protection against zoonotic infections; and use of personal protective equipment (ppe) as preventive measures when performing veterinary procedures. the sub-themes encompassed questions about respondents’ general knowledge of zoonotic diseases and assessed by definition of zoonosis. also, a list of infectious pathogens, diseases and syndromes was provided and respondents were asked to indicate those they believed are zoonotic, as well as the common routes for their transmission. risk perceptions were assessed in terms of respondents’ concerns on the risks of contracting zoonotic infections during veterinary activities. practices were the protection measures, especially the use of ppe, against zoonotic infections at work. however, in nigeria there is no standard guideline regarding the use of ppe against zoonotic infections. assessment of inadequate or adequate ppe use in this study was therefore based on minimal (not ideal) ppe use recommendations by the american national association of state public health veterinarians, which are overalls/gown and gloves (scheftel et  al. 2010). in addition, we included cap and face mask. the questionnaire was pre-tested prior to the study on few animal health professionals on whom the actual study was carried out and revised accordingly. the questionnaires were face-to-face administered on the respondents by the researchers. before commencement of questionnaire administration, informed consent was verbally obtained from each respondent who was assured of voluntary participation, confidentiality of his/her responses and the opportunity to withdraw at any time without prejudice in line with the helsinki declaration (wmadh 2001). a total of 529 respondents in the various animal health practices completed and returned the questionnaires. data management and statistical analysis data were summarized into microsoft excel 7 (microsoft corporation, redmond, wa, usa) spreadsheet, exported and analyzed using epi-info 3.5.3 (cdc, atlanta, usa). descriptive and analytic national diploma in animal health as basic professional qualifications), and all have had variable levels of professional training, skills and specializations. study design and sampling procedure a cross-sectional questionnaire-based survey was carried out on animal health professionals between october 2014 and september 2015 to collect information on knowledge and risk perceptions about, and preventive practices against zoonotic infections at work. the sample size was calculated using the epidat 3.1 software, with power set at 50% and margin of error at 5%. a 10% contingency was added to take care of non-response, and 582 target participants were chosen. the study population was obtained using a purposive sampling procedure because the sampling frame of the respondents in the zone was not readily available during the period of survey. the selected participants were the animal health professionals working in the livestock sub-sector (cattle, pig, sheep, goat and poultry farms), food-chain sector (slaughterhouses), veterinary clinics and hospitals, veterinary schools and research institute in the region. the procedures for this study were approved by the niger state ministry of livestock and fisheries development research ethics committee (protocol number mlfd/ngs/0672) of 19 february 2014. 0 450 900 1,800 kilometers plateau benue kogi kwara niger nassarawa federal capital territory n s w e 15°0’0” n 15°0’0” n 10°0’0” n 5°0’0” n 15°0’0” n 10°0’0” n 5°0’0” n 10°0’0” n 5°0’0” n 15°0’0” n 10°0’0” n 5°0’0” n study area figure 1. map of nigeria showing location of the study area (north‑central zone). 66 veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 risk perceptions and preventive practices in nigeria nma bida alhaji et al. veterinarians (92.0%) on the definition of zoonoses as diseases or infections transmitted from animals to humans, was significantly (p < 0.001) greater than that of para-veterinarians (32.4%). when provided a list of 11 infectious pathogens, diseases and syndromes, respondents were able to significantly classify just over half on their potentials to be transmitted from animals to humans. higher (p  <  0.05) proportions of the veterinarians gave correct classifications of dermatophytosis, bovine tuberculosis, brucellosis, rabies and highly pathogenic avian influenza as zoonoses compared to the para-veterinarians (table ii). regarding the modes of transmission of zoonotic diseases, which are through contacts with animal blood, contacts with aborted fetus and placenta, bites or scratches of animals, and contacts with carcasses/bodily fluid, the knowledge amongst veterinarians was significantly (p  <  0.05) higher compared to para-vetarinarians (table ii). risk perceptions for zoonotic infections at work animal health professionals were asked about their perceptions on the risk of exposures to zoonoses when performing veterinary procedures. there were high proportions of respondents that significantly perceived high risks of zoonotic statistics were used to analyze variables, with frequencies and proportions predominantly used to described the obtained data. independent variables were created from the questions in the questionnaire about demographic and practice specialization characteristics, knowledge, risk perceptions, and preventive practices, while levels of protective practices constituted the independent (explanatory) variables. to create dependent (outcome) variables, a unique scoring system was used for the responses. each respondent was assigned a score that reflected the stringency of his or her type of professional background. to measure responses to these independent factors, the scoring system ranged between 1 and 20 points and was converted to 100%. the score range was further categorized into ‘poor’ (≤  10 points, ≤50%) and ‘satisfactory’ (≥  11  points, ≥ 51%) to keep them as binary variables. associations between the outcome and explanatory variables were first subjected to univariable analyses using chi-square tests (dohoo et al. 2009). all factors found to be statistically significant were subsequently analyzed using likelihood stepwise backward multivariate logistic regression models to control for confounding and test for effect modification. a goodness-of-fit test using hosmer-lemeshow test was conducted and found that the final model was good. p < 0.05 was considered statistically significant in all analyses. results demographic characteristics of participants during the one-year period of the survey, 529 (91.0%) of the 582 individuals who were approached completed the questionnaires. therefore, a total of 529 animal health professionals, comprised of 25.9% (n  =  137) veterinarians and 74.1% (n  =  392) para-veterinarians, from the six states and abuja in the north-central nigeria participated in the study. median age of respondents was 38 years, with mean age of 40.06  ±  10.7 sd years. forty percent of the respondents were between ages 30-39 years, while 84.2% were males and 15.8% females. a majority (89.7%) of the respondents was married; most (36.5%) engaged in general practice, and 2.3% specialized in wildlife practice (table i). knowledge level about zoonoses among animal health professionals knowledge level about zoonoses observed in veterinarians was significantly higher than in para-veterinarians. similarly, the knowledge of table i. demographic and specialization characteristics of animal professionals in north‑central nigeria. factor frequency n = 529 (%) veterinarians n = 137 (%) para-veterinarians n = 392 (%) age 20-29 79 (15.2) 16 (20.3) 63 (79.7) 30-39 213 (40.3) 54 (25.4) 159 (74.6) 40-49 122 (23.3) 39 (32.0) 83 (68.0) 50-59 94 (17.8) 22 (23.4) 72 (76.6) 60-69 21 (3.4) 6 (28.6) 15 (71.4) gender male 443 (84.2) 102 (23.0) 341 (77.0) female 86 (15.8) 35 (40.7) 51 (59.3) marital status married 471 (89.7) 114 (24.2) 357 (75.9) single 58 (10.3) 23 (39.7) 35 (60.3) specialization small animal practice 35 (6.6) 11 (31.4) 24 (68.6) large animal practice 103 (19.4) 25 (24.3) 78 (75.7) poultry practice 85 (16.1) 32 (37.6) 53 (62.4) wildlife practice 25 (4.7) 6 (24.0) 19 (76.0) general practice 173 (32.7) 30 (17.3) 143 (82.7) abattoir worker 108 (20.45 33 (30.6) 75 (69.4) 67veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 nma bida alhaji et al. risk perceptions and preventive practices in nigeria was greater (p < 0.05) amongst veterinarians (64.2%) than para-veterinarians (32.1%). majority (76.7%) of the veterinarians reported high risk perceptions for zoonotic infections during necropsy/tissue collections, while less than half proportion (46.2%) of the para-veterinarians perceived such risk of infections for the procedures. this different perception was significant (p < 0.05) (table iii). practices used to protect against zoonotic infections at work animal health professionals that participated in the study were asked about zoonotic infection mitigation practices instituted against zoonotic infection risks at work, including use of ppe. variable proportions of them indicated that they applied significant preventive practices that are related to hand hygiene, management of waste items or devices that have edges or projections, and barrier or isolation as mitigation practices against zoonotic infections (table iv). among veterinarians, 54.0% considered hand washing before eating at work as an effective way of reducing zoonotic disease risk, while only 25.0% of the para-veterinarians considered such activity effective. a significantly higher (p  <  0.05%) proportion of veterinarians (78.8%) mentioned that they disposed needles in appropriate containers after use as a measure in reducing zoonotic disease risks, while less than one-third (29.3%) of the para-veterinarians engaged in such act. the practice of recapping needles prior to disposal was reported by a significant (p  <  0.05) higher number of veterinarians (85.4%) as mitigation measure against zoonoses risks while just 46.4% of the para-veterinarians recapped needles before infections when performing veterinary activities. over half proportion of the veterinarians (61.3%) and para-veterinarians (55.4%) indicated that they perceived low risk of zoonotic infections when handling asymptomatic animals. however, the perception of the risk of zoonotic infections when handling of animals tissues/bodily fluid/excretions table ii. knowledge levels of animal health professionals on zoonoses in north‑central nigeria: 2014‑2015. variable type of profession yes n (%) no n (%) p-value zoonosis is disease or infection transmitted from animal to human v 126 (92.0) 11 (8.0) < 0.001 p 127 (32.4) 265 (67.6) which of the following infections, diseases or syndrome is zoonosis? dermatophytosis v 75 (54.7) 62 (45.3) 0.03 p 173 (44.1) 219 (55.9) bovine tuberculosis v 112 (81.8) 25 (18.2) < 0.001 p 103 (26.3) 289 (73.7) brucellosis v 119 (86.9) 18 (13.1) < 0.001 p 141 (36.0) 251 (64.0) rabies v 102 (74.5) 35 (25.5) 0.001 p 200 (51.0) 192 (49.0) anthrax v 81 (58.4) 56 (41.6) 0.01 p 183 (46.7) 209 (53.3) hydatidosis v 78 (56.9) 59 (43.1) 0.03 p 181 (46.2) 211 (53.8) gastro-intestinal worms v 52 (38.0) 85 (60.0) 0.59 p 159 (40.6) 233 (59.4) distemper v 62 (45.3) 75 (55.7) 0.36 p 161 (41.1) 231 (58.9) fowl pox v 57 (41.6) 80 (58.4) 0.64 p 166 (42.3) 226 (57.7) infectious diarrhea v 57 (41.6) 80 (58.4) 0.63 p 172 (43.9) 220 (56.1) highly pathogenic avian influenza v 109 (79.6) 28 (20.4) < 0.001 p 169 (43.1) 223 (56.9) common routes for transmission of zoonotic diseases contacts with animal skin v 71 (51.8) 66 (48.2) 0.57 p 214 (54.6) 178 (45.4) contacts with animal blood v 112 (81.8) 25 (18.2) < 0.001 p 153 (39.0) 239 (61.0) consumption of animal products (milk and meat) v 89 (65.0) 48 (35.0) 0.001 p 191 (48.7) 201 (51.3) contacts with aborted fetus and placenta v 114 (83.2) 23 (16.8) < 0.001 p 157 (40.0) 235 (60.0) bites or scratches of animals v 119 (86.9) 18 (13.1) < 0.001 p 161 (41.1) 231 (58.9) contacts with carcasses/bodily fluid v 116 (84.7) 21 (15.3) < 0.001 p 195 (49.7) 197 (50.3) v = veterinarians; p = para-veterinarians. table iii. animal health professionals’ risk perceptions associated with zoonotic infections during veterinary procedures in north‑central nigeria: 2014‑2015. procedure type of profession low risk n (%) high risk n (%) p-value handle asymptomatic animals v 84 (61.3) 53 (38.7) 0.22 p 217 (55.4) 175 (44.6) handle clinically ill animals v 50 (36.5) 87 (63.5) 0.02 p 188 (48.0) 204 (52.0) handle dead animals v 56 (40.9) 81 (59.1) 0.001 p 155 (39.5) 237 (60.5) handle tissues/ bodily fluid/ excretions v 49 (35.8) 88 (64.2) < 0.001 p 266 (67.9) 126 (32.1) perform necropsy/ tissue collections v 32 (23.3) 105 (76.7) < 0.001 p 211 (53.8) 181 (46.2) work in areas infested with ticks v 17 (12.4) 120 (87.6) < 0.001 p 212 (54.1) 180 (45.9) v = veterinarians; p = para-veterinarians. 68 veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 risk perceptions and preventive practices in nigeria nma bida alhaji et al. interestingly, respondents reported the use of minimal ppe for seven of the twelve common practice scenarios assessed. overall, they used ppe when examining sick animals as well as when handling potentially infectious specimens. relatively high proportions of veterinarians and para-veterinarians (62.8% and 67.3, respectively) used adequate ppe when examining apparently healthy animals. however, 89.1% of the veterinarians and 58.4% of para-veterinarians consciously agreed that using adequate ppe when examining clinically ill animals was the effective way of reducing zoonotic infections. among veterinarians, a significantly (p  <  0.05) higher proportion considered use of minimal ppe for protection against zoonotic infections when handling placenta and fetal discharges, performing post mortems, and disease outbreaks investigations, respectively as inadequate, while the majority of the para-veterinarians considered minimal ppe use for these practice scenarios to be adequate. despite the likely zoonotic diseases risks, most of the veterinarians and para-veterinarians considered minimal ppe use for handling skin lesions, gastro-intestinal conditions, neurologic conditions, and animal faecal samples, as adequate for protection against risks of zoonotic infections. conversely, 67.2% and 86.1% of the veterinarians considered use of ppe for protections when handling respiratory conditions and blood samples from suspected animals, respectively to be inadequate. on the other hand, the majority of the para-veterinarians (58.9 and 51.8%, respectively) considered the ppe use for same activities to be adequate. associations of demographic and specialization characteristics with the levels of preventive practices against zoonotic infections the demographic and specialization characteristics of the respondents were compared with their overall preventive practice behaviours to determine possible associations. univariate analysis identified four independent factors and all, except gender and marital status, were significantly associated with the satisfactory preventive practices against zoonotic infection risks. at the multivariate logistic regressions, age and specialization characteristics remained significantly associated with preventive practices. however, those in age group 50-59 years were nine times more likely (or 8.99; 95% ci: 4.39, 18.44) to practice satisfactory preventive measures against zoonotic infections than those in age group 20-29 years. also, professionals that specialized in large animal practice were less likely (or 0.35; 95% ci: 0.16, 0.77) not to practice satisfactory protective measures disposal. only 13.8% of the para-veterinarians (p < 0.05) indicated they practiced sterilization of all equipment after use on affected animals to reduce risks of zoonoses unlike 48.2% of veterinarians that practiced such activity. nearly half (49.6%) of the veterinarians practiced washing and sanitizing of hands between patient contacts, conversely only one-third (p  <  0.05) of para-veterinarians (34.4%) had such behaviour. variable proportions of the respondents reported use of ppe when handling infectious procedures, isolations of suspected animals from apparently healthy ones, restriction of human contacts with suspected animals, and sterilization of all equipment that had been used on the suspected animals as mitigation measures against risks of zoonotic infections (table iv). use of ppe as protective measure against zoonotic infections at work the levels of ppe worn in twelve different veterinary procedure situations are presented in table  v. table iv. animal health professionals’ preventive practices used towards mitigating zoonotic infections at works in north‑central nigeria: 2014‑2015. practice type of profession yes n (%) no n (%) p-value washing hands with soap before eating at work v 74 (54.0) 63 (46.0) < 0.001 p 98 (25.0) 294 (75.0) washing & sanitizing hands between patient contacts v 68 (49.6) 69 (50.4) 0.001 p 135 (34.4) 257 (65.6) recapping of needles prior to disposal v 117 (85.4) 20 (14.6) < 0.001 p 182 (46.4) 210 (53.6) sterilization and reuse of syringes & needles v 53 (38.7) 84 (61.3) 0.003 p 100 (25.5) 292 (74.5) disposal of needles in an appropriate containers after use v 108 (78.8) 29 (21.2) < 0.001 p 115 (29.3) 277 (70.7) routine isolation of suspected animals from healthy ones v 118 (86.1) 19 (13.9) < 0.001 p 173 (44.1) 219 (55.9) restriction of people from having contacts with affected animals v 115 (83.2) 23 (16.8) < 0.001 p 61 (15.6) 331 (84.4) sterilization of all equipment after use on the suspected animals v 66 (48.2) 71 (51.8) < 0.001 p 54 (13.8) 338 (86.2) use of personal protective equipment when handling infectious procedures v 93 (67.9) 44 (32.1) < 0.001 p 67 (17.1) 325 (82.9) v = veterinarians; p = para-veterinarians. 69veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 nma bida alhaji et al. risk perceptions and preventive practices in nigeria indicate that a significant higher proportion (92.0%) of the veterinarians had knowledge about zoonoses as compared to the para-veterinarians (32.4%). good knowledge of zoonoses gives better understanding and perceptions of their risks. we found that considerable proportions of respondents could not significantly classify some pathogens as zoonoses. as previously reported, some gastro-intestinal worms can be zoonotic pathogens, such as toxocara canis (bingham et al. 2010). also, most respondents did not possess significant knowledge about less severe but more common zoonotic syndromes, such as infectious diarrhea. however, most (p < 0.05) participants demonstrated knowledge about the potentials of bovine tuberculosis being transmitted from cattle to human. although there were general significant knowledge levels about routes of zoonotic disease transmissions, a significant lower proportions of the para-veterinarians had knowledge about pathogen entries, which is likely to expose them to risks of contracting zoonoses at works, as they are unlikely to take proper precautions or use protective barriers when dealing with high risk conditions such as abortions and placentas. one challenge for reducing human public health diseases burdens is the understanding of variable transmission routes of zoonotic pathogens, such as against zoonotic infections than those in small animal practice. however, those that specialized in abattoir work and wildlife practices were not likely to practice significant satisfactory preventive measures against zoonotic infections (table vi). discussion knowledge and perceptions about risks of zoonotic infections among high risk groups as well as controlling their transmission are crucial to the animal health profession. this study is unique because it was the first to broadly identify the use of minimal protective equipment during veterinary procedures for protection against zoonotic infections and integrates demographics and specialization to preventive practices against zoonotic diseases in nigeria. the results of this study table v. use of minimal ppe as protective measures against zoonoses during veterinary procedures by animal health professionals in north‑central nigeria: 2014‑2015. procedure type of profession inadequate ppe kit n (%) adequate ppe kit n (%) p-value handling apparently healthy animals v 51 (37.2) 86 (62.8) 0.33 p 128 (32.7) 264 (67.3) handling clinically ill animals v 15 (10.9) 122 (89.1) < 0.001 p 163 (41.6) 229 (58.4) handling skin lesions v 60 (43.8) 77 (56.2) 0.84 p 168 (42.9) 224 (57.1) handling respiratory conditions v 92 (67.2) 45 (32.8) 0.001 p 161 (41.1) 231 (58.9) handling gastro-intestinal conditions v 57 (41.6) 80 (58.4) 0.42 p 148 (37.8) 244 (62.2) handling neurologic conditions v 44 (32.1) 93 (67.9) 0.46 p 113 (28.8) 279 (71.2) handling animal faecal samples v 68 (49.6) 69 (50.4) 0.31 p 175 (44.6) 217 (55.3) handling animal urine samples v 77 (56.2) 60 (43.8) 0.005 p 167 (57.4) 225 (22.6) handling animal blood samples v 118 (86.1) 19 (13.9) < 0.001 p 189 (48.2) 203 (51.8) handling placenta and fetal discharges v 126 (92.0) 11 (8.0) < 0.001 p 139 (35.5) 253 (64.5) performing post mortems v 111 (81.0) 26 (19.0) < 0.001 p 159 (40.6) 233 (59.4) disease outbreaks investigations v 88 (64.2) 49 (35.8) < 0.001 p 121 (30.9) 271 (69.1) v = veterinarians; p = para-veterinarians. minimal ppe (personal protective equipment) are coverall clothing, hand gloves and boots. table vi. animal health professionals’ demographic and specialization characteristics associated with preventive practices against zoonotic diseases risks in north‑central nigeria: 2014‑2015. factor poor practice 319 (60.3 %) satisfactory practice 210 (39.7 %) odds ratio 95% ci p-value age 20-29 65 (82.3) 14 (17.7) 1.00 30-39 138 (64.8) 75 (35.2) 2.52 1.33, 4.80 0.003 40-49 75 (61.5) 47 (38.5) 2.91 1.47, 5.76 0.001 50-59 32 (34.0) 62 (66.0) 8.99 4.39, 18.44 <0.001 60-69 9 (42.9) 12 (57.1) 6.19 2.19, 17.50 0.001 specialization small animal practice 15 (42.9) 20 (57.1) 1.00 large animal practice 70 (68.0) 33 (32.0) 0.35 0.16, 0.77 0.01 poultry practice 54 (63.5) 31 (36.5) 0.43 0.19, 0.96 0.04 general practice 113 (65.3) 60 (34.7) 0.40 0.19, 0.83 0.02 abattoir worker 52 (48.1) 56 (51.9) 0.81 0.37, 1.74 0.59 wildlife practice 15 (60.0) 10 (40.0) 0.50 0.18, 1.42 0.21 70 veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 risk perceptions and preventive practices in nigeria nma bida alhaji et al. avoiding recapping of needles for another use among veterinary practitioners (vaughn et al. 2004). the study found use of minimal ppe during high risk veterinary procedures, like respiratory conditions and handling of fetal discharges and placentas, to be inadequate among veterinarians. minimal ppe is dangerous for protection against emerging and re-emerging zoonotic infections because small droplets or aerosols of body fluids can be released during their handling (pappas et  al. 2005). minimal ppe use is only adequate during examinations of apparently healthy animals and perhaps clinically ill animals presented for routine check-up. veterinarians, due to the nature of training received and daily high risk veterinary procedures, are expected to use complete ppe kits (dowd et  al. 2013). adequate protective practices reduce human exposure to zoonotic pathogens as previously reported (weese 2002, odo et  al. 2015). as earlier stated, there is no official zoonotic infection control guidelines for veterinary practice in nigeria. however, these guidelines are needed for protections against emerging and re-emerging zoonotic pathogens, such as ebola and lassa viruses, which are potentially transmitted through contacts with blood. threats of emerging blood-borne pathogens should be seriously considered by the veterinary profession (bermejo et  al. 2006). it has been reported that awareness and education are significant factors that can influence the use of ppe (dowd et al. 2013). the shortfall in the use of comprehensive ppe by animal health professionals in nigeria, especially in the face of emerging and re-emerging zoonoses, should be adequately addressed through trainings. this could be given a priority at the annual continuing education programmes organized by the veterinary council of nigeria, veterinary schools and colleges of animal health in the country. despite the proportional low levels of knowledge and marginal risks perceptions achieved by the para-veterinarians in this survey, all the age groups still practice significant satisfactory protection behaviours against zoonotic infections. similarly, a significant lower number of professionals who are into large animal, poultry, and general practice specializations practiced satisfactory protective measures than those who were engaged in small animal practice. this is in consonance with the reports of a study in us in which fewer large animal and equine veterinarians were always engaged in protective practices at work than small animal practitioners (wright et  al. 2008). thus, educational initiatives that are tailored toward different specializations adopting adequate preventive measures at work are warranted. the results of this study were subject to limitations. the samples were selected from pools of animal influenza viruses, mycobacterium bovis, salmonella and e. coli, among others (meslin 1997, taylor et  al. 2001). the lack of knowledge can be alleviated by facilitating communications and inter-disciplinary collaborations on research (coulibaly and yameogo 2000). non availability of educational materials on zoonotic diseases in the veterinarians’ specialized practices has been reported to be a cause of poor knowledge on zoonoses (lipton et al. 2008). this study observed a significantly higher proportions of animal health professionals to be having high risk perceptions of zoonotic infections during examination of sick animals or when handling some products. despite the relatively variable levels of knowledge about zoonoses, these professionals still perceived high level of risks of exposure to zoonotic diseases during veterinary procedures. perception of high risks of zoonotic infections was an important driver for adequate use of preventive measures. this logically concurs with the established theories of health behaviour, such as the protection motivation theory, which suggests that perceived risk influences motivation to take protective actions (rogers 1975). our finding on high risk perceptions by respondents was in concordance with a previous report that animal health workers in tanzania perceived significant high level risks of exposure to zoonotic diseases at work (swai et al. 2010). however, a survey of 344 australian veterinarians found about half of them to perceived low risk level of exposure to zoonotic diseases (dowd et al. 2013). a study has shown that veterinarians are particularly at higher risks of exposures to emerging infectious zoonoses than other animal health workers because of their contacts with sick animals on daily basis (jackson and illaroel 2012). the present study has observed significant variable proportions of respondents’ behaviours towards preventive practices against zoonotic infections, especially the use of barriers like ppe, during veterinary procedures. most of the respondents indicated that they often wash their hands before eating at their work places. the proportion of respondents who reported always washing their hands prior to eating at work was lower among para-veterinarians; and barely half of the veterinarians reported engaging in this protective behaviour. promotion of practice policies that require hand washing and separation of eating areas from animal practice areas is required. the study found high proportion (85.4%) of veterinarians and less than half (46.4%) of the para-veterinarians engaged in recapping of needles prior to disposal. in veterinary medicine, practices of reuse of washed and recapped needles and syringes which present a preventable risk for exposures to pathogens are common. sensitization is needed to promote awareness on appropriate preventive practice of 71veterinaria italiana 2019, 55 (1), 63-72. doi: 10.12834/vetit.1048.5574.2 nma bida alhaji et al. risk perceptions and preventive practices in nigeria education on emerging and re-emerging zoonotic diseases among vulnerable professions in nigeria. most of them neither possessed adequate level of knowledge about zoonoses nor applied adequate personal protective equipment at work, which are the most challenging critical gap in the face of zoonotic infections. active methods, such as ongoing staff training, proactive role of educating clients on prevention of zoonotic infections, collaborative education relationships on knowledge and prevention of zoonotic infections between veterinarians, human public health professionals and physicians should be encouraged in nigeria. thus, stronger partnerships of animal health with public health agencies and other health professionals in this endeavour are needed in the spirit of ‘one health’. as emerging zoonoses become increasingly prevalent, it will be imperative for the veterinary profession to design standard protective practice guidelines in nigeria. in doing so, the important role of the veterinary profession as a primary line of defense against the spread of zoonotic diseases will be further highlighted. acknowledgements we thank the valuable contributions of staff of the six states and abuja veterinary directorate offices in the zone for their supports throughout the period of the survey. health professionals by non-probability approach, which may not be representative of these professionals in nigeria. although it is conventional to apply purposive sampling in epidemiological studies, it does affect the external validity of the nature of this study type. however, the distribution of respondents by state in the study zone was similar to the expected distributions across other zones in the country, suggesting that the sample size was reasonably representative of animal health professionals in nigeria. if a selection bias was present, it is expected that the presented results would represent a ‘best case’. 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especially cpv, has given rise to the progressive emergence of cpv‑2 antigenic variants, which include the type 2 (currently available only as vaccinal strain for dogs), 2a, 2b, and 2c (decaro and buonavoglia 2012, decaro et al. 2013a). a recent study found pv infection (pvi) to be the main cause of mortality in dogs according to post  mortem diagnoses in italy (eleni et  al. 2014). typical cases, consisting of puppies and kittens aged from 6 weeks to 6 months, with fever, vomiting, mucoid to haemorrhagic diarrhoea and leukopenia, are generally easy to diagnose, although with a variable prognosis (kruse et al. 2010, schoeman et al. 2013). some useful risk and prognostic factors for predicting and diagnosing pvi have been previously investigated, especially in symptomatic animals (houston et al. 1996, kruse et al. 2010, miranda et al. 2015). however, pv can have extremely variable clinical presentations, ranging from hyperacute emergencies requiring intensive care to cases characterised by the presence of few clinical signs. in the latter case, diagnosis can become challenging especially if some of the typical clinical signs are vague, or not present, or in presence of a vaccination history of the animal that suggests the existence of some level of immunity and immunoprotection (kruse et al. 2010, faz et al. 2016). for these reasons, laboratory support is necessary to reach a correct diagnosis (kruse et  al. 2010). parvoviroses are often characterised by an acute course thus, only direct tests, that are able to detect the etiological agent, are appropriate for diagnosis. several tests with different sensitivities, specificities and prices are available. these include antigenic (often in‑clinic) and 1department of veterinary medicine, university of perugia, via s. costanzo 4, 06126 perugia, italy. 2istituto zooprofilattico sperimentale of umbria and marche, via salvemini 1, 06126 perugia, italy. 3private practitioner, central italy, via s. costanzo 4, 06126 perugia, italy. *corresponding author at: department of veterinary medicine, university of perugia, via s. costanzo 4, 06126 perugia, italy. tel.:+39 0755857720, e‑mail: marialuisa.marenzoni@unipg.it. keywords cat, diagnosis, dog, misdiagnosis, parvovirus infection, pcr. summary parvoviruses (pv) can cause outbreaks with high morbidity and mortality in dogs and cats. even if typical cases exist in puppies and kittens, pv infection (pvi) can have many different clinical presentations, making the laboratory support necessary. the aim of this work was to evaluate retrospectively the frequency of misdiagnoses, particularly missed diagnoses, of pvi in 144 suspected cases (88 clinical cases and 56 necropsies) involving 96  dogs and 48 cats. a nested pcr test was chosen as the gold standard. an index of diagnostic suspicion (ids) for pvi, based on parameters reported upon submittal of the samples, was introduced to classify the initial diagnoses issued by veterinarians. the agreement between the ids of pvi and pcr results was calculated. the effect of species, age and clinical versus necroscopic presentation was evaluated by logistic regression. in 63.6% of the cases, the ids was confirmed by the pcr, whereas in 36.4% there was a missed diagnosis or a diagnosis wrongly attributed to pvi. more accurate results were obtained for dogs, animals aged < 1 year, and necropsies. parvovirus infection should be better investigated in patients with atypical or few clinical signs, in particular in cats and animals over 1 year old. maria luisa marenzoni1*, monica momesso1, maria chiara marchesi1, elisabetta manuali2, silvia pavone2, elisa sgariglia1,2, elena tordo1,3, francesco vescera3, giuseppe de nicola3, valentina stefanetti1 and chiara brachelente1 when the diagnosis of parvovirus in dogs and cats becomes challenging veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 accepted: 08.04.2018 | available on line: 31.12.2020 68 veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 possible misdiagnosis in feline and canine parvovirus infection marenzoni et al. the ids of pvi. this represents the result of the diagnostic evaluation issued by the veterinarian (clinician or pathologist), based on clinical signs, clinicopathological parameters, patient history and/ or gross examination. the cases were classified into weak or strong ids of pvi. a strong ids was defined when there were at least two of the typical clinical signs or clinicopathological biomarkers of pvi (presence of acute or hyperacute gastrointestinal signs: vomiting; haematemesis; mucoid diarrhea; haemorrhagic diarrhoea; melena; fever; leukopenia or neutropenia) or when there was a history consistent with pvi or typical gross or histological lesions (necrohaemorrhagic gastroenteritis with crypt necrosis and villous blunting; intranuclear basophilic inclusion bodies in the gastrointestinal tract; necrosis and depletion of lymphoid tissues) (decaro and buonavoglia 2012, schoeman et  al. 2013, maxie 2015). in these cases, pvi was generally considered the first, or among the first three diseases in the list of differential diagnoses. a weak ids was defined when the veterinarian considered pvi an unlikely cause of the disease and the criteria of the strong index were not satisfied (only one of the clinical signs mentioned above was present). the specimens with weak ids were generally subjected, at least initially, to other investigations. when possible, especially after a negative pcr result, the referral veterinarian was contacted in order to discuss the case and try to reach a definitive diagnosis. necroscopic examination when available, the dead animals were subjected to a thorough gross examination. tissues were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin and stained with haematoxylin and eosin for histopathological examination. the results of this last examination generally followed those of the biomolecular test. sampling specimens consisting of rectal swabs (rs) from live animals or tissues from necropsies (the gut in case of intestinal lesions, or a pool of organs including gut, liver, spleen and lung when no gross lesions were evident) were collected. rectal swabs were obtained using sterile cotton swabs, kept in tubes with 0.5 ml of phosphate buffered saline (pbs). specimens of each case were processed upon arrival and this limited the risk of contamination between samples. biomolecular investigations two hundred μl of rs or 20 mg of tissues were used biomolecular tests, such as traditional and real‑time polymerase chain reaction (pcr) (desario et  al. 2005, decaro and buonavoglia 2012, decaro et  al. 2013b, proksch et al. 2015, faz et al. 2016). however, these tests are performed only when veterinarians include pvi in the list of differential diagnoses, or when owners are willing to pay for them. this may limit the possibility of performing a correct diagnosis. therefore, considering all these aspects, misdiagnosis is possible. the consequences of a missed diagnosis can include the underestimation of the prevalence of pvi and the dissemination of the virus through a susceptible population in a clinic or animal shelter, resulting in a costly outbreak. on the contrary, a correct diagnosis is a keystone for controlling the spread of the infection and reducing pvi mortality rates. the aim of this work was to evaluate, retrospectively, the frequency of pvi misdiagnoses, particularly missed diagnoses, in 144 suspected cases (88 clinical cases and 56 necropsies) involving 96  dogs and 48 cats. a nested pcr test was chosen as the gold standard. an index of diagnostic suspicion (ids) for pvi, based on parameters reported upon submittal of the samples, was introduced to classify the initial diagnoses issued by veterinarians. a qualitative description of host factors or details of the history of the most misleading cases is given. materials and methods data collection a retrospective study was conducted on cases observed between october 2008 and december 2014. they included at least one pvi clinical sign. collected samples were subjected to biomolecular investigation for pvi. for each case, when possible, the following data were collected: specimen (number and type), identification of the animal, species, age, gender, origin (kennel, ownership, pet shop, or other), veterinary practice requiring the test, list of clinical signs, treatment, history, vaccinal status, date of vaccination (to evaluate possible vaccinal interference with maternal antibodies, in case of recent vaccination in puppies and kittens), laboratory test results (when available) and outcome. the referral clinician for clinical cases was a general practitioner or an internal medicine specialist. the referral veterinarian for necropsies was a pathologist, generally appointed by the clinician. the classification of the diagnostic suspicion of the cases subjected to biomolecular diagnosis of pvi was retrospectively summarised based on 69veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 marenzoni et al. possible misdiagnosis in feline and canine parvovirus infection in the sensitivity of both single and nested pcr protocols in the biological samples. pcr products of the expected size, originating from four samples only at the nested step, were purified using an appropriate extraction kit (qiaquick pcr purification kit, qiagen) and directly sequenced on both strands with the same primers previously described, using a dna analyser (abi 3730, applied biosystems) capillary sequencer (biofab research srl). the sequences were assembled and aligned using bioedit (2009). the sequence similarity was checked against sequences deposited in genbank using the blast (basic local alignment search tool) software (2009) to confirm the specificity of the pcr. further procedures to validate the nested protocol were performed (data not shown). statistical analysis the main diagnostic parameters of the ids compared with pcr results were estimated (world organisation for animal health 2016). cohen’s kappa test was calculated to assess the agreement between strong or weak ids of the referral veterinarian and pcr, which was considered as the gold standard. values greater than 0.8 represented an excellent agreement, values between 0.61‑0.8 represented a substantial agreement, between 0.41‑0.6 a moderate agreement, between 0.21‑0.4 a fair agreement and below 0.20 a slight agreement (landis and koch 1977). a two‑sided p value (p) of ≤  0.05 was considered statistically significant. statistical analyses were performed using statsdirect software, version 2.7.9, and openepi software. logistic regression was used to weight the overall effect of species, age and type of diagnosis (clinical vs post mortem examination) of the case on performing a correct diagnosis. the variables were examined separately for their association with the missed diagnoses (cases with weak ids and positive pcr, considered false negative) or with incorrect pvi diagnoses (cases with strong ids and negative pcr, considered false positive) evaluated as the outcomes. the effect of age was analysed separately, grouping to extract dna using a commercial kit (dneasy tissue kit, qiagen, milan, italy). the concentration and purity of the extracted dna was quantified using a nanodrop spectrophotometer (nanodrop 2000, thermo fisher scientific, milan, italy). a conventional pcr, targeting a fragment of 583 bp of the vp2 capsid protein‑encoding gene of pv, was used to detect pv dna, including both fpv and the variants of cpv (table i, buonavoglia et  al. 2001). this protocol was able to amplify the dna of all the pv strains, including the mboii restriction site, which is able to recognise the mutation at position  426, which characterises the type 2c and some 2a variants of cpv (buonavoglia et al. 2001, demeter et al. 2010). this allowed to recognise the presence of vaccinal interference, in case the dna of vaccinal (variant 2 or 2b) and wild (variants 2a, 2b, and 2c) strains were present simultaneously. moreover, a nested pcr protocol was developed to increase the sensitivity of the test: two internal primers inside the fragment amplified by the previously published primer pair (buonavoglia et al. 2001) were designed using the primer3 software ( h t t p : / / b i o i n fo. u t . e e / p r i m e r 3 ‑ 0 . 4 . 0 / p r i m e r 3 / ) (table i). however, this nested protocol was not able to distinguish pv strains, as it does not amplify the variant‑specific site. an aliquot of 10 μl of rs dna or 100 ng of dna from tissues was tested in duplicate in a pcr assay (microtech, italy). twenty‑five µl of reaction mixture contained 10x buffer, 3 mm mgcl 2 , 200  µm each deoxyribonucleotide triphosphate, 1 µm each primer (sigma‑genosys), 0.5 u taq dna polymerase (microtech, italy), and dna as described above. one  µl of dna from the first test was used for the nested protocol developed in this study. cycling conditions are given in table i. in each set of reactions a positive (cpv cornell strain) and a negative control (negative dna sample), as well as a negative reaction mix control, were included. the dna of a feline cell culture (crandell feline kidney), infected with 100 tcid 50 /100 µl of the cornell strain, was subjected to serial 10‑fold dilutions, ranging from 100 ng to 0.01 fg, to determine the difference table i. pcr test protocol used for the biomolecular detection of parvovirus infection. pcr assay target gene primer sequence (5'-3') amplification profile product size (bp) pcr type referencesinitial denaturation denaturation annealing extension final extension 555 f vp2 caggaagatatccagaagga 94 °c, 5 min 94 °c, 15 sec 40 cycles 72 °c, 45 sec 72 °c, 5 min 583 single buonavoglia et al. 2001555 r ggtgctagttgatatgtaataaaca 53 °c, 30 sec parvofn vp2 caccagtttatccaaatggtca 94 °c, 5 min 94 °c, 30 sec 35 cycles 72 °c, 45 sec 72 °c, 5 min 211 nested the current studyparvorn cctttccaccaaaaatctgag 60 °c, 30 sec f = forward primer of the first round of pcr; r = reverse primer of the first round of pcr; fn = forward primer of the second round of pcr; rn = reverse primer of the second round of pcr. 70 veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 possible misdiagnosis in feline and canine parvovirus infection marenzoni et al. ≤  15 days (n  =  16), cases where identification of pv strains was not possible due to the risk of vaccinal interference (n = 3), and cases with non‑repeatable pcr results (n  =  3) were excluded (figure 1). thus, 156 cases, 51 from cats (32.7%) and 105 (67.3%) from dogs, were eventually counted in the study. the flow chart of the included cases is reported in figure 1. pcr was requested by 15 different veterinary practices (clinical cases) and 2 diagnostic laboratories (necropsies). due to a special pricing arrangement during the study period, one veterinary practice sent to the laboratory not only the regular pvi suspected cases (n = 32), but also those presenting only a single clinical sign (n = 12). of the 156 cases included in the study, 93 (59.6%) were from clinical cases whereas 63 (40.4%) from post mortem examination. table ii reports the age and species of the animals examined. unfortunately, data were not available for all cases. complete blood or biochemical analyses were not always available, as well vaccinal status was unknown in 59 cases (37.8%, 36 cats and 23 dogs). forty‑three animals (28.8%, it into three classes: animals younger than 6 months, from 6 to 1 year and older that 1 year. the list of the single clinical signs was not statistically evaluated. variables scoring p  ≤  0.20 in an early univariate analysis, or considered to be biologically relevant, were included in the regression model. odds ratios (ors) and corresponding 95% confidence intervals (95% ci) were obtained by means of logistic regression. data were analysed by commercial software r, version 2.8.1 (r, development core team 2007). a value of p ≤ 0.05 was considered statistically significant for the analysis. results data collection between october 2008 and december 2014, 180  samples were tested by pcr for pvi regardless of the ids. however, cases from members of the canidae family (wolf and fox, n  =  2), puppies aged cases of dogs and cats submitted to pcr (n = 180) records eligible for the ids classi�cation (n = 156) cases classi�ed by ids (n = 144) cases excluded for missing data (n = 12) cases excluded for inconclusive results, non-dog and non-cat species, or age under < 15 days of life (n = 24) necropsies (n = 88) clinical cases (n = 56) figure 1. flow chart of the cases analysed. table ii. distribution of positive (+) and negative (-) results of the cases, based on species and age, submitted to biomolecular diagnosis for parvovirus infection. cases pcr results total≤ 6 months > 6 months-1 year > 1 year + + + dogs 63 (41.4%) 8 (5.3%) 13 (8.6%) 2 (1.3%) 10 (6.6%) 8 (5.3%) 104 (68.4%) cats 18 (11.8%) 5 (3.3%) 9 (5.9%) 0 (0%) 13 (8.6%) 3 (1.9%) 48 (31.6%) total 81 (53.2%) 13 (8.6%) 22 (14.5%) 2 (1.3%) 23 (15.2%) 11 (7.2%) 152* (100%) *the age was not recorded in 4 cases (1 positive pcr dog, 1 positive pcr cat, and two negative pcr cats). 71veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 marenzoni et al. possible misdiagnosis in feline and canine parvovirus infection nineteen (82.6%) samples were positive on pcr testing for pv; in 10 cases the presence of pv was associated with comorbidities. they consisted of 2 dogs with a history of chronic diarrhoea, 2 dogs with canine distemper virus coinfection, 1 dog with megaesophagus, 1 cat with respiratory signs, 1  cat with pyometra, 1 cat with an obstructive lower urinary tract disease, 1 cat with a perforated gastric ulcer, and 1 cat with severe cardiac hypertrophy. in other 20 cases which were negative to pv pcr and without a diagnostic suspicion issued by the veterinarian, it was not possible to reach a definitive diagnosis because either the owner did not allow further testing or the animal died and necropsy was not possible. in other 5 pcr negative cases, diagnosis of coccidiosis (3 kittens and 1 puppy) and severe nephropathy (1 dog) was made. in 20 cases (including 11 dogs, 2 of them with weak ids, and 9 cats, 5 of them with weak ids, all confirmed by pcr positive results), case history reported the death within a few days of another animal belonging either to the same owner or to the same shelter. however, there was also a case of a pv pcr positive puppy with weak ids which came from a litter in which the other puppies were healthy. cases with weak ids and positive pv pcr, also included 3 dogs with intestinal stasis and one dog subjected to surgery for acute abdomen. eighteen cases (10 dogs and 8 cats) with weak ids and pcr positive results presented vomiting episodes or non‑haemorrhagic diarrhoea. two kittens, one with fever and another with leukopenia, were 39 dogs and 3 cats) just begun the vaccine protocol while 54 (37.5%, 26 cats and 28 dogs) were not vaccinated. vaccinal interference was considered as probable in 17 cases; however, the presence of cpv 2c or 2a variants was subsequently identified for 14 of them by enzymatic digestion. in 74 (47.4%) cases, it was possible to know the pvi clinical outcome. sixty four animals died while 10 survived. of the 144 cases for which it was possible to express an ids, 98 were characterised by strong and 46 by weak ids (table ii). biomolecular investigations conventional pcr was able to detect 0.1  ng extracted from dna of the cell culture infected with 100 tcid 50 /100 µl of cornell strain, while nested pcr 0.01 ng. the new developed nested pcr also proved to be highly specific as the amplified pcr product sequences showed 100% similarity with fpv or cpv. when the 156 cases were tested by conventional and nested pcr assays, 111 (71.2%) resulted positive to both tests, whereas 17 cases (10.9%) were positive to nested pcr only. ids classification results of the classification of the 144 cases based on ids and pcr are reported in table  iii. as shown, it was possible to reach a definitive diagnosis in 124  out of 144 cases (86.1%). one hundred and table iii. performances of the index of diagnostic suspicion (ids) of parvovirus infection (pvi) and corresponding 95% confidence interval (95% ci) on total cases, and the subgroups of clinical cases and necropsies, subjected to biomolecular diagnosis for pvi. parameter number of cases (%) estimate (95% cl) total cases clinical cases necropsies strong ids and positive pcr 83 (57.6%) 51 (57.9%) 32 (57.1%) weak ids and negative pcr 10 (6.9%) 3 (3.4%) 7 (12.5%) strong ids and negative pcr 15 (10.5%) 13 (14.8%) 2 (3.6%) weak ids and positive pcr 36 (25%) 21 (23.9%) 15 (26.8%) sensitivity 69.75% (60.98 -77.28) 70.83% (59.49 80.06) 68.09% (53.83 79.6) specificity 40% (23.4 59.26) 18.75% (6.6 43.01) 77.78% (45.26 93.68) positive predictive value 84.69% (76.27 90.5) 79.69% (68.29 87.73) 94.12% (80.9198.37) negative predictive value 21.74% (12.26 35.57) 12.5% (4.34 31) 31.82% (16.3652.68) accuracy 64.58% (56.4971.92) 61.36% (50.9270.86) 69.64% (56.6680.1) positive likelihood ratio 1.162 (1.01 1.34) 0.8718 (0.74 1.03) 3.064 (1.12 8.4) negative likelihood ratio 0.76 (0.53 1.07) 1.556(0.08 28.97) 0.4103 (0.33 0.51) diagnostic odds 1.54 (0.63 3.74) 0.56 (0.14 2.17) 7.467 (1.38 40.34) cohen’s kappa 0.07 (-0.080.22) -0.09 (-0.29 0.11) 0.29 (0.07 0.51) sensitivity = the proportion of positives that are correctly identified as such; specificity = the proportion of negatives that are correctly identified as such; positive predictive value = the proportion of true positive tests out of the overall positive tests; negative predictive value = the proportion of negative positive tests out of the overall negative tests; accuracy = the proportion of correctly classified subjects among all the results; positive likelihood ratio (lr+) = sensitivity / (1 − specificity); negative likelihood ratio (lr-) = (1 − sensitivity) / specificity; diagnostic odds = lr+/lr72 veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 possible misdiagnosis in feline and canine parvovirus infection marenzoni et al. and 1 with weak ids) reported the possibility that the pvi originated in their structures, as various animals with different owners, which came to the practice for routine procedures, had pvi clinical signs just few days after their admission to the clinic. statistical analysis overall, in 93 out of 144 cases (64.6%) the referral veterinarian correctly identified pvi cases, as confirmed by pcr, but failed to diagnose pvi in the remaining 36.4% of the cases. as in some cases data could not be obtained, some final subgroups used for statistical analysis were inconsistent. even if the analyses were performed with both, the conventional and the nested pcr assays, and the results obtained from both tests did not significantly differ, data used for statistical analyses were from nested pcr. the agreement between ids and nested pcr assay was moderate. necropsies appeared to have a better diagnostic prediction (69.6%) than clinical cases (61.4%) (table iii). the pv infection was more easily diagnosed in animals aged ≤  6  months (72.9%) compared to animals aged >  6  months‑1  year (69.6%) and in animals over 1 year (36.4%) (table iv). the diagnostic accuracy of pvi in dogs was higher than in cats (71.9% vs. 50%, respectively, table v). the final model of the logistic regression based on cases with missed diagnoses (false negative, n = 36) found that cats had an or = 2.37 (95% ci 0.99‑5.68, p = 0.05), if the model was adjusted with a cutoff of ≤ 6 months of age. on the other hand, the effect of initially suspected to be affected by feline infectious peritonitis. as reported by the veterinarians, when a blood test was performed, cases with weak ids generally had all the parameters within the normal range. interestingly, some clinical cases subjected to necropsy included cases in which sudden death occurred (n = 8, 4 dogs and 4 cats) and cases in which the initial suspicious was poisoning (n = 5, 3 cats and 2 dogs). sudden deaths included 3  cases showing haemorrhagic gastro‑enteritis at gross examination, 4 cases in which the histological examination supported the strong ids, which was assigned after necropsy, and one case without specific clinical signs. in this case, the gross examination revealed an intestinal stasis with haemorrhagic exudate in the gut. the poisoning suspected cases consisted of 3 stray cats from colonies with repeated deaths, one hunting dog and a puppy, which had eaten rotten food. a veterinary practice with which a special price was arranged, wanted to process 44 cases, 32 with strong and 12 with weak ids (with vomiting or non‑haemorrhagic diarrhoea or a low increase in body temperature). thirty one cases with strong ids and all cases with weak ids were positive to pv pcr. the veterinarian of this practice reported that these weak ids cases would not have been sent for laboratory testing without having the incentive of the special price offer that was specifically arranged during this project. three veterinary practices (3 cats, 2 with strong ids table iv. performances of the index of diagnostic suspicion (ids) of parvovirus infection (pvi) and corresponding 95% confidence interval (95% ci) on the subgroups of different ages, subjected to biomolecular diagnosis for pvi. the sum of the subgroups is under 144, as the age was not recorded in 3 cases. parameter number of cases (%) estimate (95% cl) aged ≤ 6 months aged > 6 months-1 year aged > 1 year strong ids and positive pcr 60 (70.5%) 15 (65.2%) 7 (21.2%) weak ids and negative pcr 2 (2.4%) 1 (4.3%) 5 (15.15%) strong ids and negative pcr 9 (10.6%) 1 (4.3%) 5 (15.15%) weak ids and positive pcr 14 (16.5%) 6 (26.2%) 16 (48.5%) sensitivity 81.08% (70.71 88.38) 71.43% (50.04 86.19) 30.43% (15.6 50.87) specificity 18.18% (5.13747.7) 50% (9.453 90.55) 50% (23.66 76.34) positive predictive value 86.96% (77.03 92.98) 93.75% (71.67 98.89) 58.33% (31.95 80.67) negative predictive value 12.5% (3.5 36.02) 14.29% (2.6 51.31) 23.81% (10.63 45.09) accuracy 72.94% (62.66 81.24) 69.57% (49.13 84.4) 36.36% (22.19 53.38) positive likelihood ratio 0.99 (0.79 1.24) 1.43 (0.19 10.69) 0.6087 (0.22 1.70) negative likelihood ratio 1.04 (0.01 98.47) 0.57 (0.06 5.62) 1.391 (0.83 2.33) diagnostic odds 0.96 (0.184.9) 2.5 (0.1346.78) 0.4375 (0.1 2.01) cohen’s kappa -0.01 (-0.21 0.2) 0.1006 (-0.210.41) -0.15 (-0.420.12) sensitivity = the proportion of positives that are correctly identified as such; specificity = the proportion of negatives that are correctly identified as such; positive predictive value = the proportion of true positive tests out of the overall positive tests; negative predictive value = the proportion of negative positive tests out of the overall negative tests; accuracy = the proportion of correctly classified subjects among all the results; positive likelihood ratio (lr+) = sensitivity / (1 − specificity); negative likelihood ratio (lr-) = (1 − sensitivity) / specificity; diagnostic odds = lr+/lr73veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 marenzoni et al. possible misdiagnosis in feline and canine parvovirus infection we can't exclude that the sampling method used to enrol cases could have introduced a bias in this study, as a specific subpopulation of cases was selected. considering that all cases submitted had at least one clinical sign and no healthy animals were analysed, the assay characteristics should be cautiously evaluated. logistic regression was used to weight all these aspects. in this study, pvi diagnoses was more accurate in dogs and young animals or following necroscopy examination. necropsies had better diagnostic prediction than clinical cases, probably because of the increased diagnostic sensitivity and specificity due to findings of gross and histological examinations. however, cases subjected to necropsy generally were those atypical. they didn't have a pvi clinical suspicion and required gross and histological examination for a definitive diagnosis. conversely, pvi with typical clinical signs is normally diagnosed by using lower expensive tests. in this study, many necropsies with strong ids were not initially considered as pvi because they were hyperacute or atypical cases of pvi. accordingly, the correct diagnosis of pvi could also be overestimated in this study. pvi diagnosis was more accurate in young rather than adult animals, as confirmed also by logistic regression. although animals aged from 6 weeks to 6 months are the typical target of pvi (houston et al. 1996, kruse et al. 2010, miranda et al. 2015), cases of pvi in adult dogs have also been reported (decaro and buonavoglia 2012). in this study, diagnosis was missed in a significant proportion of animals aged > 1 year. this was in line with the results obtained by faz species lost significance (p = 0.08) when the age > 1 year is considered. both dogs and cats over 1  year had a 7‑fold greater probability of a missed diagnosis (or = 7.17, 95% ci 2.53‑20.29, p < 0.0001). the type of presentation (clinical vs. necropsy cases) was left out of the model, since it did not affect it. the logistic regression that referred to incorrect pvi diagnoses (false positive, n = 15) found that only the type of presentation had a limited significance, with clinical presentation having an higher probability (or = 8.92; 95% ci 1.03‑77.2, p = 0.05) to overestimate the infection. discussion this study confirms the results of a previous study (faz et  al. 2016) and highlights the concern that veterinarians often don't suspect parvovirus infection in dogs and cats with atypical presentation. the consequences of this can be catastrophic for kennel facilities, shelters or even veterinary clinics, because pv are highly contagious. in this study, nested pcr was chosen to confirm the diagnostic suspect because of its high sensitivity and specificity. it reduced the rates of false negative results derived from in‑clinic tests in case of low viral load or the presence of antibodies and improved the diagnostic accuracy, in the cases with few clinical signs and better prognosis (desario et  al. 2005, decaro et  al. 2013b, decaro et  al. 2014a, proksch et  al. 2015, faz et  al. 2016). moreover, nested pcr was capable to identify the majority of cases with vaccinal interference, and it has a medium cost between in‑clinic tests and real‑time pcr. however, table v. performances of the index of diagnostic suspicion (ids) of parvovirus infection (pvi) and corresponding 95% confidence interval (95% ci) on subgroups of species, subjected to biomolecular diagnosis for pvi. parameter number of cases (%) estimate (95% cl) dogs cats strong ids and positive pcr 63 (65.7%) 20 (41.6%) weak ids and negative pcr 6 (6.3%) 4 (8.4%) strong ids and negative pcr 9 (9.4%) 6 (12.5%) weak ids and positive pcr 18 (18.6%) 18 (37.5%) sensitivity 77.78% (67.58 85.46) 52.63% (37.26 67.52) specificity 40% (19.82 64.25) 40% (16.82 68.73) positive predictive value 87.5% (77.92 93.28) 76.92% (57.95 88.97) negative predictive value 25% (12 44.9) 18.18% (7.31 38.52) accuracy 71.88% (62.1779.89) 50% (36.39 63.61) positive likelihood ratio 1.296 (1.03 1.63) 0.88 (0.58 1.33) negative likelihood ratio 0.5556 (0.73-7.43) 1.18 (0.51 2.75) diagnostic odds 2.33 (0.73 7.43) 0.74 (0.18 3.1) cohen’s kappa 0.14 (-0.05 0.3) -0.05 (-0.29 0.19) sensitivity = the proportion of positives that are correctly identified as such; specificity = the proportion of negatives that are correctly identified as such; positive predictive value = the proportion of true positive tests out of the overall positive tests; negative predictive value = the proportion of negative positive tests out of the overall negative tests; accuracy = the proportion of correctly classified subjects among all the results; positive likelihood ratio (lr+) = sensitivity / (1 − specificity); negative likelihood ratio (lr-) = (1 − sensitivity) / specificity; diagnostic odds = lr+/lr74 veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 possible misdiagnosis in feline and canine parvovirus infection marenzoni et al. the discrepancy observed between ids and pcr results was the most informative for understanding when veterinarians misdiagnose a case of pvi. for example, according to referred clinical signs, veterinarians often identify pvi based on haemorrhagic diarrhoea, conversely they tend to exclude it from the list of differential diagnoses in case of non‑haemorrhagic diarrhoea. logistic regression confirmed that pvi is generally overestimated by clinicians, mostly because they misdiagnosed bloody diarrhea with pvi. in this study, 4 cases (3  kittens and 1 puppy) with haemorrhagic diarrhoea were initially considered pvi, but later they were found to be coccidiosis. this parasitic infection has already been reported as the cause of diarrhoea in puppies that were recently vaccinated for pv (decaro et al. 2007a). in another case, an adult dog having haemorrhagic diarrhoea was suspected to have pvi, whereas it had a severe nephropathy. on the other hand, other infectious agents, such as canine circovirus, canine coronavirus, canine distemper, canine adenovirus, clostridium spp., salmonella spp. can cause clinical signs similar to pvi including haemorrhagic diarrhoea (decaro et al. 2007b, zappulli et al. 2008, decaro et al. 2014, merck veterinary manual 2016). moreover, pvi was also often excluded by the clinicians in cases in which vomiting was the only clinical sign present, confirming the results of a recent study (faz et al. 2016). a normal blood count was another clinicopathological parameter that made pvi appear less likely to be suspected by the veterinarian, as reported by faz and colleagues (faz et  al. 2016). actually, leukopenia is a prognostic and not a diagnostic factor (scheman et  al. 2013, houston et  al. 1996, kruse et  al. 2010). lastly, comorbidity, when present, can modify clinical presentations, clinicopathological parameters and, thus, complicate the diagnosis. a factor not included in the analysis, that might have influenced the results, was the amount of money the owner wanted to spend for the diagnosis. this could have affected the choice of the test and, consequently, the performance of the diagnosis. for example, the 12 cases with weak ids which were sent by the veterinary practice to the laboratory because of the special economic arrangement, were confirmed as cases of pvi by pcr. it is likely that, under normal conditions, these samples woudn't be sent to the laboratory for testing as they belonged to animals with aspecific or few clinical signs that recovered within a few days. as the identification of these atypical cases is essential to limit the disease spread, costs of the laboratory tests become an important limiting factor not only for diagnosis but also for implementing surveillance and control program in the future. in such a context, any pcr positive case, even in the absence of a consistent and colleagues (faz et al. 2016). on the other hand, even though in a limited number of confirmed cases, young age was also confounding. it was the case of one puppy which was referred as the result of the suspected ingestion of rotten food, a young dog which underwent surgery for acute abdomen with a suspicion of ingestion of a radiolucent foreign body or two kittens, in which the main suspect was feline infectious peritonitis, frequently observed in cats under 16 months (pedersen 2014). anamnestic information was also an important diagnostic bias in this study. the death of several cats sharing the same place, or of a hunting dog was erroneously considered as a result of poisoning. in another case, a weak ids was issued for a pv pcr positive sick puppy, because it was the only sick puppy of a healthy litter. it is likely that different titers of maternal antibodies were passively transmitted from the bitch to the puppies during lactation, giving to each puppy a variable protection (decaro et al. 2005). vaccinal history was reported mostly for the youngest animals and dogs. vaccinal interference was recognised in 14 out of 17 confirmed cases of pvi, whereas in the 3 non‑identified cases, the more expensive real‑time pcr was necessary to solve any doubt (decaro et  al. 2005, decaro et  al. 2006a). vaccinal history was recently reported by faz and colleagues (faz et  al. 2016) as a factor leading veterinarians to rule out pvi from the list of differential diagnoses. veterinarians should instead consider that incomplete vaccine protocol doesn't protect young animals from the risk of developing pv clinical signs. to better estimate the protection level against pv in a vaccinated animal, the clinician should know how long ago the animal was vaccinated, which protocol was used, and also whether the animal had completed the protocol as indicated in the manufacturer's guidelines (day et al. 2016). moreover, our results also confirmed that the number of cats vaccinated against pv is lower than the number of dogs (diez et  al. 2015), suggesting that this species could be more susceptible to viral infection. dogs had better diagnostic results than cats that had a significant number of missed diagnosis. the reason could be related to the fact that, generally, in dogs pvi typical clinical signs are more frequent and appear at an earlier stage than cats. moreover, cats are often taken to the veterinarian in critical clinical conditions when they frequently develop hypothermia and lose an important sign of pvi, which is fever. dehydration can also be more severe in cats, as well as in small dogs. this also makes it difficult to collect blood to investigate clinicopathological parameters, further reducing the possibility of reaching a correct diagnosis. 75veterinaria italiana 2020, 56 (2), 67‑76. doi: 10.12834/vetit.1415.7682.1 marenzoni et al. possible misdiagnosis in feline and canine parvovirus infection or non‑haemorrhagic diarrhoea should also be systematically tested for pvi. some cases subjected to necropsy are probably hyperacute or atypical cases of pvi that are originally misdiagnosed at clinical presentation. the challenge given by the different clinical manifestation of the disease and overall costs to perform a diagnosis of pvi should be taken into consideration in the future, especially if a surveillance system is to be devised for this infection. acknowledgements the authors acknowledge mr. carlo sanesi and massimo arcangeli for their skilful technical assistance. a special thanks is reserved to dr. mark rishniw for his careful and considerable revision of the manuscript. clinical diagnosis, becomes epidemiologically important: since many pvi 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and have vast diversity of species world‑wide (khalil et  al. 2018, rasul and akhtar 1975, wall and shearer 2008). there are nearly 860  species which have been reported throughout the globe (horak et al. 2003). ticks are classified into four families i.e. ixodidae, argasidae, laelaptidae and nuttalliellidae (anderson and magnarelli, 2008). members of the ixodidae and argasidae are considered the most important vectors of pathogens causing diseases in humans and animals (gosh et al. 2007, omer et al. 2007). ixodidae (hard ticks) contain 650 while the argasidae (soft ticks) includes 150 species (anderson and magnarelli 2008). pakistan alone presents huge diversity of ticks due to its geographical location in subtropical region (rasul and akhtar 1975). in the country, the most commonly distributed genera are rhipicephalus, boophilus, dermacentor, amblyomma, hyalomma, haemaphysalis, argas and ornithodoros (audouin 1826, hoogstraal and varma 1962). hard tick species have been documented to constitute threat for the human and animal life (dantas‑torres 2008, walker 2003) as they act as a carrier of diseases like theileriosis, anaplasmosis, babesiosis and crimean‑congo haemorrhagic fever (cchf) (jabbar et al. 2015, ramzan et al. 2018). checklists are important source of information for any faunal group. it also provides information about its spatial distribution in a particular region or country. no comprehensive check list for tick species of pakistan is available so far, except a combined list of india, pakistan and bangladesh ticks (gosh et  al. 2007) that lacks complete information about tick fauna of the country. therefore, there is a need for an updated and comprehensive checklist of tick species in pakistan to serve as a ready reference 1state key laboratory for biology of plant diseases and insect pests, institute of plant protection, chinese academy of agricultural sciences, beijing 100193, china. 2institute of plant protection, mns‑university of agriculture, multan, pakistan. 3research center for advanced materials science (rcams), king khalid university, p.o. box 9004, abha 61413, saudi arabia. 4unit of bee research and honey production, faculty of science, king khalid university, p.o. box 9004, abha 61413, saudi arabia . 5biology department, faculty of science, king khalid university, p.o. box 9004, abha 61413, saudi arabia. *corresponding author at: institute of plant protection, mns university of agriculture, multan, pakistan. e mail: naeem1130@yahoo.com. keywords argasidae, ixodidae, synonyms, hosts, pakistan. summary in developed and underdeveloped countries, ticks are important vectors transmitting various pathogens that cause diseases of veterinary and public health importance, like babesiosis, theileriosis, crimean‑congo haemorrhagic fever (cchf) and many more. many species of ticks have been reported in scientific literature from pakistan, which need to be listed for ready reference. for this purpose, a checklist of tick species recorded in pakistan is presented here after comprehensive review of the available literature on the subject. overall, nine genera and 53 species of ticks infesting animals in pakistan are presented in this checklist. muhammad ramzan1,2, unsar naeem‑ullah2*, syed haroon masood bokhari2, shafia saba2, khalid ali khan3,4,5 and shafqat saeed2 checklist of the tick (acari: argasidae, ixodidae) species of pakistan veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 accepted: 18.06.2019 | available on line: 31.12.2020 222 tick species in pakistan ramzan et al. veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 authorship and year of publication are written using normal characters. host animals and references are then given (each after one “tab” space). at the very next line, synonyms (where available) including binomial names with authors and year of publication are added. checklist of tick species at pakistan level previous checklist of ticks which has been published in 2007, enlists only 23 species from pakistan (ghosh et  al. 2007). in the present list, 53 species under 9 genera, from the country along with their documented hosts are presented. and a guide to the research community. the aim of this paper is to provide an updated checklist of tick species identified in pakistan. materials and methods the current checklist has been prepared after a comprehensive and thorough review of literature on the subject with the help of google scholar. it is being written in following pages, according to style and format given below. in the checklist, genus names are written in bold while author names and year of publication are not in bold. from very next line, with an interval given by one “tab”, species names are given also in bold, table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references superfamily ixodoidea birula 1894, leach 1815, banks 1894 family argasidae canestrini 1890 genus argas latreille 1795 abdussalami poultry ghosh et al. 2007, iqbal 1971, hoogstraal & mccarthy 1965 persicus poultry abbasi et al. 2017, ghosh et al. 2007, kakarsulemankhel 2011, oken 1818, ramzan et al. 2008, rafique et al. 2015 synonyms rhynchoprion persicus oken 1818 argas mauritianus guérin‑méneville 1844 argas firmatus neumann 1896 argas persicus dissimiles oken 1818 reflexus poultry, fowl kaiser & hoogstraal 1964, ghosh et al. 2007, fabricius 1794, who 1976 synonyms acarus reflexus fabricius 1794 acarus columbarum shaw 1793 acarus marginatus fabricius 1794 rhynchoprion columbae hermann 1804 ixodes espagnol brebisson 1827 ixodes hispanus brebisson 1827 argas reflescus rondelli 1930 vespertilionis poultry latreille 1796, ali 1986, murray 1877, kaiser & hoogstraal 1964, rafique et al. 2015 synonyms argaspipistrellae audoin 1832 genus ornithodoros koch 1844 thokozani mégnin 1882 poultry, donkey razzak & shaikh 1969 family ixodidae murray 1877 genus amblyomma koch 1844 pomposum buffalo, cattle, goat, sheep donitz 1909, rehman et al. 2004 synonyms amblyomma nocens amblyomma superbum amblyomma variegatum nocens amblyomma variegatum pomposum continued 223veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 ramzan et al. tick species in pakistan table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus amblyomma variegatum cattle, buffalo fabricius 1794, fuller 1899, perveen 2011, kakersulemankhel 2011, shah et al. 2015, razzak & shaikh 1969 synonyms acarus variegatus fabricius 1798 amblyomma elegans guérin‑méneville 1844 amblyomma variegatum variegatum amblyomma variegatus fabricius 1798 amblyomma venustum haemalastor elegans guérin‑méneville 1844) hyalomma venustum ixodes elegans guérin‑méneville 1844 ixodes variegates fabricius 1798 genus boophilus curtice 1891 sharifi cattle buffalo kishida 1939, ghosh et al. 2007, kishida & nakamura 1939 synonyms uroboophilus sharifi minning 1934 annulatus sheep, goat, buffalo, cow say 1821, ghosh et al. 2007, rana et al. 2014, kakarsulemankhel 2010, riaz & tasawar 2017, sajid et al. 2007 synonyms ixodes annulatus say 1821 haemaphysalis rosea koch 1844a ixodes indentatus gamgee 1869 ixodes calcaratus birula 1894 boophilus balcanicus minning 1934 boophilus congolensis minning 1934 boophilus palestinensis minning 1934 boophilus schulzei minning 1934 genus dermacentor koch 1844a andersoni cattle, sheep duges 1834, stiles 1908, karim et al. 2017 synonyms dermacentor venustus banks 1908 circumguttatus buffaloes, cattle neumann, 1897, rehman et al. 2004 synonyms amblyocentor circumguttatus dermacentor circumguttatus circumguttatus dermacentor circumguttatus cunhasilvai marginatus dog, cattle, goat, horse, sheep sulzer 1776, ghosh et al. 2007, uzakov 1964 synonyms acarus marginatus sulzer 1776 ixodes marmoratus risso 1826 dermacentor dentipes koch 1844a dermacentor parabolicus koch 1844a dermacentor gynaecoides olenev 1927 dermacentor longicoxalis olenev 1927 dermacentor rotundicoxalis olenev 1931b, olenev 1929a dermacentor lacteolus schulze 1933 dermacentor antrorum reznik 1950 raskimensis horses, deer, cattle shah et al. 2015, pomerantsev 1946 continued 224 veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 tick species in pakistan ramzan et al. table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus dermacentor koch 1844a rhinocerinus buffaloes, cattle denny 1843 synonyms amblyocentor rhinocerinus amblyomma rhinocerinus dermacentor rhinocerinus otis dermacentor rhinocerinus schillingsi dermacentor rhinocerotis arangis dermacentor rhinocerotis permaculatus dermacentor rhinocerotis rhinocerotis de geer 1778 ixodes rhinocerinus genus haemaphysalis koch 1844 aciculifer buffalo, cattle warburton 1913, reznik 1950 synonyms haemaphysalis aciculifer aciculifer bispinosa horse neumann 1897, risso 1826 synonyms haemaphysalis bispinosa bispinosa punctata cattle, sheep, goat, horse hasselquist 1762, hoogstraal & varma 1962, neumann 1897 flava sheep neumann 1897 synonyms haemaphysalis flava armata haemaphysalis flava flava neumann 1897 haemaphysalis watanabei houyi buffalo, cattle nuttall & warburton 1915, kamensky 1928 haemaphysalis calcarata houyi kashmirensis hoogstraal & varma 1962 parmata buffalo, cattle neumann 1905 sulcata sheep canestrini & fanzago 1878, canestrini & fanzago 1978 ixodes viperarum koch 1844b herpetobia sulcata canestrini 1890 haemaphysalis nicollei larrousse 1925 haemaphysalis angorense schulze & schlottke 1927 haemaphysalis cholodkovskyi olenev 1928 haemaphysalis montana kamensky 1928 haemaphysalis sewelli sharif 1928 haemaphysalis montana pospelova‑shtrom 1935a, pospelova‑shtron 1935b haemaphysalis cretica senevet & caminopetros 1936 haemaphysalis beneditoi gil collado 1938 haemaphysalis recta oswald 1941 hyalomma sulcata mamikonyan 1950, airapetyan et al. 1960 haemaphysalis cretica feldman‑muhsam 1952 genus hyalomma koch 1844a anatolicum anatolicum buffalo, sheep, goat, cattle, dog linnaeus 1758, iqbal 1971, khalid et al. 1991, ahmad 1991, durrani 1992, ahmad et al. 2012 synonyms hyalomma depressum schulze 1919 hyalomma cicatricosum schulze & schlottke 1930 continued 225veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 ramzan et al. tick species in pakistan table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus hyalomma koch 1844a anatolicum (anadolu kenesi) cattle, sheep, goat, camel, horse koch 1844b, donitz 1905, ramzan et al. 2020 synonyms hyalomma aegyptium aegyptium brunnipes hyalomma aegyptium excavata hyalomma aegyptium excavatum hyalomma aegyptium mesopotamium hyalomma aegyptium ornatipes hyalomma anatolicum anatolicum hyalomma armeniorum hyalomma depressum hyalomma detritum albipictum ornatipes hyalomma lusitanicum depressum hyalomma marginatum balcanicum brunnipes hyalomma marginatum marginatum brunnipes hyalomma mesopotamium hyalomma pavlovskyi schulze & schlottke 1929 hyalomma pusillum hyalomma pusillum alexandrinum hyalomma pusillum ornatipes hyalomma pusillum pusillum hyalomma savignyi armeniorum hyalomma savignyi exsul hyalomma savignyi mesopotamium hyalomma savignyi pusillum aegyptium cattle linnaeus 1758 synonyms acarus aegyptius linnaeus 1758 acarus testudinis hasselquist1762 cynorhaestes aegyptius linnaeus 1758 hyalomma aegyptium syriacum hyalomma aegyptius linnaeus, 1758 hyalomma affine hyalomma syriacum hyalomma syriacum punctata schulze 1920 hyalomma syriacum punctatum schulze 1920 hyalomma testudinis hasselquist 1762 ixodes aegyptius linnaeus 1758 ixodes testudinis leydig 1855 dromedarii buffalo, cattle, goat, sheep, dog, camel, horse koch 1844b synonyms ixodes trilineatus lucas 1836 ixodes cinctus lucas 1840 hyalomma margaropoides senevet 1922 hyalomma canariense schulze and schlottke 1930 hyalomma persiacum olenev 1931 hyalomma yakimovi olenev 1931 hyalomma delpyi schulze & gossel 1936 continued 226 veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 tick species in pakistan ramzan et al. table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus hyalomma koch 1844a detritum camel schulze 1919, schulze 1920 synonyms hyalomma schulze & gossel 1936 excavatum buffalo koch 1844 synonyms acarus aegyptium linnaeus 1758 hyalomma syriacum koch 1844 ixodes cornuger kolenati 1857 hyalomma affine neumann 1899 hyalomma punctata schulze 1919 hyalomma suriacum oswald 1941 hussaini cattle, buffalo sharif 1928, feldwan‑muhsam 1951 synonyms hyalomma hussaini typica sharif 1928 impeltatum cattle, camel schulze & schlottke 1930 synonyms hyalomma brumpti delpy 1946 hyalomma dromedarii leptosoma hyalomma erythraeum hyalomma fezzanensis hyalomma impeltatum impeltatum hyalomma leptosoma hyalomma savignyi impeltatum hyalomma sinaii isaaci sharif, 1928 cattle, buffalo ghosh et al. 2007 synonyms hyalomma aegyptium isaaci hyalomma dromedarii indosinensis hyalomma marginatum isaaci hyalomma sharifi isaaci kumari sharif, 1928 cattle, buffalo ghosh et al. 2007 marginatum buffalo koch 1844 synonyms hyalomma aegyptium marginatum hyalomma cypriacum hyalomma dentatum, hyalomma marginatum bacuense hyalomma marginatum balcanicum schulze & schlottke 1929 hyalomma marginatum brionicum hyalomma marginatum caspium hyalomma marginatum espanoli hyalomma marginatum hispanum fabricius 1787 hyalomma marginatum marginatum hyalomma marginatum olenevi hyalomma plumbeum nigricum hyalomma rufipes glabratum hyalomma steineri codinai hyalomma transcaucasicum marginatum toranicum koch 1844a, durrani & kamal 2008 continued 227veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 ramzan et al. tick species in pakistan table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus hyalomma koch 1844a rufipes koch 1844 buffalo, cattle, sheep, goats koch 1844a synonyms hyalomma aegyptium impressum rufipes koch 1844a hyalomma aequipunctatum hyalomma impressum rufipes koch 1844a hyalomma marginatum impressum hyalomma marginatum rufipes koch 1844a hyalomma plumbeum impressum hyalomma rufipes rufipes koch 1844 hyalomma savignyi impressa rousselot 1946 schulzei camel olenev 1931 scupense buffalo, cattle, sheep, goat, dog, cat, poultry schulze 1919, sharif 1928 synonyms hyalomma aegyptium ferozedini hyalomma dardanicum hyalomma detritum hyalomma detritum albipictum hyalomma detritum annulatum hyalomma detritum damascenium hyalomma detritum dardanicum hyalomma detritum detritum hyalomma detritum mauritanicum hyalomma detritum perstrigatum hyalomma detritum rubrum hyalomma detritum scupense hyalomma mauritanicum hyalomma mauritanicum annulatum hyalomma scupense detritum hyalomma scupense scupense hyalomma sharifi schulze and schlottke 1929 hyalomma steineri hyalomma steineri enigkianum hyalomma steineri steineri hyalomma uralense hyalomma verae hyalomma volgense turanicum buffaloes, dog, cat, poultry, goat, cattle, sheep pomerantse 1946 synonyms hyalomma aegyptium impressum transiens hyalomma impressum luteipes hyalomma impressum planum hyalomma impressum planum rhinocerotis schulze and schlottke 1929a hyalomma impressum transiens hyalomma lewisi schulze 1936e continued 228 veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 tick species in pakistan ramzan et al. table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus hyalomma koch 1844a turanicum buffaloes, dog, cat, poultry, goat, cattle, sheep pomerantse 1946 synonyms hyalomma planum hyalomma planum rhinocerotis schulze and schlottke 1929a hyalomma rhinocerotis schulze and schlottke 1929a hyalomma savignyi typica rousselot 1946 hyalomma transiens hyalomma zambesianum hyalommina lewisi schulze 1936e genus ixodes latreille 1795 ricinus cattle, sheep linnaeus 1758, lucas 1844 a, b, leydig 1855, iqbal et al. 2014 synonyms acarus ricinoides de geer 1778 ixodesreduvius latreille 1806 acarus caraborum acarus reduvius linnaeus 1758 acarus ricinus crotonus ricinus cynorhaestes hermanni cynorhaestes megathyreus cynohraestes reduvius linnaeus 1758 cynorhaestes ricinus euixodes reduvius linnaeus 1758 euixodes ricinus ixodes areolaris, ixodes bipunctatus ixodes fodiens ixodes fouisseur ixodes fuscus koch 1844b) ixodes lacertae ixodes megathyreus ixodes nigricans ixodes obscurus neumann 1899 ixodes pustularum ixodes reduvius linnaeus 1758 ixodes reticulatus koch 1856 ixodes ricinus onchorhyncha ixodes rufus ixodes sciuri koch 1844c ixodes sulcatus koch 1844a ixodes trabeatus ixodes vulgaris fabricius 1805 phauloixodes rufus rhipicephalus rufus continued 229veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 ramzan et al. tick species in pakistan table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus rhipicephalus koch 1844b appendiculatus cattle, buffalo, dog neumann 1901 synonyms eurhipicephalus appendiculatus arnoldi buffalo, cattle theiler and zumpt 1949 decoloratus buffalo, cattle koch 1867 synonyms boophilus annulatus decoloratus boophilus capensis massey 1908 boophilus decoloratus boophilus florae boophilus scheepersi eurhipicephalus decoloratus margaropus annulatus decoloratus margaropus decoloratus palpoboophilus decoloratus rhipicephalus annulatus decoloratus evertsi buffalo, cattle neumann 1897, neumann 1899, neumann 1996, neumann 1905 synonyms eurhipicephalus evertsi rhipicephalus evertsi albigeniculatus rhipicephalus evertsi evertsi rhipicephalus evertsi mimeticus rhipicephalus mimeticus haemaphysaloides sheep, cattle, goat supino 1897, irshad et al. 2010 synonyms rhipicephalus expeditus rhipicephalus haemaphysaloides expedita rhipicephalus haemaphysaloides expeditus rhipicephalus haemaphysaloides haemaphysaloides rhipicephalus haemaphysaloides niger rhipicephalus haemaphysaloides ruber rhipicephalus ruber kochi buffalo, cattle dönitz 1905, dönitz 1907a synonyms rhipicephalus neavei longus buffalo, cattle razzak and shaikh 1969, neumann 1907 synonyms rhipicephalus capensis longus rhipicephalus confusus santos dias 1956c rhipicephalus falcatus microplus cattle, camel canestrini 1888, farooqi et al. 2017, karim et al. 2017, asim et al. 2009, sultana et al. 2015 synonyms margaropus microplus canestrini 1888 margaropus annulatus microplusrohr margaropus annulatus australis (fuller) boophilus annulatus argentinus boophilus annulatus australis fuller 1899 boophilus annulatus caudatus boophilus annulatus microplus boophilus argentinus, boophilus australis fuller 1899 continued 230 veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 tick species in pakistan ramzan et al. table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus rhipicephalus koch 1844b microplus cattle, camel canestrini 1888, farooqi et al. 2017, karim et al. 2017, asim et al. 2009, sultana et al. 2015 boophilus caudatus, boophilus cyclops boophilus distans, boophilus microplus boophilus microplus annulatus boophilus minningi boophilus sharifi siddiqi & jan 1984a rhipicephalus sharifi siddiqi & jan 1984 haemaphysalis micropla margaropus annulatus argentinus margaropus annulatus australis fuller 1899, fabricicus 1798 margaropus annulatus caudatus margaropus annulatus mexicanus margaropus annulatus microplus margaropus argentinus margaropus australis fuller 1899 margaropus caudatus margaropus micropla margaropus microplus palpoboophilus brachyuris palpoboophilus minningi rhipicephalus annulatus argentinus rhipicephalus annulatus australis fuller 1899 rhipicephalus annulatus caudatus rhipicephalus annulatus microplus rhipicephalus argentinus rhipicephalus caudatus uroboophilus australis fuller 1899 uroboophilus caudatus uroboophilus cyclops uroboophilus distans uroboophilus indicus uroboophilus microplus pravus sheep, goat, pig,camel, dog, donkey dönitz 1910 synonyms rhipicephalus pravus pravus sanguineus buffalo, cattle,goat, camel, dog, horse latreille 1806, ahmad 1991, atif et al. 2012, bashir et al. 2009, durrani & shakoori 2009, hussain & kumar 1985, roy et al. 2018, shah et al. 2015, theiler 1947 synonyms ixodes sanguineus latreille 1806 ixodes linnaei audouin 1826, audouin & milne‑edwards 1832 ixodes plumbeus donitz 1910, duges 1834 ixodes dugesi gervais 1844 rhipicephalus limbatus koch 1844b rhipicephalus linnei koch 1844b continued 231veterinaria italiana 2020, 56 (4), 221‑236. doi: 10.12834/vetit.1721.9077.1 ramzan et al. tick species in pakistan table i. checklist of tick species of pakistan. —cont’d species authors and year of publication host animals references genus rhipicephalus koch 1844b sanguineus buffalo, cattle,goat, camel, dog, horse latreille 1806, ahmad 1991, atif et al. 2012, bashir et al. 2009, durrani & shakoori 2009, hussain & kumar 1985, roy et al. 2018, shah et al. 2015, theiler 1947 synonyms rhipicephalus rutilus koch 1844b rhipicephalussiculus koch 1844b rhipicephalus carinatus frauenfeld 1867, warburton 1910 rhipicephalus rubicundus frauenfeld 1867, warburton 1910 rhipicephalus stigmaticus gerstacker 1873 rhipicephalus bhamensis supino 1897 rhipicephalus brevicollis neumann 1897 rhipicephalusflavus supino 1897 boophilus dugesi donitz 1907 eurhipicephalus sanguineus stephens & christophers 1908 rhipicephalus texanus banks 1908 rhipicephalus breviceps warburton 1910 rhipicephalus dugesi neumann 1911 a, b rhipicephalus macropis schulze 1936 turanicus dog, cattle, sheep pomerantzev 1946, manan et al. 2007 synonyms rhipicephalus secundus feldman‑muhsam 1952 rhipicephalus turamicus uzakov 1964 acknowledgments the authors would like to appreciate support 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(ixodidae). zeitschrift für parasitenkunde, 8, 619‑637. schulze p. 1936. zwei neue arten der gattung hyalomma und die morphologische bedeutung der analbeschilderung der ixodiden. zoologischer anzeiger, 114, 187‑192. senevet g. 1922. les espèces algériennes du genre hyalomma. archives de l’institut pasteur, afrique du nord, 2, 393‑418. senevet g. & caminopetros j. 1936. une nouvelle variété de l’haemaphysalis punctata. archives de l’institut pasteur d’algérie, 14, 24‑29. shah a., khan m., iqbal z., sajid m. & akhtar m. 2006. some epidemiological aspects and vector role of tick infestation on layers in the faisalabad district (pakistan). world's poultry science journal, 62, 145‑157. shah a., shah s.r., rafi m.a., noorrahim m.s. & mitra a. 2015. identification of the prevalent ticks (ixodid) in goats and sheep in peshawar, pakistan. sharif m. 1928. a revision of the indian ixodidae with special reference to the collection in the indian museum: calcutta. 275 centro di referenza nazionale per gli interventi assistiti con gli animali, istituto zooprofilattico sperimentale delle venezie, viale dell'università 10, 35020 legnaro (pd), italy * corresponding author at: centro di referenza nazionale per gli interventi assistiti con gli animali, istituto zooprofilattico sperimentale delle venezie, viale dell'università 10, 35020 legnaro (pd), italy. tel.: +39 049 8084281, e‑mail: lfarina@izsvenezie.it. marta de santis, laura contalbrigo, martina simonato, mirko ruzza, marica toson and luca farina* veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1226.6831.1 accepted: 10.04.2017 | available on line: 31.12.2018 parole chiave interventi assistiti con gli animali, animali in società, terapie complementari, realtà italiane iaa, animali da compagnia keywords animal assisted interventions, animals in society, complementary therapies, italian aai providers, pets. riassunto nel corso degli ultimi cinquant'anni, gli interventi assistiti con gli animali (iaa) hanno avuto una notevole diffusione nei paesi occidentali, attirando l’attenzione della comunità scientifica e del pubblico: un’evoluzione accompagnata dall’incremento del numero di associazioni pertinenti. qual è lo stato attuale degli iaa in italia? quante realtà hanno a che fare con gli iaa? quali sono i professionisti e gli animali coinvolti? i risultati del questionario riportati in questo articolo hanno l'obiettivo di rispondere a queste domande, fotografando la distribuzione e le principali caratteristiche degli iaa nel territorio italiano. secondo i 208 intervistati, il settore ha visto una notevole espansione negli ultimi venti anni. le realtà che si occupano di iaa, nella maggior parte dei casi associazioni o centri specializzati che lavorano soprattutto con cani ed equidi in attività assistite con gli animali (aaa) rivolte a disabili e a bambini, si trovano principalmente nel nord e nel centro del paese. il quadro del settore appare ancora frammentato, in particolare per quanto riguarda l’équipe dei professionisti coinvolti e la loro formazione; tuttavia, la recente messa a punto di linee guida nazionali da parte dello stato, delle regioni e delle province autonome di trento e bolzano ha l'obiettivo di uniformare il campo e definire le migliori pratiche per ciascun tipo di intervento. summary animal assisted interventions (aai) have become increasingly popular in western countries during the last fifty years, attracting a lot of attention both from the general public and the scientific community. in italy, similarly to other countries, this evolution has been accompanied by the diffusion of associations delivering aai. what is the current state of aai in italy? how many realities are dealing with aai? what professionals and animals are involved? the results of the questionnaire here reported have the objective to answer these questions, outlining a snapshot of the distribution and the main features of aai within italian territory. according to the 208 respondents, the sector has seen a remarkable expansion over the last twenty years. aai providers are located mainly in the north and centre of the country, the majority of them are arranged in associations or aai specialized centers and work mainly with dogs and equids in animal assisted activity (aaa) programs addressed to disabled people and children. the picture of the sector still appears fragmented in particular regarding team of professionals involved and their training; nonetheless, the recent set up of national guidelines by the italian authorities has the objective of standardizing the field and defining best practices for each type of intervention. interventi assistiti con gli animali: la realtà italiana animal assisted interventions in practice: mapping italian providers 276 ministers (dpcm 20031) and subsequently, in 2009, it established the national reference center for animal assisted interventions (nrc aai) with the mandate of promoting research into standardized operating protocols, strengthening collaborations between human and veterinary medicine, enhancing knowledge on the applicability of the interventions in given categories of patients, organizing and managing training pathways, collecting data and disseminating information about aai among the international scientific community. moreover, in 2015 an agreement between the italian government, the regional authorities and the autonomous provinces of trento and bolzano was sanctioned, setting up guidelines on aai (italian national guidelines for animal assisted interventions 2015). these guidelines aim at recording and guiding the development of aai sector through a dialogue among the institutions, all the stakeholders and the scientific world, in order to make the most of the resources and the interest which is blooming throughout all these levels. to achieve this goal, these guidelines foresee a) specific aai training for each professional involved in the design and realization of aai (veterinarian, animal handler, etc.); b) the establishment of a regional register of traders and facilities; c) health, welfare and behavioral requirements for the animals involved; d) the evaluation of the results of projects carried out, where possible, by scientifically validated indicators. at present this agreement is being transposed and implemented by every italian region. to our knowledge, there are no other countries in the world having regulated aai at national level. in this framework, since 2013 the nrc aai has conducted an investigation on aai italian providers, through its website, with the aim of supporting the accessibility of data about aai practitioners, aai centers’ location and services to general public. thanks to this initiative, it was possible to collect through a questionnaire some information about each provider, allowing us to outline the current state of aai in italy, the realities dealing with aai, the professionals and animals involved. the main objective of this paper is to outline a snapshot of the distribution and the main features of aai on the italian territory. materials and methods participants the sample comprised 208 aai italian providers who introduction animal assisted interventions (aai), generally defined ‘pet therapy’ or médiation animale in france, have become increasingly popular during the last fifty years in western countries, attracting a lot of attention both from the general public and the scientific community (michalon 2014). currently, aai are often used to reach therapeutic purposes as effective supports during rehabilitation processes, leading to physical, psychological and social benefits (muñoz lasa et al. 2011) or exploited in education, prevention and community efforts (beetz 2013, komorosky and o’neal 2015). since boris levinson’s article ‘the dog as a co‑therapist’ (levinson 1962) was published, the benefits achievable through the human animal relationship have been largely investigated for several categories of patients, as for example children with autism spectrum disorder (borgi et al. 2016, gabriels et  al. 2015, o’haire 2013), elderly patients affected by dementia or psychiatric disorders (bernabei et  al. 2013, majić et  al. 2013, virués‑ortega et  al. 2012), and alcohol/drug addicted inmates (allison and ramaswamy 2016, contalbrigo et  al. 2016, mercer et  al. 2015). therefore, aai are now introduced in many different settings, including schools, nursing homes, hospitals, prisons, daycare centers and social farms (cirulli 2013, julius et  al. 2014), even though a need for more evidence based research still persists (fine and beck 2010). from the social point of view, this strong development of aai is characterized by “a push by enthusiastic advocates rather than a pull by prescribing physicians” as commented by palley et al. (2010) on animal assisted therapy (aat) in human medicine: a sort of bottom‑up dynamic has run over this field, in which the growing interest about the topic among the general public have elicited the necessity to regulate and structure the sector, taking concern of many issues about the involvement of animals in activities related to human health and wellbeing and stressing ethical (italian national committee for bioethics 2005), safety (bert et al. 2016) and economic arguments (clower and neaves 2015). hence, at international level, some associations and organizations have developed and established standards and best practices for aai, as in the case of the white paper of the international association of human‑animal interaction organizations (iahaio 2014), or the animal‑assisted interventions code of practice for the uk, edited by the society for companion animal studies (scas 2013). similarly to other western countries, also italy has recently experienced an increase in the diffusion of associations delivering aai. therefore, in 2003, the italian ministry of health made a first step towards the legitimization of the animals’ role in human emotional life and their therapeutic value through an official decree of the president of the council of animal assisted interventions in italy de santis et al. veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1226.6831.1 1 d.p.c.m. 28 febbraio 2003, recepimento dell'accordo recante disposizioni in materia di benessere degli animali da compagnia e pet‑therapy [transposition of the agreement related to the welfare of pet animals and pet therapy]. off j, 52, 4‑3‑2003. 277 results the results here reported refer to 208 italian aai providers that filled in the questionnaire until 30 june 2016. concerning their geographical distribution, the regions with higher number of providers are: lombardy with 33 realities (16%), veneto with 26 (13%) and piedmont with 22 (11%) (figure 1). respondents were mostly associations (n  =  118) and aai specialized centers (n  =  62) that represent together the 87% of aai providers, while the others are freelance, public health services, care farms and other (figure 2). most of providers have started their activity in aai from 2000 to 2015, with a peak of 74 realities initiated from 2005 to 2010 (figure 3). according to the respondents, 77 (37%) of them have a partnership with local health services, while 115 do not and 16 did not respond to this question. pearson chi‑squared test showed no association (p‑value  =  0.68) between the typology of provider (association, aai center, etc.) and the collaboration with local health services. moreover, within the respondents, 100 declared to be members of national reference associations; when asked for more details about these reference associations, it resulted a high variability, but the most frequently volunteered to complete an on‑line questionnaire. all the data collected have been treated in accordance with the current legislation in order to guarantee the security and privacy. materials a four section questionnaire was developed to collect information about aai italian providers. the first section picked up contact details of the respondents, type of organization and year of start‑up. the second section comprised questions pertaining to their structures: whether or not they have residential animals, what species and the number of animals involved in the activities. the third section was composed by forced‑choice questions which investigated professionals involved, and whether or not they have had specific training in aai. open choice questions were formulated to deepen the type of training followed by each professional. the fourth section comprised questions pertaining projects: typology (animal assisted activity: aaa, animal assisted education: aae, aat ), number of projects within the last two years, clients/patients’ categories, and forced‑choice questions about the affiliation to national reference associations in this field, collaboration with local health authorities and presence of a rate table. procedure data were collected via an online questionnaire between january 2013 and june 2016. a link to the questionnaire was posted in the nrc aai website (http://www.izsvenezie.it/temi/ a l t r i ‑ te m i / i nte r ve nt i ‑ a s s i s t i t i ‑ co n ‑ g l i ‑ a n i m a l i / censimento‑nazionale/): the compilation of the questionnaire gives the opportunity to be displayed in the map on the nrc aai website, which shows aai italian providers and their contacts. the initiative was publicized in 2013 through nrc aai website, and the invitation to participate to the questionnaire was sent through the direct newsletter to all subscribers to the site. moreover, the opportunity to participate to the study has been disclosed during these years in all public contexts in which the nrc aai was present. data analyses all data collected were stored, validated and analyzed using excel and stata 12.1. a descriptive analysis has been performed, calculating frequencies for categorical variables. pearson chi‑squared test has been calculated to evaluate the association between categorical variables. de santis et al. animal assisted interventions in italy veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.1226.6831.1 figure 1. distribution of aai providers throughout italian regions (n = 208 respondents). nick title first author et al. 278 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx horses), and are located in veneto, piedmont, lazio and abruzzo regions. realities that have between 30 and 50 animals, both residential and non‑residential, are 14. according to the respondents, of 201 realities, 93% declared to do aaa projects, 82% aae, 70% aat. seven providers did not specify the typology of aai delivered. we asked the respondents to specify to what categories of participants their interventions are addressed. it resulted that aaa is addressed mostly to people with disability and school aged children, aae to school and developing aged children, aat to people with disability and children in developing age (figure 5). the total amount of projects delivered is 1,165, that can be split off into 544 aaa, 345 aae, 276 aat projects. concerning aat, we divided the projects according to patients’ category and the principal species involved (dogs, horses, donkeys, rabbits and cats). the most common aat interventions result with dogs for disabled people (78 projects) followed by aat with dogs for developing age indicated were csen (http://www.csencinofilia. it/), apnec (http://www.apnec.it/) and siua (http:// www.siua.it/). according to the respondents, dogs are involved most frequently in aai in general, followed by horses and donkeys (figure 4). looking at the providers that declared to have residential animals, 115 realities have animals in their structures (55.29%), 82 have not (39.42%) and 11 did not respond (5.29%). within them, 28 declared to involve also visiting animals. most of residential animals are again dogs (n = 60), horses (n = 58) and donkeys (n = 55). concerning the distribution of the animals involved through italian regions, the number of animals for each region results higher in veneto (n  =  316), piedmont and lazio. aai providers with residential animals have 13 animals on average, while there are only four providers that declared to have more than 50 residential animals (mostly dogs, donkeys and association 57% aai center 30% not compiled 6% freelance 3% other 2% public health services 1%care farms 1% figure 2. percentages per typology of aai providers (multiple choice, n = 208). other: residential educational community for children, social enterprise, nursing home, spinal unit. 12 9 9 36 74 58 1 0 10 20 30 40 50 60 70 80 ≤1 99 0 19 91 -1 99 5 19 96 -2 00 0 20 01 -2 00 5 20 05 -2 01 0 20 10 -2 01 5 20 16 figure 3. number of italian aai providers according to years of institution (classes of 5 years). 134 62 24 61 31 34 0 50 100 150 dog horse cat donkey rabbit other figure 4. frequency per type of animal involved in responding italian aai providers (multiple choice, n = 192). other: guinea pig, chicken, goat, ferret, pig, sheep, bee, duck, chinchilla, gerbil, goose, parrot, turtle, fish. 45 114 91 26 0 50 100 150 elderly disability developing age hospital patients figure 5. number of responding italian realities using aat per category of participants (n = 145, 3 not specified). first author et al. nick title veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx 279 implementation of aai legislation, promulgating regional laws and official documents concerning the field. on the other hand, this distribution could be affected by the geographic proximity of the nrc aai (which is in veneto), therefore providers located in these areas could have known more easily about the opportunity to register themselves. regarding the typology of aai providers, more than half of them (57%) are associations involved in social promotion or amateur sport clubs, often non‑profit; this feature reflects the nature of aai phenomenon in italy, that is often animated by people providing volunteer work. another relevant portion of providers (30%) is classified as aai specialized centers: these are facilities with both residential and non‑residential animals, in which aai take place routinely. in this framework, 37% of providers collaborate with local health services, revealing that aai are considered by the italian national health system as possible steps of rehabilitative processes. the scenario of italian aai providers is further enriched by their attitude towards aai national networks: these are represented by some big italian associations that have been historically dealing with human/animal interaction, e.g. siua, apnocs, lapo, etc., or with dog lover/equestrian sports field (csen, apnec, fise). approximately, half of the respondents refer to them as a clear sign that aai providers need to aggregate and build dynamic networks to exchange experiences and challenges at national level. indeed, only few of them indicate affiliations with international associations such as animal assisted intervention international (aaii), iahaio, pet partners suggesting the predominance of a national perspective in the field. concerning the species involved, dogs result to be the most frequently engaged in aai. of course, in our country, dogs are 48.2% of the total amount of pets living in italian families (6.9 million), exceeded only by cats with 7.5 million (assalco‑zoomark 2016), therefore children (58  projects) and therapeutic horseback riding addressed to disabled people (41 projects), as shown in figure 6. then we listed the professionals working in aai italian realities and, as shown in figure 7, animal handlers (86%), psychologists (76%), animal (dog/ horse) trainers (74%) and veterinarians (72%) are most frequently represented. 35.17% of providers performing aat and 67.06% of providers performing aae declared to make use of a multidisciplinary team composed by all the professionals laid down by the italian national guidelines. notably, 63 providers out of 145 (43%) declared to deliver aat with the support of a specialized medical practitioner. as for aai training, the respondents declared that 91% of animal handlers and trainers, 75% of psychologists and 66% of medical practitioners/ physicians followed a specific training in aai. finally, more than half of the respondents (55%) declared to have a rate table for interventions (n = 196). discussion aai have strongly developed both at italian and international level during the last decades. since 2000, italian aai providers have increased throughout the country, especially in some regions of the north and centre. on the basis of our study, the territories with the highest number of providers are lombardy (16%), veneto (13%) and piedmont (11%), followed by tuscany (9%), lazio (9%) and emilia romagna (7%). this distribution could reflect the great sensitivity of these territories towards aai: their regional authorities have been historically involved in aai development within their communities and they were particularly active in the 37 5 10 7 6 78 41 29 16 16 58 35 25 16 10 20 10 10 5 3 dog (134) horse (62) donkey (61) rabbit (31) cat (24) elderly disability developing age hospital patients figure 6. number of aat projects delivered by italian aai realities (n = 201), according to the animals involved and the categories of participants. in brackets: number of providers working with the species. 80 151 158 132 155 179 0 50 100 150 200 250 sp ec ial ize d do ct or ve te rin ar ian ps yc ho lo gi st pr of es sio na l ed uc at or do g/ ho rse tra in er an im al ha nd ler yes no not answered figure 7. personnel involved in aai in responding italian realities (n = 208). nick title first author et al. 280 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx institutionalized frail elderly patients assigned to a cat assisted therapy program. however, yet, little scientific research has focused on cats in the field of human animal interaction and the results are sometimes conflicting (da silva garcia and martins 2016). on the other hand, rabbits are particularly appreciated for their small size and toy appearance, even if particular attention should be paid to their welfare during interventions (loukaki et al. 2010). the five species mentioned above (dog, horse, donkey, cat, rabbit) are cited in the italian national guidelines and they are at present the only ones admitted to aat and aae in our country, besides specific training courses are mandatory for their handlers. however, according to the respondents, there are other species that are traditionally involved in aai (guinea pigs, chicken, goats, ferrets, etc.): these animals still require particular evaluations by national authorities concerning their safety and welfare before they could become eligible for aat and aae. finally, the fact that more than a half (55.29%) of aai providers declared to hold residential animals alerts to the need for adequate attention to their housing and husbandry, in order to ensure animal health and well‑being, as well as users’ safety. therefore the italian national guidelines set up specific structural and management requirements for aai centers with residential animals, which have to get a medical clearance by local health authorities. concerning the typology of aai, 93% of providers declared to do aaa, 82% aae, 70% aat. according to the italian guidelines, aat should be characterized by a medical prescription and therapeutic objectives, while aae has specific educational goals; both are monitored and assessed by means of precise tools under the guidance of specific professionals. on the other hand, aaa has recreational and socialization objectives: the reduced need for planning and designing due to their relatively simple aims may explain the predominance of aaa programs among the respondents. we also speculate that the number of aat and aae projects could be overestimated, in light of the fact that the classification of the various types of interventions was only recently (2015) introduced by the national guidelines and so it may have not yet become part of the common mentality as a substitute for the more familiar, but too vague expression ‘pet therapy’. the target population of aai project is various, but disability and scholar age are the most cited. according to the respondents, the most frequent aat programs are canine assisted therapy addressed to disables and children, and equine assisted therapy for disabled people. this trend corresponds to the international scientific literature with the prevalent involvement of children with physical and mental disabilities: for example, children with autism they are largely represented, but their role in aai is supported by their symbiotic relationship with humans, which seems to date back to 18,000 years ago (thalmann et  al. 2013). indeed, dogs’ ability to respond to human directions is nowadays exploited in various contexts, including security work, moving livestock, and assisting humans with disabilities (payne et  al. 2015). not by chance, the aforementioned boris levinson’s 1962 article, which is considered to be the first benchmark of pet therapy, is entitled ‘the dog as a co‑therapist’. as internationally, also in italy aai projects and researches involving dogs are widely documented, in particular with children in hospitals (vagnoli et al. 2015, calcaterra et  al. 2015, palestrini et  al. 2016), with adolescents (stefanini et al. 2015) and geriatric patients (berry et al. 2012, mossello et al. 2011). among the most involved species in aai, horses and donkeys are placed in second and third place, respectively, but the difference is very limited. the human‑horse relationship, too, has a long history and horseback riding is getting very popular in therapeutic riding programs during the last years (hausberger et  al. 2008). equine‑assisted interventions include hippotherapy, educational riding and vaulting, sport riding for the disabled, driving and equine‑facilitated psychotherapy. in italy, therapeutic horseback riding has been used in rehabilitation since 1972, but in the last twenty years it has seen a great development at organizational, scientific and formative levels (pasquinelli 2009). as for donkeys, this species has seen a new interest in recent years, due to the development of onotherapy as confirmed in our study: since the 50s, the number of donkeys bred in italy has fallen drastically because of the increasing use of machinery in agriculture and the depopulation of rural areas. in the field of aai, donkeys are particularly valued for their unique characteristics: as stated by rose and colleagues (rose et al. 2011), their size and physical structure, together with the neotenic aspect, make them an unavoidable but not intimidating interlocutor with a physically welcoming acceptance. moreover, in front of a new situation, donkeys seem to be instinctively curious, rather than impulsive or anxious. a study from borioni and colleagues (borioni et  al. 2012) evaluating the efficacy of equestrian rehabilitation and onotherapy on physical and psycho‑social performances of subjects affected by intellectual disability, concluded that there is an improvement in autonomy and social integration for subjects undergoing horse and donkey therapy. onotherapy, thus, is presented as a suitable alternative to equestrian therapy. according to the respondents, cats and rabbits result to be involved less frequently in aai. a study by stasi and colleagues (stasi et  al. 2004) reported an improvement in depressive symptoms and a significant decrease in blood pressure values in first author et al. nick title veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx 281 294 providers, collected through a previous survey conducted by the italian nrc aai in the 2012: a comparison with our data could be useful to give a glance to the spread of aai in italy during the last four years. this study confirms our results showing that lombardy (14%) and veneto (12%) are the regions with the highest number of aai providers. considering the typology, the majority of them are endorsed to be associations, with a small percentage of public health authorities involved. author conclusions underline the heterogeneity of aai italian realities and the necessity for the setting up of guidelines and standardized protocols. this path has been effectively followed during subsequent years, culminating with the aforementioned agreement of 25 march 2015. another survey was conducted in the emilia romagna region (cirulli et  al. 2007). it is difficult to compare our results with that report, because of different sampling and methods, but in accordance with our study, dog resulted to be the most involved species; the professionals involved were educators, veterinarians and psychologists above all, and the patients were mainly children and elderly with disabilities. a research of international literature showed no similar studies in other countries. this study therefore seems to be the first with these characteristics. there are, indeed, some surveys conducted in specific aai contexts: for example, a recent study by schuurmans and colleagues (schuurmans et  al. 2016) investigated aai in dutch nursing homes and found that 76% of nursing homes respondents (n = 165) used aai in one way or another. similarly to our study, the species that is most involved in dutch nursing homes is dog, but horses and donkeys were not considered in that survey due to the evident difficulty to introduce these big animals in that kind of setting. moreover, data of a survey carried out in france in 2011, revealed that 10% of nursing homes (765 out of 7,725 interviewed) have aai carried out with dogs and that 2,408 establishments (31%) have a constant presence of animals within the premises (kohler 2010). other studies were conducted earlier specifically in the field of pet‑assisted psychotherapy (mason and hagan 1999, rice et  al. 1973). as it can be concluded from this comparison, the species, the personnel and the patients/clients involved strongly depend on the context in which aai are delivered, which makes the comparison between different territories – and cultures – very difficult. finally, it is worth mentioning a master thesis by schlote (schlote 2009) that tackles the topic with a thorough survey on animal‑assisted therapy and equine‑assisted therapy/learning in canada. the ‘state of art’ presented for canada in 2009 coincides, for some aspects, with the results of our study: for example, the animal species involved in aai or, more generally, some considerations on this field. in fact, spectrum disorders (davis et  al. 2015, berry et  al. 2013), with or at risk for mental health problems (hoagwood et  al. 2016), hospitalized children (chur‑hansen et  al. 2014, vagnoli et  al. 2015), or in pedagogical/educational programs (bone 2013). as for the professionals involved, aai providers declared to rely mostly on animal handlers (86%), psychologists (76%) and animal (dog/horse) trainers (74%), while the involvement of physicians is still small. even among those who claimed to deliver therapy, less than half declared to collaborate with physicians, whose only 66% followed a specific training for aai. in a survey on italian medical practitioners’ attitude towards aai, the majority of practitioners (85%) stated that they would like to attend training or refresher courses on aai (pinto et al. 2015). these results open the discussion to some considerations on the multidisciplinary team that is required for aai. depending on the delivery model used, professionals can play different roles in the aat/ aae setting. as stated by brooks (brooks 2006) we can distinguish a diamond model and a triangle model: in the diamond model, the medical, psychological or educational professional works in partnership with the animal handler, while in the triangle model he works without the assistance of an animal handler. therefore, the triangle model requires the professional to assume the roles and responsibilities on both sides of the balance. as for aat/aae, the national guidelines are formulated on the basis of the diamond model, so they provide the presence in the setting of both the animal handler and the professional who is referent for the patient/client. this model guarantees more safety on the setting, since there is one person that is responsible for the human and one person responsible for the animal side. we are now witnessing the transition from a field that was often based on forms of voluntary assistance to structured interventions with specific therapeutic or educational targets and the involvement of a multidisciplinary team with specific skills. as shown by our data, only 35.17% of aat and 67.06% of aae providers have a team with the composition set by the italian guidelines. therefore the involvement of medical, educational or psychological professionals should increase. indeed, the diamond approach raises some concerns about the economic sustainability of interventions for the general public and a reflection on the opportunity that they could be partially tap into the health system funds. for the moment, some more structured associations (just over 50%) already have a tariff for their performances. comparison with other studies very little comparable research is available. as for italy, siliprandi (siliprandi 2013) published data from nick title first author et al. 282 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx another limit is given by the period of time for data collection: since we refer to a period of about 3 years, some of the questions and answers could have become obsolete over time. future research could focus on a comparison of the italian situation after the implementation of the national guidelines on aai, since they foresee an official data flow from the providers to nrc aai, through the regional authorities. finally, it would be interesting to compare aai in italy with other national contexts, in order to bring out similarities and differences in the approach to this discipline. conclusions this paper outlines a snapshot on the current status of aai in italy: over the last twenty years, the sector has seen the flourishing of associations and centers that provide aai mainly with dogs and horses and addressed especially to people with disabilities and children. this development was accompanied by a growing attention by researchers and institutions in the effort to standardize the field through national guidelines. one of the biggest challenges is currently represented by the definition of the responsibilities and competences that each professional involved in the multidisciplinary team should have, depending on the type of intervention (therapeutic, educational or recreational), to safeguard the health and welfare of both patients/users and animals involved. the implementation of the guidelines at regional level is likely to lead to a further evolution of the sector, which will be interesting to analyze in detail and compare with other international experiences. schlote concludes in her work that “the field of aai in canada is still in flux. similar to the situation in the united states, the field is fragmented, disjointed, unmonitored, lacking of any clear direction, and facing a number of challenges that many believe to be impeding its evolution into a discipline that is more widely recognized and accepted.” although this fragmentation and the state of constant change are also evident in the italian context, in recent years the stage has been set for a change of approach and a recognition of this discipline. limitations of the study and future research this study has several limitations because it was realized to make available a practical and useful instruments to general public, to find aai centers and associations located in italy and not to collect data for a survey. therefore, the representativeness of the sample could be discussed since the questionnaire is based on voluntary participation through an internet page, it is inevitably limited firstly to individuals with access to the internet, and secondarily to those who heard about the study. on the other hand, it must be underlined that participants are still distributed throughout italy and the initiative was advertised in every public situation where nrc aai participated, just to ensure the widest possible dissemination of information. nonetheless, a more systematic gathering of population demographics would be necessary in order to know if the sample of this study is really representative of the target population. 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poster session presented at the meeting of iahaio, paris. rice s.s., brown l.t. & caldwell h.s. 1973. animals and 47 introduction leishmaniasis is a vector-borne disease caused by flagellated protozoans of the genus leishmania (ready 2010). canine leishmaniasis (canl) due to leishmania infantum is an endemic zoonosis in the mediterranean basin, whose dogs are the main reservoir (miranda et  al. 2008). l.  infantum is transmitted by blood-sucking phlebotomine sand flies (miro et  al. 2012). phlebotomus spp. (syn. lutzomyia spp.) is the primary vector, domestic dogs, rodents, sloths, and opossums play the role of reservoir hosts (pigott et  al. 2014). l.  infantum infection in dogs is characterized by a chronic subclinical infection (esteve et al. 2015). some dogs may present with one or few clinical symptoms and others with multiple clinical manifestations, which are called polysymptomatic dogs (saridomichelakis 2009). clinical signs in dogs include generalized lymphadenomegaly, hepatomegaly, splenomegaly, fever, diarrhea, lethargy, and progressive weight loss (solano-gallego et  al. 2012). furthermore, the majority of dogs shows skin lesions. common biochemical abnormalities include hyperproteinemia with hypergammaglobulinemia and hypoalbuminemia (naucke and lorentz 2012). age seems to be a risk factor that influences the development of the disease. clinical disease has two peaks, one in young dogs, between 2 and 4 -years old, then again in older dogs, older than 7 years of age (sykes and greene 2012). the most useful diagnostic approache for investigation of infection in sick and clinically healthy infected dogs is the detection of specific antibodies by several serological techniques and the demonstration of the dna of the parasite in tissues by molecular techniques (solano-gallego et  al. 2009). currently, the data regarding canl in kosovo is scarce. in this study, we aimed at determining the seroprevalence of canl in stray dogs in the southwestern region of kosovo. materials and methods the study was conducted between april/september 2016 in the shelter ‘straycoco’ located in nagavc, rahovec. some municipalities in kosovo have indeed established shelters for the control of stray 1university of prishtina ‘hasan prishtina’, faculty of agriculture and veterinary, kosovo. 2national institute of public health of kosovo, kosovo. *corresponding author at: university of prishtina ‘hasan prishtina’, faculty of agriculture and veterinary, bulevardi ‘bill clinton’, p.n. 10000 prishtinë, kosovo. e‑mail: kurtesh.sherifi@uni‑pr.edu. keywords canine leishmaniasis, elisa, leishmania infantum, kosovo, stray dogs. summary canine leishmaniasis caused by leishmania infantum is endemic illness in mediterranean countries. the disease represents an important public health issue in kosovo which reported several cases in previous years. in this study we performed a clinical and serological surveillance of canine leishmaniasis in dogs within the southwestern region of kosovo. blood samples were collected from stray dogs in four municipalities including prizren, gjakova, rahovec and deçan. a total of 125 samples were collected between april/september 2016, and antibodies of leishmania infantum were detected by competitive enzyme-linked immunosorbent assay (elisa igg). out of 125  serum samples, 23 dogs (18.4%, 95%ci 12.6-26.1) tested positive for leishmania infantum. three of the 23 positive dogs (13%) showed typical clinical signs of canine leishmaniasis including skin and ocular lesions, decreased appetite, lameness, diarrhea, lethargy and progressive weight loss. the present study confirmed that leishmaniasis is endemic in the southwestern part of kosovo and emphasized the need for establishing a stronger surveillance and control. betim xhekaj1, mentor alishani1, agim rexhepi1, xhevat jakupi2 and kurtesh sherifi1* serological survey of canine leishmaniasis in southwestern region of kosovo veterinaria italiana 2020, 56 (1), 47-50. doi: 10.12834/vetit.1345.7407.5 accepted: 19.02.2018 | available on line: 24.04.2020 48 veterinaria italiana 2020, 56 (1), 47-50. doi: 10.12834/vetit.1345.7407.5 canine leishmaniasis in kosovo xhekaj et al. table i. results of tested samples for canl in stray dogs in four municipalities of southwestern kosovo, 2016. municipality prizren gjakova rahovec deçan total no. of samples 60 17 35 13 125 age 1‑3 years 38 11 20 8 84 3‑5 years 22 6 15 5 41 elisa positive 4 pos. (1‑3 y) 1 pos. (1‑3 y) 2 pos. (1‑3 y) 0 pos. (1‑3 y) 7 pos. (1‑3 y) 9 pos. (3‑5 y) 2 pos. (3‑5 y) 4 pos. (3‑5 y) 1 pos. (3‑5 y) 16 pos. (3‑5 y) elisa negative 34 neg. (1‑3 y) 10 neg. (1‑3 y) 18 neg. (1‑3 y) 8 neg. (1‑3 y) 70 neg. (1‑3 y) 13 neg. (3‑5 y) 4 neg. (3‑5 y) 11 neg. (3‑5 y) 4 neg. (3‑5 y) 32 neg. (3‑5 y) prevalence (%) 21.6% 17.6% 17.1% 7.6% 18.4% 95%ci 13.1‑33.6 6.1‑41.0 8.1‑32.6 1.3‑33.3 12.6‑26.1 until analysis. age estimation and clinical visit of the dogs were performed at the moment of blood sampling, and then during their stay in the shelter. the dogs showing skin and ocular lesions, decreased appetite, diarrhea, lethargy and lameness were identified as suspected cases for canl. a serodiagnosis was conducted by detecting specific dogs through a catch-neuter-release project. blood samples were collected from 125 stray dogs that were captured in four municipalities including prizren, gjakove, rahovec and decan. a total volume of 5  ml blood sample was collected from each dog by cephalic venipuncture and sera were separated at the laboratory and kept at -  20  °c orahovac montenegro peja istog decani 7.6% albania fyrom serbia 25 0 25 50 75 100 km gjakova 17.6% rahoveci 17.1% prizren 21.6% canine leishmaniasis 21.6% prizren 17.6% gjakova 17.1% rahoveci 7.6% decani dragashi klina zubin potok leposavic mitrovicazveçan skenderaj malisheva suhareka gllogovci vushtria podujeva prishtina kamenica gjilani novo bërda vitia kaçaniku shtërpca ferizaj lipljan shtime obilliç fuskë kosove figure 1. prevalence rate of canl in the southwestern region of kosovo. 49veterinaria italiana 2020, 56 (1), 47-50. doi: 10.12834/vetit.1345.7407.5 xhekaj et al. canine leishmaniasis in kosovo origin of study regions are showed in table i and figure 1, respectively. discussion canl is a major zoonotic disease, endemic in tropical and subtropical countries and fatal in humans and dogs. the first surveillance of canl in kosovo was done in 2008, when the percentage of canl was 3.3% positive (lazri et al. 2008). although the limitations of the sample size, the results of this survey confirm the high prevalence of canl in stray dogs of kosovo. the infection rate of canl was higher in municipalities in the southwestern region of kosovo, bordering albania. this area includes prizren (21.6%), gjakova (17.6%), rahovec (17.1%) and deçan (7.6%) (alten 2014, unpublished data). the competent vectors of canl phlebotomus spp. were identified in the different municipalities of kosovo, and found that they are especially high in abundance of phlebotomus (lar.) major s.l. that were found in southwestern part of the country. the present study highlighted the need for increasing control capacity of canl in kosovo. reasonably, further studies are warranted in order to establish the prevalence of the diseases in other areas of kosovo as well as a deep entomological survey. antibodies against l.  infantum using competitive enzyme-linked immunosorbent assay (elisa igg), according to manufacturer instructions (nova tec immundiagnostica gmbh, germany), at the institute of veterinary and agriculture in kosovo. results antibodies of canl were detected in 23/125 stray dogs representing the 18.4% (95% ci 12.6-26.1) of tested samples. the highest number of positive samples were detected in the prizren municipality. after correction of sensitivity (95%) and specificity (96%) of the test, the highest estimated true prevalence was 21.6% (95%ci 13.1-33.6) in prizren (13 out of 60 samples), followed by gjakova 17.6% (95%ci 6.1-41.0) (3 out of 17 samples), rahovec 17.1% (95%ci 8.1-32.6) (6  out of 35 samples) and deçan 7.6% (95%ci 1.3-33.3) (1  out of 13 samples). in three out of 23 positive dogs (13%), clinical signs of canl were observed. suspicion of canl was made through observation of dogs skin and ocular lesions, decreased appetite, lameness, diarrhea, lethargy and progressive weight loss. dogs between 3 and 5 years represented the 70% of the canl positive dogs (16/23). the remaining 30% (7/23 dogs) were dogs with age between 1 to 3 years old. results and 50 veterinaria italiana 2020, 56 (1), 47-50. doi: 10.12834/vetit.1345.7407.5 canine leishmaniasis in kosovo xhekaj et al. esteve l.o., saza s.v., hoseind s. & solano-gallegoa l. 2015. histopathological findings and detection of toll-like receptor 2 in cutaneous lesions of canine leishmaniosis. vet parasitol, 209, 157-163. lazri t., duscher g., edelhofer r., bytyci b., gjino p. & joachim a. 2008. infektionenmitarthropodenübertragenen parasitenbei hundenim kosovo und in albanienunterbesonderer berücksichtigung der leishmanieninfektionen. wien klin wochenschr (in german), 120 (4), 54-58. miranda s., roura x., picado a., ferrer l. & ramis a. 2008. characterization of sex, age, and breed for a population of canine leishmaniosis diseased dogs. res vet scien, 85, 35-38. miró g., checa r., montoya a., hernández l., dado d. & gálvez r. 2012. current situation of leishmania infantum infection in shelter dogs in northern spain. parasites & vectors, 5, 60. naucke t.j. & lorentz s. 2012. first report of venereal and vertical transmission of canine leishmaniosis from naturally infected dogs in germany. parasites & vectors, 5, 67. pigott d.m., bhatt s., golding n., duda k.a., battle k.e., references brady o.j., messina j.p., balard y., bastien p., pratlong f., brownstein j.s., freifeld c.c., mekaru s.r., gething p.w., george d.b., myers m.f., reithinger r. & hay s.i. 2014.global distribution maps of the leishmaniases. elife, 27, 3. ready p.d. 2010. leishmaniasis emergence in europe. euro surveill, 15, 10. saridomichelakis m.n. 2009. advances in the pathogenesis of canine leishmaniosis: epidemiologic and diagnostic implications. vet dermatol, 20, 471-489. solano-gallego l., miró g., koutinas a., cardoso l., pennisi m.g., ferrer l., bourdeau p., oliva g. & baneth g. 2012. leishvet guidelines for the practical management of canine leishmaniosis. parasites & vectors, 4, 86. solano-gallego l., koutinas a., miró g., cardoso l., pennisi m.g., ferrer l., bourdeau p., oliva g. & baneth g. 2009. directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. vet parasitol, 165, 1-18. sykes j.e. & greene c.e. 2012. infectious diseases of the dog and cat, 4th ed. (saunders, eds.). elsevier, 1376 pp. sykes j.e. 2014. canine and feline infectious diseases (saunders, eds.). elsevier, 928 pp. 275 veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 accepted: 23.01.2020 | available on line: 31.12.2021 1college of veterinary sciences and animal husbandry, section microbiology, abdul wali khan university, mardan-khyber pakhtunkhwa pakistan. 2department of animal health, the university of agriculture, peshawar-khyber pakhtunkhwa pakistan. 3center of microbiology and biotechnology, veterinary research institute, peshawar-khyber pakhtunkhwa pakistan. 4college of veterinary medicine, nanjing agricultural university, nanjing, 210095, pr china. 5department of clinical veterinary medicine, college of veterinary medicine, china agricultural university, beijing, p.r. china. *corresponding author at: college of veterinary sciences and animal husbandry, section microbiology, abdul wali khan university, mardan-khyber pakhtunkhwa pakistan. e-mail: sadeeq@awkum.edu.pk. sadeeqe ur rahman1*, nazeer muhammad2, tariq ali3, umer saddique2, shakoor ahmad2, muhammad shafiq4 and bo han5 keywords esbl-producing e. coli, ndm-1, genotypes, peshawar pakistan, poultry, oxa-48, ctxm, poultry meat. summary antimicrobial resistance in food-producing animals has not yet judiciously been reported from pakistan. here, we report on the isolation rate of poultry-associated multidrug resistant extended spectrum β-lactamase (esbl) -producing escherichia coli in peshawar, pakistan. a total of 200 samples, 50 from retail-poultry meat, 50 from sick birds, 50 from the boiler farm-environment, and 50 from human beings working on or exposed to poultry were analyzed for isolation of esbl-producing e. coli, esbl-encoding genes and antimicrobial susceptibility. a total of 81 e. coli isolates [(50.0% phylogroup-a, 33.3% d and 16.7% phylogroup b2)], were recovered, 36 (44.4%) of them were found to be esbl-producers. pcr revealed that bla ctxm was the most prevalent (14/36 = 38.9%) esbl-encoding gene followed by bla shv2 (9/36  =  25%). strikingly, co-occurrence of multiple esbland/or carbapenemase-encoding genes in a single isolate was observed, and combination of bla ctxm + bla shv2 was the most predominant (19.4%) followed by bla ctxm + bla ndm1 + bla oxa-48 (11.1%) and bla ctxm + bla oxa-48 (8.8%). all these esbl producers were found to be multidrug resistant (mdr) and were carrying either integron 1 (48.5%) or 2 (51.5%). finally, 14 of the 36 isolates were also found positive for variable region and insertion sequence common region 1, which was found linked to esbl/carbapenemase encoding genes in 5/14 isolates suggesting its role in dissemination. genotypic characterization of multidrug resistant escherichia coli isolates reveals co-existence of esbland carbapenemaseencoding genes linked to iscr1 2013, ur rahman et al. 2018b). carbapenemases on the other hands are enzymes that can inactivate carbapenem drugs-the most effective and last resort of antibiotics. esbls and carbapenemases are predominantly produced in enterobacteriaceae, mainly in escherichia coli that is utilized as crucial resistance mechanism against cephalosporins and carbapenems (nordmann et  al. 2011, ur  rahman et al. 2018b). furthermore, generally, e. coli encoding for esbl and carabepenemases are resistant to more introduction antimicrobial resistance (amr) is a global emerging threat. particularly, resistance to β-lactams and carbapenems is quite alarming as these drugs are proven safe and efficient, but are losing its effect due to emerging amr. bacteria achieve resistance to β-lactams through production of extended spectrum β-lactamases (esbl) that inactivate antimicrobials including third-, fourth-generation cephalosporins and monobactams (el salabi et  al. 276 veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 genotypic characterization of esbl-producing e. coli in pakistan ur rahman et al. cephalosporinasesthe ambler class c have also been described with slight expanded-spectrum activity against carbapenems (giske et al. 2008). infections due to β-lactamases-producing enterobacteriaceae have serious implications both for public health and infection control strategies. such kind of infections are difficult to treat increasing the risk of infection-related mortality by 5 fold (kumar et al. 2009). finally, the overwhelming ability of the β-lactamases-producing enterobacteriaceae to persist and speedily disseminate with the help of mobile elements in different clinicaland community-settings, and their frequent association with mdr phenotypes presents a huge challenge (el salabi et  al. 2013, ali et  al. 2016, nordmann and poirel 2002). mdr escherichia coli carrying esbl and carbapenemases is increasingly disseminating all over the world. the situation is more alarming in the developing countries partly due to excessive use of antibiotics and absence of surveillance reports. the persistent and excessive use of antibiotics for treatment and as growth promoter in poultry industry in pakistan is more likely providing impetus for the emergence of novel mechanisms of resistance including resistance to β-lactams in enterobacteriaceae. thus, this study was designed with the objectives to determine the occurrence of extended-spectrum β-lactamase (esbl)and carbapenemase-producing e.  coli in broiler and poultry farm environment in peshawar-pakistan and characterize these isolates for the types of esbl genes and susceptibility common antimicrobials. materials and methods ethics proper and prior ethical approval was obtained from the supervisory and technical committee of the university of agriculture peshawar and abdul wali khan university mardan. all work described in here is carried out according to the local institutional and national guidelines and legislations for human and animal samples for research purpose. prior consent was obtained from owners of the poultry farm/bird or human beings along with consent for publication of the resultant data. study area and samples peshawar is the capital city of khyber pakhtunkhwa situated in the northwest of pakistan with 34.0150° n, 71.5805° e coordinates. samples for study were collected in september 2016 to april 2017. a total of 200 samples including 50 samples from healthy chickens, 50 from apparently looking sick chicken, than one classes of antimicrobials and hence are multidrug resistant (mdr), and thus presenting a serious challenge in healthcare settings. in addition to implications of antimicrobials for prophylaxis and as growth promoters, recurrent bacterial infections encourages excessive and constant usage of these antimicrobials thereby resulting emergence and development of resistance against these compounds (ur rahman et al. 2018b). emergence of resistance against carbapenem and cephalosporins is particularly more worrisome as limited options of treatment are left for patients infected with these resistant microbes. three major categories, bla ctxm , bla shv and bla tem , of esbl-encoding genes have been described well so far with bla ctxm as the most prevailing genotype, which has been further divided into five subgroups (bla ctxm-1 , bla ctxm-2 , bla ctxm-8 , bla ctxm-9 , bla ctxm-25 ). the families of each three categories have been expanded so much that almost more than 172 variants have been described for each type of esbl (http://www.lahey.org/studies) (chong et  al. 2018). with obvious geographical variations among the esbl subtypes, bla ctxm-15 genotype has been reported frequently from asia (d'andrea et  al. 2013), and has even frequently been isolated from food-producing-animals (ali et  al. 2016, ali et  al. 2017) as well as from community (abrar et  al. 2017, brigante et  al. 2005). e.  coli carrying bla ctxm-15 often carry many other antibiotic resistance genes and often causing community acquired infections (chong et  al. 2018). it has been shown that esbl carrying e.  coli spread through contaminated food chain or water thereby resulting spread of resistance elements harboring by these bacteria through the help of mobile elements on conjugative plasmids such as integron and insertion sequence common region 1 (iscr1) (ahmed et  al. 2016, ali et  al. 2016, huang et al. 2014). carbapenemases are highly versatile family of β-lactamases recognizing almost all hydrolysable β-lactams, and are resilient against almost all available commercial β-lactamase inhibitors (lolans et  al. 2005, nordmann and poirel 2002). carbapenemase-encoding genes are not naturally found in e.  coli, however, these genes have been acquired largely from other related or environmental bacteria when found associated with mobile genetic elements such as conjugative plasmids (queenan and bush 2007). the first carbapenemase producing e.  coli was reported in 1993 encoded by nmca (naas and nordmann 1994). since then and particularly over the last decade, increasing number of carbapenemase encoding genes have been described in enterobacteriaceae, enlisting them into 3 classes of β-lactamases such as the ambler class a, b, and d β-lactamases (queenan and bush 2007). moreover, rare chromosomally-encoded 277veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 ur rahman et al. genotypic characterization of esbl-producing e. coli in pakistan for e.  coli growth, (ii) macconkey agar media plate with having cefotaxime (2 µg/ml) which was used as selective media for esbl-producing e. coli growth and (iii) macconkey agar media plate with having meropenem (0.5 µg/ml) which was selective for growth of e.  coli producing carbapenemase. the plates were incubated overnight at 37 °c after inoculation. pink color colonies that grew on (ii) and (iii), and presented morphology related with e.  coli were candidates for esbl-producing and carbapenemase-producing phenotypes and further biochemical tests were performed for e. coli identification. e.  coli species were confirmed by using api kits (biomérieux, marcy i’etoile, france). biochemically confirmed e.  coli isolates were also genotyped by specific pcr as described earlier (tantawiwat et  al. 2005). confirmed e.  coli isolates and suspected esbl producers were stored in bhi broth containing 30% glycerol at 80 °c. presumptive esbl-producing e. coli were confirmed phenotypically for esbl production by double-disc synergy test in accordance with recommendations of the clinical and laboratory standards institute (clsi 2014), using antimicrobial discs of cefotaxime (30 μg), cefotaxime plus clavulanic acid (30/10 μg), ceftazidime (30 μg), ceftazidime plus clavulanic acid (30/10 μg) (becton dickson, sparks, md usa) for esbl production. the test was declared positive for esbl production when the zone of inhibition of cefotaxime plus clavulanic acid or ceftazidime plus clavulanic acid was ≥ 5 mm larger than their respective single discs (clsi 2014). for positive control, klebsiella pneumoniae atcc 700603 (esbls-positive strain) was used. esbl and carbapenemase encoding genes identification identification of esbl and carbapenemase encoding genes was achieved by regular pcr. total dna was extracted by using standard boiling method (johnson and woodford 1998) pcr was first performed for bla ctx-m , bla shv , and bla tem of esbl-producing isolates (table i). variants, bla ctx-m-1 and bla ctx-m-9 , of bla ctx-m were also determined through pcr using specific primers as described in table i. all esbl producers were also subjected to pcr for carbapenemase-encoding genes like bla oxa , and bla ndm . the primers, amplicons expected size and references are described in table  i. the polymerase chain reaction was performed as described earlier (ali et  al. 2016, sartor et  al. 2014). amplified pcr products were evaluated by 1% agarose gel electrophoresis. antibiotic susceptibility testing mueller-hinton agar (difcotm) was used to perform 50 from poultry rearing environment within poultry farms and finally 50 samples from human beings dealing with poultry in the farms or purchase points were collected from peshawar. samples from sick broiler chickens were obtained from poultry postmortem section of veterinary research institute, peshawar, where farmers from various cities including nearby peri-urban areas of peshawar city bring sick birds for disease diagnosis or/and treatment. samples from healthy broiler chicken were obtained from live bird market at peshawar city at sales point, while environmental samples were obtained from open shed poultry farms of peshawar district that provides broiler to these sales points. these samples included drinking water (n  =  20), swabs from floor (n  =  10), utensils (n  =  10) and humid walls (n  =  10). fecal swab samples or swab samples from hands, nose and feet or blood samples of human beings (n = 50) dealing with these broiler at different spots such as butchers, live bird sellers, farmers and truck drivers who transport birds from farm to market were included in this study. culturing, identification of escherichia coli, screening and confirmation of esbl production meat samples of healthy broiler chicken at retail market and sick broiler chicken from the postmortem room, were sealed in a sterile plastic bag and transported in icebox to microbiology laboratory of university of agriculture or college of veterinary sciences and animal husbandry, abdul wali khan university and placed at 2-8 °c for maximum of 10 hr until further processed. cotton swab samples from the environment of the broiler farms or humans were transported in transport medium in icebox and were processed immediately for culturing and isolation. human fecal swab samples in the transport medium or a total of 10 ml blood were aseptically obtained from people. meat samples of broiler were processed for isolation of e. coli (doy et al. 2010, paterson et al. 2010). briefly, approximately 25 mg of meat was used from each sample for culturing after mechanically mincing in 10 ml of brain heart infusion (bhi; sigma-aldrich) media followed by enrichment at 37 °c overnight (doy et  al. 2010, paterson et  al. 2010). on the next day, 50-100 µl of this broth was streaked onto macconkey agar for selective growth. in the case of swab samples a sterile cotton swab was streaked directly onto macconkey agar. screening and confirmation of esbl was performed as defined by clinical and laboratory standards institute (clsi) (clsi 2014). the media plates used for each type of sample included (i) macconkey agar plate with no antibiotics which was control 278 veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 genotypic characterization of esbl-producing e. coli in pakistan ur rahman et al. (xdr) if found resistant to all commonly used drugs, multi-drug resistant (mdr)-if found resistant against more than two different kinds of antibiotics tested (magiorakos et al. 2012). determination of phylogroups of e. coli phylogenetic analyses was performed on esbl producing-e.  coli isolates to place them into one of the four phylogenetic groups: phylogeneticgroup a, group b1, group b2 and group d. for this purpose, a triplex pcr assay targeted chua, yjaa genes and the dna fragment tspe4 was used as described previously by clermont and colleagues (clermont et al. 2000). antibiotic susceptibility of esbl carrying isolates. a total of 9 different types of antibiotic discs (becton dickison, sparks, md usa) following standard kirby-bauer disk diffusion method according to recommendations of the clsi (clsi 2014) were tested. both, β-lactam and non-β-lactam antibiotics, were tested such as ampicillin (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), aztreonam (30 μg) and meropenem (10 μg), and tetracycline (30 μg), gentamicin (10 μg), tetracycline (20 μg), meropenem (10 μg) and norfloxacin (10 μg), respectively. e.  coli atcc 25922 (esbl-negative) and klebsiella pneumoniae atcc 700603 (esbl-positive) were used as quality control strains (clsi. 2014). the esbl e. coli were marked declared as extensively-drug resistant table i. primers used in this study. primer name target gene sequence (5’-3’) size -bp ref β -lactamases ctx-m –f bla ctxm atggttaaaaaatcactgcg 873 paauw et al. 2006 ctx-m –r aaaccgttggtgacgat ctx-m9-f bla ctxm tggtgacaaagagagtgcaacg 875 paauw et al. 2006 ctx-m9-r tcacagcccttcggcgat shv –f bla shv ggg tta ttc tta ttt gtc gc 567 chang et al. 2001, yao et al. 2007shv -r ttagcgttgccaagtgctc ctxm1-f1 bla ctxm-1 gct gtt gtt agg aag tgt gc 490 shibata et al. 2006 ctxm1-r cca ttg ccc gag gtg aag tem-f bla tem-1, -52, -71, -104 -105, -138, -151, -152 ata aaa ttc ttg aag acg aaa 1086 yao et al. 2007 tem-r gac agt tac caa tgc tta atc ndm-f bla ndm1 agctgagcaccgcattag 720 poirel et al. 2011 ndm-r cggaatggctcatcacgatc oxa-f bla oxa-48 gcgtggttaaggatgaacac 438 poirel et al. 2011 oxa -r gttgtcatccttgttaggcg integrons and integron variable region inti1-f inti1 cct ccc gca cga tga tc 280-bp dillon et al. 2005 inti1-r tcc acg cat cgt cag gc inti2-f inti2 aaa tct tta acc cgc aaa cgc 439-bp dillon et al. 2005 inti2-r atg tct aac agt cca ttt tta aat tct a inti3-f inti3 agt ggg tgg cga atg agt g 599-bp dillon et al. 2005 inti3-r tgt tct tgt atc ggc agg tg inti1-vr-f inti1 variable region tca tgg ctt gtt atg act gt variable white et al. 2000 nti1-vr-r gta ggg ctt att atg cac gc specific to e. coli ual uida tgg taa tta ccg acg aaa acg gc 147-bp tantawiwat et al. 2005 uar acg cgt ggt tac agt ctt gcg e. coli phylogrouping chua-f chua gac gaa cca acg gtc agg at 279-bp clermont et al. 2000 chua-r tgc cgc cag tac caa aga ca yjaa-f yjaa tga agt gtc agg aga cgc tg 211-bp clermont et al. 2000 yjaa-r atg gag aat gcg ttc ctc aac tspe4c2-f tspe4c2 gag taa tgt cgg ggc att ca 152-bp clermont et al. 2000 tspe4c2-r cgc gcc aac aaa gta tta cg iscr1 iscr1 cgc cca ctc aaa caa acg 469-bp kiiru et al. 2013 gag gct ttg gtg taa ccg f = forward; r = reverse. 279veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 ur rahman et al. genotypic characterization of esbl-producing e. coli in pakistan the farming environment and 24% (12/50) from risk associated to humans. chi-square test indicated a significantly (p < 0.05) higher e.  coli positivity rate from sick chicken as compared to healthy chicken and between the human and environmental samples. screening of esbl phenotypes by double disc synergy test indicated a total of 36 (44.4%) presumptive esbl-producing e. coli isolates, majority (50%; 15/30) of these recovered from diseased chicks followed by healthy chicken (47.6%; 10/21), chicken rearing environment (38.8%; 7/18) and human (16.6%; 2/12) (table ii). distribution and diversity of esbl‑and carbapenemase‑encoding genes all esblproducing isolates were further assessed for the types of esbl-encoding genes and results are presented in table iii. our results indicated that bla ctxm was found to be the most prevalent (38.9%; 14/36) esbl-encoding gene followed by shv2 (25%; 9/36). strikingly, no bla tem and bla ctxm9 genes could be amplified from any of the isolates under study. interestingly, these all isolates were also screened for common carbapenemase-encoding genes which showed that bla ndm1 was found to be the most prevalent gene (16.7%; 6/36) followed by bla oxa-48 (13.9%; 5/36). chi square test indicate significant association (p < 0.05) of bla ctx-m , bla shv2 and bla oxa-48 gene with diseased chicken and healthy chicken while between human being and environment significance association was observed in bla ctx-m-1 and bla shv-2 genes. interestingly, there were co-occurrence of multiple esbl-encoding genes in a single isolate, and combination of bla shv2  + bla ctxm was found as dominant combination with a frequency of 19.4%. of note, bla ndm1 -carrying isolates (n  =  4) were also found positive for bla ctx-m indicating an alarming combination or favorable co-occurrence. four isolates were carrying a combination of three different genes viz., bla ctxm + bla ndm1 + bla oxa-48 , while a single isolate was carrying four different resistance genes (bla ctxm + bla shv2 + bla ndm-1 + bla oxa-48 ) showing co-occurrence of different esbl-encoding genes in a single isolate. frequency of esbl or carbapenemase encoding genes is mentioned in table iv. detection of integrons and variable region for identification of the integrons (class 1-3) in all esbl-producing e.  coli isolates using integron-integrase gene specific primers, inti1, inti2 and inti3 as described (dillon et  al. 2005). the variable regions of class 1 integrons were detected in all inti1 positive esbl-producing e. coli using pcr amplification with primers targeting the flanking regions (white et al. 2000). statistical analysis data was compiled with the help of microsoft excel and analysed through chi-square test at p  <  0.05 probability level using spss 16.0 analysis software. results escherichia coli isolates and esbl phenotypes a total of 81 e. coli isolates were recovered from 200 samples tested. frequency of sample positivity of e. coli was 60% (30/50) from disease broiler chicken, 42% (21/50) from healthy chicken, 36% (18/50) from table ii. occurrence of drug resistant escherichia coli (n = 200). sample nature total no samples sample positivity n esbl producers n mdr n chisq sick chicken 50 30 (60.0%) 15/30 (50.0%) 17/30 (56.7%) 52.00* healthy chicken 50 21 (42.0%) 10/21 (47.6%) 12/21 (57.1%) environment 50 18 (36.0%) 7/18 (38.9%) 9/18 (50.0%) 1.000* human 50 12 (24.0%) 4/12 (33.3%) 5/12 (41.7%) total 200 81 (40.5%) 36/81 (44.4%) 43/81 (53.1%) * indicates statistical significance difference in the diseased chicks vs healthy chicks and environmental vs human sample. table iii. frequency prevalence of extended-spectrum β-lactamase encoding genes (n = 36). name of gene diseased chick healthy chick chi-sq environment human being chi-sq total n ctx-m 7/15 (46.7%) 3/12 (25.0%) 1.44* 2/7 (28.6%) 2/2 (100.0%) 1.24 14/36 (38.9%) ctx-m -1 1/15 (6.7%) 1/12 (8.3%) 1.100 2/7 (28.6%) 1/2 (50.0%) 1.110* 5/36 (13.9%) shv-2 4/15 (26.7%) 2/12 (16.7%) 1.100* 1/7 (14.3%) 2/2 (100.0%) 1.130* 9/36 (25.0%) oxa-48 2/15 (13.3%) 1/12 (8.3%) 78.00* 1/7 (14.3%) 1/2 (50.0%) 68.00 5/36 (13.8%) ndm-1 2/15 (13.3%) 2/12 (16.7%) 1.12 1/7 (14.3%) 1/2 (50.0%) 1.22 6/36 (16.7%) tem 0/15 (0.0%) 0/12 (0.0%) 1.22 0/7 (0.0%) 0/2 (0.0%) 1.32 0/36 (0.0%) * indicates statistical significance difference in the diseased chicks vs healty chicks and environmental vs human sample. 280 veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 genotypic characterization of esbl-producing e. coli in pakistan ur rahman et al. present in a total of 17 isolates (51.5%) followed by integron 1, which was found in 16 (48.5%) isolates. notably, integron 3 could not be amplified from any of the isolates. interestingly, a combination of integron 1 and 2 was also observed in a total of 7  isolates (21.1%) that was found mainly in isolates recovered from sick chicken. only 3 isolates recovered from healthy chicken carrying class 1  integron, while 2 isolates of them were carrying class 2. of the 36 isolates, 14 were also found positive for the presence of variable region with amplicon size ranging from ~  0.25 kb to 2  kb. furthermore, insertion sequence iscr1 was also pcr-amplified from a total of 14 isolates (42.4%). finally, association of the esbl genes with the iscr1 was assessed by primer combination targeting iscr1 and an esbl gene, indicating a total of 7 isolates produced an amplicon of the expected size showing association with the insertion sequence and more likely role in dissemination of these resistance encoding genes. overall, our results showed esbl producing multidrug resistant e. coli carrying diverse esbl and carbapenemase encoding genes with ctx-m in dominance followed by shv-2. discussion over the last couple of years, esbland carbapenemase-producing e.  coli have been frequently reported from food-producing animals presenting a global challenge for public health and food security (ali et al. 2016, ali et al. 2017, ali et al. 2018, seiffert et al. 2013). quite often, these isolates exhibiting mdr phenotypes further complicating its eradication during a disease condition by reducing the number of choices of drugs available against them (ur  rahman et  al. 2018, nordmann and poirel 2002). antimicrobial resistance (amr) is even more serious in developing countries like pakistan where usage of antimicrobials are not strictly regulated, antimicrobial susceptibility patterns esblproducing isolates were subjected to drug susceptibility testing by standard disc diffusion method and results are presented in table v. results indicated that almost all isolates under study were found resistant to first generation cephalosporins (cefalexin), while resistance to third generation cephalosporins, cefotaxime and ceftazidime was noted to be 100% and 79.4%, respectively. of note, 82.4% isolates were resistant against aztreonam, 58.8% against gentamicin and 11.8% against meropenum. moreover, 29.4% were found resistant to norfloxacin and 52.9% were resistant against tetracycline. altogether, all of the isolates were found mdr by showing resistance against at least two classes of antimicrobials tested. phylogenetic analysis a triplex pcr based phylogenetic analysis of the esbl-producing e.  coli was carried out and results are depicted in table vi. our results showed that the commensal phylogroup a was found to be the most prevalent (50.0%), followed by virulent extra-intestinal group d (33.3%) and phylogroup b2 (16.7%), respectively. integron, variable region and insertion sequence iscr1 all esbl-producing isolates were further characterized for the type of integrons, presence of variable regions and insertion sequence common region 1 (iscr1) and results are presented in table vi. overall, our results showed that integron  2 was table iv. distribution of extended-spectrum β-lactamase-encoding genes (n = 36) β-lactamase gene total number of isolates frequency (%) ctxm 14 38.9 ctxm-1 5 13.9 shv2 9 25.0 tem 0 0.0 ndm-1 6 16.7 oxa-48 5 13.9 ctxm + shv2 7 19.4 ctxm + ndm-1 4 11.1 shv2 + oxa48 2 5.6 shv2 + ndm-1 3 8.3 oxa-48 + ndm-1 3 8.3 oxa-48 + ctxm 4 11.1 ctxm + shv2 + ndm-1 2 5.6 ctxm+ ndm+ oxa-48 4 11.1 ctxm + shv2 + oxa-48 + ndm-1 1 2.8 table v. antibiotic sensitivity profile of e. coli isolates (n =36). drug concentration (µg) resistant % intermediates % susceptible % clr 30 94.1 5.9 0.0 am 10 88.2 11.8 0.0 ctx 30 100.0 0.0 0.0 caz 30 79.4 8.8 11.8 azt 30 82.4 11.8 5.9 gm 10 58.8 29.4 11.8 te 20 52.9 14.7 32.4 mpn 10 11.8 14.7 73.5 nor 10 29.4 32.4 38.2 clr = cefalexin; am = ampicillin; ctx = cefotaxime; caz = ceftazidime; azt = aztreonam; gm = gentamicin; te = tetracycline; mpn = meropenem; nor = norfloxacin. 281veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 ur rahman et al. genotypic characterization of esbl-producing e. coli in pakistan provides a lion share of 28% of total meat produced in the country (pakistan 2016). unfortunately, the use of antibiotics in poultry industry is not strictly regulated (mitema 2010), raising concerns about the possible emergence of antimicrobial-resistant microorganisms (naeem et  al. 2006, shahid et  al. 2007). we designed this study to identify the prevalence of esbland carbapenemase-producing e. coli isolated from poultry meat and risk-associated human beings. the results showed a prevalence of 41.9% of esbl particularly in animals, triggering emergence of amr. this phenomenon corroborates with bacterial response of toxin production in response to signals in the surroundings of microbes (ur  rahman and van ulsen 2013, ur  rahman et  al. 2014, van ulsen et  al. 2014, piet et  al. 2016). furthermore, lack of surveillance and monitoring data regarding usage of antimicrobials and emergence of drug resistance further complicates the scenario in developing countries. poultry industry in pakistan is one of the biggest and dynamic industry with over 200 billion pak. rs. (2 bill. $) investment, and this sector table vi. frequency prevalence of extended-spectrum β-lactamase encoding genes (n = 36). id source p.g esbl genotype carbapenemase integron typing vr iscr1 isr1 + esbl r/i phenotypes ctxm ctxm1 shv2 tem oxa-48 ndm-1 int.1 int.2 int.3 1 pd 20 dc d + + + + + + + clr, am,gm,te, nor 2 pd 30 dc d + + + + clr, am,gm, c,te 3 ph 3 hc a + + + + + + clr,gm,te, c, am, nor 4 o hc d + + + clr,te, am 5 pe 32 e a + + + + clr, am 6 pd 21 dc d + + + + clr, am, gm,te, nor 7 pd 46 dc a + + + clr, am,te 8 b hc a clr 9 pd 33 dc d + + + + + clr, am,gm,te 10 ph 4 hc a + + clr, gm, c, 11 pd 32 dc d + + + + + clr, am, c,te 12 pd 28 dc d + + + 13 pe 33 e a + + + clr,te,c 14 pd 25 dc d + + + + clr 15 pe 43 e a + 16 pd 24 dc d + + clr, am 17 pd 34 dc d + + + + clr 18 pd 1 dc a + clr ,te 19 pd 31 hc a + clr 20 pe 31 e b2 + + + + + + + clr,gm,te 21 pd 41 dc a + + clr,te 22 pd 5 dc a + clr, am 23 phh 44 hc a + + + clr, am 24 hl 13 hc a + + + + clr, am, gm,te, c 25 pe 47 e a + + clr 26 pe 5 e d + clr 27 pd 39 dc a + + + + + + clr, mpn,am,gm, nor, c,te 28 ph 45 hc a 29 pd 29 dc a + clr, am 30 phb 3 human b2 + + + + + + clr,te, am,nor,c 31 phb 17 e b2 + + + clr,te, nor,c 32 phh 40 hc b2 + + + clr 33 phb 11 human b2 + + + + + + clr,te,gm, mpn,nor,c 34 phb 37 hc a + clr,te 35 phb 25 human b2 clr,te 36 phb 20 human d clr,te, nor, c dc = diseased chicken; hc = healthy chicken; e = environment; + = present; = absent; int.1 = integron class 1; int.2 = integron class 2; int.3 = integron class 3; vr = variable region; p.g = phylogenetic groups; clr = cephalexin; c = chloramphenicol; gm = gentamicin; te = tetracycline. 282 veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 genotypic characterization of esbl-producing e. coli in pakistan ur rahman et al. first generation cephalosporins and cefotaxime, while almost 50% of isolates are found resistant to other antibiotics tested, except carbapenems. this observed lower susceptibility of these isolates to majority of antibiotics tested is most probably indicative of consistent use of these antibiotics that might provide selective pressure for the resistant bacteria. finally, the majority of these isolates were found to be mdr that belonged to commensal phylogroup a. although resistance determinants were also found to be carried by virulent groups of e.  coli (d/b2), however, acquiring these elements by commensal groups is highly worrisome given the increasing chances of dissemination of these resistant elements. this is suggesting the need for an improvement of the rationale use of antibiotics in poultry production. mobile elements such as integron and insertion sequences are considered crucial for the dissemination of resistance conferring elements and emergence of mdr bacteria. previous reports suggested an overwhelming presence of clinical class  1 integron in esbl-positive e.  coli (dillon et  al. 2005, gu et  al. 2008). integron of class 1 generally carries a variable region comprising a gene cassette arrays encoding different resistance elements. our results of identification of class 1 integron in the majority of isolates along with variable regions (table  v) suggesting its involvement in dissemination of resistance elements. strikingly, we determined that majority of bla ctx-m genes were associated with iscr1. these results corroborate with previous findings of our lab (ali et  al. 2016) and other studies from different countries of the world (kar et al. 2015, kiiru et al. 2013, xu et al. 2015). notably, our findings of the most pre-dominant esbl genotype (ctx-m) and its strong association with the iscr1 indicated that they are more likely mobilized by iscr1 elements. conversely, only one carbapenemase encoding gene (oxa-48) was found linked to iscr1. conclusions our study concludes a high prevalence of esbland carbapenemase-producing e.  coli in the region. taken together, the current high occurrence of multidrug resistant esbl-producing e.  coli carrying clinical class 1 integron and its association with iscr1 is quite worrisome. this calls for an efficient control policy with restriction on the consumption of extended-spectrum cephalosporins for long term use in poultry industry in the area. funding this study was partially supported by natural producing e.  coli. this is a higher incidence rate of esbl producing e.  coli recovered from poultry as compared to previously published report of 32% in peshawar pakistan (ahmad et  al. 2018), 30% in poultry in bangladesh (hasan et al. 2012) and 10.7% in france (girlich et  al. 2007). the higher incidence rate of esbl-producing e. coli is possibly due to over use or consistent usage of antibiotics during poultry production. contrary to our findings, however, even higher incidence rate of esbl-producing e. coli have been reported from other parts of the world such as 93% in spain (egea et al. 2012) and in 81% in the netherlands (blaak et al. 2015). esbl-producing e. coli has been widely reported from human patients that were hospitalized (abrar et  al. 2017, rahman et  al. 2016, ullah et  al. 2017) as well as from community and environment in pakistan (ullah et  al. 2009). however, results of our study cannot be generalized as we analyzed a limited number of samples from a single district, i.e. peshawar of khyber pakhtunkhwa province, pakistan. due to limited resources, we did not extend our study further to investigate the occurrence and trend of esbl-producing e.  coli in different mechanized and open shed poultry farms in the whole district. also, we did not investigate the occurrence of esbland carbapenemase-production in bacteria other than e. coli. our study indicates that bla ctx-m remained the predominant genotype in esbl-producing e.  coli. these results are in agreement with other contemporary findings from pakistan (khan et  al. 2010, mirza et  al. 2006), china (ali et  al. 2016, ali et  al. 2017) and india (upadhyay et  al. 2015), etc. this strongly suggests that bla ctx-m is the most dominant genotype of esbl. this goes along with higher occurrence of shv genotype in our study and as reported by others (habeeb et  al. 2013). more worrisome is the combination of esbl with carbapenemase encoding genes such bla oxa-48 and bla ndm-1 (table iii). co-occurrence of esbl and carbapenemase encoding genes have also been reported earlier from pakistan (ullah et  al. 2017, ur rahman et al. 2018a, younas et al. 2019) from other districts from human patients and poultry. isolation of e.  coli harboring resistance elements encoding for esbl and carbapenemase enzymes from poultry meat and its environment is in fact highly alarming posing threats to public health as these resistance elements can finally be acquired by ham pathogens. combination of bla ctx-m + bla shv + bla oxa-48 and bla ndm-1 is quite challenging. such combinations of different resistance conferring-genetic elements would certainly have an impact on the severity of resistance to different antibiotics and play a role in the development of mdr. this is partly reflected by phenotypic resistance to most of the antibiotics tested in this study (table v). our results show that most of the isolates are found resistant to 283veterinaria italiana 2021, 57 (4), 275-285. doi: 10.12834/vetit.1780.9397.4 ur rahman et al. genotypic characterization of esbl-producing e. coli in pakistan acknowledgments the authors acknowledge the support from technical staff of khyber teaching hospital during collection of samples from targeted human patients. national science foundation of china under the project “international young scientist award” awarded to dr sadeeq ur  rahman (-project no. 31550110200) and relief international under the umbrella of usaid program “fighting zoonosis” awarded to dr sadeeq. the authors specially thanking staff members of khyber teaching hospital for help during collection of human samples. abrar s., vajeeha a., ul-ain n. & riaz s. 2017. distribution of ctx-m group i and group iii β-lactamases produced by escherichia coli and klebsiella pneumoniae in lahore, pakistan. microb pathog, 103, 8-12. doi:org/10.1016/j. micpath.2016.12.004. ahmad k., khattak f., ali a., rahat s., noor s., mahsood n. & somayya r. 2018. carbapenemases and extended spectrum β-lactamase-producing multidrug-resistant escherichia coli isolated from retail chicken in peshawar: first report from pakistan. j food prot, 81, 1339-1345. ahmed h.a., el-hofy f.i., shafik s.m., abdelrahman m.a. & elsaid g.a. 2016. characterization of virulence-associated genes, antimicrobial resistance genes, and class 1 integrons in salmonella enterica serovar typhimurium isolates from chicken meat and humans in egypt. foodborne pathog dis, 13 (6), 281-288. doi:10.1089/fpd.2015.2097. ali t., zhang l., shahid m., zhang s., liu g., gao j. & han b. 2016. esbl-producing escherichia coli from cows suffering mastitis in china contain clinical class 1 integrons with ctx-m linked to iscr1. front microbiol, 7, 1931. doi:10.3389/fmicb.2016.01931. ali t., ur rahman s., zhang l., shahid m., han d., gao j., zhang s., ruegg p.l., saddique u. & han b. 2017. characteristics and genetic diversity of multi-drug resistant extended-spectrum beta-lactamase (esbl)-producing escherichia coli isolated from bovine mastitis. oncotarget, 8 (52), 90144-90163. doi:10.18632/ oncotarget.21496. blaak h., van hoek a.h., hamidjaja r.a., van der plaats r.q., kerkhof-de heer l., de roda husman a.m. & schets f.m. 2015. distribution, numbers, and diversity of esbl-producing e. coli in the poultry farm environment. plos one, 10 (8), e0135402. doi:org/10.1371/journal. pone.0135402. brigante g., luzzaro f., perilli m., lombardi g., colì a., rossolini g.m., amicosante g. & toniolo a. 2005. evolution of ctx-m-type β-lactamases in isolates of escherichia coli infecting hospital and community patients. int j antimicrob agents, 25, 157-162. chong y., shimoda s. & shimono n. 2018. current epidemiology, genetic 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chiave african horse sickness virus, cavalli, culicoides, subclinico, sud-africa. riassunto tra il 2013 e il 2014 è stato condotto uno studio per determinare la prevalenza del virus della peste equina (ahsv) nei culicoides e l'incidenza dell'infezione causata dal virus in 28 cavalli di due stabilimenti equini dell'east rand, nella provincia di gauteng, sud africa. ogni due settimane, in ciascun stabilimento, sono stati catturati i culicoides e raccolti i campioni di sangue dai cavalli; è stata testata la presenza di ahsv rna mediante pcr real-time quantitative reverse transcription. pur trattandosi di infezioni subcliniche, durante il periodo di studio sono stati infettati da ahsv nove cavalli immunizzati. il virus della peste equina è stato anche identificato in un gruppo di culicoides catturati sul campo. le osservazioni ricapitolano dati pubblicati in precedenza per un contesto diverso, in cui sono state condotte ulteriori indagini per determinare quale ruolo svolge l'infezione subclinica nell'epidemiologia delle malattie. virus della peste equina (ahsv) nei cavalli e nei culicoides nell'east rand, nella provincia di gauteng, sud africa keywords african horse sickness virus, culicoides vectors, horses, south africa, subclinical. summary a prospective study was undertaken during 2013 and 2014, to determine the prevalence of african horse sickness virus (ahsv) in culicoides midges and the incidence of infection caused by the virus in 28 vaccinated resident horses on two equine establishments on the east rand, gauteng province, south africa. field caught culicoides midges together with whole blood samples from participating horses were collected every two weeks at each establishment. culicoides midges and blood samples were tested for the presence of ahsv rna by real-time quantitative reverse transcription polymerase chain reaction. nine immunised horses became infected with ahsv during the study period, although infections were subclinical. african horse sickness virus was also identified from a field-collected midge pool. the observations recapitulate previously published data in another setting, where further investigation is warranted to determine what role subclinical infection plays in the diseases epidemiology. veterinaria italiana 2019, 55 (1), 91-94. doi: 10.12834/vetit.1160.6400.3 accepted: 12.03.2018 | available on line: 31.03.2019 1department of veterinary tropical diseases, vector and vector-borne diseases research programme, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110, south africa. 2private veterinary surgeon, south africa. 3equine research centre, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort 0110, south africa. 4college of public health, medical and veterinary sciences, james cook university, australia *corresponding author at: department of veterinary tropical diseases, vector and vector-borne diseases research programme, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110, south africa. e-mail: af.craig83@gmail.com. anthony f. craig1*, glenn c. packer2, alan j. guthrie3 and estelle h. venter1,4 evaluating african horse sickness virus in horses and field-caught culicoides biting midges on the east rand, gauteng province, south africa short communication 92 veterinaria italiana 2019, 55 (1), 91-94. doi: 10.12834/vetit.1160.6400.3 evaluating ahsv in horses and culicoides craig et al. enterprise with a poor infrastructure and dilapidated stabling facilities. preventative measures were haphazard and the equines lived outside, free to roam the farm including low lying water catchment areas providing an adequate breeding habitat for the culicoides. vaccination was not regularly part of the management regime in previous ahs seasons, however, vaccination was administered by the owner prior to the start of the study using the obp polyvalent live attenuated vaccine as was used at establishment a. during the study the 28 horses, 15 from establishment a and 13 from establishment b, were clinically examined for any signs of ahs; rectal temperatures were recorded by digital thermometer and whole blood was drawn by jugular venepuncture in ethylene diamine tetra-acetic acid (edta) vacutainer® tubes from each horse on a two-weekly basis during the 2013-2014 ahs season (december-may). blood was stored at 4  °c until analysed for the presence of ahsv by real-time quantitative reverse transcription polymerase chain reaction (rt-qpcr) as previously published by quan and co-workers (quan et  al. 2010). all blood samples were classified as positive with a 95% limit of detection, when the cycle threshold (ct) value of the samples was < 37.14. a total of nine (9/28) immunised horses showed rnaaemia during the study where all horses were subclinical and showed no change to their habitus and had no rise in rectal temperature, appearing clinically normal on the day of sampling. as the assay does not distinguish between vaccine and wild virus, it was important to consider the date of vaccination in relation to the start of the study. we found that three (3/28) horses showed rnaaemia at the start of the study where we draw the inference that these three could be vaccine reactions and not natural field infection as vaccination was performed 2-3 weeks earlier. the remaining six horses (6/28; one from establishment a and five from establishment b) showing rnaaemia during the course of the study were negative for ahsv rna at the start, which implied these six were naturally infected with wild virus under field conditions during the ahs season. in order to further evaluate the prevalence of ahsv in the immediate area, culicoides midges were collected using the 220 v onderstepoort downdraft suction light traps operating with an 8  w uv-light tube (arc-institute of agricultural engineering, south africa) as previously described by venter and colleagues (venter et  al. 2009). insect collections were stored in 70% ethanol at 4  °c until midge separation and identification, where collections were then differentiated and pooled into groups of 200 according to collection dates, relevant species, gender and parity status. pools contained only african horse sickness virus (ahsv) has an intense and negative impact in equine veterinary science both within south africa and internationally. this non-contagious arthropod borne disease is endemic to sub-saharan africa, with periodic outbreaks having occurred in turkey, cyprus, lebanon, syria, iraq, afghanistan, iran, pakistan, india, egypt, morocco, spain and portugal (lubroth 1988, mellor and hamblin 2004). african horse sickness (ahs) having a mortality rate of up to 90% with the potential for rapid international spread, further illustrates its profound economic importance (guthrie and quan 2009). transmission of the virus occurs via the bite of an infected haematophagous culicoides midge vector, of which the most significant is culicoides (avaritia) imicola kieffer, followed closely by culicoides (avaritia) bolitinos meiswinkel (meiswinkel et al. 2000). throughout most of the endemic range within south africa, the climate is suitable for adult culicoides midges to remain active throughout the year, therefore suggesting a continuous transmission cycle is possible, where the virus may circulate over-winter either in subclinically infected hosts like donkeys and/or zebras as well as in culicoides midges and associated equine populations (venter et  al. 2014). since there is no curative treatment for ahs, vaccination and vector control are performed to prevent and control this disease limiting horse / vector exposure thus reducing viral infection in susceptible horses (guthrie and quan 2009). during 2013 and 2014, samples were collected from two equine establishments (establishment a and b) to determine the prevalence of ahsv in culicoides midges and the incidence of infection caused by the virus in 28 resident horses. the two establishments were located on the east rand, gauteng province, south africa, approximately 20 km from each other, where the east rand is a summer rainfall area with dry winters and occasional frost, resident to an abundance of smallholder equestrian facilities. the selection of each establishment was closely linked to; management, stabling facilities, vector control and vaccination procedures. establishment a was selected as the control establishment, where good record keeping and reporting systems were in place to detect any abnormality, horse’s rectal temperatures were recorded twice daily, insecticides were applied regularly and insect proof accommodation was made priority in an attempt to lower midge numbers in stable blocks; together with eliminating any insect breeding sites through general establishment cleanliness. vaccination protocols followed were those recommended by the vaccine manufacturer onderstepoort biological products soc (ltd) (obp) and facility veterinarians. in contrast, establishment b was an old farming 93veterinaria italiana 2019, 55 (1), 91-94. doi: 10.12834/vetit.1160.6400.3 craig et al. evaluating ahsv in horses and culicoides meal was ingested. following a 13-day incubation period, these midges were then able to transmit the virus. furthermore, the rate of transmission of bluetongue virus (btv) a closely related orbivirus, was evaluated by baylis and colleagues (baylis et al. 2008), with the same species of culicoides as used by mellor and colleagues (mellor et al. 1975), verifying experimentally that a single midge with a viral titre > 102.9 tcid 50  / ml could reliably transmit the btv to susceptible sheep. whether these findings can be extrapolated to ahsv in the african context requires further investigation as no threshold value has yet been determined for a subclinically infected horse to infect a smaller midge species such as c. imicola or c. bolitinos (weyer et al. 2013). we hypothesised that the incidence of ahs would be far higher on the poorly managed horse establishment b. our objective being to compare and contrast disease incidence on a well and poorly managed horse establishment. the management of establishment b however, unexpectedly improved their prophylactic measures by vaccinating. this hindered our objective as vaccinating would prevent virus amplification in the horses, reducing the potential for secondary vector spill over and build-up of virus in midges on establishment b as it would on establishment a (bird et  al. 2011). the incidence of ahs on establishment b was low during the study where mortality rates had been high in the past as attested by the attendant veterinarian. vaccination however could not be the sole constituent when evaluating the reduction in the number of clinical cases evident during the study period. we need to emphasize and evaluate the several confounding factors at play, including the continuous herd exposure to the virus in previous seasons, where it has been shown that natural infection of horses with wild-type virus will induce a broader and stronger cross-reactive immunity than that observed with the live-attenuated vaccine (blackburn and swanepoel 1988). this could incorporate greater herd immunity where no clinical manifestation of disease was evident during the study and therefore this together with vaccination, increased the probability of protection against ahs, presenting with lower disease incidence. although the incidence of infection was low during the study, the observations confirm that on both establishments immunised horses became infected subclinically with ahs. although subclinical cases found are substantially fewer than the clinical cases, they are still a concern in terms of spread of disease (grewar et  al. 2013). how such cases influence viral transmission warrants further investigation, where answering how the virus alters its capacity to infect horses either clinically or subclinically can be used to decrease the dissemination of this disease, all the more with evidence of genome re-assortment and c. imicola and c. bolitinos of which are the most significant vectors. upon analysis of the field-caught culicoides midge collections, the total sum of c. imicola and c.  bolitinos midges trapped at both establishments during the collection period was 11,157 sorted into 91 like pools. in an attempt to understand the seasonal disease prevalence on both participating establishments; pooled midge collections were evaluated for the ahsv rna by using the same rt-qpcr as used for evaluating the blood samples, where samples were considered positive when the ct value was < 40 (guthrie et al. 2013). of the 91 pools collected (55 from establishment a and 36 from establishment b), only 1 parous pool (1/91) from establishment b revealed a positive result by rt-qpcr, for the presence of ahsv rna with an observed field infection prevalence of 2.7%. we cannot however determine if this was due to a single positive midge or multiple midge infections. notably, ahsv could be identified from a field-collected midge pool suggesting that vector competent populations are present. in this study the number of midges collected was significantly lower than those reported in larger studies by scheffer and co-workers (scheffer et  al. 2011). the prevalence of the disease is dependent on a build-up of virus in midge populations which must first reach a critical level, thereafter spill over will occur into associated equine populations (venter et  al. 2006). this could be one likely explanation for the low infection prevalence detected in field-collected midges on each establishment. another likely explanation to the low prevalence of infection detected in the midges collected from each establishment was due to the lower number of ahs cases reported around the study area during the study period. this lower number of viraemic horses will be directly proportional to the lower field infection prevalence found in field-collected midges, subsequently reducing amplification of the virus in vector populations (venter et al. 1997, venter et al. 2006). although midge populations were reduced in number, ahsv was present in the area where subclinical infection in horses was evident producing a detectable viraemia using pcr. weyer and colleagues (weyer et  al. 2013) reported that horses that had been vaccinated, as in the case of establishment a and b, could still become infected with the ahsv subclinically, and speculated that they could be a source of virus for infecting midges. in a study by mellor and colleagues (mellor et  al. 1975), a minimum experimental viral titre of 104-104.5  mid 50  /  0.02 ml blood, was sufficient for laboratory colonies of c. sonorensis to become infected with ahsv when a blood 94 veterinaria italiana 2019, 55 (1), 91-94. doi: 10.12834/vetit.1160.6400.3 evaluating ahsv in horses and culicoides craig et al. grant support the research was supported by the department of agriculture, forestry and fisheries and funded by the equine research centre of the university of pretoria and agriseta. statement of animal rights materials used in these experiments posed no health risk to researchers and no vertebrate animals were harmed. research was conducted in accordance with the institutional animal ethics committee guidelines. project approval number v066-13. the reversion to virulence of live attenuated ahs vaccine viruses (weyer et al. 2017). acknowledgements we thank the equine research centre and department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, for supporting this work and the two equine establishments for making their stabling facilities and horses available. baylis m., meiswinkel r. & venter g.j. 1999. a preliminary attempt to use climate data and satellite imagery to model the abundance and distribution of culicoides imicola (diptera: ceratopogonidae) in southern africa. j south african vet ass, 70, 80-89. bird b.h., maartens l.h., campbell s., erasmus b.j., erickson b.r., dodd k.a. & nichol s.t. 2011. rift valley fever virus vaccine lacking the nss and nsm genes is safe, nonteratogenic, and confers protection from viraemia, pyrexia, and abortion following challenge in adult and pregnant sheep. j virol, 85 (24), 12901-12909. blackburn n.k. & swanepoel r. 1988. observations on antibody levels associated with active and passive immunity to african horse sickness. trop animal health prod, 20, 203-210. grewar j.d., weyer c.t., guthrie a.j., koen p., davey s., quan m., visser d., russouw e. & bührmann g. 2013. the 2011 outbreak of african horse sickness in the african horse sickness controlled area in south africa. journal of the south african veterinary association, 84, 1-7. guthrie a.j. & quan m. 2009. african horse sickness. in infectious diseases of the horse (t.s. mair & r.e. hutchinson, eds). equine veterinary journal, fordham, cambridgeshire, 72-82. guthrie a.j., maclachlan n.j., joone c., lourens c.w., weyer c.t., quan m., monyai m.s. & gardner i.a. 2013. diagnostic accuracy of a duplex real-time reverse transcription quantitative pcr assay for detection of african horse sickness virus. j virol methods, 189, 30-35. lubroth j. 1988. african horse sickness and the epizootic in spain 1987. equine pract, 10, 26-33. meiswinkel r., baylis m. & labuschagne k. 2000. stabling and the protection of horses from culicoides bolitinos (diptera: ceratopogonidae), a recently identified vector of african horse sickness. bull entomol res, 90, 509-515. mellor p.s., boorman j. & jennings m. 1975. the multiplication of african horse-sickness virus in two references species of culicoides (diptera, ceratopogonidae). arch virol, 47, 351-356. mellor p.s. & hamblin c. 2004. african horse sickness. vet res, 35, 445-466. quan m., lourens c.w., maclachlan n.j., gardner i.a. & guthrie a.j. 2010. development and optimisation of a duplex real-time reverse transcription quantitative pcr assay targeting the vp7 and ns2 genes of african horse sickness virus. j virol methods, 167, 45-52. scheffer e.g., venter g.j., joone c., osterrieder n. & guthrie a.j. 2011. use of real-time quantitative reverse transcription polymerase chain reaction for the detection of african horse sickness virus replication in culicoides imicola. onderstepoort j vet res, 78, 344. venter g.j., nevill e. & van der linde t.c. 1997. seasonal abundance and parity of stock-associated culicoides species (diptera: ceratopogonidae) in different climatic regions in southern africa in relation to their viral vector potential. onderstepoort j vet res, 64, 259-271. venter g.j., koekemoer j.j.o. & paweska j.t. 2006. investigations on outbreaks of african horse sickness in the surveillance zone in south africa. rev sci tec, 25 (3), 1097-1109. venter g.j., labuschagne k., hermanides k.g., boikanyo s.n.b., majatladi d.m. & morey l. 2009. comparison of the efficiency of five suction light traps under field conditions in south africa for the collection of culicoides species. vet parasitol, 166 (3), 299-307. venter g.j., labuschagne k., majatladi d.m., boikanyo s.n.b., lourens c., ebersohn k. & venter e.h. 2014. culicoides species abundance and potential over-wintering of african horse sickness virus in the onderstepoort area, gauteng, south africa. j south african vet ass, 85 (1), 1-6. weyer c.t., quan m., joone c., lourens c.w., maclachlan n.j. & guthrie a.j. 2013. african horse sickness in naturally infected, immunised horses. equine vet j, 45 (1), 117-119. 5 parole chiave operatori di canile, canile, benessere degli operatori di canile, stress professionale, cura degli animali. riassunto la legge nazionale italiana “sugli animali da compagnia e sul controllo delle popolazioni di cani randagi” vieta l'eutanasia dei cani randagi a meno che essi non siano pericolosi o seriamente sofferenti. i cani vaganti vengono catturati e alloggiati in canili a lungo termine fino al momento del reinserimento, dell’adozione o della morte. in questo scenario, il benessere dei cani randagi è diventato una questione di interesse scientifico comunitario, mentre sono disponibili solo poche informazioni relativamente alla sfera umana di coloro che lavorano nei canili. l'obiettivo di questo studio è, pertanto, quello di valutare il rapporto sociale tra i cani alloggiati nei canili italiani e gli operatori (dipendenti e volontari) che vi lavorano e l'impatto che questo lavoro ha sulla qualità della loro vita. a tal fine è stato sviluppato da un gruppo multidisciplinare di esperti un questionario rivolto agli operatori dei canili. il questionario è stato strutturato in tre parti principali: informazioni generali, competenze degli operatori e benessere degli operatori e sfera emotiva. questo questionario è stato incluso nel protocollo shelter quality ed è stato utilizzato per valutare il benessere dei cani ospitati in canili a lungo termine ed è stato distribuito in 64 canili italiani. è stata condotta un'analisi descrittiva dei dati. i risultati del presente studio mostrano che, in generale, gli operatori dei canili italiani hanno una percezione positiva del loro lavoro anche se questo ha un impatto stressante sulla loro vita. relazione cani-operatori dei canili: impatto sulla qualità della vita umana e sulla sua sfera emozionale keywords shelter operators, shelter dogs, shelter operators wellbeing, occupational stress, animal care. summary the italian national law on companion animals and stray dog population control prohibits euthanasia of shelter dogs if they are not dangerous or seriously suffering. free roaming dogs are captured and housed in long-term shelters (lts) until rehomed, adopted or dead. in this scenario, the sheltered dogs’ welfare has become a community of scientific interest but few information is available about the human sphere in dogs’ shelters. the aim of this study was to evaluate the social relationship between dogs and shelter operators (employees and volunteers) in italian shelters and the impact of their job on their quality of life. a questionnaire addressed to shelter operators was developed by a multidisciplinary group of experts and it was structured in three main sections: general information, operators’ skills and operators’ welfare and emotional sphere. the questionnaire was distributed in 64 italian shelters during the field application of the shelter quality protocol for the assessment of dogs’ welfare in lts (izs 04/13 rc funded by ministry of health) that was used to assess the welfare of dogs housed in lts. a descriptive analysis was carried out. these results show that italian shelter operators have a positive perception of their job despite a stressful impact on their lives. veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 accepted: 08.04.2018 | available on line: 31.03.2019 1human-animal relationship and animal welfare laboratory, istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. 2istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. *corresponding author at: human-animal relationship and animal welfare laboratory, istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861332632, e-mail: m.nardoia@izs.it. maria nardoia1, laura arena1, greta berteselli1, paolo migliaccio1, lejla valerii2, ludovica di giustino2 and paolo dalla villa1 development of a questionnaire to evaluate the occupational stress in dog's shelter operators 6 veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 dog-shelter operators relationship and human quality of life nardoia et al. in italy there are currently 9,819,199 owned dogs and it has been estimated that the number of dogs that entered italian shelters amounted to 100,194 in 2015 (ministy of health 2015). sheltered and free-roaming dogs may experience poor health and welfare and can pose a significant threat to human health becoming a vehicle of serious zoonotic disease such as rabies, echinococcosis, toxocariasis, leishmaniasis, toxoplasmosis and bartonellosis (fico 1994, matter and daniels 2000, slater 2005). in addition to disease transmission, dog bites, urine and faeces in the environment and the possibility to cause road accidents, also pose a risk to human well-being (fico 1994, matter and daniels 2000, slater 2005). several measures (movement restriction, reproduction and habitat control, removal, rehoming, trap-neutered-release) to control dog population are used in different areas. in many countries, such as usa and australia, the euthanasia is a routine practice to control overpopulation, with a rate of 30%-60% (marston et al. 2004, bartlett et al. 2005, rogelberg et al. 2007, mohan-gibbons et al. 2014). the euthanasia of dogs and the prolonged exposure to suffering, combined with negative public perception of this work, work-to-family conflict, lack of support by friends and family, work environment (i.e. smells and mess) etc., can generate traumatic stress reactions with a negative impact on shelter workers’ psychological and emotional well-being issues in those caring for animals (figley and roop 2006, foster and maples 2014). a study from rogelberg and colleagues (rogelberg et  al. 2007) highlighted that euthanasia of dogs is perceived as greater stressful among employees due to the ‘senseless killing of healthy animals’, particularly when alternative approaches of care or rehabilitation have not been explored (arluke 1994). research addressing occupational stress in animal shelter workers is almost exclusively limited to discussion of euthanasia related strain. however, unlike usa and australia, some european countries such as italy, greece, austria and germany forbid the euthanasia of stray dogs except for those that are seriously suffering or dangerous. in these countries the adoption of this “no kill” policy, besides to be economically demanding, can led to the confinement of thousands of dogs in shelters that can become their final home, if not adopted. in this situation shelter workers spend significant amounts of time caring for and building a bond with these dogs almost in a surrogate owner role (roberts 2015). the impact of non-euthanasia related stressors on the psychological and emotional sphere and introduction dog is one of the most popular animal thanks to his significant role as companion animal, hunting partner, working assistant (i.e. drug dog) and also as model in comparative medicine for understanding human disease such as in oncology field where the dogs, considered as environmental sentinels, can help in the knowledges on tumor biology and in therapeutic trials (porrello et al. 2006, serpell, 2016). several studies have been carried out on the physical and psychological benefits of pet ownership and the human-companion animal interactions, reporting the capacity of pets to buffer the stress on their owners, to be an emotional support or to increase physical activity (walsh 2009, wells 2009). alzheimer patients or people affected by psychiatric disorders can benefit from the contact with pets through assisted animal activity (baun and mccabe 2003, cevizci et  al. 2013). moreover, there are evidences that the relation between children and animal increases their capacity to feel empathy (barker and wolen 2008). furthermore, the positive effect of human contact on the welfare and behaviour of dogs has been documented (valsecchi et al. 2007, normando et al. 2009). the relationship between human and dog is characterized by emotions. each individual feels pleasure and security when are together, distress when separated. dog-human relationship can be considered as an affectional bond (prato-previde et  al. 2003, walsh 2009). human interaction may be an effective means of reducing the stress level of dogs in shelter environment (hennessy et  al. 1999, coppola et al. 2006, bergamaco et al. 2010), in particular the training activity with sheltered dogs increases their adoptability (luescher et al. 2009). despite dog plays a special place in the society and a strong bond develops between human and dog based on love and affection, many factors can negatively affect this relationship with consequently abandonment of the dog (mondelli et  al. 2004). aggressive behaviours, owner moving, too high cost of care, lack of time or dogs may not meet the owners’ expectations: that are the main reasons for dogs’ abandonment (patronek 1996). the uncontrolled reproduction of these dogs, which become free roaming, due to their promiscuous nature and permitted by human beings who take care of them, combined with the irresponsible ownership, lead to canine overpopulation (ortega-pacheco and jimenez-coello 2011). as consequence, these dogs are confined in shelters with, in many cases, a negative impact on their welfare, due to poor environment, social deprivation, overcrowding and inappropriate management (taylor and mills 2007, moesta et al. 2015). 1 italian law n. 281/1991. legge quadro in materia di animali di affezione e prevenzione del randagismo. off j, 203, 30.08.1991. 7veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 nardoia et al. dog-shelter operators relationship and human quality of life performed by the shelter operators and information on the quality of human-animal relationship and shelter operator wellbeing (welfare and emotional sphere). in particular, the first part of questionnaire included not only demographic information such as nationality, workplace type, gender, age, occupation, education level, and number of years of involvement in this work but also their general experience in an animal shelter, their perception of the dog shelter role, owning dog or other pets. the questionnaire investigated the activities in the shelter (work schedule, main tasks, number of dogs to look after); the emotional sphere of operators and their relationship with dogs; their perception on physical and emotional stress, and satisfaction; the impact of the work on their life. this part of questionnaire, included structured questions, uses a likert scale from 1 to 5 (1 means not at all and 5 means completely) to evaluate the level of physical or emotional stress, the level of satisfaction about specific aspects of their work in the shelter, the feeling at the end of the day, and the type of relationship with sheltered dogs. the questionnaire ended with two open-ended questions to give participants the opportunity to add anything else related to their work in shelter, or add comments and suggestions on the questionnaire. data analysis the answers of the questionnaires were insert in a database in order to perform a descriptive analyses followed by exploration of frequencies. results general information a total of 260 people working in 34 dog shelters (over the 64 involved in the shelter quality project) participated and completed the questionnaire. about the type of shelters, twelve were public animal shelters managed by animal protection associations, six were private animal shelters managed by animal protection associations, five public and five private shelters, three owned by animal protection associations and three classified as ’other’. the majority of respondents (78%) were volunteers and females (70%). participants’ age ranged from 18  (19%) to over 50 years, with the latter being the majority (31%) (figure 1). the results showed that 54% of participants attended a training course on a voluntary basis in most cases (60%). seventy-five percent of interviewees owned at least one dog that in the 74% of the cases was adopted from a shelter. the majority of shelter operators (79%) said wellbeing of operators working in dog shelters has been under-represented. shelter operators have to face with an intense emotionally demanding job that put them at special risk to develop two severe forms of traumatic stress, namely compassion fatigue and perpetration-induced traumatic stress (macnair 2002, figley and roop 2006, potter et al. 2010, bride et  al. 2007) that are referred to as “costs of caring”. the costs of caring can lead to burnout (kowalski 2002) namely physical, emotional and mental exhaustion (white 2006) with signs of anxiety, panic, depression, hypersensitivity. the information available in the literature on occupational stress experienced by personnel working in shelter workplace not directly related to euthanasia is even limited. the researches on occupational stress, compassion fatigue, and post-traumatic stress disorder in animal care community has been largely performed in the united states and to a lesser extent in the united kingdom. there is still less literature that focuses on the positive aspect, including the emotional rewards or gratification received through the empathy and compassion, of those working in animal health care. to date, no studies have been carried out in italy in this field. the purpose of the present work is to investigate the social relationship between dogs and shelter operators (employees and volunteers) working in dog shelters located in italy and the impact of their job on their quality of life. materials and methods participants the questionnaire was distributed previously via e-mail to the operators and the volunteers worked in all shelters which have joined to the shelter quality project for the assessment of dogs’ welfare in long-term shelters (izs 04/13 rc funded by ministry of health). the participation was voluntary. the filled questionnaires were sent back via e-mail or in some cases, they were recollected at the moment of the visit in the shelters for animal welfare assessment. questionnaire design the questionnaire was developed through a multidisciplinary approach, with the collaboration of vets, psychologists and sociologists, and through a review of literature of the general occupational stress and with special attention on stress among animal shelter operators. the semi-structured questionnaire, containing both open-ended and closed ended questions, included thirty questions divided into three main parts: general information of the participants, information on the main tasks/skills 8 veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 dog-shelter operators relationship and human quality of life nardoia et al. furthermore, employees spend more hours in the shelter respect to volunteers; in fact, 67% of employees pass from 6 to 8 hours working in the shelter, while 62% of volunteers only from 3 to 5 hours. in general, the majority of shelter volunteers takes care of a low number of dogs (from 1 to 10), the number of dogs cared by employees seems to be higher compared to that of voluntaries (figure 5). human‑animal relationship shelter operators wellbeing and emotional sphere the majority of respondents (75%) declared to have a friendly/social relationship with the majority/totality of dogs and 83% of operators, when approaching the pen, perceived that the emotional state of dogs was happiness (figure 6). concerning the exposure to risks, 43% of participants was bitten by a dog in the shelter and 46% of them needed medical care because of the injury. in relation to this episode, 72% of respondents felt indifferent, 13% sad, 8% frustrated and 7% worried. after this episode, 33% that entered the profession motivated by a love for animals (figure 2). the majority of respondents (54%) worked in the shelter for more than 3 years (figure 3). only 14% of interviewed had another working experience in a shelter and, among them, 38% affirmed to spend more than 3 hours in this shelter (figure 4). when asked to the participants what is the main role of dogs’ shelters in the society, the 80% answered the nowadays a lts is basically addressing stray dog control and animal abandonment issues, while respect to the question ‘what role would you like shelter had in the society’, 50% of answers highlighted that shelters should play an educational role for the community and a place to promote a human-animal relationship. information about work in the shelter and main tasks performed the main tasks performed with dogs by the shelter workers in the present study were: walking at leash, cleaning pens and feeding dogs. 18-30 19% 31-40 26% 41-50 24% >50 31% figure 1. age distribution of dog-shelter operators responding to the questionnaire. do not have animals at home 2% study 2% social volunteering 9% job opportunity 8% love for animals 79% figure 2. main reasons for working in a shelter as resulting from a questionnaire amongst dog-shelter operators. 16% 7% 12% 10% 54% 2% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% < 6 months 6-12 months 1-2 years 2-3 years > 3 years na figure 3. time spent in the current shelters as resulting from a questionnaire amongst dog-shelter operators. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% < 6 months 6-12 months 1-2 years 2-3 years > 3 years 11% 24% 16% 11% 38% figure 4. time spent f in other shelters as resulting from a questionnaire amongst dog-shelter operators. 9veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 nardoia et al. dog-shelter operators relationship and human quality of life perceived both in female (88%) and male (81%) interviewed (figure 8). figure 9 shows that in all respondents (100%) physical stress increased with the increasing in the number of dogs. an high emotional stress was perceived by 40% of shelter employees and a similar percentage of employees felt medium emotional of participants declared a changed in attitude toward dogs giving more attention and being more careful to dogs behaviour. shelter operators felt sad or frustrated in relation to presence of sick dogs (92%), abnormal behaviour (77%), euthanasia (75%), facilities’ inadequacy (75%), presence of aggressive dogs (70%), dogs confinement (64%) and social restriction in relation to their ethological needs (62%). respondents felt angry or sad respect to unsuccessful adoptions in 66% of cases and respect to the presence of puppies in the shelter in 45%. successful adoptions (95%), social activities with dogs (94%) and sterilizations (71%) are sources of satisfaction for the most of respondents. volunteers appeared to be more satisfied in relation to their job rather than employees, in fact 89% of volunteers affirmed to felt high satisfied respect to 69% of employees. both emotional and physical stress appeared to be higher in employees (42%) rather than in volunteers (25%) (figure 7). however, emotional stress appeared to be higher in female (32%) rather than in male operators (23%) while there were no relevant differences for the physical stress both in male (20%) and female (17%) workers (figure 8). an high level of satisfaction was 2% 25% 23% 27% 13% 4% 4% 2% 9% 71% 14% 4% 1% 1% 0% 1% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 1-10 11-50 51-100 101-200 201-300 >300 na employees volunteers figure 5. number of dogs cared by shelter operators per day. 83% 2% 2% 1% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% happiness indi�erence/ neutrality fear aggression figure 6. dogs emotional state when shelter operators approach to the pen. 69% 36% 49% 27% 2% 4% 18% 33% 26% 31% 8% 22% 13% 36% 25% 42% 90% 74% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% volunteers employees volunteers employees volunteers employees physical stress emotional stress satisfaction high medium low figure 7. emotional impact of dog-shelter operators according to their role in the shelter. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% male female male female male female physical stress emotional stress satisfaction high medium low 57% 62% 51% 42% 3% 2% 23% 21% 26% 26% 16% 10% 20% 17% 23% 32% 81% 88% figure 8. emotional impact of dog-shelter operators according to their gender. 60% 47% 45% 26% 20% 3% 2% 20% 24% 20% 0% 23% 42% 40% 7% 25% 20% 16% 33% 100% 33% 32% 40% 90% 73% 60% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 to 50 51 to 200 > 200 1 to 50 51 to 200 > 200 1 to 50 51 to 200 > 200 physical stress emotional stress satisfaction high medium low figure 9. emotional impact of dog-shelter operators according to their role in the shelter. 10 veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 dog-shelter operators relationship and human quality of life nardoia et al. of information in italy concerning stress in animal shelter workers and this study wants to be a first step to fill that gap and some interesting results deserve to be discussed in depth. the participants’ age of this study, indicated that this job is not perceived as particularly physical demanding and therefore is not affected by an early retirement or changing of profession, contrarily to foster and maples (foster and maples 2014) which found a declining number of the veterinary support staff older than age 50, probably because of the physical and emotional demands of the job. in the present study the majority of respondents worked for more than 3 years in the current or other shelters indicating therefore a low turnover among shelter workers and a low rate of job leaving. at the same time, the length of employment is a sign of enthusiasm for this work. these results are consistent with those reported by foster and maples (foster and maples 2014) which found that 52% of participants were involved in their job for more than 5 years. the dedication and deep passion for the work were underlined also by the participation to training courses on a voluntary bases in many cases. this may depend on whether in both employees and volunteers the desire to care for dogs, and not only the financial reward, was the motivation to enter this profession and work with animals. this result is confirmed by taylor (taylor 2010) who pointed out that the motivation to work in a dog shelter is ’for the animals’. stress when the number of assisted dogs increased at more than 200 (figure 9). at the same time and coherently with these data, the level of satisfaction decreased with the increasing of the number of dogs to assist. shelter employees (90%) felt high satisfaction when they had to take care of a low number (1-50) of dogs, 73% when had to assist from 50 to 200 animals and 60% felt satisfaction when dogs were more than 200 (figure 9). the daily activities carried out by shelter operators having negative impact on their emotional stress appeared to be mainly adoptions’ management and public relations (45%) and administrative part (40%) (figure 10). despite this aspect, data show that the level of satisfaction in shelter operators was high in all activities they carry out with dogs (figure 10). discussion and conclusions the stress in animal shelter worker is studied in general with the assumption that euthanasia is the most stressful part of this job. however, considering the prohibition of euthanasia in some countries, researchers suggested other kind of stressor such as negative public perception of their work, negative media, lack of understanding among friends and family, conflict among colleagues, and poor physical working conditions, could negatively affect the wellbeing of shelter workers (reeve et al. 2005, figley and roop 2006). to date, there is still a lack 68% 57% 54% 57% 63% 60% 54% 50% 42% 37% 35% 30% 1% 2% 3% 4% 5% 0% 20% 23% 24% 25% 21% 25% 22% 25% 30% 32% 24% 25% 7% 12% 13% 4% 11% 9% 12% 20% 22% 18% 16% 15% 25% 25% 28% 31% 40% 45% 92% 85% 84% 92% 84% 91% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% w al ki n g /p h ys ic al a ct iv it ie s fe ed in g pe n s h yg ie n e te ra p ie s/ m ed ic at io n tr ai n in g /b eh av .r eh ab ili ta ti o n a d o p ti o n /p u b lic r el at io n s w al ki n g /p h ys ic al a ct iv it ie s fe ed in g pe n s h yg ie n e te ra p ie s/ m ed ic at io n tr ai n in g /b eh av .r eh ab ili ta ti o n a d o p ti o n /p u b lic r el at io n s w al ki n g /p h ys ic al a ct iv it ie s fe ed in g pe n s h yg ie n e te ra p ie s/ m ed ic at io n tr ai n in g /b eh av .r eh ab ili ta ti o n a d o p ti o n /p u b lic r el at io n s physical stress emotional stress satisfaction high medium low figure 10. emotional impact of dog-shelter operators according to the main activities carried out. 11veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 nardoia et al. dog-shelter operators relationship and human quality of life activities. in the present study, many participants revealed to be injured by a dog and needed medical care but, unlike to what reported in literature, the event was accepted as a commonplace risk of this job, since the majority of them felt indifferent in relation to this episode and only a little part of them changed the attitude toward dogs giving more attention to their behaviour in order to prevent other bite accidents. these preliminary results show that, in general, italian shelter operators have a positive perception of their occupation and duties and enjoy them. this aspect results important because mentally and physically healthy workers are more productive workers (davis et al. 2005). the interviewed participants’ satisfaction was associated with activities with animals. indeed, the positive interaction and bond with dogs might help to balance the negative effects of work in shelter (i.e. caring for the intensely ill or traumatized dogs) (stamm 1999). volunteers appear more satisfied than employees even if they spend, on average, less hours (3-5) working in shelters than employees (6-8) (data not shown) and are, therefore, less involved in those interactions with animals under their care that lead to satisfaction. the choice to work voluntarily in the shelter could explain the great satisfaction of volunteers, there is probably a kind of gratification resulting from the activity carried out in the shelter (taylor 2010). the less satisfaction of emplyees could be explained by the study of figley and roop (figley and roop 2006), which found that the main causes of sadness and frustration in shelter operators are the low adoptions rate, dogs confinement and social restriction, the presence of abnormal behaviour over time, or the inadequacy of environment. all these causes could represent personal failures. in our study, employees spend more hours in the shelter respect to volunteers, developing as consequence a strong relationship with dog. however, this strong relationship might have a double-sided and play a double role intensifying the stressors, besides providing positive feelings. in addition, the increase of workload pressures decreases the level of satisfaction with a corresponding increase in stress. these findings support the idea that there are several other shelter stressors, besides euthanasia decision, experienced by animal shelter workers that could have an impact on their quality of life, wellbeing and mental health status (scotney et al. 2015). the present study revealed that women are more affected by emotional stress than men and might be at greater risk of secondary traumatic stress (rohlf and benner 2005). literature findings showed that there are sex differences in the perception of post traumatic events (pte’s). however, much more research needed the majority of participants believes that nowadays a lts is basically addressing stray dog control and animal abandonment issues and 50% of them affirmed also that shelters should play an educational role for the community and should be a place to promote a human-animal relationship. in italy, there is the prejudice that dog shelters are merely places with ugly dogs having behavioural problems such as hyperactivity, destructiveness, excessive barking (martson et  al. 2004, menchetti et  al. 2015). it is however a fact that spatial and social restriction in shelters leads to stress related behaviours (beerda et al. 1999) reducing accordingly the adoption success (patronek et  al. 1996). to increase adoption rate and change public opinion on shelter dogs’ characteristics, several programs of socialization were implemented in italy and other countries (kogan et  al. 2002, normando et  al. 2009, braun 2011, mohan-gibbons et  al. 2014, menchetti 2015) encouraging people to visit, walk and play with dogs. the promotion of contact with human and dog training in these programs has been shown to positively affect welfare and increase desirable behaviours (e.g. sitting, being quiet, making eye contact) of dogs living in shelters (protopopova et  al., 2012; herron et  al., 2014), making them more attractive to visitors (wells and hepper, 2000; conley et al., 2014). many times, the reluctance in adopting sheltered dogs derive also from a scarce knowledge of dog behaviour and physiology (herron et al., 2007; mohan-gibbons et  al., 2014). provide an education on how to manage dog behavioural problems, on reproductive physiology and breed characteristics could also help to decrease the number of returns after adoptions. all these issues should be covered by the shelters as expected by the most of italian operators involved in this study. in the current study, the positive reaction of shelter dogs towards care givers when they come to the kennel, is sign of a good quantity and quality of interactions with staff, such as shared activities (bennett and rohlf 2007, arhant et al. 2010). a positive attitude and positive handling increase the willingness of dogs to approach an unknown person (arhant et  al. 2014). in contrast, aversive training methods are instead correlated with aggressive or fearful dog behaviour toward humans (hiby et  al. 2004, herron et  al. 2009, arhant et  al. 2010) and, consequently, the animals are less interacting with strangers (rooney and cowan 2011). in addition, exploration of an unknown person by dogs can be considered as a positive emotional response and an indicator of dogs’ good welfare (boissy et  al. 2007, araujo et al. 2010). literature reports that caretakers who have been bitten or attacked by animals feel uncomfortable (chang and hart 2002) and tend to limit their care 12 veterinaria italiana 2019, 55 (1), 5-14. doi: 10.12834/vetit.1476.7954.2 dog-shelter operators relationship and human quality of life nardoia et al. of quantitative measures in the questionnaire is advantageous, compared to the only qualitative interview. it overcomes some limitations associated to the possibility to capture the thoughts and feelings of respondents only at one moment in time or to the influence of contest on the respondents. therefore, such kind of interview format provides opportunity for comparisons. acknoledgements a special thanks goes to all shelter operators that, on volunteer base, took part to this study enthusiastically. to be done in this sense regarding how sex acts as a vulnerability factor (tolin and foa 2006). the sample used in the current study may not be representative of the entire population of animal shelter workers. therefore, this kind of limitation should be considered before drawing conclusions from the results of this study. the information from this study represents an initial effort that would provide preliminary data on animal shelter worker stress in italy. the development of this questionnaire protocol could provide also groundwork for future research in the area of animal shelter worker stress in italy. the inclusion araujo j.a., de rivera c., ethier j.l., landsberg g.m., denenberg s., arnold s. & milgram n.w. 2010. anxitane® tablets reduce fear of human beings in a laboratory model of anxiety-related behavior. j vet behav, 5, 268-275. arhant c. & troxler j. 2014. approach behaviour of shelter dogs and its relationships with the attitudes of shelter staff to dogs. appl anim behav sci, 160, 116-126. arhant c., bubna-littitz h., bartels a., futschik a. & troxler j. 2010. behaviour of smaller and larger 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2007. the effect of the kennel environment on canine welfare: a critical review of experimental studies. anim welfare, 16, 435-447. taylor n. 2010. animal shelter emotion management: a case of in situ hegemonic resistance? sociology, 44, 85-101. tolin d. & foa e. 2006. sex differences in trauma and posttraumatic stress disorder: a quantitative review of 25 years of research. psychol bull, 132, 6, 959-992. 375 1istituto zooprofilattico sperimentale delle venezie, legnaro (pd), italy. 2national center for global health, istituto superiore di sanità, roma, italy. 3istituto zooprofilattico sperimentale della lombardia e dell’emilia romagna ‘bruno ubertini’, brescia, italy. *corresponding author at: istituto zooprofilattico sperimentale delle venezie, legnaro (pd), italy. e-mail: pmulatti@izsvenezie.it. parole chiave bovini da latte, mycobacterium spp., sorveglianza, tubercolosi bovina, zoonosi. riassunto con decisione 2008/404/ce, la regione veneto è stata dichiarata ufficialmente indenne (ui) da tubercolosi bovina ai sensi della direttiva 64/432/cee. a settembre 2015 è stato identificato un focolaio di tubercolosi bovina in un allevamento di 69 bovini da latte della provincia di padova. sottoposti a controlli ufficiali, 24 di essi (34.78%) hanno reagito positivamente alla prova intradermica. mycobacterium caprae è stato isolato in campioni appartenenti a 22 capi e identificato attraverso tecniche biomolecolari. le pcr utilizzate hanno rilevato la presenza del sottotipo allgäu, e non è stata riscontrata nessuna variazione tra i diversi isolati. l’indagine epidemiologica ha evidenziato che 16 bovine erano state introdotte dall’austria tra la fine del 2011 e l’inizio del 2015. tuttavia le analisi biomolecolari hanno evidenziato una stretta correlazione tra il m. caprae isolato in questo studio e il ceppo identificato negli anni 2007-2009 nella provincia di trento, sebbene nessun contatto a rischio sia stato individuato. il m. caprae è un agente zoonotico che rappresenta un grave pericolo per la salute pubblica e risulta indispensabile che i piani di controllo si basino su un’attenta valutazione del rischio di possibile introduzione anche nei territori dichiarati ufficialmente indenni da tubercolosi bovina . descrizione di un focolaio di mycobacterium caprae in un allevamento da latte della regione veneto keywords bovine tuberculosis, dairy cattle, m. caprae, mycobacterium spp., surveillance, zoonoses. summary veneto region, northeast italy, has been declared officially free from bovine tuberculosis since 2008, although the disease is sporadically detected in association with cattle trade. in september 2015, bovine tuberculosis was detected in a dairy cattle farm of the region, in a holding with 69 animals. the herd underwent single intradermal tuberculin testing as part of the regional surveillance plan, and 24 animals resulted positive. mycobacterium caprae was evidenced in 22 samples, further genotyped by pcr-based assays as allgäu type. epidemiological investigation reported that sixteen animals were introduced from an officially tuberculosis free member state in previous years. nevertheless, spoligotyping and multilocus variable tandem repeat analysis (mlva) indicated that m. caprae was strictly related to the strain circulating in 2007-2009 in trento province (trentino-alto adige region, northeast italy), although no at-risk contacts were described. m. caprae is a zoonotic pathogen and further analyses are warranted in order to control its spread and impact on public health and animal trade. laura amato1,2, paolo mulatti1*, maria pacciarini3, eliana schiavon1, mariagrazia zanoni3, and lebana bonfanti1 mycobacterium caprae in a dairy farm in northeast italy veterinaria italiana 2019, 55 (4), 375-379. doi: 10.12834/vetit.1350.7441.2 accepted: 14.01.2018 | available on line: 31.12.2019 case report 376 m. caprae in a dairy farm amato et al. veterinaria italiana 2019, 55 (4), 375-379. doi: 10.12834/vetit.1350.7441.2 at the slaughterhouse. in lactating cows, also supramammary lymph nodes need to be collected. anatomo-pathological inspections of the lymph nodes and tonsils, histological investigations, and ziehl-neelsen staining are performed at the local territorial laboratory (istituto zooprofilattico sperimentale delle venezie izsve). bacteriological culture and pcr for is6110, an insertion element found exclusively within the members of the mycobacterium tuberculosis complex, are performed at the national reference laboratory (nrl) at the istituto zooprofilattico sperimentale della lombardia e dell’emilia romagna (izsler), followed by molecular test for identifying, genotyping and differencing mycobacteria subtypes (domogalla et  al. 2013, boniotti et  al. 2014). genotyping is performed through spoligotyping and mycobacterial interspersed repetitive units (miru) variable number of tandem repeats (vntr) analyses, as described by boniotti and colleagues (boniotti et al. 2014). when positive sit reactors are detected, investigations need to be also carried out on animal handlers. latent infections are routinely identified by sensitization to tb antigens (mantoux test), while pulmonary lesions can be detected by x-ray body scans. results a total of 305,969 residential dairy cattle were examined through sit in veneto region in 2015 (source: national animal database). in september, a single farm tested positive to sit, and further epidemiological and diagnostic investigations were performed (table i). the holding was a family-run dairy farm located in padua province. a total of 69  dairy cattle of all ages were present in the farm, 30  of which were currently lactating. the examination of the site of sit injections allowed the detection of 24 positive animals. nine animals (37.50%) had increased skin thickness (> 4 mm) only, eight (33.33%) showed increased skin thickness (> 4 mm) and pain at palpation, and seven (29.17%) presented also at least one other sign including: extensive oedema, necrosis at the injection site, or inflammation of the regional lymph nodes. following the high intra-herd prevalence of sit positive animals (p = 34.78%), the local veterinary services decided to cull all the cattle present in the farm. two sit-negative calves underwent on-farm emergency slaughter due to health issues not directly related to btb, and no further analyses were performed. at the slaughterhouse, lymph nodes and tonsils were collected as indicated in chapter 2.4.6 of oie terrestrial manual, 2016, and in the annex b of eu directive 64/432/eec. introduction bovine tuberculosis (btb) is a well-known infectious disease of cattle that may pose a serious threat to public and animal health. the etiological agents of the disease are members of mycobacterium tuberculosis complex (mtc), including mycobacterium bovis and mycobacterium caprae. while m. bovis has been widely studied and is acknowledgedly associated with btb, m.  caprae has only recently been classified as a separate species (aranaz et  al. 2003), and included in the mtc (pérez-lago et al. 2014). the main hosts for btb are cattle and ruminants, although the disease can affect a broad range of domestic and wild animals (pérez-lago et al. 2014). bovine tb represents a threat for human health in developing, and industrialized nations (pérez-lago et  al. 2014), although the main agent of human tb is considered m.  tuberculosis. however, a substantial number of zoonotic tb human cases are characterized by extra-pulmonary manifestation (prodinger et al. 2014). in italy, btb is subjected to national control activities since the 1960s (zanardi et al. 2013). since 2008 veneto region has been declared officially tuberculosis free (otf) (decision n. 2008/404/ ec), and a multiannual regional control plan has been implemented, in concert with risk-based surveillance activities, to maintain the otf status. in the last four years, few cases were identified, at the slaughterhouse, in nearly all animals introduced from non-otf member states or italian regions. in 2015, surveillance activities revealed a single farm, in veneto region, positive for btb. in this manuscript, we describe an autochthonous btb case case and provide data obtained by the regional surveillance plan for btb. materials and methods the regional control programme for btb includes active and passive activities. cattle are screened by the local veterinary services at the slaughterhouse, and notification of lesions consistent with btb is mandatory. risk-based surveillance activities consist in testing cattle originating from areas considered at high risk for btb, sampled-based tests on beef cattle, and cadenced tests on residential dairy cattle. all animals older than six weeks have to be inspected with single intradermal tuberculin test (sit), as indicated in the oie manual of diagnostic tests and vaccines for terrestrial animals 2016 (chapter 2.4.6) and in the annex b of eu directive 64/432/ eec. the site of injection has to be examined after 72 hours and positive reactions identified. in case of positive tests, retropharyngeal, tracheobronchial, mediastinal, mandibular, mesenteric, and hepatic lymph nodes and tonsils need to be collected 377 amato et al. m. caprae in a dairy farm veterinaria italiana 2019, 55 (4), 375-379. doi: 10.12834/vetit.1350.7441.2 22  samples, and identified by molecular tests. the panel of pcrs described by domogalla and colleagues (domogalla et  al. 2013) revealed the presence of subtype allgäu containing the complete rd4 region sequence. the combined spoligotyping and mlva with 12 miru-vntr markers (order of markers: etra, etrb, etrc, etrd, etre, miru26, vntr2163a, vntr2163b, 3155, 4052, 1895, 3232) was: sb0418, 4, 3, 5, 3, 4, 5, 5, 2, 3, 2, 4, 15. no variation was found in the isolates of the reported case. with exception of vntr3232, the profile reliably matched with the genotype isolated in 2007-2009 in the outbreaks of trento province (chin et al. 2009). five people were tested twice with the human sit (mantoux) in september and november 2015; the other two were subjected to thoracic x-rays scans. laboratory investigations did not reveal any positive results. epidemiological investigations on the affected farm allowed to track back cattle that were introduced up to the identification of btb. sixteen animals were introduced from austria between the last quarter of 2011 and the first quarter of 2015, 14 of which were first introduced in lairage facilities in trento province. of the two remaining cattle, one was moved to a dairy farm in bolzano province and then to the lairage facility in trento, before arriving in padua. the last cattle remained in a dairy farm in trento between november 2009 and october 2013 before being introduced in the affected farm in padua. of the remaining 53 heads present at the time of m.  caprae detection, 44 were born in the farm, while nine were introduced from other farms located in padua province where m.  caprae was never detected. discussion in past years only sporadic cases of m.  caprae were detected in individual animals in veneto region; this pathogen also circulated in other northern italy areas in late 2000s (chin et al. 2009). tracing activities of animal movements permitted to attribute most of the previous m. caprae detections to the introduction of cattle from austria and germany, which at that time were already classified as otf. epidemiological investigations on the case we reported, revealed that 16 animals were introduced from austria between the last quarter of 2011 and the first quarter of 2015. nine of the lactating cows testing positive to sit originated from austria, and seven of them had lesions consistent with btb, with isolation of m.  caprae. austria has been an otf member state since 1999 (decision n.  1999/467/ec), although nowadays btb has been evidenced in some areas of the country. m.  caprae was previously evidenced in wild deer (cervus twenty-four out of 67 inspected animals (35.82%) showed lesions consistent with btb, of these nineteen tested also positive to sit (table i). seven animals (7/24, 29.17%) had multifocal, coalescing granulomatous pneumonia involving most part of the lungs. in other six cases (6/24, 25.00%), multifocal to confluent caseating granulomas were identified in tracheobronchial and mediastinal lymph nodes. the remaining eleven heads (11/24, 45.83%) had non-coalescent granulomas in the tracheobronchial, mediastinal, mesenteric and/or retropharyngeal lymph nodes. further histological examinations revealed nodular lesions with a necrotic mineralized center surrounded by macrophagic and lymphocytic infiltration, with rare giant multinucleated cells and fibro-connective host reaction, consistent with btb. the ziehl-neelsen staining confirmed the presence of acid-resistant bacteria in six animals with histological lesions (6/24, 25.00%) (table i); five of these (83.46%) resulted positive for m. caprae also by culture. however, the acid-resistant bacteria are hardly identified in old lesions because of the presence of cells and fibrosis around the center of the lesion, de facto enclosing the microorganisms and preventing the staining (mcgavin and zachary 2007). of the five animals which tested positive by sit with no evidence of gross lesions, one presented histological lesions and resulted positive to the ziehl-neelsen test; one had histological findings in addition to the positivity to sit, and one was positive by microbiological culture (table i). the last two sit positive individuals were negative to all further tests performed. organ samples were delivered to the national reference laboratory (nrl) at the istituto zooprofilattico sperimentale of lombardy and emilia romagna (izsler) for bacteriological culture and molecular test. m.  caprae was isolated from table i. comparison between the results of the single intradermal tuberculin test (sit) and other tests. single intradermal tuberculin test1 + total gross lesions + 19 5 67 5 38 ziehl-neelsen + 5 1 6714 4 n.a.2 5 38 pcr + 17 5 67 7 38 1 sit was carried out on all of the animals present in the farm (n = 69); two sit-negative calves undergone emergency on-farm slaughter and no further analyses were performed. 2 ziehl-neelsen test is performed only on samples that resulted positive to the gross lesions and histological examinations (n = 24). 378 m. caprae in a dairy farm amato et al. veterinaria italiana 2019, 55 (4), 375-379. doi: 10.12834/vetit.1350.7441.2 in domestic and wild animals also in other eu countries (pérez-lago et  al. 2014). cases in humans were also reported in austria and germany, stressing the potential zoonotic risk of m. caprae (kubica et al. 2003, prodinger et al. 2002). in particular, in germany, the pathogen had been reported as the cause of more than 30% of the human tb cases in 1999-2001(kubica et  al. 2003). furthermore, as diagnostic tools able to discriminate m.  bovis and m.  caprae have been developed only recently, early m. caprae cases might have been underestimated (kubica et  al. 2003). sits have been indeed developed specifically for m. bovis and underestimation of m.  caprae could also be related to the fact that the directive n. 64/432/eec1 provides mandatory notification and restrictive measures for m. bovis only. the cases of btb in otf countries represents a remarkable threat to public health and international trade. accordingly, an elevated level of alertness should be kept also in otf areas, and risk-based surveillance programmes are needed to promptly detect any btb introduction and spread to domestic or wild animal populations. this is also corroborated by the detection of m.  caprae in italy in 2015, which indicated the effectiveness of the regional surveillance plan. elaphus) populations in the austrian alps (fink et al. 2015). furthermore, m.  capraerelated tuberculosis cases had been repeatedly notified in austria in recent years (prodinger et  al. 2002, promed 2016, schoepf et  al. 2012). the allgäu subtype has been detected in cattle and red deer of specific austrian alpine regions (rettinger et  al. 2017). however, by genotyping, it was not possible to connect m. caprae detected in this study with that circulating in austria. in fact, the spoligotyping indicated that the m. caprae identified in 2015 was related to the strain circulating in 2007-2009 in trento province (chin et  al. 2009). nevertheless, no at-risk contacts was highlighted between the farm in padua province and the one resulted positive in trento. moreover, the epidemiological scenario is further complicated by the fact that the cattle introduced from austria, which was temporarily located in a lairage facility in trento province before being moved to the farm in padua, tested negative to all screening tests. overall, more data are needed to assess the origin of this m.  caprae strain. more recent methods, such as whole genome sequencing (wgs), could help identifying the actual origin of the pathogen. since its first identification in 1999 in spain (pérez-lago et al. 2014) m. caprae has been detected 379 amato et al. m. caprae in a dairy farm veterinaria italiana 2019, 55 (4), 375-379. doi: 10.12834/vetit.1350.7441.2 aranaz a., cousins d., mateos a. & domínguez l. 2003. elevation of mycobacterium tuberculosis subsp. caprae aranaz et al. 1999 to species rank as mycobacterium caprae comb. nov., sp. nov. int j syst evol microbiol, 53 (pt 6), 1785-1789. boniotti m.b., gaffuri a., gelmetti d., tagliabue s., chiari m., mangeli a., spisani m., nassuato c., gibelli l., sacchi c., zanoni m. & pacciarini m.l. 2014. detection and molecular characterization of mycobacterium microti isolates in wild boar from northern italy. j clin microbiol, 52 (8), 2834-2843. chin f., farina g., moresco a. & tezzele r. 2009. tubercolosi bovina in provincia di trento. l’osservatorio, 6, 4-10. domogalla j., prodinger w.m., blum h., krebs s., gellert s., müller m., neuendorf e., sedlmaier f. & büttner m. 2013. region of difference 4 in alpine mycobacterium caprae isolates indicates three variants. j clin microbiol, 51 (5), 1381-1388. fink m., schleicher c., gonano m., prodinger w.m., pacciarini m., glawischnig w., ryser-degiorgis m.p., walzer c., stalder g.l., lombardo d., schobesberger h., winter p. & büttner m. 2015. red deer as maintenance host for bovine tuberculosis, alpine region. emerg infect dis, 21 (3), 464-467. kubica t., rusch-gerdes s. & niemann s. 2003. mycobacterium bovis subsp. caprae caused one-third of human m. bovis-associated tuberculosis cases reported in germany between 1999 and 2001. j clin microbiol, 41 (7), 3070-3077. mcgavin m.d. & zachary j.f. 2007. pathologic basis of veterinary disease. 4th ed., elsevier health sciences, st. louis, missouri. 526-527. references pérez-lago l., navarro y. & garcía-de-viedma d. 2014. current knowledge and pending challenges in zoonosis caused by mycobacterium bovis: a review. res vet sci, 97, s94-s100. prodinger w.m., eigentler a., allerberger f., schönbauer m. & glawischnig w. 2002. infection of red deer, cattle, and humans with mycobacterium bovis subsp. caprae in western austria. j clin microbiol, 40 (6), 2270-2272. prodinger w.m., indra a., koksalan o.k., kilicaslan z. & richter e. 2014. mycobacterium caprae infection in humans. expert rev anti infect ther, 12, 1501-1513. program for monitoring emerging diseases (promed). 2016. bovine tuberculosis austria: (vo, tr) bovine, m. caprae archive number: 20160311.4085658. http:// www.promedmail.org/. rettinger a., broeckl s., fink m., prodinger w.m., blum h., krebs s., domogalla j., just f., gellert s., straubinger r.k. & büttner m. 2017. the region of difference four is a robust genetic marker for subtyping mycobacterium caprae isolates and is linked to spatial distribution of three subtypes. transbound emerg dis, 64, 782-792. schoepf k., prodinger w.m., glawischnig w., hofer e., revilla-fernandez s., hofrichter j., fritz j., köfer j. & schmoll f. 2012. a two-years’ survey on the prevalence of tuberculosis caused by mycobacterium caprae in red deer (cervus elaphus) in the tyrol, austria. isrn vet sci, 1-7. zanardi g., boniotti m.b., gaffuri a., casto b., zanoni m. & pacciarini m.l. 2013. tuberculosis transmission by mycobacterium bovis in a mixed cattle and goat herd. res vet sci, 95, 430-433. 227 veterinaria italiana 2021, 57 (3), 227-232. doi: 10.12834/vetit.1700.8999.4 accepted: 13.03.2019 | available on line: 31.12.2021 1istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. 2albrecht daniel thaer-institut for agricultural and horticultural sciences, breeding biology and molecular genetics, invalidenstrasse 42, 10115, berlin, germany. *corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. e-mail: a.chiaverini@izs.it. alexandra chiaverini1*, shijie lyu2, giuliano garofolo1, elisabetta di giannatale1 and giacomo migliorati1 keywords meat traceability, parentage test, pig, microsatellite, inbreeding. summary the origin of meat and meat products can be traced by verifying the identity of an offspring from its parents’ genotypes. although there are many microsatellite panels applicable to swine population, efficiency of parental testing decreases when the population consists of consanguineous animals. the aims of the present study were to develop a new microsatellite panel for traceability using parentage test in inbreed pig population and to assess how hybridization can influence the efficiency of parental testing. a new genotyping assay, based on 20-microsatellite assay, was performed in 304 individuals consisting of related and unrelated animals. the results showed that the microsatellites used in this study display high level of polymorphism ensuring a parentage assignment of 100%. this genotyping panel can be a useful tool to test a ’parent-to-fork’ traceability system based on 20 microsatellite loci and can overcome technical limitations in inbreed population. assessment of a new microsatellites panel for traceability in italian inbreed pigs using parentage test parentage analysis relies on a process of exclusion. the genotype of candidate parent is compared to the offspring's genotype and is excluded as parent if a mismatch occurs at one or more loci (jamieson and taylor 1997). during the last decades, the selection of high-performance pig breeds in combination with the displacement of extensive production systems has led to a dramatic decrease in the gene pool, resulting in a reduction of genetic variation among populations (michailidou et  al. 2014). therefore, the effectiveness of a traceability system based on parentage test is linked to availability of an efficient panel of microsatellites, able to produce accurate results for both, pure breed pigs and commercial hybrids with high level of inbreeding. although there are many microsatellites sets to be used in the swine population (blasi et  al. 2003, costa et  al. 2012, guastella et  al. 2010, putnová et  al. 2003, lin et  al. 2014, oh et  al. 2014, nechtelberg et  al. 2001), there is no data on the influence of inbreeding on the performance of microsatellite array. over the past decades, there has been a growing public interest in enhancing food traceability and transparency in food production. for these reasons, the eu’s general food law was introduced in 2002 and made traceability mandatory for all food chain (reg. ec 178/2002). dna-based techniques, such as microsatellites, appear to be an ideal tracking tool to assess the origin of meat and meat products and allow the identification of the animal producer through a molecular fingerprint (individual tracking) (scarano and rao 2014, orrù et al. 2006). in contrast to the situation in cattle, where cows have on average three calves in their life, a sow produces about 40-60 piglets during her reproductive lifetime. thus, it is not cost effective to record each pig in order to enable tracing of meat products by direct dna fingerprints. in a real scenario, where the pig production is based on intensive breeding, it is therefore preferable to use the parentage test. specifically, it is common to compare the maternal genotypes because very often the paternal lineage is unavailable, due to husbandry practices (menéndez et  al. 2015). a simple approach to short communication 228 veterinaria italiana 2021, 57 (3), 227-232. doi: 10.12834/vetit.1700.8999.4 microsatellite panel for traceability in inbreed pig chiaverini et al. dneasy mini spin column (qiagen) according to the manufacturer’s instructions. individual genotyping was performed using twenty microsatellite markers fluorescently labelled divided in 3 multiplex pcr (table  i), using type-it microsatellite pcr kit (qiagen). the first 14  microsatellite markers are suggested by nechtelberg and colleagues (nechtelberg et  al. 2001) and reported in fao guidelines (fao 2011); while the last six markers are present in the usda marc database1. the pcr conditions were as follow: 1) denaturation at 95 ˚c for 15 minutes; 2) 35 cycles of 95  ˚c for 30 seconds, 60  ˚c for 90 seconds and 72  ˚c for 30 seconds; 3) last step on 72  ˚c for 10 minutes. fragment analysis was carried out with capillary electrophoresis on the abi 3500 genetic analyzer (applied biosystems, foster city, ca) using genescan™ 600 liz® size standard. the allele size was assigned using the genemapper  4.0 software (applied biosystems, foster city, ca) and the allele nomenclature was standardized using reference samples according to international society for animal genetics (isag). allele frequencies, number of alleles (na) and polymorphism information content (pic) were assessed by excel microsatellite toolkit (park 2001). genepop package (raymond and rousset 1995) was utilized to calculate observed heterozygosity (ho), expected heterozygosity (he), hardy-weinberg equilibrium (hwe) and f-statistics for each locus (weir and cockerham 1984). the new assay was compared with the previous panel suggested by nechtelberg and colleagues (nechtelberg et  al. 2001) to verify if the efficiency of the parentage test is directly linked to the number of microsatellites used. the probability of non-exclusion for one candidate mother was assessed in case the genotype of both parents was not known (1ex) and if only the parent’s genotype of the opposite sex was known (2ex) according to the formula of jamieson and taylor (jamieson and taylor 1997). further, the non-exclusion probability for identity analysis of unrelated and related individuals was calculated as previously described (waits et  al. 2001). the mother assignments were performed using cervus 3.0 software (marshall et al. 1998, kalinowski et al. 2007). the summary statistics results calculated for the assay used in this study were reported in table ii. the total number of alleles for the twenty markers panel were 164 with a mean number of 8.2 alleles per locus, ranging between 4 (swr153) and 15  (s0005) alleles. the expected heterozygosity (he) and observed heterozygosity (ho) mean the aims of this study were to test a traceability system ‘from parents to fork’, using new microsatellite markers and to verify how inbreeding may affect the efficiency of parentage test in this typology of breeding. the study included 304 animals consisting of related animals (71 sows and 71 adult offspring) and 162 unrelated animals from different farms. these animals were commercial crossbreeds between italian duroc, italian large white and italian landrace. dna was extracted from blood using table i. list of microsatellite primers sequence. locus primer sequence (5'-3') chr. reference multiplex n. 1 s0005 f: tccttccctcctggtaacta-fam r:gcacttcctgattctgggta 5 [13] s0090 f:ccaagactgccttgtaggtgaaa-vic r:gctatcaagtattgtaccattag 12 [13] s0101 f:gaatgcaaagagttcagtgtagg-pet r:gtctccctcacacttaccgcag 7 [13] s0155 f: tgttctctgtttctcctctgtttg-fam r:gttaaagtggaaagagtcaatgat 1 [13] s0355 f:tctggctcctacactccttcttgg -ned r:gtttgggtgggtgctgaaaaatagga 15 [13] s0386 f: gaactcctgggtcttattttcta-ned r:gtcaaaaatctttttatctccaacagtat / [13] sw24 f: ctttgggtggagtgtgtgc -fam r:atccaaatgctgcaagcg 17 [13] sw240 f: agaaattagtgcctcaaattgg-vic r:aaaccattaagtccctagcaaa 2 [13] sw857 f:tgagaggtcagttacagaagacc-pet r:gatcctcctccaaatcccat 14 [13] sw951 f: tttcacaactctggcaccag-ned r:gatcgtgcccaaatggac 10 [13] multiplex n. 2 sw72 f: atcagaacagtgcgccgt -pet r:gtttgaaaatggggtgtttcc 3 [13] sw936 f: tctggagctagcataagtgccfam r:gtgcaagtacacatgcaggg 15 [13] sw911 f: ctcagttctttgggactgaacc-hex r:catctgtggaaaaaaaaagcc 9 [13] s0228 f: ggcataggctggcagcaaca -hex r:gttccgccctcacagacccaaat 6 [13] multiplex n. 3 sw1370 f: agagcagtggtctgctaagatg-ned r:gaattgcctaaatttacttgtcc 2 [15] sw1035 f: tatgggggccctaaaaagac-pet r:aacggccttaacctcctcag 16 [15] swr153 f: ccacgttctcctttttgaggvic r: atgagttgtggtgtaggtcgc 4 [15] sw2038 f: gccgagaaacccttcacc -vic r:tagcctgttcagtgccacc 14 [15] s0017 f: ctaggagaaaatctgaggttfam r: gtttgaatggaggtgctgta 8 [15] sw1823 f: caggtcattgctgtagtgaagg-ned r:gagccttgggctacgtagtg 6 [15] 1 http://www.genome.iastate.edu/ pigs/maps/marc.html. 229veterinaria italiana 2021, 57 (3), 227-232. doi: 10.12834/vetit.1700.8999.4 chiaverini et al. microsatellite panel for traceability in inbreed pig table ii. allele frequencies and f-statistics of 20 microsatellite loci used in this study. locus na he ho pic hw f(null) fis fst fit s0005 15 0.8355 0.8905 0.8795 ns 0.0275 0.0164 0.0815 0.0966 s0090 7 0.7072 0.6851 0.6344 ns 0.0166 0.0867 0.088 0.009 s0101 5 0.4671 0.4764 0.4464 ns 0.0097 0.0011 0.0332 0.0343 s0155 6 0.6678 0.6517 0.5892 ns 0.0181 0.0987 0.117 0.0298 s0355 8 0.5362 0.5056 0.5056 ns 0.0476 0.1195 0.926 0.0159 s0386 7 0.4803 0.5556 0.4748 ns 0.0746 0.1144 0.0434 0.1529 sw24 12 0.6875 0.7748 0.7371 * 0.0578 0.0395 0.1317 0.1659 sw240 13 0.6842 0.7188 0.6903 ns 0.0247 0.0143 0.0613 0.0748 sw857 9 0.7961 0.8115 0.7865 *** 0.0072 0.088 0.0494 0.0411 sw951 5 0.4934 0.5474 0.4875 ns 0.0495 0.0946 0.0086 0.1023 sw72 14 0.7138 0.6819 0.6456 ** 0.0278 0.0957 0.0686 0.0205 sw936 9 0.7697 0.7283 0.678 ns 0.0287 0.1066 0.0794 0.0187 sw911 8 0.6513 0.6271 0.5684 ns 0.0211 0.0641 0.043 0.0183 s0227 7 0.773 0.7265 0.6864 ns 0.0346 0.1064 0.062 0.0341 sw1370 7 0.5592 0.6602 0.6251 ** 0.0821 0.1265 0.0547 0.1742 sw1035 5 0.7928 0.726 0.6791 ns 0.0477 0.1484 0.0864 0.0492 swr153 4 0.5066 0.5796 0.5354 ns 0.0574 0.101 0.0502 0.1462 sw2038 7 0.6842 0.7961 0.766 ** 0.0746 0.1054 0.0701 0.1681 s0017 7 0.4737 0.735 0.691 *** 0.2167 0.3431 0.0351 0.3662 sw1823 9 0.4803 0.8 0.7712 *** 0.2469 0.3764 0.0674 0.4185 all 8.2 0.6379 0.6839 / / / 0.0298 0.0681 0.0959 na = number of alleles per locus; ho = observed heterozygosity; he = expected heterozygosity; pic = polymorphic information content; hw = deviation from hardy-weinberg equilibrium; f(null) = frequencies of null alleles; fis = inbreeding coefficient; fst = fixation index; fit = overall inbreeding coefficient; ns = not significant; *significant at the 5% level; **significant at the 1% level; ***significant at the 0.1% level. the results of the comparison between the new assay and the previous panel suggested by nechtelberg and colleagues (nechtelberg et  al. 2001) are shown in figure 1. the probability of non-exclusion in executing the parentage test if there are no data on parental genotypes (1ex) was 5.84e-04 for the 20  microsatellites assay showing a high performance; the other panel showed a lower performance (figure  1a). we found similar results among the panels in the case of probability of assigning the putative parent incorrectly and knowing the genotype of the other parent (2ex) as shown by the trend of the graph (figure 1b). the results of simulation of parentage test confirmed the effectiveness of the microsatellites assay for establishment of parentage in inbreed pig population. the identity test showed different results along the panels tested, whereas the related test with the 20-microsatellites assay performed well and showed improvement of the relationship assignment compared with the other panel (figure 1c). the probability of non-exclusion for identity test using 14 microsatellite markers was 8.25e-13 and for 20  markers was 1.83e-18. the non-exclusion probability for full-sib test using values were 0.6839 and 0.6379, respectively. three loci (s0101, s0386 and sw951) showed a value of polymorphism information content (pic) lower than 0.5. the probability of finding null alleles is significant for three loci (sw24, s0101, sw857), these values were highly influenced by inbreeding of sub structured populations. the locus sw24 showed a significant deviation from hardy-weinberg equilibrium at 5% significance level after bonferroni correction; the loci sw72, sw1370 and sw2038 at 1% significance level and sw857, s0017 and sw1823 at 0,1%. deviations from hardy-weinberg equilibrium at many loci might be caused by the inbreeding. eleven out of 20 markers showed positive inbreeding coefficient (fis) value indicating that exist of inbreeding in these loci. furthermore, two loci s0017 and sw1823 showed very high fis value of 0.3431 and 0.3764, respectively. the overall fis coefficient for the loci was 0.0298, indicating a significant (p < 0.001) excess of homozygotes in the whole samples. meanwhile, the overall inbreeding coefficient (fit) value of an individual relative to the total population was 0.0959. the fixation index (fst) value for all samples was 0.0681, confirming the presence of inbreeding in our study group. 230 veterinaria italiana 2021, 57 (3), 227-232. doi: 10.12834/vetit.1700.8999.4 microsatellite panel for traceability in inbreed pig chiaverini et al. table iii. results of the mother's assignments using the 14 and 20 microsatellite panels. 20 markers 14 markers correct assignment wrong assignment no assignment correct assignment wrong assignment no assignment unrelated animals / / 162 / 13 149 piglets 71 / / 68 2 1 using the 20-microsatellite panel, it was possible to correctly assign each of the 71 offspring to their mother and none of the external individuals were attributed to the group of mothers (table iii). when we analysed the same group of animals using the 14 microsatellites, we found that two offspring were incorrectly assigned, and a son-mother combination remained unresolved, while 13 individuals, from the unrelated population, were wrongly assigned to putative mothers (table iii). these results confirmed that the offspring attribution to their mother using 14 markers only are insufficient and can result in incorrect traceability, as individuals outside are included in the related population. in conclusion, in the populations where genetic variability is limited, an accurate traceability which would be based exclusively on parentage test, is possible only by using a very large number of markers. our results support the efficacy of the described 20-microsatellites assay as a valuable tool for parentage testing in inbreed pigs. although many microsatellites have been described for domestic pigs, in a population in which there is a high level of 14  and  20  microsatellite markers was 1.39e-05 and 8.00e-08, respectively (figure 1d). the different results obtained from the probability values in our study confirmed that the new marker set should have good discriminatory power to resolve any situation, including parentage test with multi-putative mothers. based on our observational data, we tested 233  putative mother-offspring relationships (table  iii). we have compared the results obtained in our study with those derived from only 14 microsatellites, as suggested by nechtelberg and colleagues (nechtelberg et al. 2001), and verified the accuracy of assigning the mother to each offspring in real conditions, because probabilities of parentage in animals with certain degree of inbreeding is lower than the probabilities calculated (putnòva et  al. 2003). in both of cases, a strict confidence (95%) level has been applied and the results of the assignments of parentage were reported in table iii. in addition to 162 unrelated animals, we tested 71 offspring and their respective mothers, assuming an unknown relationship. markers 14 7.00e-03 6.00e-03 6.34e-03 5.00e-03 4.00e-03 3.00e-03 2.00e-03 1.00e-03 0.00e+00 15 16 17 18 19 20 4.68e-03 3.22e-03 2.64e-03 1.52e-03 1.03e-03 5.84e-03 a markers 14 2.50e-07 2.30e-07 2.00e-07 1.50e-07 1.00e-07 5.00e-08 0.00e+00 15 16 17 18 19 20 8.00e-08 3.00e-08 1.00e-08 2.82e-09 8.93e-10 1.84e-10 b markers 14 7.00e-13 9.00e-13 8.00e-13 6.00e-13 5.00e-13 4.00e-13 3.00e-13 2.00e-13 1.00e-13 0.00e+00 15 16 17 18 19 20 c markers 14 1.60e-05 1.40e-05 1.20e-05 1.39e-05 6.39e-06 1.00e-05 8.00e-06 6.00e-06 2.00e-06 4.00e-06 0.00e+00 15 16 17 18 19 20 2.67e-06 1.38e-06 5.10e-07 2.10e-07 8.00e-08 d 8.25e-13 1.25e-13 1.52e-14 1.83e-18 2.70e-172.37e-163.35e-15 figure 1. graphic representations of the comparison between the new assay and the previous panel suggest by nechtelberg et al. the following graphs report the non-exclusion probability if the parental genotype is unavailable (1ex). 231veterinaria italiana 2021, 57 (3), 227-232. doi: 10.12834/vetit.1700.8999.4 chiaverini et al. microsatellite panel for traceability in inbreed pig acknowledgements the authors thank dr marzia di silverio, dr davide di federico and dr ugo ciavattella for technical support. statement of animal rights the samples analyzed in this study were taken along the national eradication program for the swine vesicular disease. therefore, the farm animal-welfare bodies didn’t point out any animal welfare issue and considered unnecessary the authorization from the ethics committee. consanguinity, a percentage of kinship testings may not be resolved by using a limited set of loci, and for this reason we recommend to amplify the panel with more microsatellites markers. since the offspring in question shared common alleles across all loci, the comparison with maternal genotypes only might be challenging. the new set of microsatellite loci shown in this study overcomes these technical limitations and therefore has a potential to become a new more effective alternative for a reliable ‘parent-to-fork’ traceability system in inbreed populations. 232 veterinaria italiana 2021, 57 (3), 227-232. doi: 10.12834/vetit.1700.8999.4 microsatellite panel for traceability in inbreed pig chiaverini et al. blasi m., lanza a., varlotta c. & rosati a. 2003. pig ham genetic traceability. italian j animal sci, 2 (s1), 82-84. costa v., pérez-gonzález j., santos p., fernández-llario p., carranza j., zsolnai a., anton i., buzgó j., varga g., monteiro n. & beja-pereira a. 2012. microsatellite markers for identification and parentage analysis in the european wild boar (sus scrofa). bmc res notes, 5, 479. european communities (ec).2002. 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tests and ecumenicism. j heredity, 86, 248-249. scarano d. & rao r. 2014. dna markers for food products autenthication. diversity, 6, 579-596. waits l.p., luikart g. & taberlet p. 2001. estimating the probability of identity among genotypes in natural populations: cautions and guidelines. mol ecol, 10, 249-256. weir b.s. & cockerham c.c. 1984. estimating f-statistics for the analysis of population structure. evolution, 38 (6), 1358-1370. 329 veterinaria italiana 2021, 57 (4), 329-334. doi: 10.12834/vetit.1769.9341.2 accepted: 03.09.2019 | available on line: 31.12.2021 1postgraduate program in heath and animal production on the amazon (ppgspaa), federal rural university of the amazon (ufra), belem, brazil. 2faculty of veterinary medicine, federal university of para (ufpa), belém, brazil. 3national primate center (cenp), health surveillance secretariat, ministry of health, ananindeua, brazil. 4primate research institute. kyoto university. inuyama, aichi, japan. *corresponding author at: postgraduate program in heath and animal production on the amazon (ppgspaa), federal rural university of the amazon (ufra), belem, brazil. e‑mail: silva.filho@ufra.edu.br. taianara tocantins gomes almeida1, ednaldo silva filho1*, maria vivina monteiro barros2, aline amaral imbeloni3, wellington bandeira silva3, michael alan huffman4 and frederico ozanan barros monteiro1 keywords hematology, biochemistry, tamarin, neotropical primates. summary the spix’s saddleback tamarin, leontocebus fuscicollis is widely distributed across the amazon region, but is endangered. this species is serving an important role in biomedical research in captivity. however, reference values for hematological and biochemical parameters are required for the proper characterization of the species. it was therefore the objective of our research to establish these parameters taking into consideration sex and body mass differences in healthy adult spix’s saddleback tamarins. collecting 2 ml of blood from each individual, 20 animals were examined (7 males, 13 females), and hematological and biochemical parameters were determined using commercial kits. of the sixteen variables measured, only red blood cell (rbc), hemoglobin (hb) and hematocrit (ht) values were significantly higher in males (7.12 ± 0.98 106/mm, 14.98 ± 1.25 g/dl and 48.71 ± 4.91%, respectively), while red cell distribution width (rdw) was higher in females (14.58 ± 1.89%). of the biochemical parameters measured, only gamma-glutamyl transferase (ggt) enzyme showed higher activity in females (8.08 ± 4.87 u/l), and a high glucose concentration range was observed (102.0 to 521.0 mg/dl) for both sexes. these parameters established with reference ranges for healthy adults provide a reliable reference source for the interpretation of laboratory housed saddleback tamarin. hematological and biochemical parameters of spix's saddleback tamarin (leontocebus fuscicollis) raised in captivity (platyrrhini or neotropical monkeys) are easier to handle and require lower costs for breeding, compared to old world primates (catarrhini). however, they are more remotely related to humans, therefore, less likely to constitute optimal animal models for the study of some topics, especially infectious human diseases, (abee et  al. 2012). tamarins and marmosets (callitrichidae family), are small neotropical monkeys that have become more popular than others for providing two offspring per pregnancy (more prolificity) and low-cost colonies. also, they can be models to study diseases whose species-specificity is low, such as bacterial and parasitic infections. the proper management of these animals in captivity favors the production of introduction the spix’s saddleback tamarin (leontocebus fuscicollis) has a wide geographical distribution, across the amazon, including brazil (acre, amazonas, mato grosso, and rondônia states), bolivia, colombia, ecuador, and peru. this species is included under the category ‘least concern’ by the international union for conservation of nature (iucn 2016). however, some species of the genus are endangered, due to increasing human pressures resulting in habitat fragmentation (iucn 2016). non-human primates are used in biomedical research because of their phylogenetic similarities with humans (silva et al. 2013). new world primates 330 veterinaria italiana 2021, 57 (4), 329-334. doi: 10.12834/vetit.1769.9341.2 hematological and biochemical parameters of spix's saddleback tamarin almeida et al. an assistant using leather gloves, restraining them by the scruff of the neck, captured each individuals at time from the enclosure. before starting physical examinations, each animal was transferred to a small cloth bag to measure body mass, determined by a filizola® scale, using the preset tare device (indústrias filiziola s/a, são paulo, sp, brazil), with a range of 0.05 kg minimum up to 40  kg maximum. clinical evaluation was performed by general inspection, palpation and the measurement of heart and respiratory function. cardiac and pulmonary functions were measured by listening to accurately identify sounds of the heart and lungs, using a classic ii pediatric stethoscope (3m™ littmann®, usa), at the apex of the heart and lung area. rectal temperature was recorded using a digital thermometer (bd-brazil; são paulo, sp) inserted into the rectum for approximately 1 minute. blood samples (2 ml separated into two tubes of 1 ml each) were taken from the femoral vein using sterile syringes and needles. for the hemogram, tubes containing the anticoagulant ethylenediaminetetraacetic acid (edta) were used. for biochemical analysis, blood was collected in vacuum tubes without anticoagulant, left at room temperature for clot retraction and centrifuged at 2,000 g for 10 min. the sera obtained were frozen at - 20 °c until the time of analysis. hematological analises were done at the cenp laboratory. leucocyte counts was carried out using 100 cells in a blood smear colored with panoptic fast (newprov produtos para laboratório ltda, pinhais  pr). biochemical determinations were performed using commercial kits (doles® and labtest®) and a bs-120 automatic biochemical analyzer (shenzhen mindray bio-medical eletronics®, germany). the following values were determined; total protein, albumin, high-density lipoprotein (hdl), triglycerids, urea, creatinine, aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (fa) and gamma glutamyl-transferase (ggt) enzymes. all parameters were analyzed using the komolgorov-smirnov test to determine the normality distribution. descriptive statistics including the average, standard deviation and minimal-maximum values were also used to summarize the data. to avoid the effect of sex on these biochemical and hematological variables, we used the tukey test with a normal distribution. parameters without normality were compared using the mann-whitney test. the significance level was set at p < 0.05. results all subjects were in excellent health status according to the clinical and laboratory exams. individuals with high genetic and health quality for use in biomedical research. in this context, studies aimed to establish reference values for hematological and biochemical parameters in neotropical primates should be encouraged. this is justified by the environmental variation, health, and nutrition, inherent in each breeding system. these data may be used in clinical practice and as tools for research regarding the health of laboratory animals. reference values are generated from healthy animals, by applying standard statistical methods and ratings that represent the estimates within which 95% of clinically determined normal healthy individuals are found (george et  al. 2010). however, there have been very few studies aimed at determining hematological and biochemical parameters of the genus leontocebus. the hematological and biochemical effect of sex and age class have been described in some phylogenetically close species of leontocebus fuscicollis, such as saguinus oedipus (shukan et al. 2012) and saguinus leucopus (fox et al. 2008), but the species in question has thus far not been described. this study aimed to establish the hematological and biochemical parameters of leontocebus fuscicollis considering the effect of sex and body mass in healthy adult animals raised at the national primate center (cenp), pará state, brazil. the prediction is that, under normal handling at cenp, these parameters can influence blood variables. material and methods the spix’s saddleback tamarin, formerly known as saguinus fuscicollis was redescribed and taxonomically reassessed in 2015 based on morphometric and molecular genetic analyses (sampaio et  al. 2015). it was found to differ significantly enough from other members of saguinus that it was assigned to the genus leontocebus. the subjects were kept in captivity at the cenp (ananindeua, pará, brazil, latitude 1°38’26” and longitude 48°38’22”). twenty adult, 3-10 year old, saddleback tamarin (leontocebus fuscicollis; 7  males, 13 females) were evaluated using clinical and laboratory examinations. animals were housed alone or in couples in enclosures (1.5m x 2m x 3m) that were positioned in a north-south orientation to receive 12 h of natural light each day. the average temperature was 33 °c and humidity was 85%. the animals’ diet contained various fruits and vegetables, eggs, milk, and commercial primate food with 180 g/kg crude protein (callitrichidae p25 megazoo, rações megazoo, betim, minas gerais, brazil). water was offered ad libitum. 331veterinaria italiana 2021, 57 (4), 329-334. doi: 10.12834/vetit.1769.9341.2 almeida et al. hematological and biochemical parameters of spix's saddleback tamarin differences were noted (table i). regarding biochemical values, ggt enzyme activity was significantly higher for females (p < 0.05). there were no other statistically significant differences between males and females for any other parameters measured (table ii). discussion hematological parameters have already been established for some old world primate species used in biomedical research (harewood et al. 1999, ibeloni body mass and rectal temperatures showed no significant statistical differences (p > 0.05) between males (0.449 ± 0.027 kg; 40.46 ± 0.27 °c) and females (0.433  ±  0.002 kg; 40.43  ±  0.44 °c), respectively. during the capture process, animals showed no visible signs a stress (urination or defecation). all individuals were born in captivity and were used to frequent handling. values of red blood cells (rbc), hemoglobin (hb) and hematocrit (ht) were significantly higher in males (p < 0.05), while red blood cell distribution width (rdw) was higher in females (p < 0.05). in the other hematological parameters no significant sex table i. hematological parameters expressed as the mean ± standard deviation (sd) and as the range for 20 healthy leontocebus fuscicollis categorized by sex. parameters distribution n sex mean ± sd min.‑max. p value rbc (106/mm) gaussian 7 ♂ 7.12 ± 0.98 5.12‑8.16 0.01 13 ♀ 5.80 ± 0.87 4.22‑6.98 hemoglobin (g/dl) no gaussian 7 ♂ 14.98 ± 1.25 12.80‑16.40 0.01 13 ♀ 12.60 ± 2.09 9.25‑14.80 hematocrit (%) gaussian 7 ♂ 48.71 ± 4.91 38.70‑53.40 0.01 13 ♀ 41.36 ± 6.18 31.90‑50.35 mcv (fl) gaussian 7 ♂ 68.48 ± 3.64 65.40‑75.60 0.30 13 ♀ 71.56 ± 6.08 58.50‑78.60 mch (pg) gaussian 7 ♂ 21.27 ± 2.18 19.50‑25.00 0.55 13 ♀ 21.91 ± 2.31 16.70‑24.50 mchc (g/dl) gaussian 7 ♂ 30.87 ± 1.63 29.30‑33.20 0.66 13 ♀ 30.56 ± 1.36 28.60‑33.10 rdw (%) no gaussian 7 ♂ 12.90 ± 0.61 12.20‑13.80 0.01 13 ♀ 14.58 ± 1.89 12.30‑20.00 platelets (103/mm) no gaussian 7 ♂ 384.71 ± 143.85 206.00‑613.00 0.21 13 ♀ 507.76 ± 214.23 307.00‑934.00 wbc (103/µl) gaussian 7 ♂ 8.76 ± 7.702 2.40‑20.00 0.69 13 ♀ 7.78 ± 3.16 2.70‑13.60 segmented (103/µl) gaussian 7 ♂ 3.85 ± 3.09 0.74‑8.97 0.38 13 ♀ 4.95 ± 2.33 1.16‑9.02 neutrophis (103/µl) gaussian 7 ♂ 0.00 0.00 1.00 13 ♀ 0.00 0.00 lymphocytes (103/µl) no gaussian 7 ♂ 2.73 ± 3.04 1.21‑9.55 0.50 13 ♀ 2.06 ± 1.27 0.38‑4.76 monocytes (103/µl) no gaussian 7 ♂ 0.76 ± 0.86 0.13‑2.60 0.38 13 ♀ 0.72 ± 0.52 0.00‑1.76 eosinophils (103/µl) no gaussian 7 ♂ 0.05 ± 0.15 0.00‑0.40 0.41 13 ♀ 0.02 ± 0.04 0.00‑0.13 basophils (103/µl) gaussian 7 ♂ 0.07 ± 0.07 0.00‑0.20 0.19 13 ♀ 0.05 ± 0.05 0.00‑0.14 mpv (fl) gaussian 7 ♂ 9.84 ± 2.39 7.70‑13.40 0.53 13 ♀ 10.50 ± 2.13 6.24‑13.90 min.‑max. = minimum‑maximum; rbc = red blood cells; n = individual number; ♂ = male; ♀ = female; sd = standard derivation; mcv = mean corpuscular volume; mch = mean corpuscular hemoglobin; mchc = mean corpuscular hemoglobin concentration; rdw = red cell distribution width; wbc = white blood cell. 332 veterinaria italiana 2021, 57 (4), 329-334. doi: 10.12834/vetit.1769.9341.2 hematological and biochemical parameters of spix's saddleback tamarin almeida et al. primates (nhp), including aotus (monteiro et  al. 2009, takeshita et al. 2011, lins et al. 2012) and cebus apella (riviello and wirz 2001, wirz et  al. 2008). for one callitrichid, s. oedipus, shukan and colleagues (shukan et al. 2012) evaluated the same parameters, and also found significant differences between males and females, with females always having lower values. hormonal effects can most likely explain this, e.g. androgens are stimulants of erythropoiesis, while estrogens are inhibitors (harewood et al. 1999, takeshita et al. 2011). red blood cell distribution width (rdw) indicates the degree of anisocytosis of the erythrocyte; its high levels suggest an increase of heterogenity of red blood cell size (comar and silva 2009). in humans, rdw is a useful measure to differentiate several kinds of anemia, for example, regeneration of anemias, due to an increase of reticulocyte number, elevates this index (grotto 2009). our findings demonstrated high rdw values for females, similar to those observed in cebus apella (shukan et al. 2012) and chlorocebus aethiopis (imbeloni et al. 2016). however, the observed difference was small, and possibly of no clinical relevance. et  al. 2016). however, there is little information for neotropical primates (riviello and wirz 2001), especially in the genus leontocebus. there are studies about hematological and biochemical parameters for s. leucopus (fox et al. 2008, castañeda et al. 2013) and s. oedipus (shukan et  al. 2013). the european association of zoos and aquariums (eaza 2010) has determined hematological and biochemical parameters for l. fuscicollis. body temperature is an indicator of animal stress and the absence of fever reflects good general health status (nakamura 2011). in a study with 27 callithrix penicillata, rectal temperature varied between 38.1-40.1  °c and no significant sex effect was observed (pereira and barros 2016), which was similar to the present study. the same authors correlated rectal temperature with hematological values and observed that temperature only influenced lymphocyte count; however, in our analysis, we found no such correlation. in the present study, red blood cells and hematocrit values were higher in males then females (p < 0.05). the influence of sex on these parameters has also been observed in other neotropical non-human table ii. biochemical parameters expressed as the mean ± standard deviation (sd) and as the range for 20 healthy leontocebus fuscicollis categorized by sex. parameters distribution n sex mean ± sd min.‑max. p value ldh (mg/dl) gaussian 7 ♂ 72.71 ± 28.86 32.00‑105.00 0.52 13 ♀ 82.16 ± 32.84 31.00‑123.00 triglycerides (mg/dl) gaussian 7 ♂ 217.71 ± 97.41 119.00‑421.00 0.38 13 ♀ 258.46 ± 95.83 129.00‑495.00 alt (u/l) gausian 7 ♂ 74.57 ± 18.55 52.00‑108.00 0.39 13 ♀ 67.75 ± 15.04 38.00‑86.00 ast (u/l) gaussian 7 ♂ 67.71 ± 34.73 12.00‑111.00 0.36 13 ♀ 83.16 ± 34.42 0.00‑126.00 creatinine (mg/dl) gaussian 7 ♂ 0.94 ± 0.18 0.70‑1.30 0.64 13 ♀ 0.87 ± 0.34 0.00‑1.27 fa (u/l) gaussian 7 ♂ 246.33 ± 18.46 220.00‑272.00 0.07 13 ♀ 358.91 ± 137.97 71.00‑601.00 ggt (u/l) no gaussian 7 ♂ 2.14 ± 1.57b 0.00‑4.00 0.01 13 ♀ 8.08 ± 4.87 0.00‑15.00 glucose (mg/dl) gaussian 7 ♂ 216.85 ± 52.66 173.00‑309.00 0.21 13 ♀ 287.23 ± 137.70 102.00‑521.00 tp (mg/dl) gaussian 7 ♂ 7.68 ± 0.53 6.90‑8.40 0.54 13 ♀ 7.38 ± 1.19 5.26‑10.0 albumin (mg/dl) no gaussian 7 ♂ 2.91 ± 1.10 0.5‑3.60 0.40 13 ♀ 3.17 ± 0.65 1.79‑3.88 urea (mg/dl) gaussian 7 ♂ 30.56 ± 5.97 23.30‑39.60 0.27 13 ♀ 27.4 ± 5.78 18.10‑38.10 min.‑max. = minimum‑maximum; n = individual number; ♂ = male; ♀ = female; sd = standard derivation; alt = alanine aminotransferase; ast = aspartate aminotransferase; ggt = gamma‑glutamyl transferase; alp = alkaline phosphatase; tp = total protein; ldh = lactate dehydrogenase. 333veterinaria italiana 2021, 57 (4), 329-334. doi: 10.12834/vetit.1769.9341.2 almeida et al. hematological and biochemical parameters of spix's saddleback tamarin their investigation of 27 adult s. leucopus, reported significant differences between males and females for total protein, albumin, hemoglobin, hgm, glucose and alkaline phosphatase. these results were also consistent with the present study. conclusions our results can be useful as a reference tool for interpreting the health of callitrichids being used in laboratory conditions, especially using l. fuscicollis in captivity. however, it is necessary to evaluate these markers in different conditions of nutrition and climatic stress, since these factors correspond to challenges of an animal’s adaptation to its environment in captivity. further investigation will help to promote the best welfare conditions to help perpetuate the species survival. acknowledgments we are grateful to the national primate center (cenp), the evandro chagas institute (iec), the coordination for support from the improvement of higher level personnel (capes) program, and the national council of technological and scientific development (cnpq). statement of animal rights all procedures that use animals were in accordance with the norms established by the national council of control of animal experimentation (concea) of brazil. all experimental procedures were registered on the biodiversity authorization and information system of the chico mendes institute of biodiversity (sisbio/icmbio, protocol number 47969-1) and was approved by the ethics committee of animal use at the evandro chagas institute (ceua/iec, protocol number 17/2015). the global leucocyte values were similar to those found in c. penicillata (boere et  al. 2005). in the present study, leucocyte count did not differ between males and females, as previously observed in the same species by boere and colleagues (boere et  al. 2005) and in c. jacchus by cunha and colleagues (cunha et  al. 2005). in general, sex did not influence leucocyte count. the alterations of global leucometry occur, principally, in response to bacterial and virus inflammations, allergies, stress and hematological neoplasms, such as leukemia (mcpherson 2013). in c.  jacchus, stress significantly increased leucocyte count in both males and females (pereira and barros 2016). regarding biochemical parameters, significant sex differences were only found for ggt. the results reported by riviello and wirz (riviello and wirz 2001) and wirz and colleagues (wirz et al. 2008) for c. apella showed significant sex differences for ast, ggt, urea and creatinine. however, these authors did not discuss the influence of sex on the activity of these parameters. beyond sex differences, age, nutrition, management conditions and housing also should influence the biochemical parameters of different nhp species (mcpherson 2013). the average value of glucose in the animals studied (225 ± 121.21 mg/dl) was higher than other studies of f. fuscicollis (173.00 ± 66.00 mg/dl, eaza 2010) and other species of callitrichids, such as s. leucopus (134.27 ± 54.59 mg/dl, castañeda et  al. 2013) and c. jacchus (192.00 ± 52.00 mg/dl, clarke 1994). however, some studies detected average glucose concentrations higher than reported in this study, where we can cite s. oedipus (266.00  ±  93.64  mg/ dl, shukan et  al. 2012) and c. penicillata (228.55 ± 50.37 mg/dl, boere et al. 2005). our results are consistent with all of the above mentioned studies; reporting no significant differences between males and females with regards to these biochemical parameters. fox and colleagues (fox et  al. 2008), in 334 veterinaria italiana 2021, 57 (4), 329-334. doi: 10.12834/vetit.1769.9341.2 hematological and biochemical parameters of spix's saddleback tamarin almeida et al. abee c.r., mansfield k., tardif, s. & morris t. 2012. nonhuman primates in biomedical research: biology and management. 2nd ed. elsevier, san diego. acevedo-garcés y.a., álvarez-cardona j., vargas-valencia v., hernández-castro c., garcía-montoya g.m. & soto-calderón i.d. 2014. clinical and parasitological evaluation of white-footed tamarins (primates: cebidae: saguinusleucopus) from two free-range populations located in 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parameters in owl monkeys infected and uninfected with trypanoxyuris sp. j helminthol, 83, 225-229. nakamura k. 2011. central circuitries for body temperature regulation and fever. am j physiol regul integr comp physiol, 301, 207-228. riviello m.c. & wirz a. 2001. haematology and blood chemistry of cebus apella in relation to sex and age. j med primatol, 30, 308-312. rogers l.b., kaack m.b., henson m.c., rasmussen t., henson e., veazey r.s., krogstad d.j. & davison b.b. 2005. hematologic and lymphocyte immunophenotypic reference values for normal rhesus monkey (macaca mulatta) umbilical cord blood; gravidity may play a role in study design. j med primatol, 34, 147-153. sampaio r., röhe f., pinho g., silva-júnior j.s., farias i.p. & rylands a.b. 2015. re-description and assessment of the taxonomic status of saguinus fuscicollis cruzlimai hershkovitz, 1966 (primates, callitrichinae). primates, 56, 131-144. shukan e.t., boe c.y., hasenfus a.v., pieper ba. & snowdon c.t. 2012. normal hematologic and serum biochemical values of cotton-top tamarins (saguinus oedipus). j am assoc lab anim sci, 51, 150-154. takeshita r.s.c., monteiro f.o.b., de miranda lins e lins f.l., da silva g.a., faturi c., coutinho l.n., monteiro m.v.b., kugelmeier t., de castro p.h.g. & muniz j.a.p.c. 2011. hematological, hepatic, and renal evaluation in aotus azarai infulatus. j med primatol, 40, 104-110. wirz a., truppa v. & rivielo m.c. 2008. hematological and plasma biochemical values for capitive tufted capuchin monkeys (cebus apela). am j primatol, 70, 463-472. 137 short communication even if prohibited in the european union by the council directive 96/23/ec1, illicit treatments in beef cattle still represent a health safety problem. the rapid metabolism and elimination of these substances and the use of undetectable molecules can sometimes render the chemical investigations inconclusive (imbimbo et  al. 2012). histological examination, introduced in italy as screening test as part of the national residues plan (pnr ‑ ministry of health)2, still plays a key role to detect illegal treatments.it provides proof of morphological and functional alterations of the thymus caused by the illegal use of corticosteroids with relatively low costs and in a short time (castagnaro et al. 2006). treatments with corticosteroids cause early involution of the thymus, identifiable as both gross and microscopic lesions (cannizzo et  al. 2008). thymus atrophy is considered a lasting indirect biomarker for the detection of treated animals (vascellari et  al. 2012), but recent findings seem to indicate that the cortex/medulla ratio (c/m) is strongly related to steroidal treatment in both veal calves and beef cattle, and that it is a more reliable 1laboratory of veterinary and comparative histopathology, istituto zooprofilattico sperimentale dell’umbria e delle marche ‘togo rosati’, via g. salvemini 1, 06126 perugia, italy. 2igiene degli allevamenti e delle produzioni zootecniche, azienda sanitaria locale umbria 1, piazza del tabacchificio 8, 06083 bastia umbra, italy. 3department of agricultural and environmental sciences, food and environmental sciences, university of perugia, via borgo xx giugno 74, 06121 perugia, italy. *corresponding author at: laboratory of veterinary and comparative histopathology, istituto zooprofilattico sperimentale dell’umbria e delle marche ‘togo rosati’, via g. salvemini 1, 06126 perugia, italy. tel.: +39 0753431, fax +39 075343286, e‑mail: e.manuali@izsum.it. keywords chianina cattle, thymus, cortex/medulla ratio. summary chianina is an italian cattle breed appreciated for its meat and resilience skills. no standard values are present in literature regarding chianina thymic involution. a possible early physiological involution has been reported during the italian national residue plan screening tests. the aim of this work was to perform an anatomo‑histopathological study of the thymus in chianina cattle to improve knowledge about thymic involution in this breed. forty chianina bulls (16‑24 months old), never treated with corticosteroids and regularly slaughtered in the umbria region (italy), were enrolled. animals aged 19‑21 months which received score 3 thymic atrophy had a prevalence of 0.15 (ci 95%: 0.02‑0.45%), while the prevalence was 0.29 (ci 95%: 0.10‑0.56%) among animals aged 22‑24 months. the thymus/ carcass weight and thymic cortex/medulla ratio resulted close to those reported in cattle experimentally challenged with corticosteroids. results suggest that the chianina breed could be characterized by a physiological premature involution of the thymus gland in comparison to other breeds. these results represent a starting point to increase the reliability of the national residue plan histological screening test. martina sebastianelli1, silvia pavone1, claudio forte1, stefano mezzasoma2, maurizio bordoni2, andrea onofri3, anna fratto1 and elisabetta manuali1* preliminar histological study of the thymus in regularly slaughtered chianina beef cattle to improve methods for the detection of illicit treatments veterinaria italiana 2020, 56 (2), 137‑140. doi: 10.12834/vetit.1624.8712.1 accepted: 01.03.2019 | available on line: 31.12.2020 1 council directive 96/22/ec of 29 april 1996 concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of ß‑agonists, and repealing directives 81/602/eec, 88/146/eec and 88/299/eec. 2 ministero della salute: relazione finale. piano nazionale residui 2009. roma, italy: 1‑63. www.salute.gov.it/imgs/c_17_pubblicazioni_1296_allegato.pdf. 138 veterinaria italiana 2020, 56 (2), 137‑140. doi: 10.12834/vetit.1624.8712.1 thymic involution in chianina beef cattle manuali et al. the thymus were examined at low magnification (5x) using a nikon ds‑fi1 digital camera (nikon corporation, tokyo, japan) connected to the microscope leica dmr (leica microsystems, milan, italy), using nis‑elements br‑2 as software. the cortex/ medulla ratios (c/m) were calculated as reported by vascellari and colleagues (vascellari et al. 2012). statistical analysis was carried out using r 2.7  version software. descriptive statistics and box plots to graphically depict the groups of numerical data were performed. kruskal‑wallis followed by pairwise comparisons made with wilcoxon's rank‑sum test were used to compare histological and morphometrical data. the p value of 0.05 was the assumed significance level. a beta regression model was used to determine correlation between thymus weight/carcass weight ratio, age and atrophy score. no differences were observed through the beta regression model when comparing weight data with the degrees of thymic atrophy of the samples. a negative correlation between age and t/c ratio was registered, in line with the normal physiologic pattern of thymic involution (table i). the average value of 0.164 observed for thymus weight/carcass weight ratio in younger animals (16‑18 months) is lower than that found in 5 months old veal calves experimentally treated for 21 days with dex (t/c  =  1.75) (cannizzo et  al. 2011); in 17‑22‑month‑old charolaise beef cattle challenged with dex for 40 days, the average t/c value was 0.29 (cannizzo et al. 2011). comparing this value with the result (t/c  =  0.134) obtained on the same age class (19‑21 months), it is clear that the value showed by untreated chianina bulls is still lower than that of the treated charolaise, confirming a remarkable thymic involution in the untreated chianina bulls included in the study. a total of 21 samples (52.5%) showed slight thymic atrophy due to mild infiltration of adipose tissue (score 1) (figure 1a), 12 samples (30%) showed moderate fatty infiltration and initial replacement of the cortex (score 2) (figure 1b), and 7 samples (17.5%) showed severe adipose infiltration and marked biomarker than the evaluation of fatty infiltration in the thymic parenchyma (bozzetta et al. 2011). knowledge concerning the modifications of the thymus in beef bulls treated with corticosteroids is still fragmentary (cannnizzo et  al. 2010), and there is an absolute lack of studies on the employment of these molecules in the national breeds. the experience gained during the pnr highlighted the need to clarify the meaning of the microscopic appearance of the thymus in young beef bulls belonging to the chianina breed. severe atrophy of the thymus is frequently observed in chianina bulls even though illicit treatments with corticosteroids have always been excluded by liquid chromatography tandem mass spectrometry (lc‑ms/ms). it is possible to suspect that early physiological involution of the thymus in chianina bulls is likely related to genetic factors. the aim of this work was to perform an anatomo‑histopathological study of the thymus in regularly slaughtered chianina beef cattle in the umbria region (italy), improve general knowledge about the physiological pattern of thymic involution in this breed and to increase the reliability of the pnr histological screening test. a total of 40 chianina beef bulls aged between 16 and 24 months, slaughtered in the umbria region (italy), were enrolled in this study. the animals came from two selected farms included in the pnr that resulted always negative for residues of corticosteroids when periodically checked. from documental analysis, conducted in the farms’ treatment registers and veterinary prescriptions, it can be asserted that animals have never been subjected to corticosteroid or antimicrobic therapy. at slaughtering, cervical and thoracic portions of the thymus of each animal were collected and weighed. the thymus weight/carcass weight ratio was calculated as suggested by biolatti and colleagues (biolatti et  al. 2005). the central area of the thoracic thymus of each animal was collected and fixed in 10% neutral buffered formalin. the tissue samples were paraffin‑embedded and stained with haematoxylin and eosin (he). the morphology of the thymic parenchyma was evaluated for adipose tissue infiltration associated with cortical atrophy, according to bozzetta and colleagues (bozzetta et  al. 2011), and vascellari and colleagues (vascellari et  al. 2012), and a score from 1 to 3 (mild, moderate, severe) was attributed, as pnr guidelines request. the scoring system was blindly performed by three independent observers and inter‑laboratory agreement was evaluated by k cohen test (k  cohen  =  0.84), confirming the histological grading assigned. for the morphometrical evaluation, sections of table i. chianina beef bulls. average values of thymus weight, carcass weight, and t/c ratio for each age and relative sem and p. age (months) thymus weight (g) carcass weight (kg) t/c 16-18 600.60 a 370.10 a 0.164 a 19-21 537.20 ab 408.00 ab 0.134 ab 22-24 494.18 b 460.81 b 0.108 b sem 25.865 20.851 0.008 p 0.0495 0.0154 0.0007 t/c = thymus weight/carcass weight ratio; sem = standard error of mean. 139veterinaria italiana 2020, 56 (2), 137‑140. doi: 10.12834/vetit.1624.8712.1 manuali et al. thymic involution in chianina beef cattle (ci  95%: 0.02‑0.45%), while the prevalence of score 3 among animals aged 22‑24 months was 0.39 (ci 95%: 0.10‑0.56%). the results of the morphometric investigations showed a mean value of thymic c/m diameter ratio of 0.6 for score 3 samples. this value was similar or lower than that reported in animals experimentally treated with corticosteroids (0.53 in charolaise beef cattle, vascellari et al. 2012; 1 for veal calves, bozzetta et  al. 2011). even if the negative results derived from chemical and documentary investigations carried out during the official checks excluded the hypothesis of illicit treatments, considering the histological screening tests currently in use, 17.5% cortex atrophy that often reduced the medullary framework, up to the complete replacement of the lymphoid tissue by fat (score 3) (figure 1c). the distribution of the degrees of atrophy according to age is shown in table ii, while descriptive statistics for c/m ratio is provided in figure 2. none of the animals aged 16‑18 months received score  3 (ci  95%: 0‑0.03%). animals aged 19‑21 months which received score 3 had a prevalence of 0.15 figure 1. a. slight thymic atrophy due to mild infiltration of adipose tissue (score 1). b. moderate fatty infiltration and initial replacement of the cortex (score 2). c. severe adipose infiltration and marked cortex atrophy that reduces the medullary framework, up to the complete replacement of the lymphoid tissue by fat. remnants of the parenchyma are represented solely by small islets of lymphocytes, positioned around the vessels that run through the adipose tissue (score 3). bars 200 µm. 1 0 1 2 3 4 5 2 thymic scores c o rt ex /m ed u lla r at io s in t h e th ym ic lo b u le 3 figure 2. correlation between atrophy score categories and cortex/ medulla ratio values. table ii. chianina beef bulls. prevalence data of thymus atrophy score for each age and relative 95% confidence interval. age (months) thymus atrophy score prevalence ci (95%) 16-18 1 0.80 0.44-0.97 2 0.20 0.03-0.56 3 0.00 0.00-0.31 19-21 1 0.54 0.25-0.81 2 0.31 0.09-0.61 3 0.15 0.02-0.45 22-24 1 0.35 0.14-0.62 2 0.35 0.14-0.62 3 0.39 0.10-0.56 thymus atrophy score: 1 mild infiltration of adipose tissue at the periphery of the lobules, 2 moderate fat interstitial infiltration with thinning and initial replacement of the cortex, 3 severe adipose infiltration of the parenchyma and marked cortex atrophy that reduces the medullary framework, up to the complete replacement of the lymphoid tissue by fat. ci = confidence interval. 140 veterinaria italiana 2020, 56 (2), 137‑140. doi: 10.12834/vetit.1624.8712.1 thymic involution in chianina beef cattle manuali et al. avoid overestimating the problem of illicit treatment with css in this breed and to report false positive cases in terms of screening. our study showed an early thymic gland involution in chianina beef cattle and provided the first data for this breed. further studies should be performed on a greater number of animals held under controlled conditions, including challenges with corticosteroids, aimed at defining the complete physiological involution process of the thymus gland in the chianina breed. of the animals included in this study may have been illegally treated with corticosteroids. we used suitable samples and proposed a valuable tool to verify the normal involution process of the thymus in the chianina breed, contributing to the identification of the different age‑dependent involution patterns that should be considered normotypical in this breed. the purpose of this study was also to improve the reliability of the histological method adopted within pnr screening tests, to biancotto g., stella r., pozza g., stefani a., lega f. & angeletti r. 2012. sub‑therapeutic treatments of bulls with dexamethasone: direct and indirect markers of treatment. food addit contam, 30 (3), 430‑442. biolatti b., bollo e., cannizzo f.t., zancanaro g., tarantola m., dacasto m., cantiello m., carletti m., biolatti p.g. & barbarino g. 2005. effects of low‑dose dexamethasone on thymus morphology and immunological parameters in veal calves. j vet med a, 52, 202‑208. bozzetta e., pezzolato m., maurella c., varello k., richelmi g.b., draisci r., ferranti c., d’angelo a. & caramelli m. 2011. development of an enhanced histopathological approach to detect low‑dose dexamethasone illicit treatment in veal calves. food addit contam, 28, 1187‑1192. cannizzo f.t., miniscalco b., riondato f., bollo e., barbarino g., giorgi p., mazzini c. & biolatti b. 2008. effects of anabolic and therapeutic doses of dexamethasone on thymus morphology and apoptosis in veal calves. vet rec, 163, 448‑452. references cannizzo f.t., capra p., divari s., ciccotelli v., biolatti b. & vincenti m. 2011. effects of low‑dose dexamethasone and prednisolone long term administration in beef calf: chemical and morphological investigation. anal chim acta, 700, 95‑104. castagnaro m. & poppi l. 2006. indirect biomarkers of illegal anabolic treatments: five years of activity under the microscope. vet res commun, 30 (suppl. 1), 105‑108. imbimbo p., castigliego l., armani a., biolatti b., cannizzo f.t., gianfaldoni d. & guidi a. 2012. a histologic study on growth promoter target organs of slaughtered beef in molise region (italy). j vet med sci, 74 (10), 1253‑1259. vascellari m., capello k., stefani a., biancotto g., moro l., stella r., pozza g. & mutinelli f. 2012. evaluation of thymus morphology and serum cortisol concentration as indirect biomarkers to detect low‑dose dexamethasone illegal treatment in beef cattle. bmc vet res, 8, 129. 43 introduction cryptosporidium is an intracellular protozoan parasite of a phylum of apicomplexa. ernest edward tyzzer first identified and reported the parasite, found frequently in the gut of the laboratory mice (tyzzer 1907). this parasite causes acute and self‑limiting gastroenteritis in humans. the parasite is detected in healthy individuals whereas persistent and fatal infection can be observed in immunocompromised individuals. it is estimated that millions of cases of disease occur annually in developing and developed countries (putignani and menichella 2010). moreover, this parasite has been reported as one of the most common pathogens in human intestine (mendonca et al. 2007, paul et al. 2009). the genus cryptosporidium includes a variety of species found in many domestic animals and human (dillingham et  al. 2002, fayer and ungar 1986, kvac and vitovec 2003, mendonca et al. 2007, o'donoghue 1995). this protozoan often detected in calves, lambs, piglets, horses, puppies and kittens, chicken and turkeys, with diarrhea. this study aimed to establish cryptosporidium prevalence and species identification in dairy cattle farms located in mashhad, northeast of iran. materials and methods sampling in this study, a cross‑sectional study with single random sampling was done. a total of 800 stool samples were collected from healthy and diarrheal (from one month before the beginning of testing sampling) holstein cattle in the city of mashhad, within different age groups (less than 6 months, between 6‑18 months and more than 18 months) from 2011 to 2015. individuals with diarrhea among those showing diarrhea for a month before sampling. direct microscopic detection the specimens were stained by modified ziehl‑neelsen method and observed with a microscope (100x). samples were then stored in a freezer at ‑ 20 °c. dna extraction genomic dna was extracted with two different procedures, a standard phenol‑chloroform procedure [h.e. mckiernan, p.b. danielson, in razi vaccine & serum research institute, agricultural research, education and extension organization, mashhad, islamic republic of iran *corresponding author at: razi vaccine & serum research institute, agricultural research, education and extension organization, mashhad, islamic republic of iran. e‑mail: a.sadr@rvsri.ac.ir. keywords cryptosporidium andersoni, cattle, pcr‑rflp, mashhad, iran. summary cryptosporidium is an intracellular and extracytoplasmic protozoan that belongs to the phylum apicomplexa. in this observational study, fecal samples were randomly collected from 800 dairy cattle, in 10 industrial dairy farms in mashhad, iran (from 2011 to 2015 years). the presence of cryptosporidium oocysts was determined by modified cold ziehl‑neelsen’s staining. results of microscopy observation showed that 23 samples (2.87%) were positive for cryptosporidium oocyst. twenty two samples were confirmed by pcr. the identification of cryptosporidium andersoni was determined by restriction digestions of pcr products, using sspi, vspi, and ddei enzymes. differences between healthy and diarrheic groups as well as between age groups were not observed. alireza sadrebazzaz molecular identification of cryptosporidium andersoni in healthy and in cattle with diarrhea of mashhad, northeast of iran veterinaria italiana 2020, 56 (1), 43‑46. doi: 10.12834/vetit.1771.9343.3 accepted: 30.07.2019 | available on line: 24.04.2020 44 cryptosporidium andersoni in cattle of iran sadrebazzaz veterinaria italiana 2020, 56 (1), 43‑46. doi: 10.12834/vetit.1771.9343.3 differences (p < 0.05) by statistix for windows, trial version 9.0 (analytical software, tallahassee, fl 32317, usa). results no differences have been observed between the two extraction methods in terms of final dna concentration. results of microscopic tests 23/800  samples were positive for cryptosporidium oocysts. analysis according to health status and age is reported in table i and ii. pcr‑rflp data out of 23 samples tested positive by zn, 22 were correctly amplified by the adopted nested pcr. the pcr products were then digested with the sspi and demonstrated similar products (385 bp and 448 bp); with the vspi enzyme showing two bands of 731 bp and 102  bp, and with the ddei enzyme showing three bands of 470 bp, 186 bp and 156 bp. all the 22  samples were identified positive for cryptosporidium andersoni (2.75%). statystical analysis no significant difference was found between age groups, and between healthy and diarrheal cows, using chi‑square test (overall x2  =  0.07, p‑value = 0.7847, d.f. = 1). it can be said that cryptosporidium andersoni causes the same infection in all age groups (overall x2 = 1.27, p‑value = 0.5292, d.f. = 2) . molecular diagnostics (third edition), 2017] and a nucleospin® tissue kit (mn), following manufacturer's instructions, from 23 positive samples by modified ziehl‑neelsen method. pcr‑restriction fragment length polymorfism (rflp) cryptosporidium oocysts were identified at species level using a nested pcr of 18s rrna gene followed by rflp (xiao et al. 1999). primary pcr dna from positive samples was amplified using the protocol described by xiao and colleagues (xiao et al. 1999). the pcr master mix was prepared by using the accupower® pcr premix kit (bioneer, korea). the pcr master mix reaction was prepared according to kit instructions in 40 µl total volume by adding 1 µl of purified genomic dna and 1 µl of 10 pmol/µl of f1 and 1 µl of 10 pmol/µl of r1 and briefly mixed. the following thermal profile was used: denaturation at 95 °c for 3 minutes, followed by 35 cycles of denaturation at 94  °c for 45  seconds, annealing at 55 °c for 45 seconds, extension at 72 °c for 1 minute, and then a final extension at 72 °c for 7 minutes. then, pcr products were loaded on 1.5% agarose gel stained with sybr safe, and electrophoresed. the bands were visualized by uv and photographed using the gel doc system. secondary pcr by using published primers (xiao et al. 1999) the following thermal profile was used: initial denaturation at 94  °c for 3 minutes, followed by 35  cycles of denaturation 94 °c for 45 seconds, annealing at 58 °c for 45 seconds, extension at 72 °c for 1 minute, and then a final extension at 72  °c for 7 minutes. rflp a total volume of 10 μl of the secondary pcr product was digested for 2 h at 37  °c with 10 u of each enzyme, sspi, ddei and vspi (fermentas) in 32 μl following manufacturer’s instruction. the digested products were separated on a 2% agarose gel electrophoresis using sybr safe staining and photographed by uv transilluminator. statistical analysis the chi‑square test was used to evaluate significant table i. prevalence of cryptosporidium spp. infection in cattle by age in mashhad, northeast of iran. age group (months) n. of cattle cryptosporidium spp. positive prevalence (%) ≤ 6 220 6 2.72 6-18 200 8 4 ≥ 18 380 9 2.36 total 800 23 2.87 table ii. prevalence of cryptosporidium spp. infection in healthy and diarrheic cattle in mashhad, northeast of iran. group n. of cattle cryptosporidium spp. positive prevalence (%) healthy 500 15 3 diarrheic 300 8 2.66 total 800 23 2.87 veterinaria italiana 2020, 56 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx 45 sadrebazzaz cryptosporidium andersoni in cattle of iran be used as suggested methods in laboratories for veterinary and medical use. the rate of cryptosporidium infections in this study was consistent with the contamination rate found in china, 5.12%, in sweden 1.8%, in wales 6.9% and in japan 1.5%, in india 12.85%, brazil 5.88%, portugal (4.5% in adult cattle), western czech republic (4.1%) (fiuza et  al. 2011, koyama et  al. 2005, kvac and vitovec 2003, mendonca et al. 2007, ondrackova et  al. 2009, paul et  al. 2009, robinson et al. 2006, silverlas et al. 2010, wang et al. 2011). the consistency of the results of the above‑mentioned studies with our data suggests that cryptosporidium andersoni has a relatively high rate in animal contamination in different countries. in iran, similar contamination rate was found in different cities, such as isfahan, tehran, tabriz, kerman and ahvaz (fotouhi ardakani 2008, nouri et al. 1995, nouri 2003). according to other studies and considering the role of cryptosporidium andersoni in cattle industry prevention methods should be considered. acknowledgment the authors thank dr. gereon schares (friedrich‑loeffler‑institut, federal research institute for animal health, institute of epidemiology, 17493 greifswald‑insel riems, germany) for valuable comments on the manuscript. we also acknowledge and thank dr lihua xiao (division of foodborne, waterborne and environmental diseases, national center for emerging and zoonotic infectious diseases, centers for disease control and prevention, atlanta, ga, united states) for valuable and practical comments. this study was financially supported by the razi vaccine and serum research institute, agricultural research, education and extension organization (areeo), mashhad, iran. nucleotide sequence accession number one of the sequences used in this study has been deposited in the genbank database under the accession no. mh395839. discussion molecular techniques for the detection and differentiation of cryptosporidium oocysts, along with conventional methods such as condensation and staining of stool specimens have significantly increased our understanding of the parasite dispersion and epidemiology. selection of molecular targets for species identification of the parasite should be performed appropriately, as the parasite genome plays an important role in the interpretation of the information obtained by the pcr method and real‑time pcr (burnet et  al. 2013, morgan et  al. 1995). molecular tools for cryptosporidium identification at species level can provide valuable information about the detection of the protozoa in different hosts, and help to recognize the epidemiology of cryptosporidiosis (blears et  al. 2000, fayer and xiao 2008, fiuza et  al. 2011, ondrackova et al. 2009, xiao et al. 2001). according to the present study, from 800 samples from dairy farms in the city of mashhad, 23  samples were positive for cryptosporidium by direct microscopy. of these 23 samples, using the pcr‑rflp procedure, as a very efficient, relatively simple and cost‑effective method, 22 samples were positive for cryptosporidium andersoni. these results confirmed the contamination rate of cows in the city of mashhad and cryptosporidium andersoni as dominant species. it is worth noting that, due to the successful dna extraction from 22 samples out of 23 positive samples, the manual dna extraction method and the mn kit have a same results for dna purity and concentration using a nanodrop spectrophotometer and can 46 cryptosporidium andersoni in cattle of iran sadrebazzaz veterinaria italiana 2020, 56 (1), 43‑46. doi: 10.12834/vetit.1771.9343.3 blears m.j., pokorny n.j., carreno r.a., chen s., de grandis s.a., lee h. & trevors j.t. 2000. dna fingerprinting of cryptosporidium parvum isolates using amplified fragment length polymorphism (aflp). j parasitol, 86, 838‑841. burnet j.b., ogorzaly l., tissier a., penny c. & cauchie h.m. 2013. novel quantitative taqman real‑time pcr assays for 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negli organi di cigni reali naturalmente infetti con i sottotipi h5n1 e h5n8 del virus dell'influenza aviaria ad alta patogenicità. l'esame è stato condotto su 22 carcasse di cigni reali morti durante i focolai di influenza aviaria in serbia nel 2006 e nel 2016-2017. il ceppo h5n8, isolato nel biennio 2016-2017, appartiene al clade genetico 2.3.4.4 gruppo  b. dopo la necroscopia, polmone, fegato, milza, pancreas, reni e tessuti cerebrali sono stati campionati per gli esami istopatologici e immunoistochimici. per rilevare l'antigene virale sono stati utilizzati anticorpi policlonali nei confronti della nucleoproteina del virus dell'influenza aviaria. le lesioni macroscopiche più significative sono state necrosi ed emorragie nel pancreas. le principali lesioni istologiche sono state necrosi multifocali nel pancreas, milza e fegato; encefalite non purulenta; congestione polmonare ed edema. in entrambi i gruppi infetti, i risultati dell'indagine immunoistochimica confermavano le lesioni istologiche del pancreas e del cervello. i risultati hanno dimostrato che il pancreas è stato l'organo più colpito in tutti i cigni reali esaminati e che oltre ad un aumento della mortalità, le lesioni patologiche rilevate nei cigni reali naturalmente infetti con virus influenzali ad alta patogenicità sottotipi h5n1 e h5n8 sono simili. lesioni macro e microscopiche rilevate in cigni reali infettati con virus influenzali ad alta patogenicità sottotipi h5n1 e h5n8 in serbia keywords h5n1, h5n8, pathological lesions, mute swans, pancreas, serbia. summary the aim of this study was to compare pathological lesions and viral antigen expression in the organs of mute swans (cygnus olor) naturally infected with highly pathogenic avian influenza virus subtypes h5n1 and h5n8. the examination was conducted on the carcasses of 22 mute swans which died during the avian influenza outbreaks in serbia in 2006 and 2016-2017. avian influenza virus subtype h5n8 isolated from mute swans in 2016-2017 was clustered within the 2.3.4.4 clade group b. after necropsy, lung, liver, spleen, pancreas, kidney and brain tissues were sampled for histopathology and immunohistochemical examination. avian influenza virus nucleoprotein polyclonal antibodies were used for detecting the viral antigen in the examined tissues. the most significant gross lesions were necrosis and haemorrhages in the pancreas. major histological lesions were multifocal necroses in the pancreas, spleen and liver, non-purulent encephalitis, lung congestion and oedema. immunohistochemical demonstration of hpaiv nucleoprotein in pancreas and brain was strongly consistent with histological lesions in both infected groups. our findings showed that pancreas was the most affected organ in all examined mute swans. in addition to increased mortality rate, similar pathological findings were detected in mute swans naturally infected with highly pathogenic avian influenza viruses h5n1 and h5n8. veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 accepted: 29.10.2018 | available on line: 31.03.2019 1scientific veterinary institute “novi sad”, rumenački put 20, novi sad, serbia. 2faculty of veterinary medicine, university of belgrade, serbia. 3veterinary specialistic institute “kraljevo”, serbia. *corresponding author at: scientific veterinary institute “novi sad”, rumenački put 20, novi sad, serbia. tel.: +381 643048001, e‑mail: biljana@niv.ns.ac.rs. biljana božić (đurđević)1*, ivana vučićević2, vladimir polaček1, nikola vasković3, tamaš petrović1, marko pajić1 and sanja aleksić-kovačević2 comparative pathological findings in mute swans (cygnus olor) naturally infected with highly pathogenic avian influenza viruses h5n1 and h5n8 in serbia short communication 96 veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 h5n1 and h5n8 infections in mute swans božić et al. to date, outbreaks of highly pathogenic avian influenza viruses (hpai) occurred twice in serbia: in 2006 and 2016. these outbreaks occurred in wild birds as well as in domestic poultry. during these outbreaks, there were no significant economic losses in the poultry industry. in february 2006, in the laboratory of the veterinary institute “kraljevo”, serbia, the presence of the highly pathogenic avian influenza virus, subtype h5n1, was confirmed in a swan carcass (vasković et  al. 2011). ten years later, increased mortality among mute swans (cygnus olor) has been detected since the end of november 2016, in vojvodina province. on november 30th, 2016 in the laboratory for virology of the scientific veterinary institute “novi sad”, serbia, the presence of the hpai subtype h5n8 was confirmed in a swan carcass. genetic analyses of hpai h5n8 showed that this virus belongs to clade 2.3.4.4 group b. this report of the outbreak was the first official notification of hpai in serbia since the epidemic of h5n1 in 2006. during these outbreaks, swans appeared to be highly susceptible and represented mainly reported affected species. also, mute swans were the only wild bird species showing neurologic symptoms, including torticollis, ataxia, and incoordination (božić et  al. 2016). the purpose of this study was to describe and compare pathological findings, presence and type of histological lesions and viral antigen distribution in mute swans that were naturally infected with hpai viruses h5n1 and h5n8, in the republic of serbia. fifteen adult mute swans (cygnus olor) that died during the hpai h5n8 outbreak have been analysed in this study. mute swan carcasses were collected during avian influenza epidemic outbreaks from november 2016 to january 2017. all of the cases occurred either in the area of special nature reserve “koviljsko petrovaradinski rit” or in nearby areas, along with the bank of the river danube in northern serbia, province of vojvodina. morphologic lesions and viral nucleoprotein expression in seven mute swans naturally infected with hpai h5n1 (tissue samples collected during the h5n1 outbreak 2006) were re-evaluated (vasković et al. 2011) in order to be compared with morphological lesions and viral nucleoprotein expression in fifteen h5n8+ swans (h5n8 outbreak, 2016-2017). the detection of h5n8 hpai virus presence was performed in pooled 10% tissue suspensions (lungs, brain, duodenum, and kidney) and selected cloacal content in pbs by molecular diagnostic methods. briefly, total rna was extracted using trizol® reagent (invitrogen, life technologies) according to the manufacturer recommendations. the presence of avian influenza virus and confirmation of virus subtype h5 was detected by identification of highly pathogenic avian influenza (hpai) viruses are considered to be a major concern worldwide because of their ability to cause significant mortality rates in the poultry industry, their pandemic potential and ability to infect humans. the diversity of influenza viruses in avian species and viral transmission between poultry and wild birds can result in genomic reassortment, making the new generations of influenza virus subtypes. currently, the influenza  a virus has been subtyped into 18  hemagglutinin (h1-18) and 11 neuraminidase (n1-11) (tong et  al. 2013). to date, all the avian influenza virus subtypes were isolated from aquatic birds, with the exception of h17n10 and h18n11 that are found in bats (shi et al. 2014). avian influenza virus can infect domestic poultry and a wide range of avian species (webster et  al. 1992, alexander 2000). wild birds of the orders anseriformes and charadriiformes are considered as natural reservoirs of low pathogenic avian influenza (lpai) viruses. lpai viruses of subtypes h5 and h7 once they are introduced into poultry flocks may evolve into hpai (highly pathogenic avian influenza) viruses. up to worldwide massive hpai h5n1 outbreaks in 2005-2006, hpai viruses have been rarely detected in wild birds, followed by high mortality rate. since these outbreaks, hpai h5n1 has continued to cause illness and death in a variety of wild birds in asia as well as in europe (feare 2010). mute swans belong to the order anseriformes and represent natural, asymptomatically infected reservoir for influenza viruses. however, during the hpai h5n1 outbreaks in europe, mute swans were one of the most frequently affected species of wild birds (nagy et al. 2007, brown et al. 2008, feare 2010). some authors characterized them as indicator species for avian influenza virus (teifke et al. 2007, terregino et al. 2006). also, reports from many european countries clearly show that the european mute swan population was predominantly affected with the hpai h5n8 infection (brown et  al. 2017). this findings suggests a significant role of mute swans in the ecology of hpai h5 viruses (nagy et al. 2007, feare 2010). currently circulating hpai virus subtype h5n8 in europe originates as a product of several reassortment events. during worldwide massive outbreaks of hpai h5n8 from 2014 to 2016, two distinct groups of h5n8 viruses were identified: group a (buan-like) and group b (gochang-like) (lee et  al. 2017). first outbreak of hpai h5n8 in europe was reported in 2014 and these viruses belong to group a. the second introduction of hpai h5n8 in europe began via the autumnal migration of wild birds in 2016 and these viruses belong to group  b. phylogenetic analyses of h5n8 clade 2.3.4.4 group b strains detected in 2016 in europe showed significant differences between viruses from h5n8 clade 2.3.4.4 group a (beerens et al. 2017, pohlmann et al. 2017). 97veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 božić et al. h5n1 and h5n8 infections in mute swans trachea and nasal cavity no macroscopic lesions were revealed. haemorrhages in subepicardium with scattered myocardial ecchymosis were present in all swans. in many cases, liver and spleen were moderately enlarged and congested. haemorrhages in the duodenal mucosa and presence of bloody mucinous exudate in the lumen of small intestine were present in five examined swans. in two swans, haemorrhagic exudate was found in the lumen of the oesophagus. haemorrhages in muscles of the neck, intercostal and pectoral muscles were present in few cases. no macroscopic visible lesions in kidneys were found. hyperaemia was clearly visible in the brain and meninges (figure 1b). histopathological examination revealed lesions in the lungs, heart, spleen, kidneys, pancreas and brain in the majority of swans infected with hpai h5n8 virus. the most prominent lesions were haemorrhages and necrosis of pancreas (15/15), as well as non-purulent encephalitis (15/15), haemorrhages in subepicardium (15/15) followed by necrosis of liver and spleen (10/15  cases). haemorrhages were observed in the lungs, heart, spleen, kidneys and brain. in addition to haemorrhages, the lungs showed evidence of congestion and oedema. in one case, interstitial pneumonia was observed. endothelial cells and macrophages in the lumen of alveoli were positive to viral nucleoprotein. in the liver parenchyma focal areas of hepatocellular necrosis and lymphocytic infiltrate and macrophages were present. presence of virus antigen was evident in the necrotic hepatocytes and in the sinusoidal endothelium of the liver. the spleen showed multifocal necrosis of the lymphoid tissues and hemosiderosis as a result of massive haemorrhages. huge amounts of anti-nucleoprotein antibody were distributed in the necrotic areas of the spleen, as well as in the macrophages and endothelial cells of blood vessels. as for the kidney, necrosis of tubules was evident. virus antigen was observed in the tubular epithelium and glomerular capillary endothelium of kidney. punctate to massive haemorrhages were located subepicardially and myocardially, too. lymphocytic infiltrate was also present in perimysium in several cases. a small number of cardiomyocytes was positive. the pancreas showed multifocal acinar necrosis with mononuclear cell infiltration (figure 2a). viral antigen was detected in the cytoplasm and nucleus of necrotic cells as well as in the macrophages (figure 2b). furthermore, histopathological examination showed neuronal satellitosis, neuronophagia and mild lymphocytic perivascular cuffing in the cerebrum. in two cases encephalomalacia was observed. the cerebellum showed massive haemorrhages, focal necrosis and lymphocytic perivascular cuffing (figure 2c). lymphocytes infiltrate meninges in almost all the the matrix (m) and h5 gene by real-time reverse transcription pcr (real-time rt-pcr) as described by spackman and colleagues (spackman et  al. 2002), and by using rna ultrasense™ one-step quantitative rt-pcr system (invitrogen, life technologies). in addition, n8 gene was detected by conventional rt-pcr technique as described by fereidouni and colleagues (fereidouni et al. 2009), by using onestep rt-pcr kit (qiagen, germany). a highly pathogenic pathotype was confirmed by sanger sequencing of the hemagglutinin (ha) gene as described by slomka and colleagues (slomka et al. 2012). tissues available for histopathology from the selected h5n8+ swans included kidney, spleen, pancreas, heart, lungs, liver and brain. collected tissues were fixed for 24 hours in 10% buffered neutral formaldehyde and processed for paraffin embedding. paraffin-embedded sections were cut (4-5 μm) and stained with haematoxylin and eosin (he). in addition, serial sections were immunohistochemically analysed to determine the distribution of influenza virus antigens in individual tissues. immunohistochemical (ihc) technique was performed using the novolink polymer detection system (novocastra, newcastle-upon-tyne, uk). antigen retrieval was achieved by heating the sections in a microwave oven at 560 w for 21 minutes in a citrate buffer (ph 6.0). endogenous peroxidase activity was blocked by incubation of the slides with novocastra peroxidase block, and nonspecific binding sites were blocked with novocastra protein block. after antigen retrieval and inactivation of endogenous peroxidase, the sections were incubated with the rabbit anti-nucleoprotein serum kindly provided by dr. jens p. teifke (the federal research institute for animal health, greifswald-insel riems, germany) diluted in phosphate buffer saline at ratio 1:1000 for 1  h in a humid chamber at room temperature. after primary and secondary antibody treatment (novolink polymer; novocastra), the immunoreaction was visualized using diaminobenzidine tetrahydrochloride (dab) solution and counterstained with mayer's haematoxylin. in general, all of fifteen mute swans infected with hpai h5n8 virus were in good body condition, with sufficient body fat reserves. noticeably, all swans showed no external gross lesions. four infected swans had diarrhoea and showed dark brown discoloured feathers around the cloaca. in three swans, bleeding from beak and nostrils was present. at necropsy, the most common lesions were multifocal, sharply demarcated necrosis and haemorrhages in the pancreas that were found in all swans (figure 1a). haemorrhages in mesenteric adipose tissue were also a consistent finding. characteristic but not present in all infected swans was a congestion and oedema of the lungs. in 98 veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 h5n1 and h5n8 infections in mute swans božić et al. during hpai outbreaks in serbia in 2006 and 2016, mute swans were the most affected wild bird species. the introduction of h5n8 clade 2.3.4.4 group b in serbia in 2016 also resulted in great proportion of diseased mute swans showing central nervous system involvement, such as incoordination and ataxia (božić et  al. 2016). these clinical signs were cases. viral antigen was detected in the cytoplasm and nucleus of neurons and glial cells of the cerebrum and cerebellum (figure 2d). major macroscopic, histological lesions and distribution of influenza virus antigen in mute swans naturally infected with hpai (h5n8 subtype) are summarized in table i. figure 1. a) pancreas, mute swan, h5n8+. multifocal necrosis and haemorrhages. b) brain, mute swan, h5n8+. hyperaemia. figure 2. a) pancreas, mute swan, h5n8+. necrosis, he, original magnification x200. b) brain, mute swan, h5n8+. haemorrhages, he, original magnification x100. c) pancreas, mute swan, h5n8+. intranuclear and intracytoplasmic staining for aiv nucleoprotein, novolink™ polymer detection system, original magnification x200. d) brain, mute swan, h5n8+. intracytoplasmic staining for aiv nucleoprotein in cortical neurons, novolink™ polymer detection system, original magnification x200. 99veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 božić et al. h5n1 and h5n8 infections in mute swans cells as well as in the macrophages, in common with previous reports (kwon et al. 2005, kim et al. 2015). subepicardial haemorrhages were dominant finding in swans infected with h5n8, while it was less common finding in swans infected with h5n1 (vasković et  al. 2011). haemorrhages of subcutaneous tissue and serosa were present in a few infected swans. lung congestion and oedema were common findings, while it was more severe in swans infected with hpai h5n8. a very striking macroscopic lesion found in mute swans infected with h5n8 was haemorrhages in mesenteric adipose tissue. these pathological lesions were not detected in swans infected with h5n1 subtype (vasković et  al. 2011). half of the investigated h5n8 and h5n1 infected mute swans had enlarged and congested liver and spleen. in both infected groups, nasal mucosa, cloaca, gonads and skin were without lesions. in swans infected with hpai h5n1, kidneys were enlarged and congested, while there was no visible macroscopic lesion in kidneys of swans infected with hpai h5n8. the histopathologic findings of renal tissue in swans infected with h5n1 and h5n8 differed. renal tubular necrosis was evident and virus antigen was observed in the tubular epithelium and glomerular capillary endothelium in swans infected with h5n8. the antigenic staining patterns were similar and lesions detected in kidneys corresponded to those observed in wild birds infected with h5n8 (kim et  al. 2015). there were no histopathologic lesions in kidneys of swans infected with h5n1, and virus antigen expression was detected in only one case. teifke and colleagues (teifke et al. 2007) and kwon and colleagues (kwon et  al. 2010) reported that was no evidence of histopathologic lesions in the kidneys of waterfowl infected with h5n1, though experimental infection not noticed in swans infected with hpai h5n1. vasković and colleagues (vasković et  al. 2011) first described pathomorphologic lesions in 7 mute swans infected with hpai h5n1 in serbia. the most frequent gross lesions included haemorrhages and necrosis in the pancreas, lung congestion and oedema, congestion and enlargement of liver and spleen, congestion of kidneys, haemorrhages in the small intestine, rectal mucosa and tonsils in the caecum. the most consistent histological lesions were non-purulent encephalitis with a perivascular accumulation of lymphocytes, lung congestion and oedema, haemorrhages and hepatocellular necrosis in liver and spleen, haemorrhages and necrosis of the exocrine pancreas cells. histological lesions were not found in kidneys. the viral nucleoprotein was most frequently detected in the pancreas (7/7), liver (5/7), lung (4/7), spleen (3/7) and brain (1/1). viral antigen was detected in the kidney tubules in one swan (1/7). comparing these two groups, it has been found that the most dominant gross lesions found in all infected mute swans were multifocal pancreatic necrosis and haemorrhages. pancreatic necrotic fields were irregularly shaped, different sizes, ranging from a few millimetres to one and a half centimetres in diameter in both infected groups. necroses of the exocrine acinar cells were detected in all tested samples of pancreatic tissue. an identical finding was obtained in swans naturally infected with h5n1 in germany (teifke et  al. 2007) as well as in wild birds naturally infected with h5n8 in south korea (kim et  al. 2015). acute necrotizing pancreatitis is a common finding of hpaiv infections as a result of the systemic replication (teifke et  al. 2007, bertran et  al. 2011, božić et  al. 2018) and possibly the main cause of death. immunohistochemical findings revealed the presence of hpai h5n1 and h5n8 virus antigen in the cytoplasm and nucleus of necrotic table i. frequency of occurrence of macroscopic, histological lesions and distribution of viral antigen it the tissues from 15 mute swans naturally infected with hpai h5n8. organ histopathological lesions ihc positive/ analyzed organs viral antigen positive cells pancreas haemorrhages multifocal acinar necrosis 15/15 acinar necrotic cells, macrophages heart haemorrhages in subepicardium, intramyocardial ecchymoses lymphocytic infiltrate in perimysium 6/15 cardiomyocytes lung congestion oedema haemorrhages 11/15 endothelial cells and macrophages spleen multifocal necrosis massive haemorrhages and hemosiderosis 12/15 macrophages and endothelial cells of blood vessels liver hepatocellular necrosis lymphocytic infiltrate 12/15 necrotic hepatocytes kidneys necrosis of tubules 13/15 tubular epithelium and glomerular capillary endothelium brain neuronal satellitosis neuronophagia lymphocytic perivascular cuffing encephalomalacia 15/15 neurons and glial cells 100 veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 h5n1 and h5n8 infections in mute swans božić et al. alexander d.j. 2000. a review of avian influenza in different bird species. vet microbiol, 74 (1-2), 3-13. beerens n., heutink r., bergervoet s.a., harders f., bossers a. & koch g. 2017. multiple reassorted viruses as cause of highly pathogenic avian influenza a (h5n8) virus epidemic, the netherlands, 2016. emerg infect dis, 23 (12), 1974-1981. bertran k., 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mute swans in the czech republic. vet microbiol, 120 (1-2), 9-16. pálmai n., erdélyi k., bálint á., márton l., dán á., deim z., ursu k., löndt b.z., brown i.h. & glávits r. 2007. pathobiology of highly pathogenic avian influenza virus (h5n1) infection in mute swans (cygnus olor). avian pathol, 36 (3), 245-249. pantin-jackwood m.j. & swayne d.e. 2009. pathogenesis and pathobiology of avian influenza virus infection in birds. rev sci tech, 28 (1), 113-136. et  al. 2007, kalthoff et  al. 2008, vasković et  al. 2011, teifke et al. 2007, kim et al. 2015). our findings also showed that pancreas was the most affected organ in all examined mute swans infected with both hpai h5n1 and h5n8 suggesting that mute swans are highly susceptible to infection with both hpai h5n1 and h5n8 viruses. acknowledgements this study was supported by the ministry of education, science and technological development of the republic of serbia, projects number tr 31011, tr 31084 and iii 46002. studies have shown that h5n1 virus infects the tubular epithelium. distinctly initiated blood vessels of the brain and meninges were consistently present in most swans infected with both h5n8 and h5n1. in the cerebrum, a large number of degenerating neurons and glial cells showed strong nuclear and cytoplasmatic reaction of virus antigen, in both infected groups. most hpai viruses replicate into endothelial cells, leading to increasing vascular permeability and causing oedema and haemorrhage in the different organs (pantin-jackwood and swayne 2009). pancreatic lesions are the most prominent finding in swans naturally and experimentally infected with highly pathogenic avian influenza viruses (pálmai 101veterinaria italiana 2019, 55 (1), 95-101. doi: 10.12834/vetit.1463.7919.2 božić et al. h5n1 and h5n8 infections in mute swans teifke j.p., klopfleisch r., globig a., starick e., hoffmann b., wolf p.u., beer m., mettenleiter t.c. & harder t.c. 2007. pathology of natural infections by h5n1 highly pathogenic avian influenza virus in mute (cygnus olor) and whooper (cygnus cygnus) swans. avian pathol, 44 (2), 137-43. terregino c., milani a., capua i., marino a.m.f. & cavaliere n. 2006. highly pathogenic avian influenza h5n1 subtype in mute swans in italy. vet rec, 158 (14), 491. tong s., zhu x., li y., shi m., zhang j., bourgeois m., yang h., chen x., recuenco s., gomez j., chen l.m., johnson a., tao y., dreyfus c., yu w., mcbride r., carney p.j., gilbert a.t., chang j., guo z., davis c.t., paulson j.c., stevens j., rupprecht c.e., holmes e.c., wilson i.a. & donis r.o. 2013. new world bats harbor diverse influenza a viruses. plos pathog, 9 (10), e1003657. vasković n., šekler m., vidanović d., polaček v., kukolj v., matović k. & jovanović m. 2011. pathomorphological lesions and distribution of viral antigen in birds infected with the pathogenic strain of h5n1 avian influenza virus. acta vet‑beograd, 61 (5-6), 591-598. webster r.g., bean w.j., gorman o.t., chambers t.m. & kawaoka y. 1992. evolution and ecology of influenza a viruses. microbiol rev, 56 (1), 152-79. pohlmann a., starick e., harder t., grund c., höper d., globig a., staubach c., dietze k., strebelow g., urlich r.g., schinköthe., teifke j.p., conraths f.j., mettenleiter t.c. & beer m. 2017. outbreaks among wild birds and domestic poultry caused by reassorted influenza  a (h5n8) clade 2.3.4.4 viruses, germany, 2016. emerg infect dis, 23 (4), 633-636 shi y., wu y., zhang w., qi j. & gao g.f. 2014. enabling the ‘host jump’: structural determinants of receptor-binding specificity in influenza a viruses. nat rev microbiol, 12 (12), 822-831. slomka m.j., to t.l., tong h.h., coward v.j., hanna a., shell w., pavlidis t., densham a.l.e., kargiolakis g., arnold m.e., banks j. & brown i.h. 2012. challenges for accurate and prompt molecular diagnosis of clades of highly pathogenic avian influenza h5n1 viruses emerging in vietnam. avian pathol, 41 (2), 177-193. spackman e., senne d.a., myers t.j., bulaga l.l., garber l.p., perdue m.l., lohman k., daum l.t. & suarez d.l. 2002. development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutini. j clin microbiol, 40 (9), 3256-3260. 229 1 istituto zooprofilattico sperimentale della sardegna, via duca degli abruzzi 8, 07100 sassari, italy. 2 ufficio sanità marittima e di frontiera (usmaf), molo teleferica, porto torres, italy. 3 these authors contributed equally to this work. * corresponding author at: istituto zooprofilattico sperimentale della sardegna, via duca degli abruzzi 8, 07100 sassari, italy. tel.: +39 0792892303, e-mail: giorgiomeloni82@gmail.com. parole chiave aedes albopictus, culex pipiens, sorveglianza entomologica, italia, sardegna, zika virus riassunto virus zika (zikv) è un virus a rna appartenente al genere flavivirus, famiglia flaviviridae, e si trasmette con la puntura delle zanzare aedes, in particolare aedes aegypti. il 1 febbraio 2016 la world health organization (who) ha dichiarato il virus zika un'emergenza per la salute pubblica di interesse internazionale e, per la presenza ormai diffusa di aedes albopictus, ha classificato l'italia a moderato rischio di trasmissione, preceduta in europa solo dalla francia. per questo motivo nel 2016 si è avviata in sardegna una sorveglianza entomologica. sono state posizionate trappole bg sentinel in 29 siti, inclusi aree urbane e punti di accesso come porti e aeroporti. le zanzare sono state catturate con cadenza bisettimanale da aprile a dicembre 2016. è stato catturato un totale di 3.089 zanzare appartenenti a 10 specie. le più abbondanti sono state culex pipiens s.l. e ae. albopictus. l’analisi con real time rt‑pcr eseguita su tutti i campioni, raggruppati in 584 pool, non ha evidenziato la presenza del virus. una sorveglianza entomologica dovrebbe essere attivata e resa permanente in prossimità di aree urbane e punti di accesso anche in considerazione della recente introduzione di specie esotiche come aedes koericus e aedes japonicus in italia e dell’ampia diffusione di ae. albopictus. sorveglianza entomologica per il virus zika in sardegna, italia, 2016 keywords aedes albopictus, culex pipiens, entomological surveillance, italy, sardinia, zika virus. summary zika virus (zikv) is a rna virus belonging to the genus flavivirus, family flaviviridae. this virus is transmitted through bite of aedes mosquitoes, in particular ae. aegypti. on february 1st 2016, the world health organization (who) has declared zikv a public health emergency of international concern. successively, considering the establishment of ae. albopictus, who has classified italy as having a moderate likelihood of local transmission of zikv, preceded in europe only by france. for this reason an entomological surveillance plan was been activated in sardinia in 2016. bg sentinel mosquito traps have been positioned in 29 sites, comprising urban areas and points of entry, as ports and airports. mosquitoes were collected fortnightly from april to december. a total of 3,089 mosquitoes were collected belonging to 10 species. the most numerous species have been cx. pipiens s.l. and ae. albopictus. all mosquitoes sampled have been assayed by real time reverse transcriptase pcr for detection of zikv rna. a total of 584 pool have been analyzed and have been reported no evidence of zikv. a permanent entomological surveillance should be implemented principally in the urban areas and points of entry, as ports and airports, because ae. albopictus, susceptible to zikv, is established in sardinia and also know the recent introduction of invasive mosquitoes species ae. koericus and ae. japonicus in italy. cipriano foxi1,3, giantonella puggioni1,3, giorgio meloni1,3*, renata rossi1, angela maria rocchigiani1, luigi vento1, antonio collovà2 and giuseppe satta1 entomological surveillance of zika virus in sardinia, italy, 2016 veterinaria italiana 2018, 54 (3), xxx‑xxx. doi: 10.12834/vetit.1303.7208.2 accepted: 09.01.2018 | available on line: 30.09.2018 230 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx zika virus in sardinia, italy, 2016 foxi et al. classified italy with a moderate likelihood of local transmission of zikv, preceded in europe only by france (who 2016b). according to these reports it is essential to assess a possible new introduction of different aedes species and to examine every autochthonous mosquito species able to transmit viruses. the aim of this work is to evaluate in sardinia: i) new introductions of invasive mosquito species, with particular attention to ae. aegypti; ii) abundance of ae.  albopictus and to define his seasonality; iii) presence of zikv in all mosquito species captured, using a specific real time rt‑pcr. materials and methods study area and mosquito sampling the study was conducted in sardinia island, located in the centre of mediterranean basin. this region is characterized by a mild winter and a hot and dry summer. during the winter, sometimes, are recorded temperatures below zero degrees, while in the summer days, temperatures often exceeding 30  °c. the period with the highest rainfall, on an average 400‑600 mm along the coast and 500‑800 mm inland, is from november to april (chessa and delitala 1997). the monitoring of the insects was carried out in urban areas of the major cities (12  sites) and in the most important border areas, ports and airports (17 sites). in details a total of 29 sites were included in this survey: 8 traps were positioned in cagliari, 4 in oristano, 2 in tortolì, 2 in nuoro, 6 in olbia, 4 in alghero, 1 in sassari, 1 in porto torres and 1  in santa teresa di gallura (figure 1). mosquitoes were collected fortnightly from april to december 2016. in the maritime and commercial ports the traps were placed close to moored ships principally represented by cruise and container ships. in the airports traps were positioned in the proximity of the airport apron, at arrivals of domestic and international flights. in urban areas traps were positioned in public services facilities, such as schools, hospitals and/or cemeteries, places with availability of larval breeding sites. a supplementary trap worked for 3 days, inside a pilothouse of a coal ship, moored in porto torres port and coming from russia. mosquitoes were collected using a bg  sentinel mosquito traps, originally developed to monitor aedes genus mosquitoes. traps, baited with a chemical lure mimicking human odor, were placed on the ground and in locations sheltered from rainfall, wind, direct sunlight and close to mosquito breeding sites. the cartridges of bg‑lure were replaced every two months. each trapping period was of 24 hours and the collected mosquitoes were taken to the laboratory as soon as possible for species identification. introduction zika virus (zikv) is a rna virus belonging to the genus flavivirus, family flaviviridae. zikv is related to the other arboviruses of public health importance, including dengue (denv) and chikungunya (chikv) viruses. zikv is transmitted through infected mosquito bites and it causes an infection characterized by non‑specific symptoms including fever, skin rash, conjunctivitis, joint inflammation and pain (calvo et al. 2016, pabbaraju et al. 2016). zikv was first isolated in uganda in 1947. only human sporadic mild cases were successively reported in east and west africa (maccrae and kirya 1982). recent studies conducted in south america have shown a link between zikv infection and congenital malformations and neurological disorders (fauci and morens 2016, oliveira melo et al. 2016). for the first time, an arbovirus has been associated with severe human congenital complications. at this regard on february 1st 2016 the world health organization (who) has declared zikv a public health emergency of international concern (who 2016a). during the last year, many imported cases have been notified in europe. just in italy, a total of 94 imported cases have been confirmed (ecdc 2017). in sardinia, one case has been notified in july. no autochthonous case of zika transmission has been reported in europe, but a secondary autochthonous case has been notified in italy due to a possible sexual transmission (venturi et al. 2016). mosquitoes belonging to the aedes genus are considered as main vectors of the zika virus (diallo et al. 2014, musso and gubler 2016). aedes aegypti is the main mosquito species implicated in outbreaks in the americas (chouin‑carneiro et  al. 2016). nowadays, the presence of ae. aegypti is confirmed in europe in island of madeira (portugal), in parts of georgia, in south western russia and a new recent introduction has been reported in holland (ecdc 2016). numerous studies indicate that aedes albopictus can be equally competent to transmit zikv (chouin‑carneiro et  al. 2016). this species is established in many countries of mediterranean basin (ecdc 2016). in italy, ae.  albopictus was reported for the first time in 1990 and in sardinia it was detected in 1994, where is currently widely established (sabatini et  al. 1990, romi 1995). more recently, new introductions of exotic mosquito species were observed in italy as ae.  koreicus and ae. japonicus (capelli et al. 2011, seidel et al. 2016). since 2014, in italy zikv has been included within a control strategy for arboviruses, combining direct and indirect control measures and providing guidelines for an appropriate management of zikv infection (ministero della salute 2014). considering established populations of ae.  albopictus, who has veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 231 foxi et al. zika virus in sardinia, italy, 2016 samples were shaking at frequency of 17/sec for 90 sec and then centrifuged in a refrigerated centrifuge at 10,000 g for 10 minutes at 4  °c. viral rna was extracted from 100 µl supernatant using the commercial kit biosprint 96 one‑for‑all vet kit (qiagen®) according to the manufacturer’s instructions. all samples were assayed by a specific real time rt‑pcr for detection of zika virus rna (lanciotti et  al. 2008). the real‑time assay was performed by using quantitect probe rt‑pcr kit (qiagen®) following the manufacturer’s protocol, with amplification in the 7900 ht fast real‑time pcr system (applied biosystems). the baseline and threshold were set using the auto‑baseline and threshold feature in sds software version 2.4 (applied biosystems®). samples were considered positive if target amplification was recorded within 45 cycles. results during the survey period, 438 catches were performed and a total of 3,089 mosquitoes belonging to 10 species collected (table i). the most abundant species were cx.  pipiens s.l. (n  =  2,473) and ae.  albopictus (n  =  481) representing 80.1  % and 15.6  % of the total catches, respectively. most of mosquitoes were represented by not engorged females (68.4 %) while engorged females two additional bg mosquito traps were used in july when a human imported case was notified, in according to the ministerial guidelines. traps were positioned in the house garden of the infected patient and 3 catches were carried out, one day a week. mosquitoes identification sampled mosquitoes were morphologically identified at the species level under a stereo microscope using taxonomic keys (severini et  al. 2009). mosquitoes were then pooled according to species, sex, presence (engorged) or absence of blood (not engorged) in the abdomen of females, collection site and date of sampling, with a maximum of 25 individuals per pool. to avoid cross‑contamination, due to lose mosquito parts, the pools were obtained by handling specimens individually with tweezers. the pooled mosquitoes were stored in 2 ml polypropylene cryotubes (eppendorf®) and frozen at ‑ 80 °c until biomolecular analysis for virus detection. detection of zika virus pools of mosquitoes were re‑suspended in 500  µl of phosphate buffered saline ph 7.3  ±  0.1 and homogenized using the tissuelyzer ii (qiagen, valancia, ca) with two 3 mm tungsten beads. figure 1. map of sardinia (italy) showing the collection sites. nick title first author et al. 232 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx the highest density from may to july (figure 2). the population density of ae.  albopictus increased from april and peaked in september, while no specimens were captured in december (figure 2). all mosquitoes collected during the study period were tested using zikv real time rt‑pcr assay. overall a total of 584 pools of mosquitoes were tested and no pool was found positive. discussion the surveillance conducted in sardinia during 2016 has shown the presence of 10 mosquitoes species indigenous currently established in the island. furthermore, no invasive mosquito species has been detected. during our monitoring cx. pipiens s.l. and ae. albopictus have been the main species reported representing over 95% of the total mosquitoes captured. both species are widely distributed in europe and represent potential vectors of arboviruses. their distribution is ubiquitously, largely diffuse in urban, sub urban and rural areas. although zikv has been detected in culex mosquito species as culex perfuscus in senegal (diallo et  al. 2014), several authors have reported that cx. pipiens is not a competent vector to zikv (boccolini et  al. 2016, heitmann et  al. 2017). in europe cx.  pipiens is considered a competent vector of several zoonotic viruses including west nile virus (wnv), usutu virus (usuv), rift valley fever virus (rvfv) and japanese encephalitis virus (jev) (lundström 1999, busquets et al. 2008, moutailler et al. 2008, ravanini et  al. 2012). in sardinia, wnv and usuv have been found in cx.  pipiens mosquitoes through real time (25.5  %) and males (6.1  %) were less abundant. culex pipiens  s.l. was the most ubiquitous species collected in 26 out of 29 collection sites. in particular 1,414 specimens (57.2 %) were captured in 10 urban sites and 1,059 (42.8  %) in 16 sites in the border areas. ae. albopictus was also captured in all regional territory and its presence was detected in 21 out of 29 trapping sites. this species was captured in all urban areas traps counting a total of 382 specimens (79.4 %), while only 99 adults (20.6 %) were captured in 9 sites situated in border areas. no mosquito was captured in the coal ship. a total of 6 mosquitoes were collected in the garden of infected patient. in particular, 2 females of ae.  albopictus, 2 females of cs. longiareolata, 1 female of an. labranchiae and 1 male of oc. caspius were recorded. culex pipiens s.l. was captured during all study period showing significant seasonal changes with table i. mosquitoes species collected in sardinia (italy) during 2016 and number of pool tested. species n. positive sites/40 collection sites males females not engorged females engorged total (%) n. of pool tested culex pipiens s.l. 37 591 1703 179 2473 (80.05) 354 aedes albopictus 22 142 336 3 481 (15.59) 146 culiseta longiareolata 17 42 17 4 63 (2.04) 43 culex spp. 5 4 35 1 40 (1.30) 11 ochlerotatus caspius 7 5 6 ---11 (0.36) 10 culex hortensis 4 3 7 ---10 (0.32) 9 ochlerotatus detritus 3 ---2 1 3 (0.10) 3 culex theileri 2 ---2 ---2 (0.06) 2 culiseta spp. 2 ---2 ---2 (0.06) 1 culiseta annulata 1 ---1 ---1 (0.03) 2 aedes spp. 1 ---1 ---1 (0.03) 1 anopheles algeriensis 1 ---1 ---1 (0.03) 1 anopheles labranchiae 1 ---1 ---1 (0.03) 1 total 787 2,114 188 3,089 584 0 2 4 6 8 10 12 apr may jun jul aug sep oct nov dec culex pipiens s.l. aedes albopictus n .o f a d u lt s/ ca tc h figure 2. seasonal abundance of cx. pipiens s.l. and ae. albopictus during 2016 in sardinia (italy). first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 233 ae.  koericus and ae.  japonicus were introduced in italy via passive transport of eggs from heavily infested areas (capelli et al. 2011, seidel et al. 2016). these mosquitoes have found favorable ecological niches and nowadays are established in italy. aedes aegypti was reported in italy up to 1972 (callot and delecolle 1972) therefore, it is worth expecting its potential reintroduction in the future, considering also its actual presence in some european regions, as madeira island, black sea coastal areas of georgia and russia and holland (who 2016b). the increase of connectivity, commercial trades and climatic changes represent a real risk of spread of exotic pathogens and disease vectors. the outbreaks of wnv, chikv and denv in europe represent an important threat to consider. epidemiological surveillance of imported cases is a critical point to evaluate the potential risk of arboviruses introduction. an imported case is a potential source of infection that in presence of suitable vector populations could determine a spread of the disease. sardinia represents a territory at high likelihood of introduction of arboviruses, because is located in the middle of mediterranean basin with high intensity of touristic and trade flows and with a well‑established ae. albopictus population. although zikv presence has not been detected during our study, a permanent entomological surveillance in urban areas and at the points of entry, as ports and airports, should be implemented to provide epidemiological information to evaluate the risk of introduction of zikv and other arboviruses in italy. acknowledgements we thank istituto superiore di sanità to provide the zika virus rna. we would like to thanks for technical assistance: environment and agricultural sector of the provinces of cagliari, oristano, nuoro, ogliastra, sassari, olbia‑tempio; management companies of the ports (cagliari, oristano, olbia, santa teresa di gallura and porto torres) and of the airports (cagliari, alghero and olbia); azienda sanitaria locale of olbia‑tempio; fire departments of nuoro and sassari; air force airport of alghero. rt‑pcr (rossi et  al. 2016). culex pipiens s.l. include two biotypes named culex pipiens pipiens and culex pipiens molestus (amara korba et  al. 2016). these forms are morphologically indistinguishable but exhibit different characteristics in both behavioral and physiology. in addition, exist a hybrid form that present biologic characteristics of both forms. in this study, no biomolecular analyses have been performed to discriminate these 3 forms. considering a previous study conducted in italy (di luca et  al. 2016b), we can suppose the presence of all three forms of cx. pipiens, included hybrid form. aedes aegypti is considered as the main vector of zikv infection in urban areas, but the viral rna has been detected also in several aedes species (diallo et  al. 2014). in sardinia are reported three species of the aedes genus: ae.  albopictus, ae. vittatus and ae.  vexans (severini et  al. 2009). aedes albopictus, highly correlated to ae.  aegypti, is considered a potential vector. in fact, field collected ae. albopictus was found zikv rna positive in gabon during chikv and denv outbreaks in 2007 (grard et  al. 2014). moreover recent experimental studies highlights that ae.  albopictus is susceptible to zikv infection (chouin‑carneiro et  al. 2016, di luca et  al. 2016a, heitmann et  al. 2017). aedes albopictus feeds primarily on wild and domestic animals included humans, and it has exophagic and exophilic behavior. currently, italy is the most greatly‑infested country in europe with well established populations in several regions including sardinia (cristo et  al. 2006). during our study, ae.  albopictus has resulted more abundant in the urban areas as reported by other authors (medlock et  al. 2012). aedes vittatus has been found naturally infected with zikv in senegal and ivory cost (diallo et  al. 2014). this species is heavily anthropophilic and bites during the daytime and night. aedes vexans, though it is not considered susceptible to zikv infection, is able to transmit other arboviruses, as rvfv and wnv (ndiaye et  al. 2016). this mosquito has a behavior resembling to ae. vittatus. other species found in low numbers during our study as culiseta longiareolata, ochlerotatus caspius and cx. theileri are capable of transmitting arboviruses (maslov 1967, moussieget 1988, lundström 1999) . similarly to ae.  albopictus, other species as nick title first author et al. 234 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx amara korba r., alayat m.s., bouiba l., boudrissa a., bouslama z., boukraa s., francis f., failloux a.b. & boubidi s.c. 2016. ecological differentiation of members of the culex pipiens complex, potential vectors of west nile virus and rift valley fever virus in algeria. 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rural da amazônia, brazil. 2universidade federal do amapá, brazil. 3universidade estadual de santa cruz, brazil. 4universidade federal do piauí, brazil. 5universidade federal do pará, brazil. 6empresa brasileira de pesquisa agropecuária-amazônia oriental, brazil. 7federal rural university of amazon, brazil. *corresponding author at: federal rural university of amazon, brazil. e-mail: silva.filho@ufra.edu.br. lorena keyse silva1, elizabeth machado barbosa2, sandra cristina becker silva3, josé elivalto guimarães campelo4, evonnildo costa gonçalves5, caio santos silva5, josé ribamar felipe marques6 and ednaldo silva filho7* keywords bubalus bubalis, lep-1620, snp, variability. summary the objective of this work was to characterize genetically some buffalo herds raised in varzea (va) and terra-firme (upland) (tf) ecosystems through polymorphism of the intron 2 of the leptin gene (lep-1620). two hundred seventy-nine animals from four distinct populations were evaluated using the pcr-rflp method for lep-1620 polymorphism (snp) of the leptin gene with restriction enzyme bsaai. the animal samples were sorted into 4 groups, according to their breed and environmental origin: mediterranean tf, murrah  tf, mediterranean va and crossbreed va. two alleles (a and g) were detected and their frequencies were analyzed. allele a frequency ranged from 0.395 (mediterranean tf) to 0.850 (murrah tf), with aa genotype ranged from 0.114 (murrah tf) to 0.700 (mediterranean tf). the observed and expected heterozygosities ranged from 0.268 (mediterranean tf) to 0.562 (murrah tf), and 0.255 (mediterranean tf) to 0.478 (murrah tf), respectively. the hardy-weinberg probabilities were greater than 0.05. the crossbred herd in varzea was the only population with significant inbreeding and the shannon index ranged between 0.423 (mediterranean tf) and 0.671 (murrah tf). polymorphism of the leptin gene in buffalo breed groups from eastern amazon and even higher latitudes, allowing their greater distribution in the world (campanile et  al. 2016). in addition, unlike cattle, these animals don’t require living in farming-like fields to convert food (plants) into meat and milk. since they can live and be very productive in natural landascapes like the lands of marajo island, their significant lower cost maintanace makes them animals of great economic  value (ramalho  et  al.  2013, rodrigues et al. 2015). the study of genetic variability has progressed in recent years with the advancement and application of molecular techniques. this enabled improving livestock productivity, since these innovations led to great amount of researches using several molecular markers associated to animal productivity traits to take place. no doubt this is good news, however, the introduction the buffaloes were introduced in brazil during the nineteenth century from asia, italy and the caribbean. they quickly adapted very well to brazil’s lands because of the similarity of the environmental conditions with their native place of origin (zetouni et  al. 2013). in brazil, the region that stands out in the production of buffaloes is the north region which has approximately 877  thousand animals, representing about 50% of the buffalo herd in this country (ibge 2014). although buffaloes show lower carcass yields compared to cattle, they present superior performance in low fertility soils, accelerated growth and ease of handling, since they can adapt well to wide climatic zones such as equatorial, tropical, subtropical, mediterranean 258 veterinaria italiana 2021, 57 (4), 257-263. doi: 10.12834/vetit.1739.9163.2 leptin gene polimorphism in buffaloes silva et al. possible that some of these snps found in this gene might be associated with productive and economic characteristics of the animals of zootechnical interest through the analysis of the candidate gene for increasing the productivity of these animals. in particular, it would be beneficial to know more about this marker because it is a potential tool to be used by ranchers for genetically improving the quality of raising and breeding buffaloes at eastern amazon region (kowalski et al. 2014). the objective of this work was to characterize some buffalo herds from varzea and terra-firme (upland) systems from eastern brazilian amazon, through polymorphism on intron 2 of leptin gene (lep-1620). materials and methods this study was submitted to the ethics committee for animal use and approved with protocol number 033/2015 of the federal rural university of amazon. fifty hairs per animal were collected with bulbs from 279 buffaloes: 164 of these animals came from two pupulations of the varzea system (49  mediterranean and 115 crossbred), and 115 animals comimg from two populations of terra firme system (10  mediterranean and 105 murrah). the varzea system population, mediterranean and crossbred, were in extensive system with native pastures, no fences and practically no sanitary, zootechnical or reproductive control of the herds, while the two populations coming from terra-firme system, the mediterranean and the murrah were in semi-extensive to extensive system with cultivated pastures and sanitary, zootechnical and reproductive control of herds. after collection, the biomaterial was stored at 2  °c until dna extraction. forty hair bulbs were selected from each animal for dna extraction. the phenolic method was used in this step, in 1.5  ml tubes, following the procedure described by sambrook and colleagues (sambrook et al. 1989). a 522 pb amplicon was obtained by polymerase chain reaction (pcr). the primers used were those described by lien and colleagues (lien et al. 1997) from the intron 2 region to the exon 3 of the buffalo leptin gene (f-5'gtc tgg agg caa agg gca gac t 3' and r-5'cca cca cct ctg tgg agt ag 3'). pcr mixture was obtained to a final volume of 15  μl, by homogenically mixing the following: 1.5 μl 10x pcr buffer, 0.6 μl 50 mm mgcl 2 , 1.0  μl  1.25  mm of dntp (invitrogen, fortaleza, ce, brazil), 0.5 μl 10 μm of each primer (foward and reverse), 0.3 μl 5u taq dna polymerase (ludwig biotec, alvorada, rs, brazil), 3.0  μl  q-solution (quiagen, valencia, ca, usa), 1.0  μl  50-100  μm of genomic dna and completed with 6.6  μl pure water. the temperature and time conditions were: initial denaturation at 94  °c for 5 min, followed number of investigations of this sort in bufflaloes is still scarce. this development allowed the use of several molecular markers related to animal productivity. therefore, the search for molecular markers that aid genetic breeding programs is of high importance. when polymorphisms are found to be associated with possible productive or reproductive traits, they contribute to improving the herd’s qualities (rodrigues et  al. 2010, marcondes and righetti 2011, zetouni et  al. 2013, barbosa et al. 2016). among the genes involved in food control and productivity, we can highlight the leptin hormone, which has a relationship with energy control, food consumption and body weight reduction. it also works as a chemical signal, communicating to the brain that the body has sufficient energy reserves to provide the beginning of puberty. in addition, it plays the role of maintenance of their entire reproductive life, regulating animal metabolism and the reproductive system (kowalski et  al. 2014, pérez-pérez et al. 2015). leptin is a protein hormone with 167 amino acids, but the active form does not present the first 21 amino acids and has a molecular weight of 16 kda, and it is expressed by the obese (ob) gene, located on the fourth autosomal chromosome, in bovines, with three exons and two introns (lara et  al. 2011). its expression is mainly in white adipose tissue, but it also can be expressed in brown adipose tissue, ovary, stomach and placenta as well. however, leptin is most active in the hypothalamus. within hypothalamic cells, after leptin binds to its specific receptors, it is brought to the cell nucleus, where it then binds to dna sequences that control the expression of appetite-enhancing neurotransmitter inhibitor genes, such as neuropeptide y (npy), and stimulate appetite-reducing genes, such as propofolomelanocotin (pomc), resulting in satiety. this hormone also acts as a signal to the hypothalamus that the animal is under food restriction due to decreased secretion of leptin, signaling the use of energy reserve, thus decreasing adipose tissue (catunda et al. 2014). several studies of the leptin gene revealed the presence of single nucleotide polymorphisms (snps) that were used as molecular markers and associated with weaning weight (souza et  al. 2010), milk composition, duration and difficulty of parturition (giblin et  al. 2010), and its implication in meat tenderness (lara et  al. 2011), mainly in cattle. although there are few works related to the leptin gene in buffalo species, one of the markers studied was lep-1620 (a/g) snp, located in intron 2 of the leptin gene. it is strongly related to milk production and characteristics such as fat and protein in buffaloes (zetouni et  al. 2013). thus, it is 259veterinaria italiana 2021, 57 (4), 257-263. doi: 10.12834/vetit.1739.9163.2 silva et al. leptin gene polimorphism in buffaloes by 30  cycles with denaturation at 94  °c for  60  s, annealing at 60  °c for 60  s, extension at 72  °c for 60  s, and ending cycle with final extension at 72  °c for 10 min. the final pcr products were visualized on 1.5% agarose gel stained with gelred (biotium/usa). a 100 bp ladder was used as reference. for restriction fragment length polymorphism (rflp) reaction, pcr products were subjected to (or digested by) the bsaai enzyme (new england biolabs, inc.) under the following conditions: 3  μl of pcr product, 0.5 μl of restriction enzyme, 1 μl of reaction buffer and 10.5 μl of distilled water. the 15 ul volume reaction was heated at 37  °c for 60 minutes. the rflp products were submitted to eletrophorese on 1.5% agarose gel stained with gelred (biotium/usa) at 90 v for 30  min. all genotypes were determined by visually analyzing the lethgths revaled by uv transluminater gel photo (or prints). the popgene software version 1.32 (yeh et al. 1997) was used to obtain all the statistical data needed for the analytical part of this study, including: the allelic and genotypic frequencies, the diversity parameters as observed (obshe), expected heterozygosities (exphe), hardy-weinberg probabilities (hwp), inbreeding coefficient (fis) and shannon index (si). the software was also used to determine the genetic identities and genetic distances of nei (nei  1972, nei 1978) among the populations studied, and it developed two dendrograms by the upgma method from the genetic distances of nei (nei 1972, nei 1978). another software, genepop version 4.6, (raymond and rousset 1995) was used to estimate the genetic and genotypic differentiations between breed groups and the f statistics (fst, fis and fit). the significant level was 5%. results we observed three digest stands (figure 1): the homozygous aa (fragment of 522 bp), heterozygous ag (fragments of 522, 439 and 83 bp), and homozygous gg (fragments of 439 and 83 bp). table i shows the allelic and genotypic frequencies, and diversity parameters. the a allele was the most frequent in the mediterranean breed (tf), mediterranean breed (va) and crossbred (va) populations, while the g allele was most frequent in the murrah (tf) population. the aa genotype was also the most frequent in the three populations where the a allele was the most frequent. however, the genotype heterozygous ag was the most frequent in the murrah (tf) population. observed and expected heterozygosities were lower in the mediterranean (tf) population and higher in the murrah (tf) population (table i). both breed groups of the terra-firme and varzea systems were within the hardy-weinberg equilibrium (p > 0.05), which indicates that the populations have imperceptible conditions of mutation, selection and migration (table i). only crossbred population of varzea presented inbreeding (table i), being genetically similar. according to the shannon figure 1. agarose gel (1.5%) demonstrating the genotype migration stands. lane 1 = aa, lane 2 = ag, lane 3 = gg and lane 4 = dna marker (100bp). fragment of 83 bp is not visible. 1 2 3 4 table i. genetic diversity parameters of the lep-1620 polymorphism in amazon buffalo groups. parameters me (tf) mu (tf) me (va) cb(va) total alleles* a 0.850 0.395 0.735 0.709 0.600 g 0.150 0.605 0.265 0.291 0.400 genotypes* aa 0.700 0.114 0.510 0.513 0.369 ag 0.300 0.562 0.449 0.391 0.462 gg 0.000 0.324 0.041 0.096 0.169 diversity heobs 0.268 0.562 0.449 0.391 0.462 heexp 0.255 0.478 0.390 0.413 0.480 hwp 0.660 0.080 0.320 0.540 0.520 fis -0.125 -0.171 -0.142 0.057 -0.108 si 0.423 0.671 0.579 0.603 0.673 *means frequencies; me (tf) = mediterranean terra-firme; mu (tf) = murrah terra-firme; me (va) = mediterranean varzea; cb (va) = crossbred varzea; heobs = observed heterozygosities; heexp = expected heterozygosities; hwp = hardy-weinberg probabilities; fis = inbreeding coefficient and shannon indexes. 260 veterinaria italiana 2021, 57 (4), 257-263. doi: 10.12834/vetit.1739.9163.2 leptin gene polimorphism in buffaloes silva et al. results of the rflp technique corroborate with those found by lien and colleagues (lien et al. 1997), who were the first to describe the substitution of a guanine (g) by an adenine (a) at the intron 2 in the leptin gene in bovine. results were also corroborated with both azari and colleagues (azari et  al. 2012), who studied the same gene when they described distributions of allelic and genotypic frequencies in three genetically different populations, including holstein cows (bos taurus), mazandarani cattle (bos indicus) and river buffaloes (bubalus bulalis), and with zetouni and colleagues (zetouni  et  al.  2013a), who studied the existence of the same polymorphism, lep-1620 (a or g), in buffaloes and their possible associations with milk, fat, protein and fat and protein percentages. in addition, the same authors found the same genotypes (aa, ag and gg) in all animals studied. the values found for he were lower than those reported by mishra and colleagues (mishra  et  al.  2009), who found average heterozygosity of 0.572 for the banni breed and 0.610 for the murrah breed when they characterized 95 animals of these 2 breeds using 24 microsatellite markers, and by marques and colleagues (marques et  al. 2011) who studied the genetic diversity of brazilian buffaloes using twenty-five microsatellite index, the highest value was found in murrah (tf) with 0.671 (table i), that is, it has greater diversity (uramoto 2005, silva et al. 2016) when compared to the lower value in mediterranean (tf). the f statistic for the leptin snp for all buffalo populations was fis = - 0.19, fit = 0.34 and fst = 0.13. gene flow (nm) estimated from fst = 0.25 (1-fst) / fst was 1.69. table ii shows the fst values (wright 1965) between populations. these values were considered high (fst  >  0.15) between the mediterranean (tf) and murrah (tf), 0.298, mediterranean (va) and murrah (tf), 0.199, and crossbred (va) and murrah (tf), 0.178. in relation to the genic and genotype differentiations among the populations (table iii), the population of crossbred (va) and the population of mediterranean (va) are the groups with the lowest divergence. table iv shows the genetic identities and genetic distances of nei among the studied populations. in this study, the populations of mediterranean (tf) and murrah (tf) were the most different, and the populations of mediterranean (va) and crossbred (va) were the most similar. to illustrate, figure  2 represents two dendrograms, drawn from these genetic distances (nei 1972, nei 1978). discussion the genetic polymorphism in discussion is on intron  2 of the leptina gene (lien et  al. 1997). the table ii. fst statistic among breed populations of buffaloes in eastern amazon. me (tf) mu (tf) me (va) cb(va) me (tf) 0.000 mu (tf) 0.298 0.000 me (va) 0.010 0.199 0.000 cb (va) 0.021 0.178 -0.006 0.000 me (tf) = mediterranean terra-firme; mu (tf) = murrah terra-firme; me (va) = mediterranean varzea; cb (va) = crossbred varzea. table iii. gene differentiations (below diagonal) and genotype differentiations (above diagonal) among breed populations of buffaloes in eastern amazon. me (tf) mu (tf) me (va) cb(va) me (tf) hs 0.363 0.222 mu (tf) hs hs hs me (va) 0.397 hs 0.692 cb (va) 0.211 hs 0.692 hs = high significant; me (tf) = mediterranean terra-firme; mu (tf) = murrah terra-firme; me (va) = mediterranean varzea; cb (va) = crossbred varzea. table iv. nei’s genetic identity (above diagonal) and genetic distance (below diagonal) among breed populations of buffaloes in eastern amazon. me (tf) mu (tf) me (va) cb(va) me (tf) 0.688 0.991 0.982 mu (tf) 0.374 0.801 0.826 me (va) 0.009 0.222 1.002 cb (va) 0.018 0.191 -0.002 me (tf) = mediterranean terra-firme; mu (tf) = murrah terra-firme; me (va) = mediterranean varzea; cb (va) = crossbred varzea. mediterranean (tf) murrah (tf) a. upgma dendogram by nei’s (1978) genetic distance b. upgma dendogram by nei’s (1972) genetic distance mediterranean (va) crossbred (va) mediterranean (tf) murrah (tf) mediterranean (va) crossbred (va) figure 2. nei’s dendrograms among breed populations of buffaloes in eastern amazon. 261veterinaria italiana 2021, 57 (4), 257-263. doi: 10.12834/vetit.1739.9163.2 silva et al. leptin gene polimorphism in buffaloes murrah (tf) breeds (table iii). similar results were found by albuquerque and colleagues (albuquerque et  al. 2006), who estimated the genetic variability with the use of rapd markers among five groups of buffaloes raised in brazil, two groups being conserved in situ, carabao and baio type, and three groups considered commercial breeds, murrah, jafarabadi and mediterranean. in this study, the population of crossbred (va) and the population of mediterranean (va) were the groups with the lowest divergence (table iii). this is explained by the fact that the two genetic groups have been submitted to crosses in the past (albuquerque et al. 2006). for nei (nei 1972, nei 1978) the determination of the genetic distance allowed to verify the genetic variability among the populations. in this study, the genetic distance values reached revealed that the populations of mediterranean (tf) and murrah (tf) are the most different, and the populations of mediterranean (va) and crossbred (va) are the most similar (table iv). these results confirmed a higher degree of kinship between the crossbred and mediterranean populations of the varzea system, possibly due to long term interbreed between these two breeds (figure 2). conclusions the lep-1620 molecular polymorphism suggested low genetic variations between the mediterranean and crossbred populations, which distinguish them from the murrah breed. both mediterranean and crossbred buffalo groups are preferred by milk production farmers in eastern amazon region. this suggests a possible selection of animals for milk production which present allele a at high frequency, specially in homozigose (aa). markers in five breeds (carabao, jafarabadi, mediterranea and murrah, plus the baio type), with expected heterozygosity between 0.532 and 0.609. in the present work, we have observed less genetic variability when compared with what was presented by these authors. shannon's index is based on information theory (ludwig et  al. 1988) which provides a principle of degree of uncertainty, and it can predict which species an individual would randomly withdraw from the population. the higher the value of this index (table i), the greater the degree of uncertainty, that is, the greater the genetic diversity (uramoto 2005). fst is an index commonly used to measure distances between the studied populations, that is, the closer the values are to 1, the greater the differentiation between populations (attia et  al. 2014). these values were lower than those found by marques and colleagues (marques et  al. 2011), with fst = 0.1998 (0.1615-2,413). fis is a parameter that expresses the occurrence of random mating in the population, that is, whether the population suffers inbreeding or not. in this study, the value of fis was less than zero, and it shows the occurrence of mating between individuals that are not related. the fit parameter expresses the difference of an individual's heterozygosity over (a or the) metapopulation. when both fis and fit values are close to zero, that means there is genetic variability in the population (barros et al. 2011). that was the case with the results found in this study. although the gene flow (nm) above 1 means that the group does not have any significant genetic differentiation, when it is below 1, it is an evidence of genetic differentiation, and when it is greater than 4, this suggests a great amount of gene exchange (wright 1965, 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buffaloes (bubalus bubalis). mol biol rep, 40, 5159-5163. wright s. 1965. the interpretation of population structure by f‐statistics with special regard to systems of mating. evolution, 19, 395-420. zetouni l., camargo g.m.f., fonseca p.d.s., monsalves f.m., 245 1universidade federal fluminense, brazil. 2universidade federal do rio de janeiro, brazil. 3fundação oswaldo cruz, brazil. *corresponding author at: universidade federal do rio de janeiro, brazil. e‑mail: panzenhagen@ufrj.br. keywords antimicrobial resistance, pfge, rio de janeiro, salmonella, swine. summary this study, conducted in the state of rio de janeiro, aimed to genetically distinguish 29  isolates of s. typhimurium isolated from 344  samples of swine carcasses by pfge (pulse‑field gel electrophoresis) and to evaluate their antimicrobial resistance profile. out of the 29 isolates, 26 (90%) were resistant to at least one antimicrobial. sulfonamides (66%), nalidixic acid (66%), trimethoprim (66%) and tetracycline (52%) were the most frequent resistant drugs. multidrug‑resistance (mdr) profile was frequent (60% of isolates). the profile eno‑na‑nxn‑fc‑c‑s‑gm‑g‑t‑te (14%), cp‑eno‑na‑fc‑c‑s‑g‑t‑te (10%) and na‑g‑t (7%) were the most frequent. five isolates within the predominant pfge pulsetype came from lymph nodes of distinct animals from multiple slaughterhouses indicating that this particular clone might be widespread in the rio de janeiro state. this paper reveals a threat to the population in the entire state and highlights the necessity of the strict control in the use of antimicrobials in swine production in the entire country. claudius cabral1, pedro panzenhagen1,2*, karina delgado1, gabriela rodrigues1, andré mercês3, dalia rodrigues2, robson franco1 and carlos conte‑junior1,2,3 genetic diversity and multidrug-resistance among salmonella typhimurium isolated from swine carcasses and slaughterhouses in rio de janeiro, brazil veterinaria italiana 2020, 56 (4), 245‑250. doi: 10.12834/vetit.1688.8954.1 accepted: 14.02.2019 | available on line: 31.12.2020 production center with slaughterhouses producing meat that supplies the local consume within the municipality or inside the state borders. however, evaluation of the microbiological quality of pork production in the rio de janeiro state is out of date (lázaro et  al. 1997, zebral et  al. 1974) and new studies need to be performed. recently, we reported 36 out of 344 (10.5%) samples from swine slaughterhouses contaminated with salmonella including carcasses, lymph nodes, ham, jowl, knives and even the cleaning water (cabral et  al. 2017). these results evidence how high is the consumer potential exposure to salmonella causing concern to public health authorities. a serious concern about salmonella strains regards their antimicrobial susceptibility profile. in severe cases of human salmonellosis, it is expected that the first‑choice antimicrobial therapy will be able to control systemic infection. because antimicrobial resistance has been increasingly introduction the foodborne pathogen salmonella is responsible for human salmonellosis, an infection that has the potential to become life‑threatening. in the united states, salmonella is the second most prevalent foodborne pathogen, and is responsible for the highest number of hospitalizations (cdc 2018). in brazil, this pathogen is the most frequent cause of foodborne diseases, and responsible for 39% of the notified and confirmed cases (finger et  al. 2019). although chicken is considered as the main reservoir of salmonella , swine is also crucial in the transmission of this pathogen and is capable of periodically shedding this bacterium through feces (mataragas et al. 2011). in brazil, several studies already revealed contamination of pork and pork‑based products with salmonella (cabral et al. 2014, silva et al. 2009, teixeira 2007). the rio de janeiro state has a pork 246 pfge and amr profile of s. typhimurium from swine cabral et al. veterinaria italiana 2020, 56 (4), 245‑250. doi: 10.12834/vetit.1688.8954.1 broth (bhi) (himedia®) with 25% glycerol. these isolates were reactivated by transferring 100  µl to a sterilized bhi broth, followed by incubation at 37  °c for 20 hours. newly grown isolates were then transferred to mueller‑hinton broth (mh broth) and incubated at 37 °c for 20 hours.  antimicrobial susceptibility test isolates were grown in mh broth to prepare the inoculum for the kirby‑bauer antimicrobial susceptibility test (ast ), which was performed as described at the clinical and laboratory standards institute ‑ clsi (clsi 2017). eighteen antimicrobials from eight classes were tested, including the most commonly used veterinary drugs in swine production and the first‑choice drugs for the treatment of human enteric infections. the diffusion disks (oxoid, basingstoke, uk) with the following drugs were used: amoxycillin/clavulanic acid (amc, 30 µg), cephalothin (cf, 30 µg), cefoxitin (cfx, 30  µg), ceftazidime (caz, 30  µg), ceftriaxone (cax, 30  µg), ciprofloxacin (cp, 5  µg), enrofloxacin (eno, 5  µg), nalidixic acid (na, 30  µg), norfloxacin (nxn, 10 µg), florfenicol (fc, 30 µg), chloramphenicol (c, 30 µg), streptomycin (s, 10 µg), gentamicin (gm, 10  µg), tobramycin (to, 10  µg), imipenem (imp, 10  µg), sulfonamides (g, 300  µg), trimethoprim (t, 5  µg), tetracycline (te, 30  µg). e. coli atcc 25922, s.  aureus atcc 25923, p. aeruginosa 10536 were tested in parallel and served as control strains according to clsi. pulsed‑field gel electrophoresis (pfge) pfge was performed at the national reference laboratory for enteroinfections at oswaldo cruz institute (fiocruz) according to the standard operating procedure for pulsenet pfge of escherichia coli o157:h7, escherichia coli non‑157 (stec), salmonella serovars, shigella sonnei and shigella flexneri, established by the centers for disease control and prevention (cdc 2013). the dna fingerprints were generated by macrorestriction with 40 u of enzyme xbai (new england biolabs, beverly, ma, usa). restriction fragments were separated in certificated 1.2% pfge agarose gels (bio‑rad, hercules, ca, usa) in tris‑borate buffer (tbe; tris‑borate 0.045 m, edta 0.001m) at 140 °c, using the chef dr iii system (bio‑rad). electrophoresis runs with an initial switch time of 2.2 s and a final switch of 63.8 s at 6v/s for 18 h. salmonella braenderup was used as standard and similarity was calculated by the dice coefficient with 1.5 to 2.0% tolerance. the generated profile assembly and dendrogram analysis were performed with bionumerics software 7.5 (biomerieux®). detected in salmonella, public health has been continually threatened (cdc 2005). another hazard is related to the possibility of the multidrug‑resistant (mdr) strains to disseminate resistance genes to non‑resistant bacteria transferring those genes between human and animal populations, mostly in the gut environment (trobos et  al. 2009). it is now well established that the selective pressure created by the inappropriate antimicrobial use in human and veterinary medicine is one of the main reasons for the increase of antimicrobial resistance (tenover 2006). within more than 2,600 salmonella enterica serovars, salmonella typhimurium is the most surveyed and frequent serovar transmitted from animals to humans worldwide (hendriksen et  al. 2011). in brazil, several studies provide evidence that this serovar has been the most frequently isolated in swine and pork‑based products (bandeira et  al. 2007, castagna et al. 2004, kich et al. 2011, michael et  al. 2002, pissetti et  al. 2012, seixas et  al. 2009, viott et al. 2013). previously, we have demonstrated that salmonella typhimurium is prevalent (55% of isolates) in the state of rio de janeiro (cabral et  al. 2017). now, the understanding of the diversity level of these isolates is indispensable to monitoring interventions strategies if outbreaks investigations are necessary (kich et  al. 2011). in this context, pulsed‑field gel electrophoresis (pfge) is eligible due to the rapid standardized protocol, parameters analysis and nomenclature, and the ability to exchange information in real‑time through internet by the center of pulsenet’s (ribot et  al. 2006). also, it is routinely used for foodborne outbreaks investigation and studies regarding animal infections and food pathogens (kich et  al. 2011, vigo et al. 2009). hence, the current study was designed to evaluate the antimicrobial susceptibility profile of 29 isolates of salmonella typhimurium, along with their molecular typing with pfge in the purpose of epidemiologically differentiate and trace the sources of those bacteria. materials and methods bacterial isolates we selected the twenty‑nine isolates of s.  typhimurium previously obtained from 344  samples of swine carcasses (intestinal faeces, mesenteric and submandibular lymph nodes, jowl, ham) and from the water for cleaning the carcasses in swine slaughterhouses (s1, s2, and s3) in the rio de janeiro state (cabral et  al. 2017). isolates were kept frozen at ‑  18  °c in brain heart infusion 247 cabral et al. pfge and amr profile of s. typhimurium from swine veterinaria italiana 2020, 56 (4), 245‑250. doi: 10.12834/vetit.1688.8954.1 results twenty‑six out of the 29 selected isolates (90%) exhibited resistance to at least one antimicrobial. also, two isolates had only intermediate resistance to at least one antibiotic and two isolates were pan‑susceptible. seventeen isolates (60%) were mdr since they showed resistance to more than three different antimicrobial classes. sixty‑six percent of the isolates were resistant to sulfonamide, nalidixic acid, and trimethoprim. also, 97% of them were susceptible to tobramycin, ceftriaxone, and imipenem (table i). pfge‑based sub‑typing was performed to determine the diversity of the isolates selected in this study, (figure 1). this analysis revealed a total of 11 different pulsetypes wherein the three predominant types were formed by at least six isolates each (clusters a and b). pulsetype 1 (cluster a) harbored eight isolates whereas pulsetype 2 (cluster b1) harbored six isolates and pulsetype 3 (cluster b2) also harbored six isolates. in the cluster c, the pulsetypes eight and nine were 92% similar. the cluster d harbored two isolates with the pulsetype 11. the use of the single xbai enzyme was able to genetically distinguish 7 out of 29 isolates (pulsetypes 4, 5, 6, 7, 8, 9 and 10) (figure 1). however, the majority of the isolates which share identical pfge profile (pulsetypes 1, 2, 3 and 11) showed different antimicrobial resistance profiles, 60 65 70 75 80 85 90 95 10 0 pfge-xbal 11 amc cf cfx caz cax cp eno na nxn fc c s gm to imp g t te 11 10 9 8 7 6 5 4 3 3 3 3 3 3 2 2 2 2 2 2 1 1 1 1 1 1 1 1 swine 1 faeces swine 5 mesenteric lymph node swine 6 mesenteric lymph node swine 7 mesenteric lymph node swine 5 jowl swine 1 mesenteric lymph node swine 6 mesenteric lymph node carcass cleaning water swine 5 mesenteric lymph node swine 1 jowl swine 1 jowl swine 1 mesenteric lymph node swine 5 ham swine 3 mesenteric lymph node swine 2 mesenteric lymph node swine 1 mesenteric lymph node swine 5 mesenteric lymph node swine 5 jowl swine 6 mesenteric lymph node swine 5 ham swine 7 mesenteric lymph node swine 4 jowl carcass cleaning water swine 7 mesenteric lymph node swine 2 ham swine 6 mesenteric lymph node swine 2 mesenteric lymph node swine 7 mesenteric lymph node swine 2 submandibular lymph node s3 s3 s1 s1 s2 s3 s3 s1 s3 s3 s3 s3 s3 s3 s3 s3 s2 s2 s3 s3 s2 s2 s2 s1 s3 s1 s1 s1 s1 c d b1 b2 a b pulsetype antimicrobial resistance pro�le sample origin slaughterhouse figure 1. pulsed‑field gel electrophoresis (pfge) dendrogram and antimicrobial resistance profile showing the genetic and phenotypic diversity among 29 isolates of salmonella typhimurium obtained from swine slaughterhouses in the rio de janeiro state. antimicrobial resistance profile abbreviations: amoxycillin/clavulanic acid (amc), cephalothin (cf ), cefoxitin (cfx), ceftazidime (caz), ceftriaxone (cax), ciprofloxacin (cp), enrofloxacin (eno), nalidixic acid (na), norfloxacin (nxn), florfenicol (fc), chloramphenicol (c), streptomycin (s), gentamicin (gm), tobramycin (to), imipenem (imp), sulfonamides (g), trimethoprim (t), tetracycline (te). grey boxes represent intermediate resistance. table i. antimicrobial susceptibility profile of 29 salmonella typhimurium isolates obtained from swine carcasses and slaughterhouses in rio de janeiro, brazil. antimicrobial susceptibility profile [n (%)] sensitive intermediate resistance resistant streptomycin 14 (48%) 10 (35%) 5 (17%) gentamicin 19 (66%) 3 (10%) 7 (24%) tobramycin 28 (97%) 1 (3%) amoxicillin + clavulanic acid 24 (83%) 4 (14%) 1 (3%) cephalothin 21 (72%) 2 (7%) 6 (21% cefoxitin 23 (79%) 2 (7%) 4 (14%) ceftazidime 27 (93%) 1 (7%) 1(3%) ceftriaxone 28 (97%) 1 (3%) imipenem 28 (97%) 1(3%) chloramphenicol 14 (48%) 15 (52%) florfenicol 14 (48%) 15 (52%) nalidixic acid 6 (21%) 4 (14%) 19 (66%) ciprofloxacin 12 (41%) 5 (18%) 12 (41%) enrofloxacin 15 (52%) 14 (48%) norfloxacin 23 (79%) 4 (14%) 2 (7%) sulfonamide 9 (31%) 1 (3%) 19 (66%) trimethoprim 9 (34%) 20 (66%) tetracycline 14 (48%) 15 (52%) 248 pfge and amr profile of s. typhimurium from swine cabral et al. veterinaria italiana 2020, 56 (4), 245‑250. doi: 10.12834/vetit.1688.8954.1 2016) reported high resistance to tetracycline (44%) and nalidixic acid (25%) in salmonella isolates obtained from pigs and multiple pork by‑products. interestingly, high frequency of isolates with resistance to tetracycline is not unexpected in brazil since this antibiotic was routinely used as a growth promoter in swine breeding. the ministry of agriculture, livestock and supply has forbidden its use as growth promoter since 1998, although it is still allowed for infection therapy. sulfonamide resistance along with gentamycin and fluoroquinolones resistance also are routinely detected among salmonella isolates, and this can be explained by their widespread use in swine breeding (silva et  al. 2009). in our study, sulfonamide and trimethoprim resistance were separately evaluated, but nowadays they are commonly used in combination due to their synergism (cotrimoxazole). trimethoprim resistance is also common, however resistance is lower when it is associated with sulfamethoxazole (lima et  al. 2016). in this study, 22/29 (76%) isolates were nalidixic acid resistant, 13/29 (44%) to ciprofloxacin and 16/29 (55%) to enrofloxacin. this last finding could be a matter of great concern to public health because, in human medicine, ciprofloxacin is the first choice drug for cases of salmonellosis, especially when dealing with septicemic strains (souza et  al. 2010). the implications of the indiscriminate use of antimicrobials in animal production on bacterial resistance have been continually reviewed (landers et  al. 2012). the uncorrect use by animal breeders and the little control by the authorities contribute to the increase of bacterial resistance. multidrug‑resistant (mdr) strains are defined as isolates resistant to three or more different antimicrobials classes (clsi 2017). despite few isolates studied here, the high frequency of mdr isolates (60%) remains alarming. in brazil, most of the studies have shown frequency of s. typhimurium in swine with mdr profile below fifty percent: almeida and colleagues (almeida et  al. 2016) 37%, lopes and colleagues (lopes et  al. 2015) 40.4%, kich and colleagues (kich et  al. 2011) 43%. mdr isolates are dangerous anywhere since they are commonly more virulent than susceptible ones, and this is a constant threat to human health (dimarzio et  al. 2013). a high variety of mdr profiles were found in our isolates even in that obtained from the same slaughterhouses. although the antimicrobial use records from the swine breeding were not available, it is possible to speculate that the breeders might have adopted different protocols and these variations may have resulted in the selection and widespread of many different multi‑resistant profiles within the isolates. pulsed‑field gel electrophoresis identified 11  distinct pulsetypes among the twenty‑nine samples. pulsetype 1 (cluster a) is the biggest one suggesting that they might be genetically different although not distinguished by the use of the single xbai enzyme. overall, amr profile was quite diverse, but four isolates (14%) shared the common profile cp‑eno‑na‑nxn‑fc‑c‑s‑gm‑g‑t‑te, three isolates (10%) shared the profile cp‑eno‑na‑fc‑c‑s‑g‑t‑te, and two isolates (7%) shared the profile na‑g‑t. no association between the antimicrobial resistance profile and pulsetype were found. however, isolates with resistance to four or more classes were in cluster b (figure 1). discussion slaughterhouses in the rio de janeiro state plays a crucial role in the economics of local municipalities as a provider of quality protein to those consumers. in 2017, we published the first study reporting that those slaughterhouses were producing salmonella‑contaminated pork (cabral et  al. 2017). here, we selected all the 29 s.  typhimurium isolated from that study to profile a phenotypically and genetically characterization by accessing the antimicrobial susceptibility profile and pfge, respectively. in brazil, few studies have evaluated the antimicrobial susceptibility profile of salmonella isolates from swine carcasses, pork or slaughterhouse environment, although those performed reported a high prevalence of isolates resistant to at least one antimicrobial. lopes and colleagues (lopes et al. 2015) reported that 76% of salmonella enterica serovars isolated from pigs and carcasses were resistant to at least one antimicrobial. lima and colleagues (lima et al. 2016) have evaluated 357 from pork and pork by‑products and found 257 isolates (72%) resistant to one or more drugs. also, almeida and colleagues (almeida et  al. 2016) reported that 17 out of 27 (63%) isolates had resistance to at least one drug. here, we reported 90% of the tested isolates showing resistance to at least one drug. although these studies did not reveal resistance patterns specifically from typhimurium serovar, they reported that drug resistance is wide‑spreading across brazilian foodborne salmonella isolates. in the present study, most of the salmonella isolates were resistant to sulfonamides, nalidixic acid, trimethoprim, and tetracycline. resistance to these four antibiotics is routinely reported in salmonella. for instance, bessa and colleagues (bessa et  al. 2007) reported high rates of salmonella resistant to sulfonamide (91%), tetracycline (85%) and streptomycin (66%). this profile was also reported by lopes and colleagues (lopes et  al. 2015) with 55%, 40%, 34% and 34% resistance to tetracycline, sulfonamide, streptomycin and nalidixic acid, respectively. lima and colleagues (lima et  al. 249 cabral et al. pfge and amr profile of s. typhimurium from swine veterinaria italiana 2020, 56 (4), 245‑250. doi: 10.12834/vetit.1688.8954.1 that isolates from the same serovar are worth to be typed since they may not belong to the same clone and consequently exhibit distinct epidemiological importance. in conclusion, the results of pfge typing along with the antimicrobial resistance profile revealed a high variety of s.  typhimurium isolates among swine samples from slaughterhouses in rio de janeiro state. the high frequency of mdr profile among these isolates indicates that pigs in that region are reservoirs with potential risk to transmit multidrug‑resistant s.  typhimurium. this paper reveals a threat to the population public health in the entire state and highlights the necessity of strict control in the use of antimicrobials in the swine production in brazil. acknowledgments the authors are thankful to coordenação de aperfeiçoamento de pessoal de nível superior ‑ capes for the financial funding. with eight isolates (figure 1). curiously, five clones in this cluster came from lymph nodes of different animals (swine  2,  6,  7) raised in the same breeder. strains isolated from lymph nodes indicate that these animals are harboring salmonella and can asymptomatically carry this pathogen since they are capable of periodically shed the bacteria through feces. according to silva and colleagues (silva et  al. 2006), infection at the farm level mainly at the finishing step is the most common event responsible for swine infection. samples within pulsetype 1 provide evidence that this particular clone is widespread in the same swine breeder. because these isolates were obtained from three different slaughterhouses (s1, s2, and s3), it is possible to speculate that they are also circulating not only in the breeders but also among the slaughterhouses in the rio de janeiro state. conversely, pfge also revealed s.  typhimurium isolates with distinct pulsetypes isolated from the same animal. for instance, we obtained isolates from different parts in swine number five belonging to the pulsetype 2, 3, 4 and 11. this finding supports the evidence almeida f., medeiros m.i.c., kich j.d. & falcão j.p. 2016. virulence‑associated genes, antimicrobial resistance and molecular typing of salmonella typhimurium strains isolated from swine from 2000 to 2012 in brazil. j appl microbiol, 120, 1677‑1690. bandeira r., da cruz payão, pellegrini d. & cardoso m. 2007. ocorrência de salmonella sp. em cortes de pernil provenientes de lotes suínos portadores ao abate. acta sci vet, 35. retrieved from http://www.redalyc.org/ resumen.oa?id=289021845010. bessa m.c., michael g.b., canu n., canal c.w., cardoso m., rabsch w. & rubino s. 2007. phenotypic and genetic characterization of salmonella enterica subsp. enterica serovar typhimurium isolated from pigs in rio grande do sul, brazil. res vet sci, 83, 302‑310. cabral c.c., conte‑junior c.a., silva j.t. & paschoalin v.m.f. 2014. salmonella spp. 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costa m. da. 2002. sorotipos de salmonella isolados em uma propriedade de suínos de terminação no sul do brasil. ciênc rural, 32, 525‑527. pissetti c., werlang g.o., biesus l.l., kich j.d. & cardoso m.r. de i. 2012. detecção de salmonella enterica e listeria monocytogenes em carcaças suínas na etapa de pré‑resfriamento. acta sci vet, 40, 1‑8. ribot e.m., fair m. a., gautom r., cameron d. n., hunter s. b., swaminathan b. & barrett t.j. 2006. standardization of pulsed‑field gel electrophoresis protocols for the subtyping of escherichia coli o157:h7, salmonella, and shigella for pulsenet. foodborne pathog dis, 3, 59‑67. 347 istituto zooprofilattico sperimentale della puglia e della basilicata, via manfredonia 20, 71121 foggia. *corresponding author at: istituto zooprofilattico sperimentale della puglia e della basilicata, via manfredonia 20, 71121 foggia, italy. tel.: +39 0881786326, e-mail: domenico.galante@izspb.it. parole chiave analisi filogenetica, colture cellulari, gene vltf-1, microscopia elettronica, orf virus, pcr. riassunto l'orf virus (orfv; family: poxviridae) è l'agente causale dell'ectima contagioso, malattia infettiva contagiosa degli ovicaprini e di altri ruminanti domestici o selvatici, con distribuzione mondiale. la malattia è endemica in italia, ma in letteratura scientifica sono disponibili pochi dati sulla sua distribuzione ed epidemiologia. in questo studio sono stati analizzati 32 campioni clinici (croste cutanee) prelevati da 27 ovini e 5 caprini provenienti da 19 focolai sospetti di ectima contagioso occorsi in puglia e basilicata tra il 2012 e il 2014. per identificare il virus sono state effettuate sia l'analisi morfologica mediante microscopia elettronica (me), sia una pcr mirata al rilevamento del gene del fattore di trascrizione tardiva (vltf-1). i ceppi virali sono stati isolati anche su linea cellulare bhk-21. la pcr è risultata essere la metodica più sensibile, evidenziando la presenza di orf virus in 28 dei 32 campioni analizzati, mentre la microscopia elettronica ha permesso di rilevare il virus solo in 26 dei 32 campioni. la maggior parte degli isolati forma un gruppo monofiletico; essi, sulla base delle sequenze del gene vltf-1, risultano essere altamente correlati a ceppi di orf virus già circolanti in sud italia in passato. identificazione ed analisi filogenetica di orf virus isolati in focolai di ectima contagioso in pecore e capre in italia keywords cell culture propagation, electron microscopy, orf virus, pcr, phylogenetic analysis, vltf-1 gene. summary orf virus (orfv; family: poxviridae) is the causative agent of contagious ecthyma, or orf disease in sheep, goats and other domestic or wild ruminants with a worldwide distribution. the disease is endemic in italy, but few data are available about its distribution and epidemiology. in the present study we analysed 32 clinical samples, obtained from crusted scab lesions of 5 goats and 27 sheep, from 19 suspected outbreaks of contagious ecthyma in apulia and basilicata regions between 2012 and 2014. negative staining electron microscopy (em) and polymerase chain reaction (pcr) targeting the late transcription factor gene (vltf-1) were used to identify the virus. isolation was also attempted on bhk-21 cell line. pcr was proved to be more sensitive than em, as it detected the virus in 28 out of 32 samples, whereas the em detected it only in 26 out of the 32 samples. the majority of isolated strains forms a monophyletic group; these isolates, according to the vltf-1 sequencing, are high related to orfv strains previously shown to circulate in southern italy. domenico galante*, maria assunta cafiero, donato antonio raele, nicola pugliese, iolanda padalino, nicola cavaliere and canio buonavoglia identification and characterization of orf viruses isolated from sheep and goats in southern italy veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 accepted: 23.04.2017 | available on line: 31.12.2019 348 nick title galante et al. veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 reported in different areas of apulia and basilicata regions, in southern italy. in most cases, clinical signs were limited to scabs on the lips, and the disease spontaneously resolved in 3-4 weeks. conversely, 2 outbreaks were characterised by high fever, oedemas of the face, and of the submandibular region, scabs on lips, tongue and teats, which led to anorexia (figure 1). in these 2 outbreaks, about the 6% of the animals died. one to 5 clinical samples (listed in table i) per outbreaks were collected from crusted scab lesions and analysed. electron microscopy the first diagnosis was achieved by em. all the crusty scab samples were mechanically homogenised in a sterile mortar with distilled water. after staining with 2% napt acid, ph 6.8 (doane and anderson 1987), the grids were observed using an electron microscope (em philips 208s, fei, eindhoven, netherland) operating at 80 kv. propagation in cell cultures the cell line used for the propagation of the virus was the baby hamster kidney (bhk-21), previously proved to be reliable for the cultivation of the orfv (kottaridi et  al. 2006a). homogenised samples were centrifuged at 3,000 x g for 5 minutes, and the supernatant was filtered through a 0.45  µm filter (merck millipore, milan, italy) and treated with antibiotics and antimycotic (namely, penicillin 100 u/ml, streptomycin 100 µg/ml, neomycin 50 µg/ ml, and amphotericin b 2.5 µg/ml). the mixture was inoculated in the cell suspension, cultivated with dulbecco’s modified eagles medium and supplemented with 10% bovine foetal serum. cell cultures were incubated at 37 °c for 96 hours, daily observed for the presence of cytopathic effects (cpes), and passaged up to 4 times. polymerase chain reaction total dna was extracted from all clinical samples by using the dneasy blood and tissue kit (qiagen, milan, italy); dna from tissues and supernatants from cell cultures were used as a template for pcr targeting the gene 045, which encodes the late transcription factor vltf-1 of ppv (delhon et  al. 2004). pcr was performed as previously described (kottaridi et  al. 2006a) by using the 045f and 045r primers. the reactions were carried out in a volume of 50 µl. the reaction mixture consisted of 25 µl pcr master mix (redtaq readymix pcr, sigma-aldrich, milan, italy), 1 µm of each primer, and 5 µl of the extracted dna. cycling conditions were as follows: initial denaturation at 95 °c for 5 minutes, followed by 40 cycles at 95  °c for 15 seconds, 57.7  °c for 30 introduction orf virus (orfv) is an epitheliotropic virus of the genus parapoxvirus (ppv), family poxviridae, which also includes the bovine papular stomatitis virus (bpsv), pseudocowpox virus (pcpv), causing milker's nodule in man, ppv of the new zealand red deer (pvnz), and other tentative species, such as the camel ppv (ccev), or seal ppv. parapoxviruses may cause disease in a wide range of wild animal species (camels, red deer, reindeer, squirrels, seals, and grey seals) (robinson and mercer 1995, scagliarini et al. 2011, tryland et al. 2005, thomas et al. 2003) as well as in humans, especially if immunocompromised (ara et  al. 2008, mercer et  al. 2014). at risk humans are those living in close contact with infected animals such as farmers, abattoir workers, veterinarians, and sheep shearers. in humans, the disease usually causes painful nodules on hands and arms and, more infrequently, on the face (kuhl et al. 2003). in zootechny, orfv has to be considered a problem for sheep industry, as it causes decrease of productions, but it also poses a risk for workers’ health (zhang et al. 2014). generally, the virus enters the host through skin lesions, and initiates a localized virus replication in the epidermal cells and keratinocytes, producing the typical clinical signs of contagious ecthyma. in sheep and goats, the disease is usually circumscribed to the skin of the lips, around the nostrils, and to the oral mucosa. sometimes, it also affects the gum and the tongue, especially in young lambs (hosamani et  al. 2009). the disease is active for 3-4 weeks and lesions heal completely in 1-2 months. despite contagious ecthyma is generally characterised by high morbidity, mortality rates are usually low. however, mortality up to 93% has been reported in younger animals (mazur and machado 1989, mazur and machado 1990). this study aimed at identifying the orfv as causative agent of the disease in 19 suspected outbreaks of contagious ecthyma occurred in apulia and basilicata regions, southern italy, between 2012 and 2014. furthermore, we compared the reliability and suitability of negative staining electron microscopy (em) propagation in cell cultures, and a polymerase chain reaction (pcr) assay for the direct identification of orfv dna from skin biopsies. finally, we characterised the strains by analysing the 045 gene, encoding the late transcription factor vltf-1, which is already used to detect genetic variations among ppvs (mahmoud et al. 2010, davari et al. 2013). materials and methods disease outbreaks and sample collection between 2012 and 2014, 19 outbreaks of suspected contagious ecthyma in sheep and goats were 349 galante et al. nick title veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 table i. clinical samples analyzed in this study. —cont’d farm relative number clinical samples place and date of collection host cell culture electron microscopy pcr 045 gene a 5467 fg foggia, puglia 07-2012 sheep + + + b 3472 fg foggia, puglia 08-2012 sheep + + + c 12821 fg foggia, puglia 08-2014 sheep + + + d 13281 fg foggia, puglia 09-2014 sheep + + e 868 fg foggia, puglia 12-2014 sheep + + f 16232/1 ba bari, puglia, 09-2013 sheep + + + f 16232/2 ba bari, puglia, 09-2013 sheep + + + g 17205/1 ba bari, puglia, 10-2013 sheep + + + g 17205/2 ba bari, puglia, 10-2013 sheep + + + h 801 ba bari, puglia, 12-2014 sheep + + + i 3869 le lecce, puglia, 09-2012 sheep + + + j 10278 pz potenza, basilicata, 06-2013 sheep + + + j 10279 pz potenza, basilicata, 06-2013 sheep + + + k 8598 pz potenza, basilicata, 06-2013 sheep + + + l 9726/19 pz potenza, basilicata, 09-2013 sheep + + + m 5174/1 matera, basilicata, 07-2013 goat + + + m 5174/2 matera, basilicata, 07-2013 goat + + + m 5174/3 matera, basilicata, 07-2013 goat + + + figure 1. clinical signs of orf virus infection in sheep in the two most severe outbreaks of the disease. nodular lesions, evolving in scabs are well evident around nostrils, on the lips (a, b), in the oral mucosa, on the tongue (c) and in some cases also around the teats (d). the oedema of the head and the hypersalivation are evident (a, b, c). continued 350 nick title galante et al. veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 the modeltest script implemented for web at findmodel2. results negative staining electron microscopy typical ppv virions were observed in 26 of the 32 samples analysed by em. the particles appeared as enveloped, ovoid virions, 220-300 nm long, and 140-170 nm wide. the surface membrane displayed regularly arranged surface tubules. in most of the analysed samples, several viral particles were visible. mature viral particles of ppv were also observed from all the suspensions of the cell cultures. cell cultures and pcr detection twenty-eight of the 32 cell cultures showed cpe (ballooning, wounding, degeneration of cells up to the complete destruction of the monolayer) starting from passage 2. for 2  samples only (13281 fg and 868  fg), cpe was evident at passage 4. vltf-1 was successfully amplified from dna purified from all 28 orfv-positive cell cultures. vltf-1 was also amplified from the corresponding infected biological samples while the other 4, shown to be negative by virus isolation, resulted negative (table i). seconds and 72 °c for 50 seconds; final extension of 72 °c for 10 minutes. amplification products were run on 2% agarose gel stained with sybr® safe (life technologies, milan, italy) and were visualised by the mean of a the molecular imager gel doctm xr+ (bio rad, milan, italy). sequencing and phylogenetic analysis amplification products obtained from dna purified from cell supernatants were purified by the qiaquick pcr purification kit (qiagen, milan, italy) and both strands were sequenced by the sanger method (big dye terminator kit, life technologies, milan, italy) using external facilities (eurofins genomics italy, milan, italy). the high quality regions were assembled by cap31. nucleotide sequences were submitted to the ncbi genbank under accession numbers from kr812113 to kr812140. sequences were compared with those present in genbank by blast to confirm orfv identification. after the identification, our sequences and other orf virus sequences submitted to the genbank, were aligned by clustalw algorithm implemented in mega 6.0 (tamura et  al. 2013) (table ii). phylogeny was inferred by maximum likelihood estimation using the phyml software (guindon and guascel 2003), with 1,000 non-parametric bootstrap replicates. the best fitting evolutionary model (gtr+g, alpha  =  0.48) was determined by using table i. clinical samples analyzed in this study. —cont’d farm relative number clinical samples place and date of collection host cell culture electron microscopy pcr 045 gene m 5174/4 matera, basilicata, 07-2013 goat + + + n 5181/1 matera, basilicata, 07-2013 sheep + + + n 5181/2 matera, basilicata, 07-2013 sheep + + + n 5181/3 matera, basilicata, 07-2013 sheep + + + n 5181/4 matera, basilicata, 07-2013 sheep + + + n 5181/5 matera, basilicata, 07-2013 sheep + + + o 6173/1 matera, basilicata, 09-2013 sheep + + + o 6173/2 matera, basilicata, 09-2013 sheep + + + o 6173/3 matera, basilicata, 09-2013 sheep + + + o 6173/4 matera, basilicata, 09-2013 sheep + + + p 1179 brindisi, puglia, 05-2013 goat q 3559 taranto, puglia, 11-2013 sheep r 145 foggia, puglia, 01-2014 sheep s 9301 potenza, basilicata, 09-2014 sheep 1 http://doua.prabi.fr/software/cap3 (latest accessed on march 4, 2016). 2 http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html, latest accessed march 4, 2016. 351 galante et al. nick title veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 for 14  nucleotides from the other sequences of cluster  a. however, no differences were shown at amino acid level. this latter sequence grouped in a distinct branch of the phylogenetic tree, with strong bootstrap support. discussion the molecular approach was confirmed to be the most sensitive for the diagnosis of orfv, and probably of other ppvs, from clinical samples. in fact, we found that pcr was able to detect orfv dna in 28 samples, while em evidenced the virus in 26 only. furthermore, orfv isolation from pcr-positive samples tissue confirmed the presence of live viral particles. reasonably this method requires more time and high-skilled personnel. as reported in other studies, the reduced sensitivity of em might be due to a low viral load in the samples, as it happens when tissues are collected from animals during the healing phase of infection (doane and anderson 1987). however, negative staining em by the drop-to-drop method has proved to be very fast, and it can be phylogeny when compared to those present in genbank, the nucleotide sequences from vltf-1 gene were 99% identical to the correspondent sequences of previously submitted orfv, thus confirming the detection of this virus in the analysed samples. phylogenetic analysis showed that all but 3 sequences were identical among themselves, and that they grouped together in cluster  a (figure  2). this cluster also included strains ov-ia82 isolated in iowa (us) from nasal secretion of a lamb (delhon et al. 2004), strain nz2, isolated in new zealand from sheep scab material (mercer et  al. 2006), and strain b029, isolated in germany from humans (friederichs et al. 2014). out of the 3 sequences not included in the cluster a, 2 (10278 and 10279) originated from sheep of the same farm (j, table i), located in potenza, basilicata. they were 100% identical, while they differed for 3 nucleotides from sequences belonging to cluster a. finally, the most divergent sequence was obtained from isolate 3472, isolated from a sheep sample collected in foggia in 2012. it differed table ii. list of strains from genbank enrolled for phylogenetic analysis. strain species host isolation place (year) genbank accession number bv-ar02 bovine papular stomatitis virus calf usa (n/a) ay386265 bv-tx09c1 bovine papular stomatitis virus cattle usa (2009) km875472 bv-tx09c5 bovine papular stomatitis virus cattle usa (2009) km875471 bv-tx09c15 bovine papular stomatitis virus cattle usa (2009) km875470 myv myxoma virus brush rabbit usa (1950) kf148065 f00.120r pseudocowpox virus reindeer finland (2000) gq329669 pcpv776 pseudocowpox virus human austria (2010) hm589037 vr634 pseudocowpox virus human n/a (n/a) gq329670 b029 orf virus human germany (1996) kf837136 d1701 orf virus sheep germany (n/a) hm133903 egypt orf virus sheep egypt (2006) eu826136 na1/11 orf virus sheep china (2011) kf234407 nz2 orf virus sheep new zealand (n/a) dq184476 ov-ia82 orf virus sheep usa (1982) ay386263 ov-sa00 orf virus human usa (n/a) ay386264 shiraz1 orf virus sheep iran (2012) kc534486 shiraz3 orf virus sheep iran (2012) kc534488 shiraz4 orf virus sheep iran (2012) kc534489 shiraz5 orf virus sheep iran (2012) kc534490 hl953 red deer parapoxvirus red deer germany (2013) km502564 sqpv squirrelpox virus red squirrel uk (n/a) he601899 n/a = information not available in genbank. 352 nick title galante et al. veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 isolates from all over the world are related to these two strains (lojkic et  al. 2010). nz2-related strains were known to circulate in central italy since the first decade of 2000’s (cardeti et al. 2005) and our study highlights its persistence. however, we also evidenced the presence, in the regions involved in the present study, of other strains, which were more distantly related to the predominant group. in particular, the vtlf-1 gene of the isolate 3472 appears well distinct from the others. interestingly, we did not observe any correlation between phylogenetic clustering and disease severity. the strains causing the most virulent outbreaks were included in the cluster a, and their vtlf-1 did not differ from the others. such finding confirms the hypothesis that the clinical features of the disease may be due to host-related factors rather than to specific pathogenic traits of the virus (gallina et al. 2008). overall, these data strongly suggest that most of the cases of contagious echtyma of apulia and basilicata are caused by a strain characterized by the same vltf-1 gene that circulates in southern italy. other strains could be present, probably imported from other countries as result of animal trade. considered a reliable option for the quick diagnosis of contagious ecthyma, especially during active outbreaks. in fact, by means of such technique, results can be achieved in 30 minutes. vtlf-1 gene is also useful for phylogenetic analysis. in the present study the gene was sequenced to check whether significant genetic differences could be found in the strains isolated in the outbreaks. while highly conserved, we found a limited variability due to point mutations, in agreement with a previous report (mahmoud et al. 2010). contagious ecthyma is a very common disease, diffused all over the world. in italy, the disease is present and it may be considered endemic in southern italy, where sheep and goat breeding is widespread. however, to the best of our knowledge, scientific literature upon orfv circulation in italy is limited (kottaridi et  al. 2006b). the present study reveals that the majority of orfv strains, which have been circulating among sheep farms of apulia and basilicata during 2012-2014, is enclosed within a monophyletic group. furthermore, they were highly related to well characterised strains, isolated 3 decades ago. in particular, the isolated strains belonged to the same group, which includes the strains ov-ia82 and nz2. importantly, several sqpv myv 3472* ov-sa00 vr634 hl953 bv-tx09c1 bv-ar02 bv-tx09c5 bv-tx09c15 f00.120r pcpv776 shiraz2 shiraz5 egypt d1701 10278* 10279* na1-11 cluster a shiraz3 shiraz4 shiraz1 885 891 figure 2. maximum likelihood phylogeny of the orf virus strains analysed in this study, based on the nucleotide sequences of the vltf-1 gene. significant (> 850/1000) bootstraps are reported at nodes. cluster a includes all the analysed strains, with the exception of 10278 pz, 10279 pz and 3472 fg. 353 galante et al. nick title veterinaria italiana 2019, 55 (4), 347-353. doi: 10.12834/vetit.1025.5477.2 ara m., zaballos p., sánchez m., querol i., zubiri m.l., simal e. & hörndler c. 2008. giant and recurrent orf virus infection in a 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diagnostic virology. a practical guide and atlas. cambridge university press, cambridge, 14-19. friederichs s., krebs s., blum h., wolf e., lang h., von buttlar h. & büttner m. 2014. comparative and retrospective molecular analysis of parapoxvirus (ppv) isolates. virus res, 181, 11-21. gallina l., scagliarini l., mcinnes c.j., guercio a., purpari g., prosperi s. & scagliarini a. 2008. parapoxvirus in goats: experimental infection and genomic analysis. vet res commun, 32, s203-s205. guindon s. & gascuel o. 2003. a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. syst biol, 52, 696-704. hosamani m., scagliarini a., bhanuprakash v., mcinnes c.j. & singh r.k. 2009. orf: an update on current research and future perspectives. expert rev anti infect ther, 7, 879-893. lojkic i., cac z., beck a., bedekovic t., cvetnic z. & sostaric b. 2010. phylogenetic analysis of croatian orf viruses isolated from sheep and goats. virol j, 7, 314. kottaridi c., nomikou k., lelli r., markoulatos p. & mangana o. 2006a. laboratory diagnosis of contagious ecthyma: comparison of different pcr protocols with virus isolation in cell culture. j virol methods, 134, 119-124. references kottaridi c., nomikou k., teodori l., savini g., lelli r., markoulatos p. & mangana o. 2006b. phylogenetic correlation of greek and italian orf virus isolated based on vir gene. vet microbiol, 116, 310-316. kuhl j.t., huerter c.j. & hashish h. 2003. a case of human orf contracted from a deer. cutis, 71, 288-290. mahmoud m., abdelrahman k. & soliman h., 2010. molecular and virological studies on contagious pustular dermatitis isolates from egyptian sheep and goats. res vet sci, 89, 290-294. mazur c. & machado r.d. 1989. detection of contagious pustular dermatitis virus of goats in a severe outbreak. vet rec, 125, 419-420. mazur c. & machado r.d. 1990. the isolation and identification of the contagious ecthyma virus of caprines in cell cultures. rev microbiol sao paulo, 21, 127-130. mercer a.a., ueda n., friederichs s.m., hofmann k., fraser k.m., bateman t. & fleming s.b. 2006. comparative analysis of genome sequences of three isolates of orf virus reveals unexpected sequence variation. virus res, 116, 146-158. robinson a.j. & mercer a.a. 1995. parapoxvirus of red deer: evidence for its inclusion as a new member in the genus parapoxvirus. virology, 208, 812-815. scagliarini a., vaccari f., turrini f., bianchi a., cordioli p. & lavazza a. 2011. parapoxvirus infections of red deer, italy. emerg infect dis, 17, 684-687. tamura k., stecher g., peterson d., filipski a. & kumar s. 2013. mega6: molecular evolutionary genetics analysis version 6.0. mol biol evol, 30, 2725-2729. thomas k., tompkins d.m., sainsbury a.w., wood a.r., dalziel r., nettleton p.f. & mcinnes c.j. 2003. a novel poxvirus lethal to red squirrels (sciurus vulgaris). j gen virol, 84, 3337-3341. tryland m., klein j., nordøy e.s. & blix a.s. 2005. isolation and partial characterization of a parapoxvirus isolated from a skin lesion of a weddell seal. virus res, 108, 83-87. zhang k., liu y., kong h., shang y. & liu x. 2014. human infection with orf virus from goats in china, 2012. vector borne zoonotic dis, 14, 365-367. zhao k., song d., he w., lu h., zhang b., li c., chen, k. & gao f. 2010. identification and phylogenetic analysis of an orf virus isolated from an outbreak in sheep in the jilin province of china. vet microbiol, 142, 408-415. 311 1department of parasitology and mycology, school of medicine, aja university of medical sciences, tehran, islamic republic of iran. 2department of pathobiology, school of veterinary medicine, shiraz university, shiraz, iran. *corresponding author at: department of pathobiology, school of veterinary medicine, shiraz university, shiraz, iran. e-mail: m.asadpour65@gmail.com. parole chiave cryptosporidium, g. intestinalis, cani, gatti, genotipizzazione, beta-giardin, ssu rrna. riassunto nel presente studio 615 campioni fecali provenienti da cliniche veterinarie sono stati esaminati per la presenza di cryptosporidium e giardia. sono state inoltre effettuate la genotipizzazione molecolare dei due microganismi mediante pcr e l’analisi di sequenza. complessivamente, sono state rilevate oocisti di cryptosporidium e giardia rispettivamente nello 0,6% (2/315) e nell'1,9% (6/315) dei cani e nello 0,7% (2/300) e 1,3% (4/300) dei gatti. i dati molecolari hanno dimostrato la presenza di c.  canis (n = 2) nei cani e c. felis (n = 2) nei gatti; inoltre nei cani sono stati evidenziati g.  intestinalis assemblage  d (n  =  2), c (n = 3) e a, sub-assemblage aii (n = 1); g. intestinalis assemblage f (n = 3) e assemblage a, sub-assemblage ai (n = 1) nei gatti. la più alta prevalenza di giardia è stata osservata nei cani di età inferiore a un anno (6/315) e in quelli con diarrea (p < 0,05). caratterizzazione molecolare e potenziale zoonotico di cryptosporidium spp. e giardia intestinalis in cani e gatti domestici di shiraz, sud-ovest dell'iran keywords cryptosporidium, g. intestinalis, dogs, cats, genotyping, beta-giardin, ssu rrna. summary in the present study, a total of 615 fecal samples from veterinary clinics were screened by microscopy for the presence of cryptosporidium and giardia oocysts. molecular genotyping of cryptosporidium and giardia were carried out using pcr and sequence analysis. overall, cryptosporidium and giardia oocysts were detected in the 0.6% (2/315) and 1.9% (6/315) of dogs and in the 0.7% (2/300) and 1.3% (4/300) of cats, respectively. sequencing revealed the presence of c. canis (n = 2) in dogs and c. felis (n = 2) in cats. moreover, g.  intestinalis assemblage d (n = 2), c (n = 3) and a, sub-assemblage aii (n = 1) were identified in dogs; g.  intestinalis assemblage f (n = 3) and assemblage a, sub-assemblage ai (n = 1) were identified in cats. the highest prevalence of giardia was observed in dogs younger than one year (6/315), and in those with diarrhea (p < 0.05). data of the study suggest that dogs and cats play a minor role in the zoonotic transmission of cryptosporidiosis and giardiasis in southwestern iran. mohamad mohsen homayouni1, seyed mostafa razavi2, minoo shaddel1 and mohammad asadpour1* prevalence and molecular characterization of cryptosporidium spp. and giardia intestinalis in household dogs and cats from shiraz, southwestern iran veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 accepted: 14.01.2018 | available on line: 31.12.2019 312 molecular characterization of cryptosporidium and giardia in iran homayouni et al. veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 iiaa15g2r1) and c.  hominis are the most common agents of cryptosporidiosis (berahmat et  al. 2017). g. intestinalis aii, biii, and biv are the most common assemblages identified in humans in iran (hooshyar et  al. 2017, kasaei et  al. 2018). the frequency of g.  intestinalis and cryptosporidium in iranian dogs and cats has been investigated mostly by microscopic methods. in a study conducted in dogs, in central iran, cryptosporidium oocysts were found in the 2.1% (3/140) in isfahan (ranjbar et al. 2017), in the 6% (4/77) in mashhad (beiromvand et al. 2013), and in the 0.4% (2/450) in zanjan (kohansal et  al. 2017). cryptosporidium was also detected in the 18% (9/50) of cats in tehran (mirzaghavami et  al. 2016). based on microscopic data, prevalence rates of g.  intestinalis in dogs and cats range from 0.6% (1/147) to 1.6% (6/450) in dogs (jafari shoorijeh et  al. 2008, kohansal et  al. 2017), and 10.7%, (15/140) to 18.9% (7/37) in cats (bahrami et al. 2011, khademvatan et  al. 2014). data upon molecular epidemiology and genetic diversity of g. intestinalis and cryptosporidium in iranian dogs and cats are scarce and poorly understood. accordingly, the aim of this study was to evaluate the prevalence and molecular diversity of cryptosporidium and g.  intestinalis in household dogs and cats from shiraz, southwestern iran. material and methods samples collection the study was carried out from july 2017 to march  2018. fecal samples were collected from 615   household dogs (n  =  315) and cats (n  =  300) referred to three veterinary clinics in shiraz, the capital of fars province, iran. the majority of dogs (304/315) and cats (202/300) were asymptomatic. metadata such as gender, breed, age, keeping conditions (indoor/outdoor), fecal consistency (diarrheic/non-diarrheic), and diet were recorded. a safe diet was defined when clean food (e.g. canned and/or packed and cooked food) and water were provided to the animal. microscopic examination and sucrose flotation all stool samples were screened for cryptosporidium oocysts using modified acid-fast staining method. furthermore, wet smears with saline and lugol’s iodine were prepared for all fecal samples for detection of g.  intestinalis oocysts. as for purification of giardia and cryptosporidium oocysts, sucrose gradient flotation technique was performed as previously described (lagapa et  al. 2009, asadpour et  al. 2018). then, the recovered introduction foodborne zoonotic pathogens are a serious public health issue and result in significant global economic losses. giardia and cryptosporidium, genera of common protozoan parasites that infect domestic and wild animals and humans, generally cause diarrhea. as for cryptosporidium, the most common species that causes human infection are the cryptosporidium parvum and cryptosporidium hominis. they are divided into many species/ genotype and subtypes using various molecular methods. these molecular tools are necessary for epidemiological purposes and understanding of the transmission of infection to humans and animals. domestic dogs and cats are frequently infected by c. canis and c. felis (itoh et al. 2014, sotiriadou et al. 2013, berahmat et al. 2017, xiao 2010). furthermore, c.  parvum and c. muris are frequently reported in domestic dogs and cats, respectively (pavlasek and ryan 2007, santín et  al. 2006). giardia is extremely common and is responsible for ~ 280 million human cases of diarrhoea every year (total giardiasis acquired by all transmission routes) and infects >  40 animal species (horlock-roberts et  al. 2017). currently eight species of giardia are accepted as valid, including the recently described giardia cricetidarum in hamsters and giardia peramelis in bandicoots (hillman et al. 2016, lyu et al.2018). giardia intestinalis infects humans and is a species complex consisting of eight assemblages (a-h) (ryan and cacciò 2013). assemblages a and b are the predominant assemblages in humans, but assemblages c, d, e and f have also been identified (cacciò et  al. 2017). within assemblage a, sub-assemblages ai, aii and aiii have been identified and of these ai and aii are commonly reported in humans and animals with subassemblage aiii reported in wild ruminants (feng and xiao 2011). assemblages c-h, are generally the host-specific giardia assemblages. assemblages c and d are widespread in dogs and assemblage f is the prevalent assemblage in cats (yang et  al. 2015). some researchers suggest that dogs and cats may play a role in zoonotic transmission of cryptosporidiosis and giardiasis (berrilli et  al. 2012, pallant et  al. 2015), while others reject this hypothesis (de lucio et  al. 2017, rehbein et  al. 2019). in iran, like in other developing countries, cryptosporidiosis and giardiasis are a public health concern with socio-economic impact. prevalence rates of g.  intestinalis and cryptosporidium have been reported in iran. g.  intestinalis was found in the 2.7% (12/450) of children in behbahan (kasaei et al. 2018) while taghipour and colleagues (taghipour et  al. 2011) reported the 2.4% of cryptosporidium prevalence in humans with diarrhea in tehran. c.  parvum (particularly subtype 313 homayouni et al. molecular characterization of cryptosporidium and giardia in iran veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 and genotyping of giardia. a 292-bp fragment of the dna sequence coding for the ssu rrna was amplified using rh4 (5’agtcgaaccctgattctccgccagg-3’) and rh11 (5’-catccggtcgatcctgcc-3’) (hopkins et  al. 1997). pcr amplification was performed with the following conditions: 25 µl of master mix, supplemented with 2µl (20 pmol) of each forward and reverse primer, 8 μl of dna (~ 40 ng) and filled to 50 µl with distilled water. pcr program was set as follows: after a primary denaturation step at 96  °c for 10 min, 35 cycles were performed consisting of denaturation at 95 °c for 30 s, annealing at 65 °c for 35 s, extension at 72 °c for 60 s, and a final extension at 72  °c for 7 min. moreover, a 753-bp fragment of the beta-giardin (bg) locus was amplified using g7 (5’-aagcccgacgacctcacccgcagtgc-3’) and g759 (5’-gaggccgccctggatcttcgagacg ac-3’) primers as described previously (caccio et al. 2002). pcr was performed in a reaction mixture containing 25 µl of master mix, supplemented with 2 µl (20 pmol) of each primer, 8 μl of dna (~ 40 ng) as template and filled to 50  µl with distilled water. pcr program consisted of an initial denaturation at 96 °c for 5 min, followed by 30 cycles consisting of denaturation at 94 °c for 35 s, annealing at 58 °c for 40 s, extension at 72 °c for 60 s, and a final extension at 72 °c for 7 min. for positive and negative controls, consisting of a g. intestinalis assemblage a (isolated from humans) and sterile water were included in all amplifications, respectively. analysis of pcr products was conducted by 1.5% agarose gel electrophoresis (fermentas, usa) and visualization using a uv transilluminator. purification of pcr products and sequencing analysis pcr products were purified by gel excision (vivantis technologies, selangor, malaysia). the recovered products were sequenced by sanger dideoxy sequencing technology. obtained sequences data were aligned by clc main workbench 6.0 software (clc bio, denmark) and clustal w mega 6 software. neighbor-joining method and bootstrap analysis over 1,500 replicates were used for reconstructing phylogenetic trees (tamura et al. 2013). statistical analysis statistical analysis was performed using spss version 21 software (spss inc. chicago, il, usa). pearson’s chi-squared (χ2) for independence and fisher’s exact two-sided tests were conducted to evaluate association between infections and host factors including gender, breed, age, fecal consistency, keeping conditions, and diet. p  <  0.05 was considered significant. oocysts were washed 3 times (1,500 rpm for 15 min) with phosphate buffer saline (pbs) (1  m, ph  =  7.4), kept in 2.5% potassium dichromate and stored at 4 ºc until further use. dna extraction total dna was extracted using stool genomic dna extraction commercial kit (bioneer, cat. no. k-3036, daejeon, korea) based on the manufacturer’s procedure with some modifications as described earlier (asadpour et al. 2018). briefly, the recovered oocyst were washed three times with tap water (2,000 rpm, 12 min), the supernatant was removed, oocyst were supplemented with 400 μl of lysis buffer and 40 μl of proteinase k (bioneer, cat. no. kb-0111, south korea), mixed gently, and incubated at 65 °c overnight. then, the supernatant was transferred to a fresh tube and centrifugated (6,000  rpm, 5  min). the remaining steps were accomplished according to the kit procedure. extracted dna (~  150  µl) was kept at - 20 °c until further use. pcr for cryptosporidium cryptosporidium -positive samples were genotyped by nested-pcr amplification of a 830-bp fragment of the dna sequence coding for the ssu rrna as described previously (xiao et  al. 2006). cryptf1 (5’-ttctagagctaatacatgcg-3’) and cryptr1 (5’-cccatttccttcgaaacagga-3’) in the first pcr, and cryptf2 (5’-ggaagggttgtatttattagata-3’) and cryptr2 (5’ctcataaggtgctgaaggagta-3’) for the second pcr reaction were used. in the present study we used a ready to use master mix (taq dna polymerase master mix red, ampliqon, denmark). each 50  µl pcr tube contained 25  µl of master mix, 2  µl (20  pmol) of each forward and reverse primer (bioneer, daejeon, south korea), 8 μl of dna (~ 40 ng) was extracted as template, filled to 50 μl with distilled water. for amplification, a bio-rad thermocycler machine (bio-rad, ca, usa) was used with an initial denaturation at 95  °c for 4  min, followed by 30 cycles, consisting of denaturation at 94  °c for 40  s, annealing at 55 °c (for primary pcr) and 58 °c (for secondary pcr) for 45 s, extension at 72 °c for 60 s. a final extension at 72 °c for 7 min was included at the end of the amplification cycles. for positive and negative controls, a c.  parvum isolate (https://www.ncbi.nlm.nih.gov/nuccore/ky410237) and sterile water were included in each reaction, respectively. in order to confirm the genotype, all secondary pcr-products were sequenced. pcr for giardia two target genes were used for molecular detection 314 molecular characterization of cryptosporidium and giardia in iran homayouni et al. veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 (tables i and ii). in this study, all examined cats belonged to the same breed (persian short hair). molecular results genotyping of cryptosporidium isolates sequencing analysis revealed the presence of c.  canis (n  =  2) in dogs and c.  felis (n  =  2) in cats, respectively. nucleotide sequences were deposited in genbank under accession numbers mg888049.1 mg888051.1 and mg889862.1, as shown in table iii. figure 1 shows the phylogenetic relatedness of the samples of this study. phylogenetic analysis showed that c. canis and the two c. felis isolates grouped in specific clusters. results prevalence rates of cryptosporidium and g. intestinalis as shown in table i and ii, cryptosporidium and g.  intestinalis were detected in the 0.6% (2 of 315) and 1.9% (6 of 315) of dogs; and in the 0.7% (2 of 300) and 1.3% (4 of 300) of cats, respectively. statistical analysis revealed a significant higher prevalence of giardia in dogs younger than one year (6/315) (p < 0.05). besides, a significant correlation resulted between g.  intestinalis infection and diarrhea in dogs (p  =  0.001). no association was evidenced between gender, breed or diet with the presence of g. intestinalis and cryptosporidium in dogs (p > 0.05) table i. potential host factors associated with cryptosporidium and giardia infections in household dogs. potential host factors no. of screened dogs (%) (n = 315) positive for cryptosporidium (n = 2) (%) p value # positive for giardia (n = 6) (%) p value# age (years) ≥ 1 143 (45.4) 2 (100) 0 (0.00) ≤ 1 172 (54.6) 0 (0.00) 0.503 6 (100) 0.034* gender male 157 (49.8) 2 (100) 0.248 3 (50) female 158 (50.2) 0 (0.00) 3 (50) 0.655 breed terrier 63 (20.0) 1 (16.66) 2 (33.33) great dane 10 (3.2) 0 (0.00) 0 (0.00) husky 53 (16.8) 0 (0.00) 1 (16.66) german shepherd 62 (19.7) 1 (16.66) 0 (0.00) dobermann 85 (27.0) 0 (0.00) 0.801 2 (33.33) 0.277 shih tzu 34 (10.8) 0 (0.00) 0 (0.00) pomeranian 8 (2.5) 0 (0.00) 1 (16.66) fecal consistency diarrheic 11(3.49) 2 (100) 0.069 6 (100) < 0.001* non-diarrheic 304 (96.50) 0 (0.00) 0 (0.00) keeping condition indoor 65 (20.63) 1 (50) 1 (16.66) outdoor 250 (79.36) 1 (50) 0.371 5 (83.33) 0.105 unsafe diet yes 145 (46.03) 2 (100) 0.801 6 (100) 0.127 no 170 (53.96) 0 (0.00) 0 (0.00) # statistical significance, p < 0.05. * the association was evaluated using fisher exact test. table ii. potential host factors associated with cryptosporidium and giardia infections in household cats. potential host factors no. of screened cats (%) (n = 300)a positive for cryptosporidium (n = 2) (%) p value b positive for giardia (n = 4) (%) p value b age (years) ≥ 1 172 (57.3) 1 (50.0) 0.672 4 (100.0) 0.139 ≤ 1 128 (42.7) 1 (50.0) 0.0 (0.00) gender male 174 (58.0) 1 (50.0) 0.664 2 (50.0) 0.142 female 126 (42.0) 1 (50.0) 2 (50.0) fecal consistency diarrheic 98 (32.7) 2 (100.0) 2 (50.0) non-diarrheic 202 (67.3) 0.0 (0.00) 0.106 2 (50.0) 0.599 keeping condition indoor 55 (18.3) 0.00 (0.00) 2 (50.0) outdoor 245 (81.7) 2 (100.0) 0.666 2 (50.0) 0.155 unsafe diet yes 258 (86.0) 2 (100.0) 0.843 4 (100.0) 0.099 no 42 (14.0) 0.0 (0.00) 0.0 (0.00) a all screened cats were persian short hire breed. statistical significance, p < 0.05. b the association was evaluated using fisher exact test. 315 homayouni et al. molecular characterization of cryptosporidium and giardia in iran veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 discussion in this study, a total of 615 stool samples were collected from household dogs and cats and screened for detection of cryptosporidium and giardia (oo) cysts. moreover, pcr methods using different targets genes were used to determine the species/genotype/assemblage of cryptosporidium and g.  intestinalis detected in this study. the prevalence rates of cryptosporidium in household dogs and cats in this study was lower compared to those reported previously (mirzaghavami et al. 2016, genotyping of giardia isolates dna sequence analysis revealed the presence of g.  intestinalis assemblages c (3/6), d (2/6) and sub-assemblage aii (1/6) in dogs. g.  intestinalis assemblage f (3/4), and sub-assemblage ai (1/4) were identified in cats. nucleotide sequence data were deposited in genbank (table iv). phylogeny is depicted in figure 2. table iii. cryptosporidium positive samples identified in household dogs and cats by nested-pcr of the ssurrna coding gene. sample name host* species genbankaccession no. 24 cat c. felis mg888051.1 111 dog c. canis mg888050.1 216 dog c. canis mg888049.1 218 cat c. felis mg889862.1 *cryptosporidium were detected in 2 dogs and 2 cats. 100 c. bovis (mg516770.1: cattle australia) c. bovis (kt922232.1: calf ethiopia) c. bovis (kp793007.1: cattle china) c. felis (mg889862.1: cat iran) c. felis (mg888051.1: cat iran) c. parvum (dq656352.1: human iran) c. parvum (ky410237.1: calf iran) c. parvum (kf830259.1: calf iran) c. suis (kj790232.1: swine china) c. suis (kj790239.1: swine china) c. ubiquitum (mg516761.1: sheep australia) c. ubiquitum (kx259133.1: deer china) c. canis (mg888050.1: dog iran) c. canis (mg888049.1: dog iran) c. baileyi (eu827305.1: chicken china) c. baileyi (gu377276.1: ostrich china) c. baileyi (gu377272.1: ostrich china) c. muris (kj469984.1: horse poland) eimeria bovis (ku052226.1: bos taurus turkey) 93 51 63 62 50 77 75 95 90 0.05 figure 1. neighbor-joining (nj) tree based on the ssu rrna coding sequences. black dots represent the cryptosporidium species detected in household dogs and cats of this study (shiraz, southwestern iran). table iv. giardia intestinalis identified in dogs and cats by pcr of the 16srrna gene. sample name host* assemblage genbankaccession no. 1 dog d mg851692.1 2 cat f mg832845.1 3 cat a mg832842.1 4 cat f mg832844.1 11 cat f mg832843.1 36 dog c mg851690.1 113 dog d mg851691.1 192 dog c mg851695.1 226 dog c mg851693.1 306 dog a mg851694.1 *giardia was detected in 6 dogs and 4 cats. 99 99 g. intestinalis (dog isolate 226) g. intestinalis (dog isolate 192) g. intestinalis (dog isolate 36) g. intestinalis (dog isolate 113) g. intestinalis (dog isolate 1) g. intestinalis (cat isolate 4) g. intestinalis (cat isolate 11) g. intestinalis (cat isolate 2) g. intestinalis (cat isolate 3) g. intestinalis (dog isolate 306) assemblage a g. muris (ef455599.1: rattus norvegicus sweden) g. intestinalis (kr051227.1: meerkat china) g. intestinalis (ku156663.1: dog china) g. intestinalis (kj888976.1: macaca mulatta china) g. intestinalis (kp899898.1: homo sapiens ethiopia) g. intestinalis (kj888974.1: monkey china) g. intestinalis (kt698974.1: cattlechina) g. intestinalis (kj188080.1: lemur china) g. intestinalis (kj188080.1: cattle bangladesh) g. intestinalis (kt698972.1: cattle china) g. intestinalis (gq919292.1: �sh australia) 53 63 100 94 97 99 58 100 57 62 57 74 98 assemblage d assemblage e assemblage f assemblage b assemblage c figure 2. neighbor-joining (nj) tree based on b-giardin coding sequences of g. intestinalis assemblages. black dots represent g. intestinalis assemblages from dogs and cats of this study (shiraz, southwestern iran). 316 molecular characterization of cryptosporidium and giardia in iran homayouni et al. veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 in zoonotic transmission of these parasites, at least in the areas under investigation. conclusions the overall prevalence of cryptosporidium and giardia in household dogs and cats was low. further studies using multiple target genes are recommended in different geographical areas of iran to provide a better understanding of the epidemiology of these two parasites. acknowledgements we appreciate aja university of medical sciences for financial support (grant no. 15/86051). the authors also would like to thank mr shahriyari, mr bahrami, and mr nabavi for their participation in sampling. we are also thankful to lucy j robertson (department of food safety and infection biology, norwegian school of veterinary science) for her kind advise on molecular techniques. ranjbar et  al. 2017, beiromvand et  al. 2013). this study indicates c.  canis and c.  felis as the prevalent species detected in dogs and cats, respectively, in this area of iran. this finding is in line with other studies (ranjbar et al. 2017, neves et al. 2014, pallant et  al. 2015, yang et  al. 2015). the overall prevalence of g. intestinalis in dogs (1.9%) was higher than that of previous studies (0/6%) (shoorijeh et  al. 2008). g.  intestinalis was detected in the 1.3% of sampled cats. these rates were also lower compared to previous studies (bahrami et al. 2011, khademvatan et al. 2017). molecular data revealed that household dogs were mostly infected with g.  intestinalis host-specific assemblages c and d. it is well established that g. intestinalis assemblages c and d are the most prevalent assemblages in dogs (ryan and cacciò 2013, sotiriadou et  al. 2013, uehlinger et al. 2013) while several studies, including this one, indicates also assemblage f capable of infecting cats (cacciò et al. 2005, santín et al. 2006, yang et al. 2015). overall, data obtained in the present study showed that household dogs and cats likely play a minor role 317 homayouni et al. molecular characterization of cryptosporidium and giardia in iran veterinaria italiana 2019, 55 (4), 311-318. doi: 10.12834/vetit.1710.9049.3 asadpour m., namazi f., razavi s.m. & nazifi s. 2018. comparative efficacy of curcumin and paromomycin against cryptosporidium parvum infection in a balb/c model. vet parasitol, 250, 7-14. bahrami a., doosti, a., nahravanian h., noorian a.m. & asbchin s.a. 2011. epidemiological survey of gastro-intestinal parasites in stray dogs and cats. aust j basic appl sci, 5, 1944-1948. beiromvand m., akhlaghi l., massom s.h.f., meamar a.r., motevalian a., oormazdi h. & razmjou e. 2013. prevalence of 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di siero prelevati durante la stagione invernale ed esaminati in elisa, 1 campione di siero prelevato da un leone (5%) è risultato positivo mentre 3 prelevati da tigri e uno da un altro leone (20%) hanno dato esito dubbio o sospetto; nel prelievo effettuato dagli stessi animali nel periodo dei monsoni, 4 campioni di siero, prelevati da 2 tigri e 2 leoni (20%), sono risultati positivi mentre un campione prelevato da una terza tigre (5%) ha dato esito dubbio o sospetto. rispetto ai negativi, gli animali risultati sieropositivi e dubbi o sospetti all’elisa hanno evidenziato valori di globulina, creatinina, azoto ureico, fosforo e creatina chinasi significativamente più elevati. analogamente, i livelli di albumina, glucosio, calcio, sodio e ferro rilevati nel gruppo sieronegativo sono risultati significativamente più bassi. la presenza di anticorpi anti-toxoplasma gondii è stato rilevata anche in un roditore catturato nelle aree adiacenti al parco. questo studio evidenzia per la prima volta l’esposizione di tigri reali del bengala e leoni asiatici al t. gondii. dimostra inoltre un’associazione significativa tra sieropositività e alterazione di alcuni parametri ematici e biochimici. presenza di anticorpi anti-toxoplasma gondii in tigri reali del bengala (panthera tigris tigris) e leoni asiatici (panthera leo persica) in india keywords biochemical parameters, haematological parameters, indirect elisa, india, toxoplasmosis, wild felids, zoological park. summary the purpose of this study was to detect the antibodies against toxoplasma gondii in royal bengal tigers (panthera tigris tigris), asiatic lions (panthera leo persica), leopards (panthera pardus), and elephants (elephas maximus indicus) residing in the mahendra chaudhury zoological park, in chhatbir, punjab (india) during winter and monsoon seasons. using indirect elisa, 20 serum samples were analysed during the winter season. results indicated that 1 lion (5%) tested seropositive, and 3 tigers and 1 lion (20%) were considered suspect. during the monsoon, 4 individuals (2 tigers and 2 lions, 20%) were seropositive, whereas only 1 tiger (5%) gave suspected results. significantly higher globulin, creatinine, blood urea nitrogen, phosphorus, and creatine kinase values were recorded in seropositive and suspected groups. levels of albumin, glucose, calcium, sodium, and iron decreased significantly in the seronegative group. results from sero-testing 40 rodents trapped in and around the park depicted the presence of antibodies against toxoplasma gondii in 1  individual. this study reveals the haemato-biochemical alterations in both seropositive and suspected wild felids for toxoplasmosis. moreover, it provides the first serological evidence of t. gondii exposure in wild felids, notably royal bengal tigers and asiatic lions, in india. veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 accepted: 05.09.2016 | available on line: 30.06.2019 guru angad dev veterinary and animal sciences university, ludhiana, punjab 141004, india * corresponding author at: department of veterinary parasitology, college of veterinary science, guru angad dev veterinary and animal sciences university, ludhiana, punjab 141004, india. e-mail: moudgil.aman@gmail.com. first record of toxoplasma gondii antibodies in royal bengal tigers (panthera tigris tigris) and asiatic lions (panthera leo persica) in india aman d. moudgil*, lachhman das singla, amrita sharma and mandeep singh bal 158 veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 toxoplasma gondii antibodies in wild felids moudgil et al. longitudes at an altitude ranging between 180 to 300  metres above main sea level. the zoological park (mahendra chaudhury zoological park, which falls under sub-mountain undulating zone of the province) considered in this study is comprised of animals and birds belonging to around 61 different species, some of which were rare or endangered. the animals targeted in this study were kept in close confinements with adequate wandering space. the wild felines were fed with buffalo meat (carabeef ) and ad libitum water; elephants were provided with green fodder and sugarcane with ad libitum water. ethical aspects and sampling frame the ethics committee for animal experiments of guru angad dev veterinary and animal sciences university granted the ethical approval (iaec/2013/27-43) to conduct this study. the blood samples from 11 rb tigers, 3 asiatic lions, 2 leopards, and 4 elephants (negative control) were collected twice (in february and august – across winter, monsoon, and summer seasons) in 2013. the wild animals were restrained in squeeze cages and blood samples were taken from the dorsal coccygeal vein. the blood samples were collected in vacutainers with anticoagulant and were stored at - 20°c directly for haematological parameters. sera were separated from the blood samples collected in the vacutainers with coagulation activators by centrifugation and stored at -  20°c for further immunological and biochemical analysis. serological screening a commercial indirect elisa (ielisa) kit from cusabio® (wuhan hi-tech medical devices park, china) was used in order to detect t.  gondii antibodies. the kit was used according to the manufacturer’s instructions. the percent positivity (%p) of samples was obtained as follows: mean od of sample _______________________ mean od of critical control x100percent positivity (%p) = od is the optical density of the sample. this is compared with the optical density of the critical control (provided in the kit), and helps in evaluating the values of percent positivity of the samples. samples with % p value less than 90% were considered seronegative. those with more than 110% were considered seropositive, and the values in-between were considered suspected. thus, the animals were divided into 3 groups, as per the infectivity pattern: seropositive, suspected, and seronegative, and further analyses of haemato-biochemical alterations were carried out among these groups. sero-testing of wild rodents (n  =  40) was also introduction toxoplasma gondii, an obligate intracellular protozoan parasite, is the etiological agent of the ubiquitous zoonotic disease, toxoplasmosis (tedesco 2004). it has the potential to infect almost all homeothermic animals (lopes et al. 2008, liu et al. 2012). approximately, one-third of the world human population is chronically infected with t.  gondii (tenter et  al. 2000). toxoplasmosis can be harmful in immunocompromised humans and pregnant women, especially if a woman is infected during the first trimester of her pregnancy (dubey and beattie 1988). it is also responsible for causing abortions in sheep (dubey and beattie 1988). domestic and wild felids are the only definitive hosts for toxoplasma gondii. they thus play the most important role in the life cycle of the detrimental parasite, as they shed environmentally resistant infective oocysts (miro et  al. 2004). the oocysts shed by infected wild felids in captivity are not only a source of infection to other non-infected captive wild felids, but also to other zoo animals, zoo keepers, zoo veterinarians, as well as to the persons visiting zoos (thiangtum et al. 2006). in india, conservation strategies for the royal bengal (rb) tigers (panthera tigris tigris) were recently implemented following a serious reduction in the rb population (banerjee 2013). though toxoplasmosis is asymptomatic in felids and rarely results in clinical signs, it can result in breeding failure in felids (thiangtum et al. 2006). serological studies are better suited to detect t. gondii infection in comparison to faecal sample examination for oocysts because wild felids shed the oocysts for only a shorter period of time (dubey and thulliez 1989). the significant role of t. gondii in the pathophysiology of wild felids and the epidemiological role of wild felids in the transmission of toxoplasmosis in india has not yet been established. this study was planned to adjudge the seropositivity of t. gondii in rb tigers, asiatic lions (panthera leo persica), leopards (panthera pardus), and elephants (elephas maximas indicus), and thus address the lack of relevant literature regarding the prevalence of toxoplasmosis in wild felids in this region. it also aims to compare the associated alterations in the haemato-biochemical parameters of the seropositive individuals to those of healthy seronegative animals. materials and methods study area and management conditions the punjab state is located between 29”  30’  n to 32”  32’  n latitude and 73”  55’  e to 76”  50’  e 159veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 moudgil et al. toxoplasma gondii antibodies in wild felids results at the beginning of the study period (winter), 1  animal was seropositive (lion) and 4 were suspected (3 tigers and 1 lion) for t. gondii by ielisa. by the end of the monsoon season, 4 animals were seropositive (2 tigers and 2 lions) and 1 (tiger) was suspected. the antibody titres of 2 tigers and 1 lion exceeded the positive threshold value by the end of the study period. the anti-toxoplasma antibody titres showed only a slight increase with the change of seasons, while the animals in seronegative titre range at the beginning of the study remained in the same range. all the elephants and leopards were seronegative in both seasons (figure 1), with percent optical density (od) values ranging between 57.54%-78.82% and 30.53%-57.07% for leopards and elephants, respectively. the serum biochemical studies targeting seropositive, carried out in order to assess their probable role in the transmission of toxoplasmosis to wild felids. the rodents were captured from the fields and jungle area around the zoological park by applying rat-catcher machines, and blood was collected by using the retro-orbital plexus puncture method. haemato‑biochemical analysis the haematological analysis of the blood samples for all of the animals (except leopards and elephants) was carried out on fully automated analyser, advia 2120 haematology system (siemens health care diagnostic inc. deerfield, il, us). analysis included haemoglobin level (hb), total erythrocyte count (tec), total leukocyte count (tlc), packed cell volume (pcv), and differential leukocyte count (dlc). biochemical parameters were analysed by using commercial kits of siemens health care diagnostics inc. il, u.s.a. these included total bilirubin (tbil), aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alkp), total protein (tp), albumin (alb), globulin (glo), blood urea nitrogen (bun), creatinine (cre), uric acid (ua), creatine kinase (ck), blood glucose (glu), sodium (na), potassium (k), chloride (cl), calcium (ca), phosphorus (p), and iron (fe). thus, haemato-biochemical alterations were studied in detail only for rb tigers and asiatic lions, as the levels of all the haematological and biochemical parameters fell within normal range in the cases of leopards and elephants. statistical analysis the analysis of variance (one-way anova) of haemato-biochemical parameters among the different groups was done using spss 16.0 software (marco et al. 2000). infected non-infected suspected 140 120 100 80 60 40 20 0 t1 t2 t3 t4 t5 t6 t7 t8 t9 t1 0 t1 1 l1 l2 l3 e1 e2 e3 e4 le op 1 le op 2 monsoon titres winter titres figure 1. antibody titres against toxoplasma gondii in royal bengal tigers (t), asiatic lions (l), indian elephants (e), leopards (leop) in mahendra chaudhury zoological park, in chhatbir, punjab, india. table i. serum biochemical values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status. biochemical parameters tbil (mg/dl) ast (u/l) alt (u/l) alkp (u/l) tp (g/dl) alb (g/dl) glo (g/dl) bun (mg/dl) cre (mg/dl) glu (mg/dl) groups group i (seropositive) 0.74±0.50 * 41±14.25* 51.2±7.79* 33.4±6.91* 7.56±0.53* 3.26±0.26* 4.3±0.43* 69.6±11.95* 3.26±0.50* 29.8±7.01* group ii (suspected) 0.66±0.34 * 45.4±18.90* 46.4±10.55* 32.8±4.21* 7.38±0.19* 3.26±0.24* 4.12±0.18* 64.2±8.67* 3.06±0.39* 23±3.74* group iii (seronegative animals) 0.96±0.38* 51.5±10.13* 46.11±10.81* 33.28±6.73* 7.36±0.46* 4.03±0.44** 3.33±0.36** 50.61±5.19** 2.08±0.29** 68.72±12.5** normal range (nr) shrivastav et al. 2011 (tigers), jani and sabapara 2010 (lions) 0.4-3.2 14.4-84.0 21.2-109.0 16.28-87.9 3.7-8.7 2.1-4.6 1.6-4.1 6.5-48.2 1.6-4.6 66-124 values indicated as mean± standard deviation; *values differing significantly at p < 0.05; tbil = total bilirubin; ast = aspartate aminotransferase; alt = alanine aminotransferase; alkp = alkaline phosphatase; tp = total protein; alb = albumin; glo = globulin; bun = blood urea nitrogen; cre = creatinine; glu = glucose. 160 veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 toxoplasma gondii antibodies in wild felids moudgil et al. recorded in animals of the seronegative group. individually, the glucose concentration values for all of the suspected and seropositive tigers and lions were below the normal range, whereas the values for seronegative individuals were within the normal range (table i). the values for ua, k, and cl did not vary significantly among any of the 3 groups. the values of ca, na, and fe in the suspected and seropositive groups were significantly (p  <  0.05) lower than the seronegative group, whereas the levels of p and ck were significantly higher in the animals of the seropositive and suspected groups than the values recorded for seronegative animals (figures 2 and 3). all the haematological parameters varied non-significantly except for tlc and neutrophils percentage, where the values recorded in the seropositive and suspected groups were significantly suspected, and seronegative groups including tigers and lions revealed no significant difference in the values of tbil, ast, alt, alkp, and tp and values of all the parameters were in the normal range in both seasons irrespective of the group (table i). the values of alb recorded in the seropositive and suspected group were significantly (p  <  0.05) lower than the values recorded in the seronegative group (table i). the globulin level showed a significant increase in the seropositive and suspected groups as compared to the seronegative group. the bun values in both seasons for both seropositive and suspected groups were significantly (p  <  0.05) higher than those recorded in the seronegative group (table  i). the creatinine levels of the animals in the seropositive and suspected groups were significantly higher than the animals of the seronegative group. the glucose levels of the seropositive and suspected groups were significantly (p < 0.05) lower than those 350 200 250 300 150 100 50 0 ck * ** * ** *** ** * ** na cl fe seropositive suspected seronegative figure 3. creatinine kinase (ck), sodium (na), chloride (cl) and iron (fe) values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status. superscripts '*', '**' and ‘***’indicate values differing significantly (p < 0.05). table ii. haematological values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status haematological parameters hb tlc tec pcv platelets n l e m groups group i (seropositive) 14.02±1.39* 18,840±2,013.34* 8.53±0.69* 41.08±4.86* 204.2±25.53* 76.2±7.22* 20.6±5.55* 2±2.34* 0.8±1.79* group ii (suspected) 13.04±2.46* 17,652±1,964.72* 8.46±1.26* 38.64±6.11* 274±46.78* 76.4±9.20* 22.4±9.21* 1.2±1.79* 0* group iii (seronegative) 14.72±1.03* 14,418±1,538.89** 8.14±0.70* 42.98±7.62* 220.78±51.34* 68.94±2.62** 29.28±2.99* 1.11±1.41* 0.5±1.15* shrivastav et al. 2011 7.8-13.8 6,200-11,050 4.66-9.15 36-45 57-75 18-35 2-6 2-6 values are indicated as mean± standard deviation; *values differing significantly (p < 0.05); hb = haemoglobin level; tec = total erythrocyte count; tlc = total leukocyte count; pcv = packed cell volume; platelets = platelets and components of differential leukocyte count i.e. n = neutrophils; l = lymphocytes; e = eosinophils; m = monocytes. 10 6 7 8 9 5 4 3 2 1 0 ua * * * * ** ** k ca p seropositive suspected seronegative figure 2. uric acid (ua), potassium (k), calcium (ca) and phosphorus (p) values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status. superscripts '*' and ‘**’indicate values differing significantly (p < 0.05). 161veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 moudgil et al. toxoplasma gondii antibodies in wild felids beattie 1988, thiangtum et  al. 2006). thus, infected raw buffalo or chicken meat offered to captive wild felids could be responsible for spreading infection. anti-toxoplasma antibodies were detected in the serum sample of 1 rodent. their possible role in the transmission of the parasite to the captive felids could therefore not be neglected. other routes of transmission might have involved mechanical transmission by flies, cockroaches, or dung beetles entering into the living area of the wild felids (thiangtum et  al. 2006). the elephants and leopards displayed negative antibody titres and the comparatively lower titre values of the elephants could be attributed to their herbivorous nature. the values of the serum biochemical parameters (alt, ast, tbil, and alkp) were within the normal range, which is consistent with the findings of mosallanejad and colleagues (mosallanejad et  al. 2007). the increased levels of the globulins in both seropositive and suspected groups could be attributed to the presence of immunoglobulins generated against an ongoing infection (sedlack and bartova 2006). the higher creatinine levels that were recorded were suggestive of t.  gondii infection in the definitive hosts (lappin 1996). toxoplasmosis is usually asymptomatic in cats, however the decreased blood glucose levels that were observed in this study can be correlated with the lethargy observed in t.  gondii seropositive felids (elmore et  al. 2010). the significantly increased creatine kinase levels in seropositive and suspected animals influenced the brain or muscles in animals belonging to these two groups and resulted in lowered response times. the decreased serum ca levels revealed the involvement of the pancreas in chronic toxoplasmosis – a condition that leads to pancreatitis in definitive hosts (advincula et al. 2010). the neutrophilic leucocytosis observed in this study is consistent with the findings of mosallanejad and colleagues (mosallanejad et  al. 2007), who considered it as a major finding relating to toxoplasmosis. other important attributes of the t. gondii infection, including anaemia and jaundice, were not observed in this study. this may be due to the chronic nature of the infection in these animals (advincula et al. 2010). this study is the first report of captive wild felids (of india) exposure to t. gondii. it indicates that captivity (in zoological gardens) fosters stress and further immunosuppression and renders the definitive hosts (wild felids) vulnerable to toxoplasmosis. the haemato-biochemical alterations may prove good indicators for the adverse aftermaths of this intestinal protozoan parasite while undergoing extra-intestinal life cycle. wild felines seropositive for t. gondii risk infecting other animals in the zoological garden as well as the individuals (zoo keepers and veterinarians) associated with the management of (p  <  0.05) higher than the values recorded in the seronegative group (table ii). the tlc values for all the groups were higher than the normal range. only 1 serum sample of wild rodent was seropositive for t. gondii (figure 4). discussion this study highlights the presence of antibodies against t.  gondii infections in captive wild felids (rb tigers and asiatic lions) living in a zoological garden. though the sero-detection of toxoplasmosis has been carried out in domestic ruminants in the past (selvaraj et al. 2007, sharma et al. 2008), there has never been a study that considers wild felids. this investigation recorded the first serological evidence of t.  gondii exposure in rb tigers and asiatic lions in india, and moreover found that seropositivity increased from winter to monsoon. this may be due to the fact that parasites were in incubation stage during the winter with no/fewer detectable anti-t.  gondii antibodies. these levels increased during the monsoon season. this increase in the number of seropositive individuals could be attributed to stressful conditions (environmental stress caused by heat and humidity) encountered by the animals during the summer and monsoon seasons, which would result in the immunosuppression of the animals that would, in turn, cause such an increase (salant and spira 2004). another factor could be the increase in moisture during the monsoon season. the survival of oocysts is prolonged in cool, moist conditions, which in turn would aggravate the infection in these animals (bisson et al. 2000). the management of conditions in the zoological gardens therefore has a direct impact on the susceptibility of animals to contract infections. the most frequent cause of toxoplasmosis infection in definitive hosts is through the ingestion of tissues from an infected intermediate hosts (dubey and infected non-infected suspected 120 100 80 60 40 20 0 r 1 r 2 r 3 r 4 r 5 r 6 r 7 r 8 r 9 r 1 0 r 1 1 r 1 2 r 1 3 r 1 4 r 1 5 r 1 6 r 1 7 r 1 8 r 1 9 r 2 0 r 2 1 r 2 2 r 2 3 r 2 4 r 2 5 r 2 6 r 2 7 r 2 8 r 2 9 r 3 0 r 3 1 r 3 2 r 3 3 r 3 4 r 3 5 r 3 6 r 3 7 r 3 8 r 3 9 r 4 0 figure 4. antibody titres against toxoplasma gondii in wild rodents (r 1 -r 40 ) captured in and around mahendra chaudhury zoological park, in chhatbir, punjab, india. 162 veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 toxoplasma gondii antibodies in wild felids moudgil et al. singh for his co-operation and help in collecting blood samples from wild felids and elephants in mczp, chhatbir, punjab. thanks are also due to dst for providing an inspire fellowship to the first author for his doctoral programme. thanks are also due to dr. kiran malhotra (associate professor of english (retd.), department of languages and haryanvi culture, ccs haryana agricultural university, hisar) for editing the manuscript for the correct use of the english language. animals. further strategies should be implemented to manage this infection. acknowledgements the authors are thankful to the dean, post graduate studies, gadvasu, ludhiana; chief wildlife warden, punjab and director, mczp chhatbir, punjab for providing every possible facility to undertake this investigation. the authors are indebted to dr. m.p. advincula jkdc., iewida syp. & salibay cc. 2010.serologic detection of toxoplasma gondii infection in stray and household cats and its haematologic evaluation. sci med, 20, 76-82. banerjee k. 2013. decadal change in surface water salinity profile of indian sunderbans: a potential indicator of climate change. j marine sci res development, s11: 002. doi: 10.4172/2155-9910.s11-002. bisson a., maley s., rubaire-akiiki c.m. & wastling j.m. 2000. the seroprevalence of antibodies to toxoplasma gondii in domestic goats in uganda. acta trop, 76, 33-38. dubey j.p. & beattie c.p. 1988. toxoplasmosis of animals and man. crc press, boca raton, fl. dubey j.p. & thulliez p. 1989. serologic diagnosis of toxoplasmosis in cats fed toxoplasma gondii tissue cysts. j am vet med assoc, 194, 1297-1299. elmore s.a., jones j.l., conard p.a., patton s., lindsay d.s. & dubey j.p. 2010. toxoplasma gondii: epidemiology, feline clinical aspects, and prevention. trends parasitol res, 26, 190-196. lappin m.r. 1996. feline toxoplasmosis: interpretation of diagnostic test results. semin vet med surg (small animal), 11, 154-160. liu q., singla l.d. & zhou h. 2012. vaccines against toxoplasma gondii: status, challenges and future directions. hum vaccin immunother, 8, 1305-1308. lopes a.p., cardoso l. & rodrigues m. 2008. serological survey of toxoplasma gondii infection in domestic cats from northeastern portugal. vet parasitol, 155, 184-189. marco i., martinez f., pastor j. & lavin s. 2000. hematologic and serum chemistry values of the captive european wildcat. j wildl dis, 36, 445-449. references miro g., montoya a., jimenez s., frisuelos c., mateo m. & fuentes i. 2004. prevalence of antibodies to toxoplasma gondii and intestinal parasites in stray, farm and household cats in spain. vet parasitol, 126, 249-255. mosallanejad b., malmasi a., mohebali m. & tabatabayi m. 2007. anterior uveitis in a kitten infected with toxoplasma gondii (tehran strain). iranian j vet res, 8, 91-93. salant h. & spira d.t. 2004. a cross sectional survey of anti toxoplasma gondii antibodies in jerusalem cats. vet parasitol, 124, 167-177. sedlack k. & bartova e. 2006.the prevalence of toxoplasma gondii igm and igg antibodies in dogs and cats from the czech republic. vet med, 51, 555-558. selvaraj j., manohar b.m., singh s. & balachandran c. 2007. seroprevalence of toxoplasma gondii in buffaloes. j vet parasitol, 21, 41-42. sharma s., sandhu k.s., bal m.s., kumar h., verma s. & dubey j.p. 2008. serological surveys of antibodies to toxoplasma gondii in sheep, cattle and buffaloes in punjab, india. j parasitol, 94, 1175-1175. tedesco r.c. 2004. ocular toxoplasmosis: the role of retinal pigment epithelium migration in infection. parasitol res, 92, 467-472. tenter a.m., heckeroth a.r. & weiss l.m. 2000. toxoplasma gondii: from animals to humans. int j parasitol, 30, 1217-1258. thiangtum k., nimsuphun b., pinyopanuwat n., chimnoi w., tunwattana w., tongthainan d., jittapalapong s., rukkwamsuk t. & maruyama s. 2006. seroprevalence of toxoplasma gondii in captive felids in thailand. vet parasitol, 136, 351-355. 203 review istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy * corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861 332249, e-mail: f.iannino@izs.it. parole chiave campylobacter, resistenza antimicrobica, cani, geni di resistenza. riassunto la resistenza antimicrobica in medicina e in agricoltura è uno dei problemi emergenti più importanti di sanità pubblica. dal 2005 la campilobacteriosi è tra le zonosi alimentari più diffuse in europa. l’infezione può essere contratta consumando cibo o bevande contaminate o entrando in contatto con individui o animali infetti. i cani sono portatori di campylobacter. possono quindi essere fonte di infezione per l'uomo o svolgere un ruolo importante come reservoir di batteri resistenti o di geni di resistenza. l’uomo, a sua volta, può essere serbatoio di campylobacter spp. per gli animali domestici. questa review analizza la letteratura corrente relativa al rischio di resistenza antimicrobica di campylobacter nell’interfaccia cane-uomo. campylobacter e resistenza antibiotica nel cane e nell’uomo: uno studio “one health” keywords campylobacter, antimicrobial resistance, dogs, resistance genes. summary increasing antimicrobial resistance in both medicine and agriculture is recognised as a major emerging public health concern. since 2005, campylobacteriosis has been the most zoonotic disease reported in humans in the european union. human infections due to campylobacter  spp. primarily comes from food. however, the human-animal interface is a potential space for the bidirectional movement of zoonotic agents, including antimicrobial resistant strains. dogs have been identified as carriers of the campylobacter species and their role as a source of infection for humans has been demonstrated. furthermore, dogs may play an important role as a reservoir of resistant bacteria or resistance genes. human beings may also be a reservoir of campylobacter spp. for their pets. this review analyses the current literature related to the risk of campylobacter antimicrobial resistance at the dog-human interface. filomena iannino*, stefania salucci, guido di donato, pietro badagliacca, giacomo vincifori and elisabetta di giannatale campylobacter and antimicrobial resistance in dogs and humans: “one health” in practice veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 accepted: 11.09.2017 | available on line: 30.09.2019 extraintestinal infections, and post-infectious complications. campylobacteriosis has been the most frequently reported zoonotic disease in humans in europe since 2005, and the annual number of notified campylobacteriosis cases has increased in many european countries in recent years (efsa 2015, tam et al. 2003). campylobacteriosis in humans is mainly caused by thermotolerant campylobacter spp., however other species including the non-thermophilic c. fetus, are also known to cause human infection. introduction notes on campylobacter infections and therapy in humans and dogs campylobacteriosis is a collective description for infectious diseases caused by members of the genus campylobacter which are ubiquitous bacteria. they are frequently found in the digestive tracts of mammals and wild and domestic birds. they commonly contaminate their surrounding environment, including water. diseases are mainly characterized by acute enteritis, 204 campylobacter and antimicrobial resistance iannino et al. veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 that the organism is a commensal, while others reported an association between infection and clinical signs (guest et al. 2007, chaban et al. 2010), particularly in younger dogs (parson et  al. 2010, burnens et al. 1992). in immune-compromised or febrile dogs, or in dogs with evidence of hemorrhagic diarrhoea, antimicrobial treatment may be indicated. in these cases, macrolides or quinolones are the antibiotics most commonly used (marks et al. 2011). risk factors at the dog‑human interface environment poor hygiene conditions may be an important source of campylobacter spp. these bacteria can survive on dry surfaces for at least 7 days (ullman and kischkel 1981), however in slurries and dirty water, campylobacter can survive for up to 3 months (nicholson et  al. 2005). most surface water sources are contaminated by animal manure, which contains campylobacter. age many studies demonstrated that younger dogs were more likely to act as carriers of campylobacter  spp. and to shed the organism (sandberg et al. 2002). this may suggest an age predisposition and immunity development (sandberg et  al. 2002, workman et  al. 2005, acke et al. 2006, parsons et al. 2010). in a study conducted in barbados, over 70% of campylobacter positive dogs were under 1-year-old, and of these, 32.8% were younger than 9 weeks old (workman et al. 2005). diarrhoea and enteric disease this topic is controversial. however, as a precautionary measure, diarrhoea should be included among the risk factors. high density housing the prevalence of campylobacter spp. is higher in dogs living in groups (for example in kennels or shelter) than in households (workman et  al. 2005, acke et  al. 2006). this is probably due to stress, increased prevalence of gastrointestinal disease, close contact with other animals, and dietary variation (table i). contact with other animals contact between dogs and other animal species could be an important risk factor. natural reservoirs the species most commonly associated with human infection are c. jejuni, followed by c. coli, c. lari, and c.  upsaliensis (wieland et  al. 2005, leonard et  al. 2011, efsa 2012). in most symptomatic cases, campylobacteriosis occurs as mild and self-limiting gastroenteritis characterised by 1-3 days of low fever, vomiting, myalgia, and headaches, followed by 3-7 days of abdominal pain with watery or bloody diarrhoea. acute infection sometimes begins with a high fever, peaking during the first days of illness. excretion ends within 10-14 days and severe complications are uncommon (altekruse and tollefson 2003, blaser and engberg 2008, bolton 2015). chronic infections or extra-intestinal infections can occur with fatal bacteraemia, hepatitis, pancreatitis, meningitis, recurrent colitis, acute cholecystitis and cystitis, cardiovascular complication, abscesses, and complications of the reproductive system (goossens et  al. 1987, manfredi et  al. 1999, acke et  al. 2009, keithlin et al. 2014). antimicrobial therapy may be required in severe cases, in immune-compromised patients, or in prolonged disease. in humans, macrolides (primarily erythromycin, or alternatively one of the newer macrolides, such as clarithromycin or azithromycin) remain the frontline agents for treating culture-confirmed campylobacter cases (ge et al. 2013). quinolones (e.g., ciprofloxacin) are also commonly used because of their common use in the empirical treatment of undiagnosed diarrheal illness such as travellers’ diarrhoea (guerrant et  al. 2001). tetracycline, doxycycline, and chloramphenicol are alternative treatments (ge  et  al. 2013). serious systemic infections should be treated with aminoglycosides such as gentamicin or beta-lactamases including carbapenem and imipenem (okada et  al. 2008). third-generation cephalosporins have not been proven effective for treating bacteremia due to the campylobacter species other than c. fetus (pacanowski et al. 2008). dogs have been identified as asymptomatic carriers of some species of campylobacter and their role as a source of infection for humans has been demonstrated (skirrow 1991, siemer et  al. 2004, karenlampi et  al. 2007, koene et  al. 2004). the high prevalence of campylobacter infection in dogs is an important topic of public health, as shown in table i. approximately 6% of human cases of campylobacteriosis are due to contact with pets (tenkate and stafford 2001, rossi et al. 2008). the role of campylobacter as an enteric pathogen in dogs is unclear. some studies did not find any significant relationship between diarrhoea and campylobacter  spp. infection (sandberg et  al. 2002, workman et  al. 2005, acke et  al. 2006), suggesting 205 iannino et al. campylobacter and antimicrobial resistance veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 table i. reported prevalence of dogs carrying campylobacter spp. in relation to isolated species, type of sample, population sampled, geography, and detection methods. bibliography population samples total dogs campylobacter spp. detection species identification method geography lópez et al. 2002 household dogs faecal samples 380 17% culture c. jejuni 12% phenotypic test argentina workman et al. 2005 household dogs rectal swabs 130 46.9% culture c. jejuni 26% pcr barbadosc. coli 4% c. upsaliensis 2% chaban et al. 2010 healthy household dogs faecal samples 70 56% pcr c. upsaliensis 43% pcr canada c. hyointestinalis 13% c. jejuni 7% c. showae 6% c. coli 0% diarrhoeic household dogs faecal samples 65 97% pcr c. upsaliensis 85% pcr c. jejuni 46% c. showae 28% c. coli 25% c. hyointestinalis 18% leonard et al. 2011 dogs from veterinary clinics faecal swabs 240 22% culture c. upsaliensis 19% pcr canada c. jejuni 3% hald & madsen 1997 healthy puppies aged between 11 and 17 weeks rectal swabs 72 29% culture c. jejuni 22.% phenotypic test denmarkc. upsaliensis 5% c. coli 1% acke et al. 2009 household dogs rectal swabs 147 41% culture c. upsaliensis 10% pcr irelandc. jejuni 30% c. coli 1% giacomelli et al. 2015 household dogs rectal swabs 100 11% culture c. jejuni 5% pcr italyc. upsaliensis 5% c. coli 1% shelter-housed dogs rectal swabs 50 26% culture c. jejuni 16% pcr italy c. upsaliensis 2% c. hyointestinalis 6% c. lari 2% mohan 2015 faecal samples from walk way area faecal samples 498 13% culture c. jejuni 5% pcr new zealand salihu et al. 2010 household dogs rectal swabs 141 28% culture c. upsaliensis 21% phenotypic test nigeria c. jejuni 6% sandberg et al. 2002 household dogs rectal swabs 529 23% culture c. upsaliensis 20% phenotypic test norway c. jejuni 3% diarrhoeic household dogs rectal swabs 66 27% culture c. upsaliensis 23% phenotypic test c. jejuni 3% engvall et al. 2003 household dogs faecal samples 91 56% culture c. upsaliensis 43% pcr sweden c. jejuni 11% c. coli 2% c. helveticus 2% c. lari 1% holmberg et al. 2015 healthy dogs under the age of 2 rectal swabs 180 37% culture c. upsaliensis 29% pcr sweden c. jejuni 4% parson et al. 2010 dogs from veterinary clinics faecal samples 249 38% culture c. upsaliensis 37% pcr uk westgarth et al. 2009 household dogs faecal samples 183 26% culture and direct pcr c. upsaliensis 25% pcr uk 206 campylobacter and antimicrobial resistance iannino et al. veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 determinants of ‘risk to human health’, towards a perspective of ‘shared risk’ between humans and animals (rabinowitz et al. 2008). the ‘one health’ approach recognises that the health of people is connected to the health of animals and the environment, and encourages physicians and veterinarians to work together. according to american veterinary medical association (avma 2008) ‘one health is the collaborative effort of multiple health science professions, together with their related disciplines and institutions (working locally, nationally, and globally) to attain optimal health for people, domestic animals, wildlife, plants, and our environment.’ initially, ‘one health’ research focused on zoonoses deriving from farm animals and wild animals. the enormous potential role of companion animals has been often disregarded. the growing number of household pets has given rise to new health issues. among these, antimicrobial resistance is an important topic to consider within the ‘one health’ approach. notes on antimicrobial resistance increasing antimicrobial resistance in both medicine and agriculture is recognised as a major emerging public health concern by various scholars and authorities, including the world health organization (moore et  al. 2002, di giannatale et  al. 2014, ozbey and tasdemi 2014, pezzotti et  al. 2003, aarestrup and engberg 2001, who 2004). this has made the clinical management of campylobacteriosis cases more complex. antimicrobial resistant enteric infections are highest in the developing world, where the use of anti-microbial drugs in animals is largely unrestricted (lengerh et al. 2013). companion animals may play an important role as a reservoir of resistant bacteria or resistance genes. furthermore, human beings may be a reservoir of pathogens for their pets (rutland et al. 2009). growing healthcare standards for an increasingly large population of household pets has led to a proliferation of geriatric animals that have extensive medical histories, which has included the administration of antimicrobial drugs. large antimicrobial use exerts selective pressure on human and animal pathogens and is considered to be a major contributor to the development of antimicrobial resistance. antimicrobial resistance can be classified into 3  groups: intrinsic, mutational, and acquired resistance. intrinsic resistance refers to the inherent resistance to an antibiotic that is a naturally occurring feature of the micro-organism. for campylobacter spp. include chicken and other poultry, wild birds, pigs, cats, sheep, cows (workman et  al. 2005), and exotic pets such as turtles (harvey and greenwood 1985) and hamsters (fox et al. 1983). the high prevalence (39.3%) reported by workman and colleagues (workman et  al. 2005) in wild birds is of particular interest, as dogs can easily encounter bird faeces in parks. food any form of homemade cooked food in a dog’s diet (or added to a dog’s diet) may increase the risk of dogs carrying campylobacter spp. (leonard et  al. 2011). raw food, especially meat, is generally considered to be a source of campylobacter  spp. (westgarth et  al. 2009). a rapid change of diet can create a predisposition to enteric dismicrobism, which could in turn favour the onset of acute diarrhoea. in this condition, pathogens like silent campylobacter spp., can take over, multiply, and exacerbate any gastroenteric symptoms. season the season can affect both the patterns of infection in dogs and the way dogs shed campylobacter spp. some authors report a higher number of isolations during the summer and autumn months (lópez 2002, mohan 2015). for example, in a study performed in cordoba (spain), carbonero and colleagues (carbonero et  al. 2012) reported that c.  upsaliensis peaked during the spring months, while c.  jejuni peaked during the summer months. this is consistent with other studies performed on other species, such as cattle and sheep, where the highest prevalence was also found during the spring and summer months (grove white et al. 2010). walking outdoors housed dogs have less opportunity to become infected. dogs that escape from their homes, or are free to access the external environment, have a higher risk of being positive for campylobacter spp. (westgarth et al. 2009). note on the ‘one health’ concept the ‘one medicine’ concept as described by schwabe (schwabe 1964) has seen unprecedented revival in the last decade. the concept has evolved into a way of thinking about epidemiology and public health that is now known as ‘one health’ (zinsstag et  al. 2009). rabinowitz suggested that, as humans, we should change the ‘us versus them’ paradigm, which implies thinking of animals as 207 iannino et al. campylobacter and antimicrobial resistance veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 on pathogenic bacteria and on commensal micro-organisms of the intestinal tract of humans and animals. resistant commensal bacteria can constitute a reservoir of resistant genes for potentially pathogenic bacteria (guardabassi et al. 2004). antimicrobial resistance in campylobacter spp. at the dog‑human interface several studies have shown that antimicrobial use in food animals contributes to the selection of antimicrobial resistance. it furthermore poses risks to humans because of the transmission of resistant zoonotic bacteria via the food chain and the indirect transfer of resistance genes from animals to man. antimicrobial resistance amongst companion animals, particularly dogs, is a complex area representing an increasing public health concern. at the crux of this critical issue is the fact that dogs often live in a close proximity to humans. close physical contact through touching, petting, and licking occurs often as a result of the perception of household pets as family members. this creates opportunities for the interspecies transmission of campylobacter  spp., including multidrug-resistant campylobacter. however, it is difficult to ascertain how antimicrobial resistance in dogs is increasing because there is little useful data on antimicrobial drug use and resistance in companion animals. furthermore, the heterogeneity of studies, different analytical methods employed for the isolation, identification, typing, and resistance assessment make the result comparison difficult. this indicates a need to harmonise and standardise diagnostic methods. in order to determine the real extent of antimicrobial resistance and to be able to compare data between laboratories monitoring resistance trends, standardised protocols for the determination of susceptibility to antibiotics should be used. table ii and figure 1 summarises the relevant literature on antimicrobial resistance in human campylobacter isolates. among fluoroquinolones, the range of ciprofloxacin varies from 16% to 86%. a rapid increase in the proportion of campylobacter strains resistant to fluoroquinolones has been reported in many countries worldwide (luangtongkum et  al. 2009). these antibiotics are usually employed for the treatment of undiagnosed diarrheal illnesses. among macrolides, the prevalence of erythromycin resistance varies from 1.5% to 28.5%. high erythromycin resistance levels were observed among strains isolated from africa (lengerh et  al. 2013). macrolides are usually employed as frontline agents for treating culture-confirmed campylobacter infection. mutational resistance occurs due to a spontaneous chromosomal mutation that produces a genetically altered bacterial population that is resistant to a drug. resistant bacteria transfer their resistance genes to a bacteria’s progeny during dna replication. acquired resistance refers to the horizontal acquisition of a genetic element that encodes antibiotic resistance from another micro-organism. this implies that genetic elements transfer from some outside source, such as other bacteria of the same species or even between different species (soares et al. 2012). horizontal transfer resistance genes can function through 3 main routes: conjugation, transformation, and transduction. conjugation is the transfer of dna fragments through a conjugative pilus or pore that forms a channel that facilitates the passage of plasmids (bacteria to bacteria). transformation is the process whereby bacterial cells take-up free dna from the environment and incorporate it into their genomes (‘free dna transfer’). transduction occurs when a bacteriophage that has previously replicated in another bacterial cell, packages a portion of the host genome (donor) into the phage head and transfers the genes to another (recipient) bacterial cell (‘bacteriophage-mediated transfer’) (huddleston et al. 2014). mobile genetic elements fall into 2 general types: elements that can move from one bacterial cell to another, which includes resistance plasmids and conjugative resistance transposons, and elements that can move from one genetic location to another in the same cell, which includes transposons and gene cassettes (bennett 2005). plasmids are the elements that move bacterial genes from one bacterial cell to another. conjugative plasmids are able to promote their own transfer and the transfer of other plasmids from one bacterial cell to another. in general, they exist separately from the main bacterial chromosome, although the majority of replication functions are provided by the host cell and carry a considerable variety of genes, including those that confer antibiotic resistance (bennett 2008). the spread of antimicrobial-resistant bacteria can occur through direct contact (petting, licking, etc.) or indirectly via the household environment, contamination of food, furnishings, etc. (guardabassi et al. 2004). when they reach the new host, resistant bacteria can either colonise and infect, or remain for only a very short period of time. during this period, the resistant bacteria can not only spread their resistance genes to other bacteria residing in the new host (commensals or pathogens), but also accept resistance genes from other bacteria (da costa et al. 2013). antimicrobial drugs exert a selection pressure 208 campylobacter and antimicrobial resistance iannino et al. veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 campylobacter  spp. isolates. the highest frequency of campylobacter resistance has been recorded for ampi-cloxacillin (88.2%), clindamycin (73%), and azithromycin (64.7%). resistance mechanisms a combination of endogenous and acquired genes underlies resistance mechanisms. in general, mechanisms of antibiotic resistance include: among aminoglycosides, gentamicin resistance varies from 0% to 18%. aminoglycosides are used for serious systemic campylobacter infections in humans. the campylobacter resistance to cephalosporins is very high (27%-100%). however, these antibiotics are limited to the treatment of c. fetus (pacanowski et al. 2008). table iii and figure 2 summarises the relevant literature on antimicrobial resistance in dog table ii. humans. relevant literature detailing campylobacter antimicrobial resistance according to detection method, antimicrobial, species isolated, breakpoints, cut-off values, and geography. — cont’d author method antimicrobial species resistant breakpoints and cut-off values and notes country liao et al. 2012 agar dilution ampicillinsulbactam campylobacter spp. 5/24 (20.8%) breakpoints in: clsi guidelines (clsi 2012) taiwan cefotaxime campylobacter spp. 21/24 (87.5%) ceftriaxone campylobacter spp. 24/24 (100%) ertapenem campylobacter spp. 3/24 (12.5%) imipenem campylobacter spp. 0/24 (0%) meropenem campylobacter spp. 0/24 (0%) doripenem campylobacter spp. 0/24 (0%) gemifloxacin campylobacter spp. 15/24 (62.5%) ciprofloxacin campylobacter spp. 15/24 (62.5%) levofloxacin campylobacter spp. 14/24 (58.3%) lengerh et al. 2013 agar disc diffusion erythromycin campylobacter spp. 10/37 (27.7%) concentration: ampicillin 30 μg amoxicillin-clavulanic acid 30 μg gentamicin 10 μg tetracycline 30 μg doxycycline 30 μg chloramphenicol 30 μg ciprofloxacin 5 μg norfloxacin 5 μg ceftriaxone 5 μg erythromycin 15 μg clindamycin 15 μg trimethoprimsulphamethoxazole 25 μg ethiopia clindamycin campylobacter spp. 18/44 (40,9%) trimethoprimsulfamethoxazole campylobacter spp. 24/44 (54.5%) ciprofloxacin campylobacter spp. 7/44 (16%) ceftriaxone campylobacter spp. 10/37 (27.7%) chloramphenicol campylobacter spp. 5/44 (11.4%) nalidixic acid campylobacter spp. 4/44 (9.1%) cephalotin campylobacter spp. 39/44 (88.6%) gentamicin campylobacter spp. 8/44 (18.2%) amoxicillin clavulanic acid campylobacter spp. 16/44 (36.4%) ampicillin campylobacter spp. 16/44 (36%) tetracycline campylobacter spp. 25/44 (56,8%) doxycycline campylobacter spp. 7/44 (15,9%) norfloxacin campylobacter spp. 6/44 (11.6 %) abay et al. 2014 disk diffusion and etest amoxicillin clavulanic acid c. jejuni 12/100 (12%) disk diffusion test breakpoints: amoxicillin clavulanic acid 30 μg ampicillin 10 μg gentamicin 10 μg nalidixic acid 30 μg streptomycin 10 μg tetracycline 30 μg etest breakpoints: ciprofloxacin ≥ 4 enrofloxacin ≥ 2 erythromycin ≥ 32 turkey ampicillin c. jejuni 44/100 (44%) gentamicin c. jejuni 0/100 (0%) nalidixic acid c. jejuni 84/100 (84%) streptomycin c. jejuni 3/100 (3%) tetracycline c. jejuni 38/100 (38%) ciprofloxacin c. jejuni 81/100 (81%) enrofloxacin c. jejuni 85/100 (85%) erythromycin c. jejuni 6/100 (6%) continued 209 iannino et al. campylobacter and antimicrobial resistance veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 agents out of bacterial cells. the best-described multi-drug efflux pump in campylobacter is cmeabc, which consists of 3 components: an inner membrane transporter (encoded by cmeb), a periplasmic fusion protein (encoded by cmea), and an outer membrane protein (encoded by cmec). cmeabc extrudes a broad range of antibiotics, dyes, heavy metals, bile salts, and detergents. other putative efflux pumps, including cmedef and cmeg, may also contribute to antibiotic resistance (akiba et al. 2006, iovine 2013, lin et al. 2002). 1. the modification of the antibiotic’s target and/or its expression (i.e., dna gyrase mutations); 2. the inability of the antibiotic to reach its target (i.e., expression of the major outer membrane protein, or momp); 3. the efflux of the antibiotic (i.e., multidrug efflux pumps such as cmeabc); 4. the modification or inactivation of the antibiotic (i.e., β-lactamase production) (iovine 2013). active efflux pump systems extrude antimicrobial table ii. humans. relevant literature detailing campylobacter antimicrobial resistance according to detection method, antimicrobial, species isolated, breakpoints, cut-off values, and geography. — cont’d author method antimicrobial species resistant breakpoints and cut-off values and notes country gaudreau et al. 2014 disk diffusion and etest erythromycin c. jejuni 16/440 (3.6%) breakpoints in: clsi guidelines (clsi, 2010) susceptibilities were assessed initially by disk diffusion and later confirmed by etest canada c. coli 7/38 (18%) ciprofloxacin c .jejuni 180/440 (41%) c. coli 19/38 (50%) tetracycline c. jejuni 274/440 (62.3%) c. coli 20/38 (52.6%) riley et al. 2015 broth microdilution ciprofloxacin c. jejuni 55/180 (30.5%) breakpoints in: clsi guidelines 2010 (clinical and laboratory standards institute, 2010) canada c. coli 16/39 ( 41%) erythromycin c. jejuni 7/180 (3.9%) c. coli 5/39 (12.8%) tetracycline c. jejuni 116/180 (64.4%) c. coli 21/39 (53.8%) stockdale et al. 2015 disk diffusion fluoroquinolone campylobacter spp. 1,710/5,890 (29.0%) / uk macrolides campylobacter spp. 129/5,881 (2.2%) thompson et al. 2015 agar disk diffusion amoxicillinclavulanic acid c. coli 0/20 (0%) breakpoints in: clsi guidelines clinical and laboratory standards institute, 2012 vietnam c. jejuni 2/44 (4.5%) ampicillin c. coli 5/20 (28%) c. jejuni 12/44 (26.5%) ceftazidime c. coli 5/20 (25%) c. jejuni 5/44 (11.3%) chloramphenicol c. coli 0/20 (0%) c. jejuni 1/44 (2,3%) ciprofloxacin c. coli 20/20 (100%) c. jejuni 30/43 (69.7%) erythromycin c. coli 5/20 (25%) c. jejuni 0/42 (0%) gatifloxacin c. coli 2/20 (10%) c. jejuni 6/44 (13.6) nalidixic acid c. coli 20/20 (100%) c. jejuni 34/44 (77.2) ofloxacin c. coli 20/20 (100%) c. jejuni 32/44 (72.7) trimethoprim c. coli 17/20 (85%) c. jejuni 32/43 (74.4%) continued 210 campylobacter and antimicrobial resistance iannino et al. veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 table ii. humans. relevant literature detailing campylobacter antimicrobial resistance according to detection method, antimicrobial, species isolated, breakpoints, cut-off values, and geography. — cont’d author method antimicrobial species resistant breakpoints and cut-off values and notes country lapierre et al. 2016 agar dilution ciprofloxacin c. jejuni 20/66 (30.3%) breakpoints: ciprofloxacin ≥ 4 μg/ml erythromycin ≥ 32 μg/ml gentamicin ≥ 16 μg/ml tetracycline ≥ 16 μg/ml chile c. coli 4/7 (57.2%) erythromycin c. jejuni 1/66 (1.5%) c. coli 2/7 (28.5%) gentamicin c. jejuni 0/66 (0.0%) c. coli 0/7 (0.0%) tetracycline c. jejuni 16/66 (24.3%) c. coli 2/7(28.5%) olkkola et al. 2016 broth microdilution ciprofloxacin c. jejuni 8/95 (8.4%) cut-off values: ciprofloxacin 0.5 μg /l erythromycin 4 μg /l tetracycline 1 μg /l streptomycin 4 μg /l gentamicin 2 μg /l nalidixic acid 16 μg /l finland erythromycin c. jejuni 0/95 (0%) tetracycline c. jejuni 2/95 (2.1%) streptomycin c. jejuni 1/95 (2.1%) gentamicin c. jejuni 0/95 (0.0%) nalidixic acid c. jejuni 8/95 (8.4%) zhou et al. 2016 agar dilution ciprofloxacin c. jejuni 176/203 (86.7%) breakpoints μg/ml: ciprofloxacin ≥ 4 μg/ml nalidixic acid ≥ 64 μg/ml doxycycline ≥ 8 μg/ml tetracycline ≥ 16 μg/ml gentamicin ≥ 8 μg/ml chloramphenicol ≥ 32 μg/ml florfenicol ≥ 8 μg/ml erythromycin ≥ 32 μg/ml. china nalidixic acid (nal) c. jejuni 176/203 (86.7%) doxycycline (dox) c. jejuni 162/203 (80.8%) tetracycline (tet) c. jejuni 186/203 (91.6%) gentamicin (gen) c. jejuni 15/203 (7.4%) chloramphenicol (chl) c. jejuni 25/203 (12.3%) florfenicol (ffc) c. jejuni 64/203 (31.5 %) erythromycin c. jejuni 4/203 (2.0%) cip-nal-dox-tet c. jejuni 151/203 (74.4%) ffc-cip-nal-doxtet c. jejuni 61/203 (29.9%) chl-ffc-cip-naldox-tet c. jejuni 21/203 (9.9%) gen-ffc-cip-naldox-tet c. jejuni 12/203 (5.9%) 0 0.2 0.4 0.6 0.8 1 d o ri p en em im ip en em m er o p en em st re p to m yc in m ac ro lid es g en ta m ic in er yt h ro m yc in g en -f fc -c ip -n al -d o xte t c h lo ra m p h en ic o l c h lff cc ip -n al -d o xte t er ta p en em n o r� o xa ci n a m o xi ci lli n c la vu la n ic a ci d c ef ta zi d im e a m p ic ill in -s u lb ac ta m g at i� o xa ci n fl u o ro q u in o lo n e ff cc ip -n al -d o xte t fl o rf en ic o l ( ff c) a m p ic ill in c lin d am yc in c ip ro �o xa ci n tr im et h o p ri m s u lfa m et h o xa zo le c ef tr ia xo n e te tr ac yc lin e le vo �o xa ci n n al d ix ic a ci d d o xy cy cl in e c ip -n al -d o xte t tr im et h o p ri m o �o xa ci n en ro �o xa ci n c ef o ta xi m e c ep h lo ti n figure 1. humans. literature detailing campylobacter antimicrobial resistance. 211 iannino et al. campylobacter and antimicrobial resistance veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 table iii. dogs. relevant literature detailing campylobacter antimicrobial resistance according to detection method, antimicrobial, species isolated, breakpoints, cut-off values, and geography. — cont’d author method antimicrobial species resistant breakpoints and cut-off values and notes country sandberg et al. 2002 e-test ampicillin c. jejuni 0/22 (0,0%) isolates from dogs and cats breakpoints: nalidixic acid ≥ 32 μg/ml streptomycin ≥ 8 μg/ml the other breakpoints are not available norway c. upsaliensis 0/20 (0,0%) ciprofloxacin c. jejuni 0/22 (0,0%) c. upsaliensis 0/20 (0,0%) chloramphenicol c. jejuni 0/22 (0,0%) c. upsaliensis 0/20 (0,0%) erythromycin c. jejuni 0/22 (0,0%) c. upsaliensis 0/20 (0,0%) gentamicin c. jejuni 0/22(0,0%) c. upsaliensis 0/20 (0.0%) nalidixic acid c. jejuni 0/22(0,0%) c. upsaliensis 1/20 (5,0%) streptomycin c. jejuni 1/22 (4,5%) c. upsaliensis 18/20 (90.0%) tetracycline c. jejuni 0/22 (0.0%) c. upsaliensis 0/20(0.0%) lee et al. 2004 e-test gentamicin c. jejuni 0/11 (0.0%) breakpoints gentamicin 16 μg/ml erythromycin 8 μg/ml ciprofloxacin 4 μg/ml tetracycline 16 μg/ml united states erythromycin c. jejuni 0/11 (0.0%) ciprofloxacin c. jejuni 1/11 (9.1%) tetracycline c. jejuni 2/11 (18.2%) tsai et al. 2007 e-test azithromycin c. jejuni 32/33 (93.9%) break point azithromycin ≥ 2 μg/ml chloramphenicol ≥ 32 μg/ml ciprofloxacin ≥ 4 μg/ml clindamycin ≥ 4 μg/ml erythromycin ≥ 8 μg/ml gentamicin ≥ 16 μg/ml nalidixic acid ≥ 32 μg/ml tetracycline ≥ 16 μg/ml taiwan chloramphenicol c. jejuni 23/33 (69.7%) ciprofloxacin c. jejuni 6/33 (18.2%) clindamycin c. jejuni 29/33 (87.9%) erythromycin c. jejuni 27/33 (81.8%) gentamicin c. jejuni 10/33 (33.3%) nalidixic acid c. jejuni 17/33 (51.5%) tetracycline c. jejuni 26/33 (78.8%) rossi et al. 2008 agar dilution erythromycin c. jejuni 0/24 (0,0%) isolates from dogs and cats breakpoints: erythromycin ≥ 8 μg/ml-1 chloramphenicol ≥ 32 μg/ml-1 gentamicin ≥ 16 μg/ml-1 ampicillin ≥ 32 μg/ml-1 tetracycline ≥ 16 μg/ml-1 nalidixic acid ≥ 32 μg/ml-1 ciprofloxacin ≥ 4 μg/ml-1 enrofloxacin ≥ 4 μg/ml-1 one c. jejuni strain was multi-drug resistant to nalidixic acid, ciprofloxacin, tetracycline, and ampicillin, 5 were resistant to nalidixic acid, ciprofloxacin, and tetracycline. italy c. upsaliensis 0/38(0,0%) c. helveticus 3/16 (18.7%). chloramphenicol c. jejuni 1/24 (4.2%) c. upsaliensis 0/38 (0,0%) c. helveticus 0/16 (0,0%) gentamicin c. jejuni 0/24 (0,0%) c. upsaliensis 0/38 (0,0%) c. helveticus 3/16 (18.7%) ampicillin c. jejuni 3/24 (12.5%) c. upsaliensis 3/38 (7.8%) c. helveticus 0/16 (0,0%) tetracycline c. jejuni 3/24 (12.5%) c. upsaliensis 0/38 (0,0%) c. helveticus 0/16 (0,0%) nalidixic acid c. jejuni 15/24 (62.5%) c. upsaliensis 3/38 (7.9%) c. helveticus 1/16 (6.2%) ciprofloxacin c. jejuni 15/24 (62.5%) c. upsaliensis 3/38 (7.9%) c. helveticus 1/16 (6.2%) enrofloxacin c. jejuni 14/24 (58.3%) c. upsaliensis 3/38 (7.9%) c. helveticus 1/16 (6.2%) continued 212 campylobacter and antimicrobial resistance iannino et al. veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 table iii. dogs. relevant literature detailing campylobacter antimicrobial resistance according to detection method, antimicrobial, species isolated, breakpoints, cut-off values, and geography. — cont’d author method antimicrobial species resistant breakpoints and cut-off values and notes country acke et al. 2009 e-test nalidixic acid c. jejuni 19/51 (37.3%) isolates from dogs and cats. breakpoints not available ireland ciprofloxacin c. jejuni 10/51 (19.6%) tetracycline c. jejuni 7/51 (13.7%) ampicillin c. jejuni 7/51 (13.7%) erythromycin c. jejuni 6/51 (11.8%) chloramphenicol c. jejuni 3/51 (5.9%) kurnar et al. 2012 disk diffusion amikacin campylobacter spp. 0/51 (0.0%) breakpoints: amikacin 30 μg amoxycillin-clavulanic acid 20 μg ampi-cloxacillin 10 μg ciprofloxacin 30 μg chloramphenicol 30 μg enrofloxacin 10 μg erythromycin 15 μg levofloxacin 5 μg streptomycin 10 μg tetracycline 30 μg india amoxycillinclavulanic acid campylobacter spp. 10/51 (19.6%) apmpi-cloxacillin campylobacter spp. 45/51 (88.2) ciprofloxacin campylobacter spp. 41/51 (80.4%) chloramphenicol campylobacter spp. 0/51 (80.0%) enrofloxacin campylobacter spp. 35/51 (68.6%) erythromycin campylobacter spp. 46/51 (90.2%) levofloxacin campylobacter spp. 0/51 (0.0%) streptomycin campylobacter spp. 0/51 (0.0%) tetracycline campylobacter spp. 45/51 (88.2%) andrzejewska et al. 2013 e-test erythromycin c. jejuni 0/2 (0.0%) erythromycin ≥ 32 μg/ml azithromycin 32 μg/ml ciprofloxacin 4 μg/ml ≥ tetracycline 16 μg/ml poland azithromycin c. jejuni 0/2(0.0% ciprofloxacin c. jejuni 2/2 (100%) tetracycline c. jejuni 1/2(50.0%) erythromycin c. coli 0/2(0.0%) azithromycin c. coli 0/2(0.0%) ciprofloxacin c. coli 1/2(50.0%) tetracycline c. coli 1/2 (50.0%) amar et al. 2014 multi locus sequence typing (mlst) and fla-typing quinolones c. jejuni 25/133 (20.9%) / switzerland c. coli 3/6 (50%) sahin et al. 2014 broth microdilution azithromycin c. jejuni 1/8 (12,2 %) breakpoints: azithromycin ≥ 8 μg/ml ciprofloxacin≥ 4 μg/ml clindamycin ≥ 8 μg/ml erythromycin ≥ 32 μg/ml florfenicol ≥ 16 μg/ml gentamicin ≥ 8 μg/ml nalidixic acid ≥ 32 μg/ml telithromycin ≥ 16, μg/ml tetracycline ≥ 16 μg/ml united states ciprofloxacin c. jejuni 1/8 (12,2 %) clindamycin c. jejuni 1/8 (12,2 %) erythromycin c. jejuni 1/8 (12,2 %) florfenicol c. jejuni 0/8 (0,0 %) gentamicin c. jejuni 0/8 (0,0 %) nalidixic acid c. jejuni 0/8 (0,0 %) telithromycin c. jejuni 1/8 (12,2 %) tetracycline c. jejuni 1/8 (12,2 %) olkkola et al. 2015 broth microdilution and agar dilution method for streptomycin erythromycin c. jejuni 0/2 (0.0%) erythromycin > 4 mg/l tetracicline > 1 mg/l streptomycin > 4 mg/l gentamicin > 2 mg/l ciprofloxacin > 0.5 mg/l nalidixic acid > 16 mg/l finland c. upsaliensis 0/24 (0.0%) tetracycline c. jejuni 0/2 (0.0%) c. upsaliensis 0/24 (0.0%) streptomycin c. jejuni 0/2 (0.0%) c. upsaliensis 19/24 (79.1%) gentamicin c. jejuni 0/2 (0.0%) c. upsaliensis 0/24 (0.0%) ciprofloxacin c. jejuni 0/2 (0.0%) c. upsaliensis 1/24 (0.2%) nalidixic acid c. jejuni 0/2 (0.0%) c. upsaliensis 1/24 (0.2%) 213 iannino et al. campylobacter and antimicrobial resistance veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 a70t, asp-203-ser, asp85tyr, asp90asn, pro104, and d90n, which are less common and do not play an important role in quinolone resistance as that which has been observed around the thr86ile mutation (luo et  al. 2003, payot et  al. 2006, bachoual et  al. 2001). multiple mechanisms for antibiotic resistance have also been reported, including active efflux pump systems and decreased outer membrane permeability (charvalos et al. 1995, taylor and tracz 2005). in addition to the mutations in gyra, the multi-drug efflux pump, cmeabc, also contributes to quinolone resistance by reducing the accumulation of the agents in campylobacter cells. this efflux pump acts synergetically with dna gyrase mutation to effect high-level quinolone resistance (iovine 2013, wieczorek and osek 2013). resistance to macrolides macrolides, and particularly erythromycin, are drugs that are used when campylobacteriosis is strongly suspected (guerrant et al. 2001). macrolides interrupt protein synthesis in bacterial ribosome by targeting the 50s subunit and inhibit bacterial rna-dependent protein synthesis. the main mechanisms of resistance to macrolides in campylobacter are target modification, efflux, and altered membrane permeability. these mechanisms might act synergistically to confer high-level macrolide resistance (iovine 2013, cagliero et  al. 2006). macrolide resistance in campylobacter is mainly associated with point mutation(s) occurring in the peptidyl-encoding region in domain v of the 23s rrna gene at positions 2074 and 2075, with the 2075 substitution being the more common position (gibreel and taylor 2006, vacher et al. 2005, multiple mechanisms of resistance can occur in a single isolate, leading to higher levels of resistance. resistance to quinolones quinolones and fluoroquinolones are broad-spectrum antibiotics used in both human and veterinary medicine and are generally considered the first choice to treat acute undiagnosed diarrhoeal illness. campylobacteriosis in humans is clinically indistinguishable from other causes of bacterial diarrhoeal illness, and so, without evidence of campylobacter infection, many cases are treated empirically with quinolones (iovine 2013). these antibiotics inhibit the synthesis of bacterial dna, causing cell death. their targets are 2 bacterial enzymes, dna gyrase and topoisomerase  iv, that act in bacterial dna replication, transcription, recombination, and repairing (wieczorek and osek 2013). dna gyrase is a tetrameric enzyme that catalyses negative dna supercoiling and consists of 2 different subunits, gyra and gyrb (encoded by the gyra and gyrb genes). campylobacter species lack topoisomerase iv, and resistance to quinolones is mainly due to amino acid(s) substitution(s) in the gyra-encoding subunit of the dna gyrase in a region identified as the quinolone-resistance determining region (qrdr) (dionisi et al. 2004, griggs et al. 2009). there are several different single gyra modifications reported to be associated with quinolone resistance in campylobacter species. the most frequently observed mutation resulting in the substitution of aminoacids is the c257t change in the gyra gene, which leads to the thr86ile substitution, and confers high-level resistance. other reported resistance-associated mutations include t86 k, 0 0.2 0.4 0.6 0.8 1 a m ik ac in a m o xy ci lli n -c la vu la n ic a ci d a m p ic ill in a m p ic lo xa ci lli n a zi th ro m yc in c h lo ra m p h en ic o l c ip ro �o xa ci n c lin d am yc in en ro �o xa ci n er yt h ro m yc in fl o rf en ic o l g en ta m ic in le vo �o xa ci n n al id ix ic a ci d q u in o lo n es st re p to m yc in te lit h ro m yc in te tr ac yc lin e d figure 2. dogs. literature detailing campylobacter antimicrobial resistance. 214 campylobacter and antimicrobial resistance iannino et al. veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 is required for intrinsic and acquired β-lactam resistance in c. jejuni. resistance to tetracyclines tetracyclines are alternative agents for antimicrobial therapy in campylobacteriosis. these are lipophilic protein synthesis inhibitors. their primary antimicrobial effect takes place by binding to the a site in the 30s subunit, thus hindering the movement of transfer rna and inhibiting peptide elongation (harms et  al. 2003). resistance to tetracycline in campylobacter principally involves a ribosomal protection protein termed tet(o), which is widely present in campylobacter isolates recovered from various animal species. this protein is part of a larger group of proteins called ribosomal protection proteins (rpps), which includes tet(m), tet(q), tet(s), tet(t), tet(w), and otra (chopra and roberts 2001). tetracycline resistance conferred by tet(o) has become highly prevalent in campylobacter worldwide. this gene is usually carried in a plasmid, although it can be chromosomally encoded (wieczorek and osek 2015, connell 2003, gibreel et  al. 2005). the gene, which encodes ribosomal protection proteins, is located on a self-transmissible plasmid, and is probably acquired through horizontal gene transfer from streptomyces, streptococcus, and enterococcus  spp. (batchelore et  al. 2004). mutations in efflux pumps can also lead to resistance to tetracyclines. resistance to aminoglycosides aminoglycoside drugs are not a priority for treating campylobacter infections but, in serious bacteremia, may be used by intravenous infusion. their bactericidal activity is due to the inhibition of bacterial protein synthesis to binding 16s rrna (mingeot et  al. 1999). aminoglicosydes exert antimicrobial activities in 2 ways: through alterations at the ribosomal binding sites, or through the production of aminoglycoside-modifying enzymes. mutations at the site of aminoglycoside attachment may interfere with ribosomal binding. this can cause resistance to streptomycin, since this agent binds to a single site on the 30s subunit of the ribosome. resistance to other aminoglycosides as a result of this mechanism are uncommon since they bind to multiple sites on both ribosomal subunits and high-level resistance cannot be selected through a single step. enzymatic modification is the most common type of aminoglycoside resistance and mechanism is of clinical importance since the genes encoding aminoglycoside-modifying enzymes can be disseminated through plasmids or transposons. the enzymatic modification decreases affinity of luangtongkum et al. 2009). these mutations confer a high-level resistance to macrolide antibiotics (erythromycin mic >128 μg/ml) in c. jejuni and c. coli (gibreel et al. 2005). these species carry 3 copies of 23s rrna gene, all of which are usually mutated in macrolide-resistant strains. however, some strains with lower mics to macrolides have been found to have only 2 mutated gene copies, suggesting a gene dosage effect (iovine 2013, vacher et al. 2005). other mutations (usually insertions) in the ribosomal proteins l4 and l22 that have lead to macrolide resistance have been described (cagliero et al. 2006). efflux is another mechanism that causes macrolide resistance in campylobacter. the cmeabc multi-drug efflux pump functions synergistically with 23s rrna mutations to effect high-level macrolide resistance (cagliero et al. 2006). in addition, the putative efflux pump cmeg may also contribute to macrolide resistance (iovine et al. 2013). other mutations (usually insertions) in the ribosomal proteins l4 and l22 that have lead to macrolide resistance have been described. these have been associated with a low level of macrolide resistance (lehtopolku et  al. 2011). macrolide resistance in c. jejuni and c. coli was conferred also from the synergy between the cmeabc efflux pump and mutations in the ribosomal proteins l4 (g74d) and l22 (insertions at position 86 or 98) (caldwell 2008). resistance to macrolides may also be caused by altered (decreased) membrane permeability that resulted from major outer membrane porin, which was chromosomally encoded by pora. (pumbwe et al. 2004) resistance to β‑lactam antibiotics β-lactam antibiotics are the most commercially available antibiotics. in 2009, beta-lactam antibiotics accounted for more than half of the total antibiotic sales globally (hamad 2010). although β-lactams are still not a drug of choice for treating campylobacter infections, it has recently been proposed that an oral combination of amoxicillin, a β-lactam antibiotic, and potassium clavulanate, a β-lactamase inhibitor, may provide an alternative therapy for campylobacter infection (elviss et al. 2009, zeng et al. 2015). these antibiotics inhibit biosynthesis of the bacterial cell wall. several β-lactam resistance mechanisms have been described, and the most widespread and threatening mechanisms are the production of β-lactamases (the enzymes that hydrolyse the β-lactam ring) and the cmeabc multi-drug efflux pump (lin et  al. 2002, alfredson and korolik 2005). another mechanism is the reduced uptake due to alteration in the outer membrane porine (iovine 2013). recently zeng (zeng et  al. 2015) described a putative lytic transglycosylase (lt) cj0843c that 215 iannino et al. campylobacter and antimicrobial resistance veterinaria italiana 2019, 55 (3), 203-220. doi: 10.12834/vetit.1161.6413.3 antimicrobial-resistant bacteria. this poses a new threat to urban hygiene. campylobacter spp. continues to be a leading cause of bacterial diarrhoea illness throughout the world. antimicrobial resistance to the drugs used to treat these illnesses can prolong the duration of illness and may compromise the treatment of patients with bacteraemia. the same antimicrobials used in dogs are used in humans. the major concern to both humans and animals is the resistance to macrolides, quinolones, and aminoglycosides such as gentamicin, which are the drugs used to treat serious campylobacteriosis. drug-resistant campylobacter can spread from humans to dogs and viceversa through direct contact or, indirectly, through the common environment. thus, an integrated ‘one health’ approach to surveillance and intervention is required. antimicrobials are essential for the health of animals and humans, but it is extremely important to apply the principles of prudent use in order to contain the development of antimicrobial resistance. it is advised that veterinarians strictly observe the following instructions from the eu-commission notice guidelines for the prudent use of antimicrobials in veterinary medicine (eu-commission notice 2015): • the prescription and dispensation of antimicrobials must be justified by a veterinary diagnosis in accordance with the current status of scientific knowledge. • where it is necessary to prescribe an antimicrobial, the prescription should be based on a diagnosis made following the clinical examination of the dog by the prescribing veterinarian. where possible, antimicrobial susceptibility testing should be carried out to determine the choice of antimicrobial. • routine prophylaxis must be avoided. • all information relating to the animals, the cause and the nature of the infection, and the range of available antimicrobial products must be taken into account when making a decision regarding antimicrobial treatment. • a narrow-spectrum antimicrobial should always be the first choice unless prior susceptibility testing – where appropriate supported by relevant epidemiological data – shows that this would be ineffective. the use of broad-spectrum antimicrobials and antimicrobial combinations should be avoided (with the exception of fixed combinations contained in authorized veterinary medicinal products). • the off-label use of the compounds in dogs aminoglycosides for the rrna a-site (wieczorek et  al. 2013, llano-sotelo et  al. 2002). multiple aminoglycoside-modifying enzymes, including 3’-aminoglycoside phosphotransferase types i, iii, iv, and vii, 3’,9-aminoglycoside adenyltransferase, and 6-aminoglycoside adenyltransferase, have been described in campylobacter infection (zhang et al. 2008). aminoglycoside resistance was first detected in c.  coli and was mediated by a 3'-aminoglycoside phosphotransferase (encoded by apha-3). this apha-3 gene remains the most common source of aminoglycoside resistance in campylobacter and is located in an insertioning sequence, is607, or is found with genes encoding streptomycin resistance (encoded by aade, a 6’-adenylyl transferase). the existence of a similar resistance cluster in enterococcus suggests that campylobacter acquired these genes through horizontal transfer (gibreel et  al. 2005). other genes that confer kanamicine resistence in c. jejuni are apha-1 and apha-7 (iovine 2013). moreover, 9 variants of gentamicin resistance genes have been identified: aph(2”)-ib, ic, ig, if, if1, if3, ih, aac(6’)-ie/aph(2”)-ia, and aac(6')-ie/aph(2”)-if2. the aph(2”)-ib, ic, if1, if3, ih, and aac(6’)-ie/aph(2”)-if2 variants were identified for the first time in campylobacter (zhao et  al. 2015). the contribution of efflux to aminoglycoside resistance is less clear, but is less important than the plasmid-borne drug-modifying enzymes described previously (iovine 2013). antimicrobial susceptibility testing methods several antimicrobial susceptibility testing (ast) methods such as disk diffusion, etest, broth dilution, and agar dilution are available to test in vitro bacterial susceptibility to antimicrobials and to provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the infected host. this type of information helps clinicians to select the appropriate antimicrobial agent. the use of genotypic approaches for the detection of antimicrobial resistance genes has also been promoted as a way to increase the speed and accuracy of susceptibility testing. when used in conjunction with phenotypic analysis, genetic tests increase sensitivity, specificity, and the speed of detection for specific resistance genes. conclusions antimicrobial resistance in campylobacter 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d'aosta, via bologna 148, 10154 turin, italy. tel.: +39 011 2686247, fax: +39 011 2686322, e-mail: claudiocaruso1986@libero.it. veterinaria italiana 2018, 54 (4), 337-341. doi: 10.12834/vetit.1006.6613.2 accepted: 19.03.2017 | available on line: 31.12.2018 claudio caruso1*, nicoletta vitale2, riccardo prato1, maria cristina radaelli3, simona zoppi1, rosaria possidente3, alessandro dondo1, laura chiavacci3, ana maria moreno martin4 and loretta masoero1 parole chiave età, fattori di rischio, genere, malattia di aujeszky, piemonte. riassunto il virus della pseudorabbia (prv) rimane una minaccia per la popolazione di cinghiali non protetti nonostante sia in corso la sua eradicazione nei suini domestici. in questo studio sono riportati i dati sulla prevalenza e l'influenza di possibili fattori di rischio in 2 popolazioni (stato libero e in area protetta) di cinghiali italiani del nord-ovest, con l'obiettivo di supportare l'implementazione di un sistema di sorveglianza basato sul rischio, utile a determinare le aree in cui è più probabile la ricomparsa della malattia di aujeszky. dal 2011 al 2015 sono stati raccolti sieri di 1.425 cinghiali selvatici; la sieroprevalenza complessiva è stata del 30,39% (433/1.425, ic 95% 28,01-32,85%). il tasso di prevalenza, invece, si è dimostrato significativamente differente tra la popolazione in condizione libera (90/902; ic 95%; 8,10-12,12%) e quella nel parco la mandria in piemonte (343/523 ci 95%; 61,51-69,65%). in entrambe le popolazioni è risultato positivo un numero significativamente maggiore di adulti e di femmine. sull'ad dovrebbero essere acquisiti, tuttavia, dati territoriali specifici da altre regioni che possano indirizzare le misure basate sul rischio al fine di ridurre la minaccia della reinfezione da ad nella modalità economicamente più efficace. il virus della pseudorabbia nei cinghiali italiani: prevalenza e fattori di rischio a supporto di un sistema di sorveglianza territoriale in piemonte summary although the eradication of pseudorabies virus (prv) in domestic pigs is ongoing, the circulation of this virus in wild boars remains a threat in the currently unprotected, ‘low prevalence’, pig population. in this study, we reported prv prevalence data and the influence of possible risk factors in 2 north-west italian wild boar populations (free and enclosed) with the goal of supporting the implementation of a risk-based ad surveillance system. sera from 1,425 wild boars were collected between 2011 and 2015 and tested by elisa for the presence of prv antibodies; the overall raw seroprevalence was 30.39% (433/1,425; ci  95% 28.01-32.85%). a significant difference was however observed between the prevalence rates of the free range population (9.98%; 90/902; ci  95%; 8.10-12.12%) and the enclosed population of la mandria park (piedmont, italy) (65.58%; 343/523; ci 95%; 61.51-69.65%). in both populations a significantly higher number of adults and females were found positive to prv elisa. specific territorial data on prv circulation in wild boars should be acquired from other regions for guiding risk-based measures in order to reduce the threat of ad re-infection in a more cost-effective manner. keywords age, aujeszky disease, gender, risk factors, piedmont, wild boar. pseudorabies virus in north-west italian wild boar (sus scrofa) populations: prevalence and risk factors to support a territorial risk-based surveillance 338 aujeszky’s disease prevalence in wild boar caruso et al. veterinaria italiana 2018, 54 (4), 337-341. doi: 10.12834/vetit.1006.6613.2 venatorio)1 and no less than 600,000 wild boar throughout italy (pedrotti et al. 2001). in order to expedite their aujeszky-free status and thus attain inclusion in annex 2 of the eu decision 2008/185/ec, all the northern alpine regions (including piedmont, lombardy, and veneto) implemented strategies and strengthened sanitary measures. with the implementation of its regional control plan for ad (nota regionale 2192 db2017), piedmont region decreased prv seroprevalence from 22.10% in 2012 to 9.84% in 2015 in pig farms, although an active serological and virological monitoring demonstrated prv circulation in wildlife (caruso et al. 2014). according to presidential decree 607 of 17/10/96, since 2003, the piedmont region also established a sanitary plan for the surveillance of diseases in wildlife. this plan also ensures permanent monitoring of occurrence, distribution, and evolution of prv in wild boars and carnivores (the latter as dead-end hosts). in accordance with this final stage of eradication, an updated analysis of the prv infection in wild boars due to contact with wild reservoirs may help to assess the potential re-incursion of prv in naive herds or ad-free areas. in this study we reported epidemiological data on the prv seroprevalence rate and its associated risk factors among wild boar populations under different management regimes: i) free-range population hunted in the piedmont region; ii) enclosed population living in a regional park la mandria, which includes 36 km2 of fenced area located in the proximity of the alpine chain, and is mainly characterised by grazing meadows, cereal fields, and deciduous forests (27% of the surface), with an estimated boar density of 18 animals/km2. overall, 1,425 wild boar serum samples were collected during 4 hunting seasons (season 1 from 2011 to 2012, season ii from 2012 to 2013, season iii from 2013 to 2014, season iv from 2014 to 2015). in total, 902 sera were from free-range wild boars while 523 were from the enclosed population. antibodies (abs) against prv were detected by elisa (enzyme linked immunosorbent assay), which was produced and provided by the italian national reference center for aujeszky disease. in order to detect abs against anti-glycoprotein b (gb), a blocking standardised elisa method was employed. briefly, this method evaluates the ability of the tested sera to inhibit specific monoclonal-labeled antibodies (2c7) to bind prv-antigen. the method works as a sandwich elisa; plates are pre-coated with a monoclonal ab specific to capturing the prv antigen that is provided in the kit and, after an incubation step, serum is added. after 3 washing steps, monoclonal-labeled ab 2c7 is added to the plate and a reaction developed with aujeszky’s disease (ad) is an economically important disease affecting wild and domestic pigs. the disease is caused by suid herpesvirus type  1, which is also known as pseudorabies virus (prv ), and belongs to the subfamily alphaherpesvirinae, genus varicellovirus ( verpoest et al. 2014). the natural hosts for prv are members of the family suidae, in which infection results in clinical or subclinical disease as well as latent infection with the possibility of viral reactivation. strict control measures and eradication programs including diva (differentiating infected from vaccinated animals) strategy were successful in many european union (eu) member countries. in other countries the prv prevalence in pig farms has drastically decreased (decision 2010/434/ eu). in ad-free countries, the vaccination of domestic pigs is forbidden. in some regions, the persisting prv circulation in wild boars (sus scrofa) is regarded as a possible threat for the currently unprotected pig population. since distinct infections and molecular differences between prv strains isolated from wild boars/hunting dogs and domestic pigs have been demonstrated, the risk of prv re-emergence and spillover from wild boar to domestic pigs is considered to be of very low concern (caruso et  al. 2014, moreno et  al. 2015). however, recent outbreaks in france and usa have been related to prv circulating in wild boars (hahn et al. 2010). in italy, the national prv-monitoring programme began in 1997 (decreto ministeriale 01/04/1997). the programme includes the application of direct prophylaxis, biosecurity measures, and vaccination; to date, even if the spread of the virus has been considerably reduced, ad has not been eradicated from pig herds yet (chiari et  al. 2015). over 80% of the italian pig production is concentrated in lombardy, emilia-romagna, piedmont and veneto. the piedmont region (45.2500° n, 7.9167° e) covers an area of 25,399 km2; pig industry is one of the most important agricultural sub-sector of the region. with a census of 1,164,095-reared animals and 2,895 farms concentrated in the provinces of cuneo and torino, it accounts for 11% of the entire italian production. pig production in this region is mainly dedicated to breeding ‘italian heavy pigs’ in order to produce protected designation of origin ham (prosciutto di parma, prosciutto di cuneo). the wild boar is the most common ungulate in italy in terms of distribution and population size. within the alpine and the prealpine areas, the presence of wild boars extends up to western italy, including the piedmont region. in the absence of an effective census methodology, the estimation of wild boar populations relies on indirect indices with an estimated regional wild boar population of about 32,000 animals (regione piedmont, piano faunistico 1 http://www.sistemapiedmont.it/fedwossfa/elenco.jsp. 339 caruso et al. aujeszky’s disease prevalence in wild boar veterinaria italiana 2018, 54 (4), 337-341. doi: 10.12834/vetit.1006.6613.2 transmission of prv . our study was set in piedmont region, whose territory is mostly characterised by the presence of alpine mountains and the seroprevalence rate in the free-range population is actually in-line with that (4.97%) found by chiari and colleagues in a low-density free-range alpine wild boar population hunted in 6 districts in the brescia province (chiari et al. 2015). unfortunately, our data were limited and fragmented, and thus we were not able to quantify a comprehensive index of abundance of wild boar population in the piedmont region district. it has recently been reported that, in contrast with other northern regions, in piedmont the wild boar population characteristics are similar to appennine populations (constant, permanent, and diffuse) (guidelines on ‘wild boar management’, ministero politiche agricole e forestali 2003). seroprevalence data according to sex and age, and relative or esteems are summarised in table  i. in the free-range population, a statistically significant difference was found between prevalence rates of juveniles and adults, with the latter being 3 times (or: 3.24, 95% ci 1.57-6.66) more probable to have abs anti-prv. these findings confirm those reported in literature (muller et  al. 1998, vicente et  al. 2005, panwitz et  al. 2011) in this study, females were nearly twice as probable to be prv-seropositive than males (or: 1.67; 95% ci 1.06-2.63). as suggested by vicente and colleagues, this is likely due to different tmb (tetramethylbenzidine); results are expressed as a percentage of inhibition. explanatory variables included in the study were: age (juveniles <  12 months vs adult >  24 months) according to caruso and colleagues (caruso et  al. 2015), gender (male vs female) and, in the free-range population, geographic districts (piedmont alpine districts of cuneo, torino, biella, vercelli, and verbania). a multivariate analysis was performed using logistic binomial regression model of fixed effects by proc logistic procedure of sas® v 9.2 (sas 2004). the likelihood ratio test was used to assess the overall significance of the model (two-tailed significance level p ≤ 0.05). the significance of each term in the model was tested by wald’s χ2. estimated odds ratios (or) and 95% wald’s ci were obtained as measures of predictor effect. hosmer-lemeshow test was performed to assess the model’s goodness-of-fit (hosmer and lemeshow 1989). descriptive analysis of the 2 samples (free range/ enclosed) showed a similar structure according to age and sex. the free range population was 51.5% female and 70% adult while the enclosed population was 57% female and 74% adult, with no statistically significant difference among the 2 populations for both variables (χ2  =  2.91, p  <  0.08; χ2  =  1.41, p  <  0.23, respectively). considering age and sex, the free-range population consisted of 71% adult females and 76% adult males, while the enclosed population consisted of 69.9% adult females and 71.2% adult males. the overall raw seroprevalence for the entire period of study was 30.39% (433/1425; ci 95% 28.01-32.85%). these results were not unexpected, since in italy the prv prevalence in wild boar populations is variable, ranging from 4% to 30%, with 2 emerging scenarios: alpine, in northern italy, where the density of wild boar population is low, and appennine, in central-southern italy where the density of wild boar population is high. the overall prevalence rate (30.39%, including free-range and enclosed population) was therefore consistent with the data reported in central and southern italy (montagnaro et  al. 2010, lari et  al. 2006, guberti et al. 2002). the prevalence rates of free-range and enclosed populations were significantly different (χ2 = 483.90, p < 0.0001). in the free-range population, prevalence was 9.98% (90/902; ci 95%; 8.10-12.12%), while in the enclosed population living in la mandria park was much higher (343/523; 65.58%; ci  95%; 61.51-69.65%) and consistent with the prevalence (56%) found by boadella and colleagues (boadella et  al. 2012). these results underline the fact that artificial conditions such as fencing and feeding lead to a significant increase of wild boar abundance and aggregation, which in turn promote interaction and table i. aujeszky’s disease seroprevalence stratified by variables and odds ratio (or) (wild boar, piedmont, italy, 2012-2016). free range population categories prevalence (ci 95%) standard error female 58.16% (47.78-68.05%) 4.98% male 41.84% (31.95-52.23%) 4.98% adult 90.82% (83.28-95.71%) 2.92% juvenile 9.18% (4.29-16.72%) 2.92% factors odds ratio 95%ci adult vs juvenile 3.236 1.573 6.659 female vs male 1.667 1.059 2.625 entire district vs cuneo 1.804 1.027 3.166 enclosure population (la mandria park) categories prevalence (ci 95%) standard error female 68.25% (61.10-74.82%) 3.39% male 31.75% (25.18-38.90%) 3.39% adult 78.84% (72.32-84.43%) 2.97% juvenile 21.16% (15.57-27.68%) 2.97% factors odds ratio 95%ci adult vs juvenile 3.724 2.251 6.161 female vs male 2.290 1.425 3.681 340 aujeszky’s disease prevalence in wild boar caruso et al. veterinaria italiana 2018, 54 (4), 337-341. doi: 10.12834/vetit.1006.6613.2 serve as mechanical carriers – indicates that a ‘zero risk’ hypothesis is not possible in this context. in this study, we did not provide any insight into what it is currently known to drive prv transmission within and between wild boar populations and, as is also noted by muller and colleagues (muller et  al. 2011), seroprevalence in populations of wild boar should be interpreted with care, since these findings may have been biased by sampling, or by investigation periods. indeed, if we focus only on the free-range population in the whole alpine region, our results are in compliance with the northern italy region, though the population density in piedmont region is higher than in most other alpine regions. nevertheless, the prv seroprevalence rate of enclosed populations drastically increased the average seroprevalence, and this fact should be carefully taken into account in the interpretations of ad epidemiological studies. our data reported a 4-year surveillance study and also included an acceptable number of serum sample. this study thus addressed the lack of data on prv in north-western italian wild boar populations and its associated risk factors. as prv could infect alpine wolves, which are currently re-introducing in northern italy, this study might also be relevant to issues relating to wild boar health and conservation or for veterinary authorities involved in ad control and eradication campaigns in italy and in piedmont region. as a possible spillover cannot be completely ruled out, data and strategies to prevent the transmission of prv from endemically infected wild boar to domestic pigs have to be investigated; additional epidemiological studies are needed, as well as studies relating in particular to the molecular characterisation of prv circulating strains in order to determine whether wildlife and domestic animals share the same strains. acknowledgements the authors wish to thank mariangela andrà, elena gobbi, and antonia sciarra (izsplv) for their skillful technical assistance. social behavioural traits and/or to age differences in sexual maturation between males and females (vicente et  al. 2005). in female wild boars – already susceptible to higher prevalence rates than males during the breeding season – social gregariousness may favour direct routes of infection, such as the respiratory route (ruiz et  al. 2007). in other studies, however, males and females were equally likely to be infected (boadella et al. 2012, pedersen et al. 2013). although the highest number of farms (56%), domestic pigs (72.6%), and ad outbreaks in 2015 (57/80; 71.25% 95% ci 60.05-80.82%, data from osservatorio epidemiologico izsplv), the prv seroprevalence in the wild boar populations hunted in the cuneo district was lower than in other areas of the region (or: 1.80; 95% ci 1.03-3.17). this finding, added evidence that prv maintenance in wildlife is not linked to the livestock. significantly higher prevalence rates were also observed in adults and females of the enclosed population. different management systems seem not to affect the influence of these variables on prv seropositivity. however, if we look at the main transmission routes of prv (venereal and oral/nasal excretion/infection) and consider the attenuated nature of wild swine prv and the low concentration of individuals in sounders compared to domestic pig holdings, aerosol transmission over long distances (no direct contact) seems to be unlikely (muller et al. 2011). as a consequence of regional control plans, increased attention was given to ad in the piedmont region and biosecurity measures were strengthened in pig farms. even if the results of this study seem to confirm that the risk of prv re-emergence and spillover from wild boar to domestic pigs could be considered of low concern, preventing direct contact between free-range wild boar and fared swine seems an appropriate risk-mitigating measure. a higher seroprevalence rate (65.58%) in the enclosed population is offset by the fact that the possibility of direct contact with pigs is minimised by limited freedom of movement. however, the risk of spillover – through rats and mice, which are moderately resistant to the infections and could also 341 caruso et al. aujeszky’s disease prevalence in wild boar veterinaria italiana 2018, 54 (4), 337-341. doi: 10.12834/vetit.1006.6613.2 boadella m., gortazar c., vicente j. & ruiz-fons f. 2012. wild boar: an increasing concern for 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veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 accepted: 27.05.2021 | available on line: 16.11.2022 1department of veterinary medicine, university of perugia, perugia, italy. 2istituto zooprofilattico sperimentale dell’umbria e delle marche ‘togo rosati’, perugia, italy. 3institute of veterinary bacteriology, centre for fish and wildlife health, vetsuisse faculty, university of bern, bern, switzerland. 4museo di storia naturale dell’università degli studi di firenze, museo ‘la specola’, university of florence, florence, italy. *corresponding author at: department of veterinary medicine, university of perugia, perugia, italy. e‑mail: marialuisa.marenzoni@unipg.it. maria luisa marenzoni1*, valentina stefanetti1, emilia del rossi1, alessia zicavo2, stefania scuota2, francesco carlo origgi3, gianluca deli1, claudia corti4, massimo trabalza marinucci1 and oliviero olivieri1 keywords mycoplasma agassizii, reintroduction, salmonella, testudinid alphaherpesvirus, testudo hermanni, free-ranging. summary testudo hermanni is included as near-threatened in the red list of the international union for conservation of nature, while t. hermanni hermanni is considered endangered in the italian red list. appropriate management of smuggled or seized wild individuals is recommended before their reintroduction into the wild. accordingly, a health monitoring study was carried out. during 2014-2016, 133 oral swabs and 121 cloacal swabs were collected from a total of approximately 180 free-ranging and rescued t. hermanni hermanni from eight different italian regions to investigate the presence of dna of testudinid alphaherpesvirus (teahv), chlamydia spp. and mycoplasma spp. in the oral cavity, and salmonella spp. isolates in the cloaca. mycoplasma spp. was detected in 52  out of 87 (59.77%) of rescued and in 1 out of 46 free-ranging (2.17%) individuals; 33 out of 53 (62.26%) mycoplasma  spp. positive samples were typed as m. agassizii by pcr. salmonella spp. was isolated from 45 out of 121 (37.19%) cloacal swabs, typed into 14 serovars, and characterized for complete antimicrobial susceptibility. a significantly different distribution of salmonella spp. isolates was found in 2016 in comparison with 2014 and 2015, without any difference between free-ranging and rescued tortoises. all the tested tortoises were negative for teahv and chlamydia spp. these results are considered a baseline information critical to monitor the dynamics of these microorganisms in free-ranging and rescued populations of t. h. hermanni, and to correctly approach the management of rescued animals and possible relocation programs. detection of testudinid alphaherpesvirus, chlamydia spp., mycoplasma spp., and salmonella spp. in free‑ranging and rescued italian testudo hermanni hermanni of nature (iucn), while the italian red list reports t. h. hermanni as endangered (rondinini et al. 2013). the balkan subspecies has been widely traded and many tortoises have returned to the wild, either by escape or by release from captivity, sometimes even hybridizing with autochthonous individuals. despite the italian distribution of t.  h.  hermanni is relatively well known, some autochthonous wild population may include captive individuals of the balkan form (corti et  al. 2013, di tizio et  al. 2016). the western subspecies (t.  h.  hermanni) occurs irregularly in the introduction the hermann’s tortoise (testudo hermanni), represented by two subspecies, is distributed in the northern mediterranean area. the nominal form (t. hermanni hermanni) is found in the western mediterranean, from catalonia to italy, while the eastern subspecies, testudo hermanni boettgeri, inhabits the balkans (mazzotti 2016). the hermann’s tortoise is considered as near-threatened in the red list of the international union for conservation 26 veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 microbiological investigation in italian testudo hermanni hermanni marenzoni et al. et  al. 2005, kabeya et  al. 2015, taylor-brown et  al. 2015, laroucau et  al. 2020). the infection has been reported both in clinically healthy and ill captive tortoises in italy (di ianni et  al. 2015). however, no conclusive characterization of chlamydiae responsible for reptile infections has been described, although c. pneumoniae and c. psittaci have been more frequently reported (kabeya et  al. 2015). recently, new species and strains of chlamydia spp. have been recognized in tortoises, suggesting also a risk of interspecies and zoonotic transmission, although their pathogenic role remains unclear (mitura et al. 2017, laroucau et al. 2020). mycoplasmas have been frequently detected in chelonians. some species are considered commensal bacteria of the host (farkas and gal 2009, di ianni et al. 2015), whereas others can cause severe diseases of the upper respiratory tract, with chronic evolution and mortality. mycoplasma  testudinis has been reported as a non-pathogenic agent of the excretory tract of t. graeca, whereas mycoplasma agassizii and mycoplasma  testudineum have been demonstrated to cause disease (upper respiratory tract disease) in both free-ranging and captive tortoises in the usa and europe (brown et  al. 1995, brown et  al. 1999, feldman et  al. 2006, lecis et  al. 2011), and they are considered a threat for the management of wild tortoise populations (jacobson et  al. 2014). m.  agassizzi has been reported in italy in rescued tortoises, but its potential role as etiologic agent of disease has not been clarified in the infected tortoises (lecis et al. 2011). moreover, the isolation of specific species of mycoplasma  has been reported for only a few species of chelonians in captivity conditions, which makes it very difficult to understand whether the virulence of the microorganisms and the severity of the infection are linked to the microbial species or to the host (kolesnik et al. 2017). therefore, their pathological role still requires clarification. salmonella spp. are ubiquitous enteric gram-negative bacteria, able to infect a wide range of host species, including mammals, reptiles, birds and insects. reptiles are generally considered carriers and intermittent shedders of salmonella  spp.; some serovars are more frequently isolated in reptiles and are therefore called reptile-associated salmonella, ras (bertrand et  al. 2008, harris et  al. 2010, pees et  al. 2013, bosch et  al. 2016). some of these have been implicated in a number of outbreaks involving humans, and especially young children (bertrand et  al. 2008, pees et  al. 2013), so that a european directive defines infection by ras as an emerging zoonosis (european commission 2003, pees et  al. 2013). although rarely, salmonella  spp. can also responsible for disease in reptiles, working as opportunistic pathogens generally associated with predisposing factors such as inappropriate environmental temperature (pasmans et  al. 2002, western mediterranean area, in particular along the coasts of spain and france. even if it is protected by national and international laws, human activities in particular those which may cause habitat loss or illegal collection of wild animals, haevily contribute to the decline of this species. rigorous monitoring, including close surveillance of infectious diseases, and appropriate management of smuggled or confiscated individuals are supported by the italian ministry of the environment (ministero dell’ambiente e tutela del territorio e del mare, mattm), as part of the efforts needed to preserve natural population. however, specific information to plan possible relocation programs, without impacting biodiversity and respecting the ecosystem, are still limited. a multidisciplinary project on free-ranging and captive italian testudo spp., with special attention to t.  h.  hermanni, has been carried out to characterize the genetic background of the tortoises candidate to be relocated, their eco-ethology, morphology, and health conditions (mattm 2019). more specifically, selected relevant infectious agents of tortoises were considered critical for the health assessment of tortoises part of the recollocation program. within this scenario, surveillance of infectious diseases becomes very important when performing relocation programs (hartley and lysons 2001). specific selection criteria for the choice of the microorganisms to detect were: 1) their ability to cause disease threatening individuals or populations either in captivity or in the wild, 2) the presence of asymptomatic carriers in their transmission cycle, and 3) the zoonotic risk for humans who handle the animals. accordingly, testudinid alphaherpesviruses (teahvs), chlamydia  spp., mycoplasma  spp., and salmonella spp. were investigated. four genotypes of teahvs are known (teahv-1, teahv-2, teahv-3, teahv-4). the genotype teahv-3 is considered one of the most relevant pathogens of chelonians, especially in t.  hermanni, because of its potential epizootic spread and mortality rates (origgi 2012, marenzoni et  al. 2018). teahv-3 is rarely associated with fatalities in t.  graeca or t. marginata, whereas it is often lethal in t. hermanni. additionally, surviving infected animals, although apparently clinically healthy, can harbor the virus and shed it following reactivation, from its dormant condition (latency), typical of herpesviruses. post-hibernation seems to be the period of major risk for reactivation (origgi et  al. 2015, marenzoni et  al. 2018). the consequences of the infection on the biodiversity could be considerable (marschang et al. 2009, martel et al. 2009). the phylum chlamydiae consists of intracellular bacteria, able to infect a wide range of animals and humans. the infection has been described both in free-ranging (mitura et al. 2017) and captive reptilian hosts, including tortoises (soldati et al. 2004, hotzel 27veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 marenzoni et al. microbiological investigation in italian testudo hermanni hermanni sampling) was applied and tortoises were sampled when found in the wild. rescued t.  hermanni included smuggled or confiscated animals housed in 10 different recovery centers, located in the eight different regions of italy, selected at national level because considered representative of other centers present in the country. the average number of animals housed in each center was approximately n  =  10, with two centers hosting much larger groups (>  100 animals); the management and the species of the hosted tortoises other than t. hermanni varied from center to center. all the animals were seized or confiscated in italy (however, based on the genetic characterization, some animals were later found to be non-italian, data not shown). information concerning for how long the tortoises had been kept in the centers before testing was not available. the os and cs samples were collected by rotating sterile polyester swabs, dipped in 500 μl of phosphate buffered saline (pbs, ph 7.2), against the mucosa of the mouth or the cloaca. all the swabs were stored at 4 °c in pbs until analysis, which was performed within 24-48 hrs. no repeated sampling was performed. biomolecular investigation total dna was extracted from 200 μl of each os using a commercial kit (qiaamp dna mini kit, qiagen, italy), according to the manufacturer’s instructions. a consensus nested pcr protocol was performed to amplify a 225 bp-long highly conserved region of the dna polymerase gene across the family herpesviridae to identify any possible herpesviruses present in the investigated tortoises (vandevanter et  al. 1996). a nested pcr was used to identify teahv-3 in case of positivity (marenzoni et al. 2018). a pcr protocol targeting the 16s rrna gene, discriminating between c. pneumoniae from c.  psittaci, was used to detect chlamydia  spp. (messmer et al. 1997). a two-step protocol based on conventional pcrs targeting a fragment of 273 bp and 1,013 bp of the gonzalez-candela et  al. 2005, sting et  al. 2013). isolates of salmonella can be used also as indicators of antibiotic-resistance because of its role and the capacity to respond to antibiotic treatments (european food safety authority and european centre for disease prevention and control 2018). the aim of the present study was to investigate the presence of teahvs, in particular teahv-3, chlamydia  spp., mycoplasma  spp., and salmonella  spp. in both free-ranging and rescued italian t.  h.  hermanni, monitored during a period of 3 years, to determine the infectious status of free-ranging tortoises and to elaborate appropriate health management guidelines for the rescued individuals to be relocated. materials and methods sample collection from april to october of the years 2014, 2015, and 2016, oral swabs (os) and cloacal swabs (cs) were individually collected from free-ranging and rescued t.  h.  hermanni. collection of os and cs was carried out only in individuals which could be sampled only with minimal restraint in compliance with animal welfare guidelines. accordingly, not all the tortoises (approximately 180 individuals) were systematically sampled for both samples. overall, 133 os (from 46 wild and 87 rescued tortoises) and 121 cs (from 84 wild and 37 rescued tortoises) were sampled. sampling type and year are described in table i. a complete clinical examination was performed for each animal at the time of sampling, including a general health assessment, inspection of visible mucous membranes, and recording of visible gross lesions. free-ranging tortoises were captured in eight different regions of insular and peninsular italy (emilia romagna, abruzzi, campania, molise, apulia, calabria, sardinia, sicily) across their distribution range. areas of sampling were identified during previous surveys carried out in the field, which are described in detail in mattm (mattm 2019). a non-probability sampling method (convenience table i. description of the type and year of sampling of free-ranging and rescued testudo hermanni hermanni. year oral swabs cloacal swabs free-ranging tortoises rescued tortoises total free-ranging tortoises rescued tortoises total 2014 22 57 79 31 27 58 2015 18 30 48 22 10 32 2016 6 6 31 31 total 46 87 133 84 37 121 28 veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 microbiological investigation in italian testudo hermanni hermanni marenzoni et al. statistical analysis the apparent prevalence of each microorganism detected in infected tortoises was defined as the proportion of positive samples (based on the applied test) out of the total number of samples analyzed. postulating the hypothetical target population as infinite, the number of animals sampled was used to estimate: 1) the maximum expected prevalence of infection with all the samples testing negative; and 2) the expected prevalence, with the corresponding 95% confidence interval (ci), if positive results were present. specific tables for sample size and calculations were used (cannon et  al. 1982, thrusfield 2005). the chi-squared test, with yates continuity correction, and fisher’s exact test were applied as appropriate to compare the proportions of positive samples based on wild or rescued origin and year of sampling for mycoplasma  spp. and salmonella  spp. a p value <  0.05 was considered statistically significant. odds ratios (ors) were calculated as a crude measure of the association between single risk factors and outcomes (isolation of salmonella  spp./ pcr positivity for mycoplasma spp.). openepi (open source epidemiologic statistics for public health, version 3.01., www.openepi.com) was used for the analysis. results none of the animal investigated showed detectable clinical signs. all os were negative for teahv and chlamydia spp. dna (0/133, 95% ci: 0.07-3.52%), consistently with an estimated prevalence lower than 2.23% for a hypothetical infinite population. mycoplasma spp. dna was detected with an estimated prevalence of 39.85% (53/133, 95% ci: 31.57-48.72%). results were reported in table  ii. prevalence was statistically different between rescued (52/87 os, 59.77%, 95% ci: 48.69-69.98%) and free-ranging tortoises (1/46 os, 2.17%, 95% ci: 0.11-12.97%, p < 0.0001), with rescued tortoises being 66.86 times (95% ci: 8.80-507.76) more likely to be positive. no differences in positivity were observed over years. test positivity was not associated with detectable clinical signs. thirty-three out of 53 (62.26%) mycoplasma spp. positive samples were typed as m. agassizii by the specific pcr-based enzymatic restriction. the only positivity to mycoplasma  spp. detected in a free-ranging tortoise was determined not to be m. agassizii. the sequences of the pcr products of both 273 bp and 1,013 bp, obtained from three randomly selected tortoises, shared 98-99% identity with the homologous 16s rrna gene sequences of m. agassizii (genbank accession nos. ky212528-ky212536, 16s rrna gene was used to detect mycoplasma spp. (vojdani and franco 1999, lierz et  al. 2007). in the case of positive samples, a pcr-based enzymatic restriction was used to confirm the presence of m. agassizii (brown et  al. 1995, brown et  al. 1999). moreover, pcr products of the expected size were purified using a commercial kit (qiaquick, qiagen, italy) and directly sequenced on both strands with the same primers as previously described (biofab srl, italy). the sequences were assembled and aligned using bioedit (bioedit 2009). the sequence similarity was checked against sequences available from genbank using the blast software (https://blast. ncbi.nlm.nih.gov/blast.cgi) to confirm the species specificity of the pcr. screening of salmonella serovars and antimicrobial susceptibility the cs were processed according to the procedures indicated by the world organization for animal health (oie) guidelines to isolate and identify salmonella  spp. and its serovars (oie 2015). briefly, each cs was used to inoculate the enrichment medium, rappaport-vassiliadis broth (oxoid, italy), and incubated at 41.5 °c for 24-48 hrs. the samples were then subcultured at 37 °c on xylose lysine desoxycholate agar (xld, oxoid, italy) and brilliant green agar (oxoid, italy) solid selective media. the results were read after 24 and 48 hrs of incubation. identification of suspect colonies was performed using composite biochemical media and the commercial biochemical test api rapid 20e (biomérieux, italy). salmonella  spp. isolates were serotyped by direct slide agglutination using specific antisera (statens serum institut, copenhagen, denmark), according to the kaufmann-white-le minor scheme. salmonella  spp. isolates were tested for antibiotic sensitivity using the standard disk diffusion method of kirby-bauer on mueller hinton agar (oxoid, italy). the following antimicrobial molecules (oxoid, italy) were tested: amoxicillin-clavulanic acid (30 μg), ampicillin (10 μg), nalidixic acid (30  μg), cephalothin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), enrofloxacin (5 μg), gentamicin (10 μg), kanamycin (30 μg), streptomycin (10  μg), sulfamethoxazole-trimethoprim (25 μg), sulfonamides (300 μg), and tetracycline (30 μg). the zone of inhibition around each disk was measured after 24 hrs incubation at 37 °c for each isolate. results were interpreted according to the clinical laboratory standards institute (clsi) guidelines (clsi 2016) and the isolates were classified either as susceptible, intermediate, or resistant. 29veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 marenzoni et al. microbiological investigation in italian testudo hermanni hermanni community for further studies at the regional reference center for pathogenic enterobacteria, istituto zooprofilattico dell’umbria e delle marche ‘togo rosati’. the distribution of salmonella  spp. isolates was significantly different according to the years of sampling, especially in  2016 (or for 2016 vs. 2014 = 2.75, 95% ci: 1.06-7.11, p = 0.03, and or for 2016 vs. 2015 = 3.12, 95% ci: 1.02-9.51, p = 0.03), whereas no statistical difference was found between free-ranging and rescued tortoises. discussion our results confirmed the presence of mycoplasma  spp. and salmonella  spp. in rescued nr025954, af060821). additionally, homologies were observed also with m. agassizii sequences detected previously in t. marginata, t. graeca, and t. hermanni in italy, although with limited coverage (different amplified intervals) (hq326165-hq326177, lecis et  al. 2011). the three sequences were submitted to genbank (accession numbers mf185252, mf185253, mf185254). salmonella spp. were isolated from 45 out of 121 cs (37.19%, 95% ci: 28.72-46.49%). the results of the isolation and typing of salmonella spp. based on year and group of tortoises are reported in table iii. the geographical origin of the isolates is reported in table iv. the isolates were collected in an official repository and have been made available to the scientific table ii. results of the pcr for mycoplasma spp. and mycoplasma agassizii in free-ranging and rescued testudo hermanni hermanni. year of sampling number of mycoplasma spp. positive total (%) number of mycoplasma agassizii* positive total (%)free-ranging tortoises (%) rescued tortoises (%) free-ranging tortoises (%) rescued tortoises (%) 2014 1/22 (4.54) 31/57 (54,38) 32/79 (40.5) 0/1 (0) 20/31 (64.5) 20/32 (62.5) 2015 0/18 (0) 21/30 (70) 21/48 (43.75) 0/0 (0) 13/21 (61.9) 13/21 (61.9) 2016 0/0 (0) ne 0/6 (0) 0/0 (0) ne 0/0 (0) total (%) 1/40 (2.5) 52/87(59.77) 53/133(39.84) 0/1 (0) 33/52 (63.46) 33/53 (62.26) ne = not executed; *pcr performed only on samples already positive for mycoplasma spp. table iii. year of sampling, number of tortoises positive for salmonella enterica subsp., apparent prevalence (pr), and list of the corresponding serovars of salmonella enterica isolated from free-ranging and rescued testudo hermanni hermanni. year of sampling number of positive free-ranging tortoises (pr) and isolated serovars number of positive rescued tortoises (pr) and isolated serovars total (pr) 2014 8 (8/31, 25.8%) 7 (7/27, 25.9%) 15 (15/58, 25.9%) abony (n = 1) abony (n = 5) hermannswerder (n = 1) miami (n = 1) langford (n = 5) newport (n = 1) wedding (n = 1) 2015 5 (5/22, 22.7%) 3 (3/10, 30%) 8 (8/32, 25%) abony (n = 1) hermannswerder (n = 2) hermannswerder (n = 1) lindern (n = 1) langford (n = 1) richmond (n = 1) salamae 17:b :enz15 (n = 1) 2016 22 (22/31, 71%) 22 (22/31, 71%) abony (n = 1) ferruch (n = 2) halle (n = 4), hermannswerder (n = 5) kottbus (n = 3) langford (n = 4) mikawasima (n = 1), newport (n = 1) zadar(n = 1) total 35 (35/84, 41.7%) 10 (10/37, 27%) 45 (45/121, 37.2%) 30 veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 microbiological investigation in italian testudo hermanni hermanni marenzoni et al. get infected with lethal outcome, often secondary to the introduction of clinically healthy carriers, including t. marginata or t. graeca (marschang et al. 1997, origgi and rigoni 2003, martel et  al. 2009, origgi 2012, marenzoni et  al. 2018). accordingly, avoiding the mixing of animals of different species and origins, as it can happen in a rescue center, is of critical importance; personnel of the rescue centers need to be educated in this direction. repeated tests and frequent health checks should be performed, in particular on rescued tortoises, in order to increase the probability of detecting the virus, which can reactivate in unpredictable manner (marenzoni et al. 2018). investigations for chlamydia  spp. suggest that chlamydiae are not a common pathogen in italian t.  hermanni. however, considering that novel chlamydia  strains have been detected (mitura et  al. 2017, laroucau et  al. 2020), the protocol used in the current study, which was designed to detect in particular c. pneumonia and c. psittaci, might not be broad enough to identify these new microorganisms. moreover, a limit of the study was that the investigations for chlamydia  spp. were restricted to oral swabs to detect respiratory disease, whereas these recent studies used successfully cloacal swabs, that maybe represent a better sample to improve the detection of chlamydia  spp. (mitura et  al. 2017, laroucau et al. 2020). detection of m. agassizii dna in rescued tortoises only, raises concerns for relocation programs. mycoplasmoses, especially those caused by m. agassizii and m.  testudineum, are considered to have contributed to the decline of some wild populations of tortoises in the usa (jacobson et  al. 2014). in this perspective, the clear difference found in the prevalence of mycoplasma spp. and m. agassizii among free-ranging and rescued animals suggests that m. agassizii is not a common component of the tortoise microbiome in the wild and its spread among free-ranging animals might have unpredictable consequences. accordingly, realease of positive animals in the wild is not recommended. a previous study reported the presence of novel mycoplasmas similar to the pathogenic m. agassizii in a group of t. graeca, t. marginata, and t.  hermanni, but it was limited to an italian recovery center; the infection was weakly associated with respiratory signs (six animals with respiratory signs, of which three were mycoplasma  pcr positive vs three mycoplasma  pcr negative) (lecis et al. 2011). further investigations are needed to determine the actual pathogenic potential of m. agassizii in t.  hermanni and the significance of this agent in t. hermanni disease ecology. the estimated prevalence of salmonella  spp. in tortoises of the present study was 37.19% (95% ci: 28.72-46.49%). however, it may have been underestimated because of the intermittent shedding of salmonella  spp., considering that italian t.  hermanni, as previously observed (lecis et al. 2011, pasmans et al. 2000, corrente et al. 2004, di ianni et  al. 2015, bertelloni et  al. 2016), and no molecular evidence of teahvs and chlamydia  spp. dna. on the contrary, it is the first description of mycoplasma spp. in free-ranging italian t. hermanni, although with a very low prevalence. as reported previously (lecis et  al. 2011), m. agassizii was detected only in rescued italian t. hermanni, but this is the first report documenting the lack of detection of m. agassizii in the free-ranging tortoises, raising biosecurity questions concerning the management of captive animals. the lack of detection of teahv dna by pcr confirmed that the tested free-ranging and rescued t.  hermanni were not shedders of teahvs. further serological investigations, not performed in this study, could be useful to complete the monitoring of the exposure to teahvs and eventually to exclude their circulation in italian free-ranging t.  h.  hermanni. in the context of a possibly naïve population of free-ranging italian tortoises, strategies to avoid virus circulation in captivity as well as the introduction of the virus into the wild must be implemented. t.  hermanni is notoriously poorly resistant to herpesvirus and highly prone to table iv. list of the serovars of salmonella enterica isolated in the present study, reporting the number of strains isolated and their geographical origin (region). serovar (n) geographical origin and number of isolates in freeranging tortoises geographical origin and number of isolates in rescued tortoises langford (10) apulia (5) calabria (2) calabria (1) calabria (1) molise (1) hermannswerder (9) apulia (5) emilia romagna (2) abruzzo (1), sicily (1) abony (8) apulia (2) sardinia (4) calabria (1) sicily (1) halle (4) campania (4) kottbus (3) campania (2) molise (1) ferruch (2) apulia (2) newport (2) apulia (1) sardinia (1) lindern (1) emilia romagna (1) miami (1) basilicata (1) mikawasima (1) molise (1) richmond (1) calabria (1) salamae, 17 :b :enz15 (1) calabria (1) wedding (1) apulia (1) zadar (1) calabria (1) 31veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 marenzoni et al. microbiological investigation in italian testudo hermanni hermanni all the salmonella  spp. isolated were completely susceptible to the antimicrobials tested, as commonly observed for salmonella  spp. isolated from wild animals (corrente et  al. 2004, percipalle et  al. 2011, rubini et  al. 2016). this supports that tortoises can be relocate into the wild with a very limited risk of introducing antimicrobial resistance into the environment through this route. the characterization of the antimicrobial susceptibility of the isolates could be a useful tool to monitor antibiotic-resistance in these animals and their environment. the results obtained with this study, that analyzed and compared the different prevalences in free-ranging and captive tortoises, contribute to formulate and update guidelines for proper sanitary management and relocation of rescued tortoises, placing particular emphasis of the risk of introduction of new microorganisms into the wild (mattm 2019). the different prevalence of relevant microorganisms (mycoplasma  spp. and m. agassizii) found between free-ranging and rescued animals and the absence of others (chlamydia  spp. and teahvs) suggest that monitoring of these infections is critical to prevent the spread of microorganisms in recovery centers and to preserve wild populations. in the future, the list of microorganisms to monitor should be extended to ranavirus, picornavirus, and ferlavirus, other known chelonian pathogens. the data regularly collected from this monitoring could be used to understand the relevance, the pathogenic potential, the host susceptibility to these infections, and draw a baseline to evaluate their possible evolution over time in these populations. only a single sampling was performed on each tortoise for practical reasons. a wide range of salmonella prevalence has been previously reported in chelonians, varying from 0%-100%, probably depending on several factors including host species, feeding habits, environment, management, location and the salmonella  serovars involved (pasmans et al. 2000, hidalgo-vila et al. 2007, lecis et al. 2011, percipalle et  al. 2011, pees et  al. 2013, marenzoni et al. 2015). the majority of the 14 serovars identified in the present study have been previously reported in italian testudo spp. (pasmans et al. 2000, corrente et  al. 2004, ebani et  al. 2005, percipalle et  al. 2011, dipineto et al. 2012, bertelloni et al. 2016, marenzoni et  al. 2015), and the presence of the same serovars has been confirmed in the 1970s (corsalini 1975) and in later studies (pasmans et  al. 2000, corrente et  al. 2004, ebani et al. 2005, percipalle et al. 2011, dipineto et al. 2012, bertelloni et al. 2016). statistical analysis showed that no difference exists in the prevalence of salmonella  spp. infection between free-ranging and rescued tortoises, whereas differences are observed across time, with 2016 being associated with a prevalence of 71%. many factors, that can stimulate differently a multiplication of bacteria, may have led to these results, such as temperature or environmental factors, or animal density, but they have not been considered herein as they were not relevant to the aim of the present work. other studies reported differences in detecting also further microorganisms in chelonians over different years (kabeya et  al. 2015, archer et  al. 2017), but the factors influencing these trends are generally difficult to identify. further studies are needed to better understand the predisposing conditions, possibly influencing these results. 32 veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 microbiological investigation in italian testudo hermanni hermanni marenzoni et al. archer g. a., phillips c. a., adamovicz l., band m., byrd j. & 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(barbieri f., doria g., & sindaco r. eds.). societas herpetologica italica. polistampa, firenze. messmer t.o., skelton s.k., moroney j.f., daugharty h. & fields b.s. 1997. application of a nested, multiplex pcr to psittacosis outbreaks. j clin microbiol, 35, 2043-2046. ministero dell'ambiente e della tutela del territorio e del 34 veterinaria italiana 2022, 58 (1), 25-34. doi: 10.12834/vetit.1915.13833.1 microbiological investigation in italian testudo hermanni hermanni marenzoni et al. detection of mycoplasma fermentans, m. hominis, and m. penetrans in patients with chronic fatigue syndrome, fibromyalgia, rheumatoid arthritis, and gulf war syndrome. j chronic fatigue syndrome, 5, 187-197. world organisation for animal health (oie). 2018. salmonellosis. http://www.oie.int/fileadmin/ h o m e / e n g / h e a l t h _ s t a n d a r d s / t a h m / 3 . 0 9 . 0 8 _ salmonellosis.pdf. epidemiology. 3rd edn. blackwell publishing, london, 228-246. vandevanter d.r., warrener p., bennett l., schultz e.r., coulter s., garber r.l. & rose t.m. 1996. detection and analysis of diverse herpesviral species by consensus primer pcr. j clin microbiol, 34, 1666-1671. vojdani a. & franco a.r. 1999. multiplex pcr for the 185 parole chiave escherichia coli, selvaggina, carne, namibia, stec. riassunto la carne di animali selvatici svolge un ruolo importante perché trasporta e trasmette i sierogruppi o157: h7 e non‑o157: h7 di shiga toxin‑producing e. coli (stec); la carne fresca di questi animali, inoltre, è una causa importante di infezioni da stec trasmesse da alimenti. scopo di questo studio è stato quello di valutare l'incidenza dei 6 maggiori ceppi stec non‑o157 (sierogruppi o26, o45, o103, o111, o121 e o145) in namibia con test per i geni shiga toxin (stx), intimina (eae) e geni specifici o‑group. da due macelli namibiani sono stati raccolti 75 campioni di carne di orice gazella (oryx gazella), 41 di springbok (antidorcas marsupialis), 5 di kudu grande (tragelaphus strepsiceros) e 5 di gnu (connochaetes taurinus) che sono stati testati per stec, utilizzando tecniche di real‑time pcr. in 94 dei 126 campioni testati è stata rilevata la presenza di stx e eae (74,6%). nei campioni positivi per stx e eae sono stati rilevati anche cinque dei maggiori 6 geni specifici del sierogruppo stec. i risultati di questo studio mostrano un'alta prevalenza di geni dei gruppi non‑o157 stec‑o nella carne di selvaggina della namibia e suggerisce la necessità di ulteriori controlli e analisi per evitare focolai di origine alimentare. prevalenza dei ceppi di escherichia coli non-o157:h7 producenti shigatossine (stec) nella carne di selvaggina in namibia keywords escherichia coli, game meat, namibia, non‑o157: h7 stec. summary large game animals play an important role as carriers and transmitters of o157:h7 and non‑o157:h7 shiga toxin‑producing escherichia coli (stec) in nature. fresh meat obtained from game animals has been identified as an important source of food‑borne stec infections. the aim of this study was to evaluate the incidence of the top 6 non‑o157 stec strains (serogroups o26, o45, o103, o111, o121, and o145) in namibian game meat based on testing for stx, eae, and o‑group‑specific genes. meat samples from gemsboks (oryx gazella) (n = 75), springboks (antidorcas marsupialis) (n = 41), greater kudus (tragelaphus strepsiceros) (n = 5), and wildebeests (connochaetes taurinus) (n = 5) were collected from 2 namibian abattoirs and tested for stec using real‑time pcr techniques. both shiga toxin (stx) and intimin (eae) virulence genes were detected in 94 out of 126 samples (74.6%). five of the top 6 stec serogroup‑specific genes were also detected in samples that were positive for both the stx and eae genes. the results of this study show a high incidence of non‑o157 stec o‑group genes in namibian game meat, which suggests that further scrutiny and testing may be necessary to avoid foodborne outbreaks. veterinaria italiana 2018, 54 (3), 185‑188. doi: 10.12834/vetit.1228.6844.3 accepted: 12.03.2018 | available on line: 30.09.2018 1 department of health sciences, namibia university of science and technology, windhoek, namibia. 2 biotechnology department, central veterinary laboratory, windhoek, namibia. 3 department of population health, school of veterinary medicine, faculty of agriculture and natural resources, university of namibia, neudamm campus, namibia. 4 department of pathobiology, school of veterinary medicine, faculty of agriculture and natural resources, university of namibia, neudamm campus, namibia. * corresponding author at: department of pathobiology , school of veterinary medicine, faculty of agriculture and natural resources, university of namibia, neudamm campus, namibia. tel.: 264 81 4635804, e‑mail: u.molini76@gmail.com. ndaindila haindongo1, johannes nkandi2, ndinomholo hamatui1, larai aku akai1, maria yvonne hemberger3, siegfried khaiseb2 and umberto molini2,4,* the prevalence of non-o157:h7 shiga toxin-producing escherichia coli (stec) in namibian game meat 186 veterinaria italiana 2018, 54 (3), 185‑188. doi: 10.12834/vetit.1228.6844.3 blesboks, elands, wildebeests, and kudus increased constantly. the aim of this study was to evaluate the prevalence of the top 6 non‑o157 stec strains in namibian game meat based on testing for stx, eae, and o‑group‑specific genes. materials and methods between may and july 2016, 126 samples of game meat were collected from 2 namibian abattoirs. the samples consisted of game meat from gemsboks (n  =  75), springboks (n = 41), greater kudus (n = 5), and wildebeest (n = 5). each sample of 325 ± 32.5 g of meat trim was homogenised with 975 ± 19.5  ml of pre‑warmed (42 °c) bax® system mp enrichment broth (dupont nutrition and health, wilmington, de) in whirlpack filter bags (nasco, fort atkinson, wi), mixed in a stomacher (seward laboratory systems, inc., bohemia, ny) for 2 minutes, and then incubated at 42 °c for 18 hours. twenty microlitres of enrichment broth were added to 200 μl of prepared bax® system lysis reagent in cluster tubes. lysis was performed by heating the tubes for 20 minutes at 37 °c and 10 minutes at 95 °c, and then cooling tubes at 4 °c for at least 5 minutes. thirty microlitres of lysate were used to hydrate tablets in polymerase chain reaction (pcr) tubes. pcr tubes were loaded into the bax® system q7 instrument, and a full process was run according to the procedure described in the bax® system user guide and analysed using the bax® system q7 software version 3.6. enrichment broths were screened for the stx (shiga toxin) and eae (intimin) genes using the bax® system real‑time pcr screening assay (du pont, wilmington, usa). only samples positive for both virulence genes were tested using the bax® real‑time pcr stec suite panel 1 (o26, o111, o121) and panel 2 (o45, o103, o145) to determine the presence of the top 6 non‑o157 stec serogroups. results among the 126 samples tested, the presence of both shiga toxin (stx) and intimin (eae) virulence genes was detected in 94 (74.6%) 95% confidence interval (ci) = 66.33‑81.39) (95% confidence interval (ci) 66.33‑81.39) samples (table i). of the 94 samples positive for both stx and eae, 1 (0.8%) (95% ci 0.19‑4.18) was positive for o45; 21 (16.7%) (95% ci 11.19‑24.16) for o103, 7 (5.6%) (95% ci 2.76‑11.03) for o121, 1 (0.8%) (95% ci 0.19‑4.18) for o145, 4 (3.2%) (95% ci 1.29‑7.87) for both o45 and o103, 19 (15.1%) (95% ci 9.89‑22.37) for o103 and o121, 3 (2.4%) (95% ci 0.86‑6.75) for o103 and o145, 4 (3.2%) (95% ci 1.29‑7.87) for both o121 and o145, 1 (0.8%) (95% ci 0.19‑4.18) for o26, o45 and o145, 1 (0.8%) (95% ci 0.19‑4.18) for o45, o121 and introduction shiga toxin‑producing escherichia coli (stec) are considered an important group of food‑borne zoonotic pathogens. they cause diarrhoea, hemorrhagic colitis (hc), and life‑threatening hemolytic uremic syndrome (hus) in humans (hussein 2007). e. coli o157:h7 is the stec strain usually associated with the most severe forms of disease (rivero et  al. 2010). more recently, it has become evident that non‑o157 stec, particularly stec serogroups o26, o45, o103, o111, o121, and o145 (referred to as the ‘top 6 non‑o157 stec’) cause illnesses similar to those caused by e. coli o157:h7 (gould et al. 2010). domestic ruminants, especially cattle, are considered to be the major reservoir of stec (karch et  al. 2005). large game animals are also recognised for playing an important role as carriers and transmitters of o157:h7 and non‑o157:h7 stec in the field (sanchez et  al. 2009). fresh meat obtained from game animals has been identified as an important source of food‑borne stec infections (magwedere et  al. 2013, rounds et  al. 2012). meat from wildlife ruminants containing stec strains has been found in belgium, germany, spain, usa, japan, and others countries (miko et al. 2009). game meat and its products are not currently subjected to any official regulation concerning microbiological contamination levels, and the data available concerning the microbiological quality of game meat for some pathogens are limited (díaz‑sánchez et al. 2012). problems related to traditional livestock farming, as well as the growth of the wildlife meat industry and the tourism sector in namibia have led to the proliferation of game‑farming units on private farmlands, including some in rural areas. between 16,000 to 26,000 tons of game meat are produced annually in namibian farmlands for regional and international export markets, local supply, and personal consumption. it is estimated that there are 2 million different food‑producing wildlife species other than fish and forest‑dwelling invertebrates in namibia (van schalkwyk et  al. 2010). the majority of namibian game meat from gemsboks (oryx gazella), greater kudus (tragelaphus strepsiceros), springboks (antidorcas marsupialis), and hartebeests (alcelaphus buselaphus) is exported to international markets as de‑boned meat (magwedere et  al. 2012). concurrent with the expansion of wildlife in namibia and a decline in domestic animal farming (particularly sheep and cattle due to a long period of drought), some export abattoirs have started to process game meat in their underutilised processing facilities during the wildlife hunting season. in the last years the demand from the international market for game meat, in particular for springboks, gemsboks, e.coli stec in namibian game meat haindongo et al. 187veterinaria italiana 2018, 54 (3), 185‑188. doi: 10.12834/vetit.1228.6844.3 moreover the detection of stx toxin gene in meat samples can be a strong indicator of the presence of stec in the meat. this, in turn, can be a threat for public health (scheutz et al. 2001). at present, little is known about the characteristics of stec strains other than o157 from wildlife meat (miko et al. 2009). stec strains were detected in game meat in belgium, usa, and germany with prevalence rates of 9‑14.8% (lehmann et  al. 2007, magwedere et  al. 2013). in the current study, the samples collected from 2 namibian abattoirs showed a prevalence rate of 74.6%, and, except for serogroup o111, all the top 6 non‑o157 stec serogroup‑specific genes were detected. the most commonly occurring serogroups were o103 and o121. the information obtained from this study shows a high prevalence of genes related to non‑o157 stec o‑groups in namibian game meat, suggesting that the meat could be contaminated by stec strains that can cause human illness. thus, changes in the way the animals are slaughtered and handled in the field and in the abattoirs are advisable. furthermore, regular scrutiny and testing of the meat for the top 6 non‑o157 stec strains may be necessary to avoid outbreaks of foodborne diseases. acknowledgements the authors wishes to thank dr. h.n. isaac and dr. g.k. marange for assisting with the sample collection. funding was provided by the ministry of agriculture, water and forestry in namibia. o145, and 7 (5.6%) (95% ci 2.76‑11.03) for o103, o121 and o145 (table ii). no samples tested positive for the o111 o‑group‑specific gene. a total of 32 springbok samples (78%) (95% ci 63.19‑87.95) tested positive for both stx and eae genes. out of 75 gemsbok samples, 52 (69.3%) (95% ci 58.13‑78.61) were contaminated: 1 sample tested positive for o45; 15 samples for o103; 1 sample for o145; 4 samples for both o45 and o103; 13 samples for both o103 and o121; 3 samples for both o103 and o145; and 1 sample exhibited the presence of o26, o45, and o145. for greater kudus, 5 samples were detected positive for the stx and eae virulence genes (100%) (95% ci 60.70‑100.00). of these, 2  samples tested positive for both o103 and o121, and 3 samples tested positive for o103, o121, and o145. five wildebeest samples tested positive (100%) (95% ci 60.70‑100.00) for o‑groups o103 (tables i and ii). discussion these results provide the first report of non‑o157:h7 shiga toxin‑producing escherichia coli genes in game meat from different species of wild animals in namibia using commercial bax® system assays. the bax® kits for non‑o157 stec have been evaluated by fratamico and colleagues (fratamico et  al. 2014) and the assays were shown to be highly specific for the stec serogroups. the sensitivity of assays for the different pcr targets was ≥ 1.23 × 103 cfu/ml using pure cultures. a previous study conducted on namibian springbok carcass samples using the pcr method described by paton and paton (paton and paton 1998) did not show the presence of stec (magwedere et al. 2013). the use of molecular methods that do not isolate stec strains or determine if all of the target genes (top 6 o‑group, eae and stx) are present in a single bacterium, is a limitation for the detection and confirmation of the presence of these pathogens. samples that tested positive for stec screening using pcr (stx/eae) methods and were positive for 1 of top 6 o‑group genes can only be reported as ‘potentially positive’ because stx and eae toxine can be present in bacteria other than the top 6 stec. haindongo et al. e.coli stec in namibian game meat table i. incidences of stx (shiga toxin) and eae (intimin) virulence genes in namibian game meat. sample origin number of samples collected positive samples (stx and eae) number percentage springbok 41 32 78% gemsbok 75 52 69.3% greater kudu 5 5 100% wildebeest 5 5 100% total samples 126 94 74.6% table ii. incidences of non-o157 shiga toxin-producing escherichia coli serogroups-specific genes in namibian game meat. ta rg et s p re se nt to ta l n um be r o f sa m pl es (% to ta l) sa m pl e or ig in sp rin gb ok ge m sb ok gr ea te r ku du w ild eb ee st o26 0 (0%) o45 1 (0.8%) 1 o103 21 (16.7%) 1 15 5 o121 7 (5.6 %) 7 o145 1 (0.8%) 1 o45, o103 4 (3.2%) 4 o103, o121 19 (15.1%) 4 13 2 o103, o145 3 (2.4%) 3 o121, o145 4 (3.2%) 4 o26, o45, o145 1 (0.8%) 1 o45, o121, o145 1 (0.8%) 1 o103, o121, o145 7 (5.6 %) 4 3 188 e.coli stec in namibian game meat haindongo et al. veterinaria italiana 2018, 54 (3), 185‑188. doi: 10.12834/vetit.1228.6844.3 díaz‑sánchez s., sánchez s., sánchez m., herrera‑león s., hanning i. & vidal d. 2012 detection and characterization of shiga toxin‑producing escherichia coli in game meat and ready‑to‑eat meat products. int j food microbiol, 160,179‑182. fratamico p.m., wasilenko j.l., garman b., demarco d.r., varkey s., jensen m., rhoden k. & tice g. 2014. evaluation of a multiplex real‑time pcr method for detecting shiga toxin‑producing escherichia coli in beef and comparison to the u.s. department of agriculture food safety and inspection service microbiology laboratory guide book method. j food prot, 77, 180‑188. gould l.h., mody r.k., ong k.l., clogher p., cronquist a.b., garman k.n., lathrop s., medus c., spina n.l., webb t.h., white p.l., wymore k., gierke r.e., mahon b.e. & griffin p.m. 2010. increased recognition of non‑o157 shiga toxin‑producing escherichia coli infections in the united states during 2000‑2010: epidemiologic features and comparison with e. coli o157 infections. foodborne pathog dis, 10, 453‑460. hussein h.s. 2007. prevalence and pathogenicity of shiga toxin‑producing escherichia coli in beef cattle and their products. j anim sci, 85, 63‑72. karch h., tarr p.i. & bielaszewska m. 2005. enterohaemorrhagic escherichia coli in human medicine. int j med microbiol, 295, 405‑418. lehmann s., timm m., steinruck h. & gallien p. 2006. detection of stec in faecal samples of free‑ranging wild and in wild meat samles. fleischwirtschaft, 4, 93‑96. magwedere k., hemberger m.y., hoffman l.c. & dziva f. 2012. zoonoses: a potential obstacle to the growing wildlife industry of namibia. infect ecol epidemiol, 2, 18365. magwedere k., dang h.a., mills e.w., cutter c.n., roberts e.l. & debroy c. 2013. incidence of shiga toxin‑producing escherichia coli strains in beef, pork, chicken, deer, boar, bison, and rabbit retail meat. j vet diagn invest, 25, 254‑258. references magwedere k., shilangale r., mbulu r.s., hemberger y., hoffman l.c. & dziva f. 2013. microbiological quality and potential public health risks of export meat from springbok (antidorcas marsupialis) in namibia. meat sci, 93, 73‑78. miko a., pries k., haby s., steege k., albrecht n., krause g. & beutin l. 2009. assessment of shiga toxin‑producing escherichia coli isolates from wildlife meat as potential pathogens for humans. appl environ microbiol, 75, 6462‑6470. paton a.w. & paton j.c. 1998. detection and characterization of shiga toxigenic escherichia coli by using multiplex pcr assays for stx1, stx2, eae, enterohemorrhagic e. coli hlya, rfbo111, and rfbo157. j clin microbiol, 36, 598‑602. sanchez s., garcia‑sanchez a., martinez r., blanco j., blanco j.e., blanco m., dahbi g., mora a., hermoso de mendoza j., alonso j.m. & rey j. 2009. detection and characterisation of shiga toxin‑producing escherichia coli other than escherichia coli o157:h7 in wild ruminants. vet j, 180, 384‑388. scheutz f., beutin l. & smith h.r. 2001. clinical detection of verocytotoxin‑producing e. coli (vtec). in verocytotoxigenic, e. coli. (g. duffy, p. garvey & d.a. mcdowell, eds). food and nutrition press inc, trumbull, connecticut, 25‑56. rivero m.a., passucci j.a., rodriguez e.m. & parma a.e. 2010. role and clinical course of verotoxigenic escherichia coli infections in childhood acute diarrhoea in argentina. j med microbiol, 59, 345‑352. rounds j.m., rigdon c.e., muhl l.j., forstner m., danzeisen g.t. & koziol b.s. 2012. non‑o157 shiga toxin‑producing escherichia coli associated with venison. emerg infect dis, 18, 279‑282. van schalkwyk d.l. mcmillin k.w., witthuhn r.c. & hoffman l.c. 2010. the contribution of wildlife to sustainable natural resource utilization in namibia: a review. sustainability, 2, 3479‑3499. 305 1 umbria regional centre of veterinary pharmacovigilance, istituto zooprofilattico sperimentale dell’umbria e delle marche, via g. salvemini 1, 06126 perugia, italy. 2 igiene degli allevamenti e produzioni zootecniche, usl umbria 1, perugia, italy. 3 igiene degli allevamenti e produzioni zootecniche, usl umbria 2, terni, italy. 4 istituto zooprofilattico sperimentale dell'umbria e delle marche via g. salvemini 1, 06126 perugia, italy. * corresponding author at: umbria regional centre of veterinary pharmacovigilance, istituto zooprofilattico sperimentale dell’umbria e delle marche, via g. salvemini 1, 06126 perugia, italy. e-mail: f.scoppetta@izsum.it. parole chiave antimicrobici veterinari, farmaco-epidemiologia veterinaria monitoraggio, one health, restistenza antimicrobica, sanità pubblica veterinaria. riassunto la diffusione della resistenza antimicrobica e la presenza di residui nei prodotti di origine animale destinati al consumo umano possono essere conseguenze dell'uso degli antimicrobici in veterinaria. i dati sul consumo sono quindi molto richiesti. gli obiettivi di questo studio sono di stimare una dose definita giornaliera regionale (dddvet_umbria) per tutti gli antimicrobici prescritti in umbria nel 2014 e di analizzare le prescrizioni per bovini, suini, piccoli ruminanti, pollame, trote arcobaleno e cavalli destinati alla produzione alimentare. le specie più trattate sono state nel 2014 i suini, il pollame e il pesce (le trote), per i quali sono stati utilizzati prevalentemente beta-lattamici. gli antimicrobici di importanza critica sono stati prescritti oltre che per suini e pollame, anche per i bovini; la colistina è risultato essere l’antimicrobico più frequentemente usato nei suini e nel pollame. superando i limiti di altri approcci proposti, questo studio indirizza la comprensione del consumo di antimicrobici negli animali da produzione alimentare. i dati sono utili per quantificare il consumo antimicrobico, identificare le fattorie problematiche e sostenere un confronto tra diverse specie animali. valutazione farmaco-epidemiologica di prescrizioni antimicrobiche nell’italia centrale, umbria 2014 keywords veterinary antimicrobials, antimicrobial monitoring, one health, antibiotic resistance, veterinary pharmaco-epidemiology. summary veterinary antimicrobial use could lead to problems such as the spread of antimicrobial resistance or the presence of residues in animal-derived products for human consumption. related to this, data on drug consumption is in strong demand. the aims of this study are therefore to evaluate a regional defined daily dose (dddvet_umbria) for all of the antimicrobials prescribed in umbria during 2014 and to analyse prescriptions for cattle, swine, small ruminants, poultry, rainbow trout, and food-producing horses. consumption, prevalence, and intensity of use indicators are calculated. swine, poultry, and fish were the most treated species during 2014. beta-lactams were the most frequently consumed antimicrobials for these species. critically important antimicrobials were mostly prescribed for swine, poultry, and cattle. colistin was the most frequently used critically important antimicrobial to treat swine and poultry. this study helps to better understand antimicrobial consumption in food-producing animals by overcoming the limitations of other proposed approaches. our data are useful for quantifying antimicrobial consumption, identifying problematic farms, and supports a comparison among different animal species. results highlight that the critical sectors in drug consumption – where the highest use of antibiotics were found – are swine, poultry, and trout farms. fausto scoppetta1*, massimo chiovoloni2, guglielmo spernanzoni3, giovanni filippini4 and marinella capuccella1 pharmaco-epidemiological evaluation of veterinary antimicrobial prescriptions for cattle, swine, small ruminants, poultry, rainbow trout, and food-producing horses in umbria in 2014 veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 accepted: 16.07.2017 | available on line: 31.12.2018 306 veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 animal species, and human (ema 2015, ema/esvac 2016). defined daily doses (dddvet) for each active ingredient per animal species and administration route are set as a reference parameter for evaluating veterinary active ingredients consumption and their comparisons across animal species and farming systems (ema 2015, postma et  al. 2015, who 2013, ema/esvac 2016). this information provides a valuable basis for decisions relating to the reduction of misuse of veterinary drugs, especially antibiotics, and may therefore be of interest to relevant health authorities. however, the list of dddvet provided by the ema/esvac for antimicrobials is reserved for swine, broilers, and cattle; and there is limited information about the registration and use of the veterinary drug handbooks in different member states, including italy (ema/esvac 2016). nevertheless, this is an important step towards a standardised approach in quantifying antibiotics in europe. the extension of the list to include other animal species and all active ingredients in veterinary medicine, through the use of information gathered from all the european countries, could improve our knowledge of drug utilisation in veterinary medicine. the aim of this study is to begin addressing some of these gaps by providing data on antimicrobial consumption in cattle, swine, small ruminants, poultry, rainbow trout, and food-producing horses in umbria, according to the methods proposed by the ema/esvac (2016). data are provided by the veterinary prescriptions received by the umbrian public health authorities in 2014. materials and methods general analysis information about the total number of farms and livestock animals per species present in umbria during 2014 was obtained from a national database, which is available at www.vetinfo.sanita.it (bdn) (table i). the bdn does not include information on antibiotic prescriptions in veterinary medicine. data on antibiotic prescriptions were obtained from the hard copies of veterinary drug prescriptions stored by the veterinary public authorities responsible for veterinary drug control; these authorities receive veterinary prescription copies from drug vendors, a mandatory procedure under both european and national italian law. copies of veterinary drug prescriptions were collected on a monthly basis and transferred to microsoft excel and microsoft access. prescriptions were split by species. the prescriptions admitted in this study were those for cattle, swine, small ruminants, poultry (defined according to council directive 2005/94/ec), rabbits, rainbow trout (fish), and food-producing horses. damaged prescriptions, off-label antibiotic drugs registered for humans, introduction veterinary antimicrobials are some of the veterinary drugs used as tools to prevent, control, and treat infections. they also protect animal welfare and health, and improve growth and production. antibiotics must be used responsibly in order to guarantee public health by reducing the spread of antimicrobial resistance and the risk of residues in animal derived food products. (economou and gousia 2015). it is well known that any antibiotic administration both in animals and in humans could lead to the selection of resistant bacteria (silbergeld et  al. 2008); this has become an increasing problem worldwide for both veterinary and human medicine (wassenaar 2005). the european union is working towards the spread of antimicrobial resistance reduction by promoting regulations and guidelines. in 2006, the use of antibiotics for growth promotion was banned (european commission 2003). recently, a statement regarding the guidelines on the prudent use of antimicrobials in veterinary medicine was issued by the european commission; it identified the need to monitor plans for various types of resistance in each production chain, as well as for pathogens, zoonotic and commensal bacteria, and for antibiotic consumption (european commission 2015). knowledge of both aspects is fundamental in assessing the relationship between resistance and the use of antimicrobials (van rennings et  al. 2015). the directive 2003/99/ec makes it mandatory to monitor antimicrobial resistance in zoonotic bacteria; furthermore, european or national plans have been implemented for commensal and pathogen germs (ministry of health 2012, efsa 2016). to date, no obligation regarding coordinated data-collection in europe (van rennings et  al. 2015) has been defined, although some countries have recently developed their own strategies (van rennings et al. 2015, aures 2013, svarm 2012, merle et  al. 2014, danmap 2014, maran 2015). since 2010, the european medicines agency launched the esvac (european surveillance of veterinary antimicrobial consumption) project, which focuses on antibiotics. specifically, the esvac initiative relates to ‘collecting and developing a coordinated approach for the collection and reporting of data on the use of antimicrobial agents in animals from the european union and european economic area member states’ (ema/esvac 2015). the ema/esvac group has provided a guideline on the standardisation of data collection and evaluation of veterinary drug consumption (ema 2015) to overcome limits shown by other proposed units of measurements (merle et  al. 2014, ema/esvac 2016). this guideline proposes ‘standardised fixed units of measurement for the reporting of data on consumption by species that take into account differences in dosing’ in order to compare consumption data between countries, veterinary antimicrobial prescriptions in umbria scoppetta et al. 307veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 • intra-mammary administration for treatment during lactation (cattle and small ruminants): the number of tubes per teat daily administered. • intra-mammary administration for dry therapy (cattle and small ruminants): the number of tubes per udder daily administered. • intrauterine administration: mg a.i./animal. dddvet_umbria was calculated for each a.i. as the average value of recommended dosage reported in the various leaflets about each prescribed drug. for those a.is. for which european medecines agency (ema) has provided, dddvet values for oral and parenteral administration for cattle, swine, and broilers, a ratio between dddvet_umbria and dddvet was calculated. prescribed ddds were evaluated by dividing the total amount of each a.i. in mg by dddvet_umbria of this a.i. consumption analysis the following indicators of antibiotic consumption were evaluated: • defined daily doses (ddd) per 1,000 animals per day: the mean number of doses consumed every day by 1000 animals (ddds/1,000 animals-die). • ddds per 1000 farms per day: the mean number of doses consumed every day by 1,000 farms (ddds/1,000 farms-die). • prevalence of use: the number of drug users (farms) divided by the overall farms present in the umbria region in 2014 (%). • intensity of use: the number of prescribed ddds divided by the number of farms with at least 1 antibiotic prescription in 2014 (ddds/ farms). analysis was performed for each productive sector. for food-producing horses and rabbits, no information was available in bdn regarding the number of animals and number of farms, while for rainbow trouts (fish), the only available information was related to farms. therefore, for food-producing horses and rabbits, only prescribed ddds were evaluated, while for rainbow trout (fish) only ddds/1,000 farms-die. results general consumption analysis the total number of prescriptions that were analysed was 10,051, which corresponded to 23,146 antibiotic records. partial or incomplete records accounted and antibiotic drugs with topical administration for which it was impossible to evaluate a dddvet_ umbria standard, were excluded from the study. for each prescription, the following information was transferred to the database: commercial name, posology, number and productive category of treated animals, days of treatment, and withdrawal periods. the active ingredient (a.i.) contained in each prescribed drug was considered a single record. analysis was performed taking into consideration each class of antibiotic and the sub-group ‘critically importance antimicrobials’ (cias), according to the world health organization (who 2011). dddvet_umbria determination and prescribed ddds evaluation dddvet_umbria values were calculated according to the ema indication (ema 2015) for each a.i., and differentiated by animal species. different dddvet_umbria values were evaluated for each administration route and for those a.is. prescribed in synergistic combination with other a.is. this means that each a.i. could have 2 or more different dddvet_ umbria values according to different animal species usage, with a different administration route, and/or in a synergistic combination with other a.i. utilisation. a database with all the prescribed drugs in 2014 in umbria was created by collecting information about the recommended dose according to each respective leaflet (including premixes for medicated feed) available from the ministry of health veterinary drug manual1. each prescription drug entry was expressed differently for each administration route, in-line with (ema/esvac 2016): • parenteral and oral route of administration: mg a.i. /kg body weight. scoppetta et al. veterinary antimicrobial prescriptions in umbria 1 https://www.vetinfo.sanita.it/j6_prontuario/public/. table i. number of farmed animals and farms in umbria in 2014. umbria region cattle farmed animals 56,694 farms 3,088 swine farmed animals 212,105 farms 3,361 small ruminants farmed animals 132,437 farms 3,510 poultry farmed animals 7,774,832 farms 261 fishes (trouts) farmed animals farms 15 total farmed animals 8,176,068 farms 10,235 308 veterinary antimicrobial prescriptions in umbria scoppetta et al. veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 was for oral administration (44.21%), followed by parenteral (40%), intra-mammary (12.63%), and intrauterine (3.16%). a comparison between dddvet_umbria and dddvet provided by ema was performed for 118  a.is. (22 for broilers, 54 for swine, and 42 for cattle). the average value of dddvet_umbria/dddvet was 1.05 (0.65 for cattle, 1.007 for poultry and 1.55 for swine). swine were the most treated animal species in umbria in 2014 (11,930.02  x  105 prescribed ddds), followed by poultry (2,853.64  x  105 prescribed ddds), cattle (275.11  x  105 prescribed ddds), rabbits (188.90 x 105 prescribed ddds), aquaculture (188.28  x  105 prescribed ddds), small ruminants (24.26  x  105 prescribed ddds), and food-producing horses (1.48 x 105 prescribed ddds) (figure 1). the results on antimicrobial consumption using for 2.47% of the total and off-label records accounted for 10.61% of the total. the number of antimicrobial records was 14,249, of which 3339 were for cias. poultry – especially game – showed the highest number of off-label records (58.35%), followed by rabbits (10.57%), swine and small ruminants (9.94%), rainbow trout (4.02%), food-producing horses (3.59%), and cattle (3.59%). the majority of prescriptions were mostly for swine (37.11%) and cattle (24.45%), followed by rabbits (17.48%), poultry (13.03%), small ruminants (6.85%), food-producing horses (0.60%), and rainbow trout (0.49%). regarding rabbits, 98.08% of evaluated prescriptions were for animals bred for home consumption. antimicrobials were the most prescribed type of drugs in 2014 (100% of prescribed records in rainbow trout, 66% in cattle, 63.72% in swine, 57.37% in rabbits, 50% in poultry, 47.17% in small ruminants, and 44.50% in food-producing horses). a total of 285 dddvet_umbria values were evaluated in this study. values referred to 65 a.is. classified in 16 antimicrobial classes2 and differentiated by animal species, administration route, and synergistic combinations. the list of a.is. classified by classes of drugs is shown in table ii. cattle (33.33%), swine (23.16%), and poultry (20.70%) showed the highest number of evaluated dddvet_umbria values; followed by rabbits (9.47%), small ruminants (8.42%), food-producing horses (3.16%), and fish (1.75%). the largest number of evaluated dddvet_umbria values 2 lists of dddvet_umbria values are available from the authors. table ii. classification of antimicrobials prescribed in 2014 in umbria. classes active ingredients aminoglycosides apramycin, amikacin, dihydrostreptomycin, streptomycin, gentamicin, kanamycin, framycetin, nemoycin, spectinomycin, paromomycin amphenicols thiamphenicol, florfenicol cephalosporins, first generation cefacetrile, cefopirin, cefazolin, cefalonium, cefalexin cephalosporins, third generation* cefoperazone, ceftiofur cephalosporins, fourth generation* cefquinome ionophore antimicrobials monensin lincosamides lincomycin macrolides* erithromycin, spiramicin, tilmicosin, tylvalosin, tulathromycin, tylosin, tildipirosin, gamithromycin nitrofurans furaltadone penicillin amoxicillin, amoxicillin + enzime inhibitor,penethamate hydriodide, phenoxymethylpenicillin, cloxacillin, dicloxacillin, benzylpenicillin, procaine penzylpenicillin, benethamine penicillin, ampicillin pleuromutilin tiamulin, valnemulin polymyxins and polipeptidic antimicrobials* colistin, bacitracin quinolones/fluoroquinolones* enrofloxacin, flumequine, oxolinc acid, danofloxacin, marbofloxacin rifaximins rifaximin sulfonamides and trimethoprim sulfadiazine, sulfadimidine, sulfadimethoxine, sulfamethoxazole, sulfamonomethoxine, phthalylsulfathiazole, sulfathiazole, sulfamethoxypyridazine, sulfamerazine, sulfaguanidine, trimethoprim tetracyclines oxytetracycline, chlortetracycline, doxycycline * critically important antimicrobials. figure 1. antimicrobials: prescribed defined daily doses (% per animal species). 309 scoppetta et al. veterinary antimicrobial prescriptions in umbria veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 quinolones/fluoroquinolones and polymixins showed a moderate utilisation for cattle. a different type of behaviour could be observed in the consumption of cias per farm (figure 2b). in this case, poultry showed the highest consumption of cias, particularly colistin, which is extensively prescribed for swine farms as well. macrolides and quinolones/fluoroquinolones were also widely prescribed for poultry; a high use of macrolides was also indicated for swine. for both, animals and farms, the lowest use of cias was found in small ruminants and for these species, colistin was the most widely prescribed cias. figure 3 shows prevalence (%) and intensity of use in reference to cias. poultry showed the highest farm prevalence, especially for macrolides, quinolones/fluoroquinolones, and polymixins. polymixins (essentially colistin) showed the highest intensity of use on farms, particularly on swine and poultry farms, followed by macrolides in swine, and quinolones/fluoroquinolones in poultry. antimicrobial consumption per animal species data on antibiotic consumption analysis per animal species are shown in table iii, table iv, and table v. ddds/1,000 animals-die confirms swine as the most treated farm animal in 2014 (15.41 x 103 ddds/1,000 animals-die), followed by cattle, poultry, and small ruminants (1.33  x  103; 0.10  x  103; 0.05  x  103 ddds/1,000 animals-die, respectively). poultry and fish farms instead showed a high consumption of antimicrobials (fish: 3,329.35  x  103; poultry: 2,995.47 x 103; swine: 972.48 x 103; cattle: 24.41 x 103; small ruminants: 1.89 x 103 ddds/1,000 farms-die). data showing consumption, prevalence, and intensity of use for each animal species are found in tables iii, iv, and v. cias were prescribed mostly for swine, poultry, and cattle (table iii, table iv, figure 2, and figure 3). in prescribed ddds, the highest percentage of cias was for swine (72.23%), followed by poultry (25.18%), cattle (1.82%), rabbits (0.73%), small ruminants (0.02%), rainbow trout (0.02%), and food-producing horses (0.001%). a large number of prescriptions for polymixins (essentially colistin) and macrolides was found in swine, which relates to the number of farmed animals (figure 2a). macrolides were also prescribed for cattle. however, in this species, third-generation cephalosporines was the most used class of cias. table iii. consumption of veterinary antimicrobials in umbria in 2014 in food-producing animals. cattle swine poultry small ruminants fishes (trout) class ddds/1000 animals‑die ddds/1000 farms‑die (x 102) ddds/1000 animals‑die ddds/1000 farms‑die (x 102) ddds/1000 animals‑die ddds/1000 farms‑die (x 102) ddds/1000 animals‑die ddds/1000 farms‑die (x 102) ddds/1000 farms‑die (x 102) aminoglycosides 148.36 27.24 290.87 183.56 0.26 76.26 6.12 2.31 0 monensin 143.47 26.34 0 0 0 0 0 0 0 first generation cephalosporins* 0.11 0.02 0 0 0 0 0 0 0 thrid generation cephalosporins* 201.23 36.94 19.58 12.36 0 0 0 0 0 fourth generation cephalosporins* 15.77 2.90 4.76 3 0 0 0 0 0 quinolones/ fluoroquinolones* 83.09 15.26 98.05 61.88 5.71 1,697.06 0.88 0.33 850 amphenicols 14.90 2.74 559.26 352.93 0.04 11.64 0 0 13,014.55 beta-lactamase inhibitors 2.98 0.55 45.33 28.60 0 0 0 0 0 lincosamides 34.87 6.40 3122.88 1,970.78 0.03 7.44 0.02 0.01 0 nitrofurans 0 0 0 0 0 0.91 0 0 0 macrolides* 105.52 19.37 1,425.45 899.57 6.51 1,935.98 1.02 0.38 0 penicillin 287.62 52.81 3,972.34 2,506.85 38.50 11,446.70 8.60 3.24 0 pleuromutilins 0 0 340.77 215.05 0.10 29.06 0 0 0 polymixins* 42.42 7.79 3,205.10 2,022.66 32.98 9,805.99 3.21 1.21 0 rifaximin 0.07 0.01 0 0 0 0 2.61 0.98 0 sulfonamides 99.20 18.21 994.92 627.87 5.13 1,525.68 0.60 0.23 71,355.70 tetraciclines 94.14 17.28 1,330.51 839.66 7.47 2,222.55 26.93 10.16 26,181.82 trimethoprim 55.72 10.23 792.27 499.98 4.09 1,216.35 0.21 0.08 70,880 * critically important antimicrobials. 310 veterinary antimicrobial prescriptions in umbria scoppetta et al. veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 compared to penicillin. a different kind of behaviour was discovered by evaluating the intensity of use, in that the highest value was shown by monensin, followed by third generation cephalosporins, tetracyclines, and macrolides. penicillin, polymixins, lincosamides, and macrolides were the most prescribed classes of drugs for swine both at animal and farm level. both prevalence and intensity of use were high in the same classes of drugs, particularly regarding the prevalence of penicillin, lincosamides, and macrolides. aminoglycosides have shown a prevalence value higher than other classes of drugs despite a low consumption expressed for both ddds/1,000 animals-die and ddds/1,000 farms-die. polymixins showed the highest value of ddds/farms, followed by lincosamides and tetracyclines. antibiotic consumption was principally related to penicillin and polymixins, both for single animals and single farms in poultry. other classes of antimicrobials showed values of ddds/1,000 animals-die and ddds/1,000 farms-die far lower than penicillin and polymixins. macrolides, penicillin, and quinolones/fluoroquinolones showed the highest values of prevalence. penicillin and polymixins with regard to antibiotics used for cattle, beta-lactams, and particularly penicillin either alone or in combinations, were the most prescribed classes of drugs and showed a high prevalence. aminoglycosides and quinolones/fluoroquinolones showed a higher prevalence than cephalosporines, despite its prevalence being generally lower when table iv. farm prevalence (%) and farm intensity of use [ddds(defined daily doses)/farms] of veterinary antimicrobials in umbria in 2014 in food-producing animals. cattle swine poultry small ruminants fishes (trout) class prevalence (%) ddds/ farms (x 103) prevalence (%) ddds/ farms (x 103) prevalence (%) ddds/ farms (x 103) prevalence (%) ddds/ farms (x 103) prevalence (%) ddds/ farms (x 103) aminoglycosides 11.89 8.37 4.91 136.48 8.43 727.80 1.94 4.35 0 0 monensin 0.45 212.07 0 0 0 0 0 0 0 0 first generation cephalosporins* 2.95 0.03 0 0 0 0 0 0 0 0 thrid generation cephalosporins* 2.40 56.27 0.51 89.17 0 0 0 0 0 0 fourth generation cephalosporins* 1.81 5.83 0.71 15.36 0 0 0 0 0 0 quinolones/ fluoroquinolones* 6.06 9.20 3.69 61.22 29.89 16,196.96 0.74 1.63 26.67 21.25 amphenicols 1.17 8.57 2.56 503.45 0.38 111.11 0.03 0.01 6.67 1,301.45 beta-lactamase inhibitors 1.85 1.08 0.66 159.50 0 0 0.03 0.01 0 0 lincosamides 2.79 8.39 4.55 1,580.18 2.68 71.00 0.03 1 0 0 nitrofurans 3.24 21.84 4.02 817.45 39.46 18,477.26 0 0 0 0 macrolides* 0 0 0 0 0.38 8.70 0.26 5.50 0 0 penicillin 21.76 8.86 11.01 824.48 30.27 109,248.92 2.76 4.29 0 0 pleuromutilins 0 0 2.26 347.13 1.53 277.34 0 0 0 0 polymixins* 1.65 17.21 3.12 2,363.17 22.61 93,589.77 0.48 9.12 0 0 rifaximin 2.33 0.02 0 0 0 0 0.06 63.08 0 0 sulfonamides 5.31 12.52 3.66 626.22 21.84 14,561.28 0.23 3.62 20 2,378.52 tetraciclines 2.9 22.65 3.60 851.29 26.05 21,212.30 3.13 11.83 33.33 523.64 trimethoprim 2.72 13.73 2.59 705.01 19.16 11,609.02 0.03 10 33.33 1,417.60 * cias table v. prescribed ddds (defined daily doses) in umbria in 2014 in horses and rabbits. prescribed ddds (x 103) horses (food producing) rabbits aminoglycosides 41.11 279.51 thrid generation cephalosporins* 3.82 0 quinolones/fluoroquinolones* 2.00 366.00 macrolides* 0 1,437.50 penicillin 54.09 0 pleuromutilins 0 3,795.57 polymixins* 0 3,375.55 sulfonamides 23.06 2,996.16 tetracyclines 3.94 3,995.94 trimethoprim 19.82 2,643.80 * critically important antimicrobials. 311 scoppetta et al. veterinary antimicrobial prescriptions in umbria veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 of antimicrobials in food-producing horses, while tetracyclines and pleuromutilins were mostly used in rabbits. third-generation cephalosporines were prescribed only for food-producing horses and were mostly prescribed for rabbits (polymixins, quinolones/fluoroquinolones and macrolides), as were other classes of cias. discussion the collection of veterinary drug use data and their descriptive analysis are important for public health (collineau et al. 2016). although the ema have recently provided dddvet values for antimicrobials for cattle, swine, and broiler, in this study we decided to use regional dddvet_umbria data for these species in order to facilitate a comparison between the animal species admitted in this study. comparing the values for swine, cattle, and broilers assigned by us, to the values assigned by the ema in 2016 (ema/esvac 2016) demonstrates the strength of this study. the showed the highest values of intensity of use. the most prescribed classes of antibiotics in small ruminants were tetracyclines, penicillin, and aminoglycosides. tetracyclines also showed the highest value of ddds/farms as referred to antimicrobials. tetracyclines and penicillin had the highest prevalence of use. sulfonamides, either alone or in combination with trimethoprim, were the most prescribed class of drugs in rainbow trout; however, tetracyclines also showed a high value of prevalence, together with trimethoprim. intensity of use was higher in sulfonamides, trimethoprim, and amphenicols. the last class of antibiotic was prescribed 100% off-labels. quinolones/fluoroquinolones were the only cias prescribed in fish and showed the lowest values of consumption and intensity of use together with a high prevalence. aminoglycosides, penicillin and potentiated sulphonamides were the most prescribed classes figure 2. focus on critically important antimicrobials. a. ddds (defined daily doses)/1000 animals-die; b. ddds/1000 farms-die. figure 3. focus on critically important antimicrobials. a. farm prevalence (%); b. intensity of use [ddds (defined daily doses]/farms). 312 veterinary antimicrobial prescriptions in umbria scoppetta et al. veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 most suitable unit of measurement to assess the relationship between antibiotic consumption and antibiotic resistance. this would be an appropriate follow-up study to the work presented here (collineau et al. 2016). beta-lactams, especially penicillin, were the most prescribed class of antimicrobials of all animal species monitored in 2014. this could be due to the lower cost of these drugs, short withdrawal periods (efsa 2016, ema/esvac 2015, de briyne et al. 2014), and broad spectrum of activity, which is sometimes obtained through the association with clavulanic acid. the use of broad-spectrum antibiotics is another critical area relating to the spread of antibiotic resistance, especially when these are prescribed without any laboratory indications about bacterium sensibility (european commission 2015). swine and cattle were the animal species with the highest number of prescriptions in 2014. together with small ruminants, these are the most bred species in umbria; however, small ruminants are treated less than other livestock, probably for economic reasons and types of farming (santman-berends et al. 2014). the analysis of prescribed ddds, ddds/1,000 animals-die and ddds/1,000 farms-die evaluated in 2014 in umbria, confirmed the high use of veterinary drugs in swine but showed a lower drug use in cattle compared to poultry and fish, especially where drug consumption concerns farms. with cattle, the low antibiotic consumptions, expressed in prescribed ddds, ddds/1,000 animals-die, and ddds/100 farms-die, could be explained by taking into account the fact that the units of measurement were influenced by the strength of each antimicrobial a.i. and in cattle, antibiotics with a high dosage were more often prescribed. swine, poultry, and aquaculture represent 3 crucial livestock industries referred to drug consumption because of group treatments, high use of medicated feed (in swine and rainbow trout), and rapid growth of animals, especially for poultry and swine. these aspects could lead to an underor over-estimation of live weight and consequently to mistakes in the drugs dosage calculation (timmerman et  al. 2006, gonzález et  al. 2010, mancini et  al. 2010, persoons et al. 2012, zonca and cagnardi 2012, di cesare et al. 2013, trauffler et  al. 2014). this problem could also influence the occurrence of antimicrobial resistance (catry et  al. 2003) in antibiotics in a positive sense. our results confirm the high use of antimicrobials in swine and poultry and the potential influence that these livestock species can have on the spread of antibiotic resistance. furthermore, swine and poultry showed a high consumption of cias, especially polymixins (colistin). other studies highlight the use of colistin in pigs and poultry as the main class of cias for curing diarrhea, especially in piglets (de briyne et  al. 2014, callens et  al. 2012). colistin differences could be attributed to the high number of registered products used by the ema for dddvet determination, which are based on the veterinary drug handbooks of nine countries, compared to our values, which are based on the registered products prescribed in only 1 italian region. it is well-known that there are differences in suggested doses provided by leaflets of commercial products which depend, for instance, on the severity and place of infection or age of the considered animals (timmerman et  al. 2006). the ema’s decision to evaluate dddvet using nine veterinary drug handbooks is an attempt to minimise the above-mentioned differences. this consideration is particularly important for cattle and swine, which have the highest number of registered products – about 3,000 for both species (https://www.vetinfo. sanita.it/j6_prontuario/public/), compared with other food-producing animals. furthermore, there are not many veterinarians working in umbria and, those who do work in the region often prescribe the same commercial products for different farms breeding the same animal species. there were few evaluated differences for poulty, which could be due to the lower number of registered products available for these animals – about 900 (https://www.vetinfo. sanita.it/j6_prontuario/public/). nevertheless, the dddvet_umbria/dddvet average value, which is 1.05, confirms the integrity of our approach. a comparison of the swine values was performed not only with the ema values but also with the ddd values assessed recently in four european countries (postma et al. 2015) and in denmark by the project danmap (danmap 2014). values for the oral administration of colistin and for the parenteral administration of ampicillin were assessed by postma and colleagues (postma et al. 2015) and were found to differ by only a few decimals from the same active ingredients assigned in our study. off-label prescriptions were mostly made for game, fish, and food-producing horses, and revealed a lack of registered veterinary products. the availability of veterinary drugs as ‘registered products’ should be increased in order to avoid problems such as mistakes in dosage and in withdrawal periods, as well as to gain a better understanding about the safety and efficacy of prescribed drugs for all bred animals. the analysis of antibiotic consumption is often evaluated by considering animal body weight. however, in this study we chose to relate antibiotic consumption to the number of farms and bred animals in umbria in 2014. this could facilitate an easier comparison with human medicine, where antibiotic consumption is expressed in ddds/1,000 inhabitants-die, which is similar to our units of measurement (ddds/1,000 animals-die and ddds/1,000 farms-die) (agenzia italiana farmaco 2016). furthermore, collineau and colleagues have recently indicated ddds/1,000 animals-die as the 313 scoppetta et al. veterinary antimicrobial prescriptions in umbria veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 should be used responsibly, in accordance with ema recommendations (ema 2013) in veterinary medicine. furthermore, the ema has recently provided information on the impact of colistin use in veterinary medicine on human medicine and antimicrobial resistance (ema 2016). data from this study could be useful to identify farms, veterinaries, and/or areas with a high consumption of cias, which in turn would be useful in guiding intervention efforts to encourage a more rational use of cias based on antibiogramme. another important class of cias, mostly used in swine and poultry in other countries in 2014, was macrolides (merle et  al. 2014, trauffler et  al. 2014, van rennings et al. 2015). this class of cias is usually used for the respiratory diseases and gastrointestinal diseases of swine (de briyne et  al. 2014), which represent the main pathologies for this species. even if macrolides were included in the cias listed by the who, because of the possibility of selecting macrolides-resistant campylobacter spp. (who 2011), their use could be preferable to colistin, which is now one of the last resources in human medicine for the treatment of different kinds of infections caused by multidrug-resistant bacteria (ema 2016). in the field of aquaculture in italy, our results confirm the high use of antimicrobials. in farmed fish, antimicrobials were the only class of drug used in umbria in 2014. this is in contrast with national and european policies, which tend to reduce antimicrobial use by increasing prevention practices such as vaccination (ministero della salute 2012, danmap 2014). one of the problems related to aquaculture is the small number of registered active ingredients; for example, neither anti-parasitic products nor florfenicol and erythromycin could be prescribed in italy in 2014. florfenicol and erythromycin can be prescribed only off-label, which is fundamental because they are elective drugs for some pathologies such as flavobacteriosis and lactococcosis (zonca and cagnardi 2012). however, it is necessary to consider that florfenicol was recently registered for farmed fish in order to guarantee animal health and production. in farmed fish, potentiated sulfonamides and tetracyclines were the most widely prescribed drugs in 2014 in umbria. these were administered by medicated feed, with possible impacts on the spread of antibiotic resistance and environmental pollution (zonca and cagnardi 2012, di cesare et al. 2013, lim et al. 2013). the same consideration could be made for quinolones/fluoroquinolones used in farmed fish in umbria, since they are classified as cias. thirdand fourth-generation cephalosporins were prescribed mostly for cattle and were probably principally related to the treatment of mastitis and/or dry-cow therapy, as has already been stated in other countries (lanza et  al. 2012, de briyne et  al. 2014, merle et  al. 2014). cephalosporins are frequently used during dry periods to prevent mastitis and this practice continues to be considered important for reducing the spread of this invalidating pathology in dairy animals (scoppetta et  al. 2016). dry-cow therapies could impact the spread of antibiotic resistance, although literature on this association is limited (rajala-schultz et al. 2009). this could represent a serious public health concern, considering that third and fourth generation cephalosporins are among the few available possible therapies for serious salmonella and e. coli infections in humans, especially children (who 2011). lack of information concerning horses in bdn and problems related to non-computerisation of veterinary prescriptions limited our evaluation to prescribed ddds only. although prescribed ddds in umbria for 2014 for food-producing horses were lower with respect to other animal species, the lack of indicators for consumption in this area lead to a lack of available data for assessing horse treatment impact on the spread of antimicrobial resistance (bowen and clegg 2015, weese 2015). the same considerations could be taken into account for rabbits, although, for this species, the most prescribed classes of antibiotics were tetracyclines and pleuromutilins, which are not considered cias. one of the biggest problems related to the quantification of drug use is linked to the differences among each national database referring to any single species. this includes a lack of information about the number of rabbit farms and the number of farmed rabbits or the number of farmed fish in each single aquaculture farm. moreover, different animal identification systems, which are more specific in cattle compared to other species – especially sheep or poultry, ensure a greater reliability of cattle bdn with respect to others. general descriptive studies regarding drug use are important as a reference point for planning further studies and strategies that address antibiotic resistance and improper drug usage. in our consideration of drug prescriptions, no information about the diagnosis stating whether antimicrobial susceptibility tests were used to select the antimicrobial of choice were reported. this made it difficult to assess the appropriateness of the prescribed therapy and its compliance with prudent principle usage (european commission 2015). conclusion the description of veterinary drug usage through a dddvet_umbria approach, allowed us to overcome the limits of the report of national sales of veterinary antimicrobials produced annually by the ema/ 314 veterinary antimicrobial prescriptions in umbria scoppetta et al. veterinaria italiana 2018, 54 (4), 305-315. doi: 10.12834/vetit.1174.6524.2 animals, and enables the identification of problem farms where controls could be carried out. our results highlight this critical area of drug consumption. moreover results of this study suggest that the spread of antibiotic resistance in veterinary medicine could include swine, poultry, and rainbow trout farms, where the highest use of antibiotics and, in particular, cias, was observed. this data could additionally be particularly useful in guiding the implementation of plans to reduce any irrational use of antibiotics and veterinary drugs, promoting prevention by 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parole chiave arbovirus, elisa, malattia di akabane, virus del sierogruppo simbu, test di neutralizzazione virale. riassunto i virus del sierogruppo simbu sono arbovirus che causano aborti, natimortalità o con malformazioni congenite. questo studio presenta i risultati di uno screening anticorpale dei virus del sierogruppo simbu in vacche da latte adulte nate prima di maggio 2012 e nelle giovenche nate dopo l'ottobre 2013; gli animali campionati in questo studio provenivano da tredici allevamenti di bovini da latte e da un gregge di pecore dislocati in cinque regioni in israele. i campioni di sieri positivi all'elisa sono stati ulteriormente testati con test specifici di neutralizzazione virale per i virus akabane, aino, sathuperi, shamonda e peaton. il rilevamento di anticorpi nelle vacche adulte in lattazione ha dimostrato che diversi virus del sierogruppo simbu hanno circolato in israele tra il 2008 e il 2014. le giovenche, inoltre, che hanno sieroconvertito nell'autunno 2014, testimoniano un'esposizione a più di un virus del sierogruppo simbu contemporaneamente. i risultati di questo studio fanno luce sulle infezioni da virus del sierogruppo simbu in israele e possono in generale contribuire ad una miglior comprensione dell'epidemiologia del sierogruppo simbu intorno al bacino del mediterraneo. virus del sierogruppo simbu in israele: prove sierologiche ne suggeriscono la circolazione keywords akabane disease, arbovirus, elisa, simbu group viruses, virus neutralization test. summary viruses of the simbu serogroup are arboviruses that are known to cause outbreaks of abortion, stillbirth and congenitally deformed neonates. this study presents the results of antibody screening of simbu serogroup viruses in heifers born in israel after october 2013, and in adult milking cows born before may 2012. thirteen dairy cattle farms in five regions, and one sheep flock, entered this study. serum samples that were found to be positive by elisa were further tested by specific virusneutralization test against a panel of simbu serogroup viruses including akabane, aino, sathuperi, shamonda, and peaton viruses. antibody detection in lactating adult cows revealed that several viruses were circulating in israel between 2008-2014. moreover, during autumn 2014 the heifers became serum-positive after being exposed to more than one simbu serogroup virus concurrently. the results of this study shed new light on simbu virus infections in israel, and may contribute to the epidemiology of the simbu serogroup around the mediterranean basin in general. veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 accepted: 12.03.2018 | available on line: 31.03.2019 1department of virology, kimron veterinary institute, bet dagan 50250, israel. 2parasitology, kimron veterinary institute, bet dagan 50250, israel. 3experimental dairy farm, volcani centre, bet dagan 50250, israel. 4kyushu research station, national institute of animal health, naro, 2702 chuzan, kagoshima 891-0105, japan. 5national institute of animal health, naro, kannodai, tsukuba, ibaraki 305-0856, japan. #these authors contributed equality to this manuscript. *corresponding author at: department of pathology, school of veterinary medicine, razi university, p.o. box: 67156-85414, kermanshah, iran. tel.: +972 50 6244374, e-mail: adib@moag.gov.il. jacob brenner1#, tohru yanase4#, tomoko kato4, shamai yaakobi3, evgeny khinich1, rita paz1, tomoyuki tsuda5 and adi behar2* serological evidence suggests that several simbu serogroup viruses circulated in israel 82 simbu serogroup viruses in israel brenner et al. veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 the aim of this study was to gain insight on the possible presence of simbu serogroup antibodies in israel. this work presents the results of antibody screening using specific virus neutralization test against a panel of five simbu group species with sera collected at the end of summer 2014, in selected regions in israel. materials and methods study design and sampling in israel, the summer and autumn are the main seasons of culicoides spp. activity, depend on the climatic conditions and the geographic locations (braverman and chechik 1996). this corresponds with viral infection of the fetus (its dam) as clinical appearance of a-h syndrome in israel usually occurs from december onward (in ovine) and around march-april (in bovine) (shimshony 1980, brenner et  al. 2004a, golender et  al. 2015, brenner et  al. 2016). moreover, the acute stage of bluetongue (bt) in sheep and epizootic hemorrhagic disease (ehd) in cattle begins in summer (yadin et al. 2009, brenner et al. 2010, brenner et al. 2011, kedmi et al. 2011, bumbarov et  al. 2012), also indicating high culicoides activity during this season. accordingly, serial serum sampling began in the first week of july until seroconversion was detected. introduction the simbu serogroup is one of the largest serogroups within the ortobuyavirus genus of the perbunyaviridae family, comprising at least 24 viruses antigenically differing, but serologically related viruses (kinney and calisher 1981, partsonson and mcphee 1985). simbu serogroup viruses are arthropod-borne, and have been primarily isolated from, or detected in, culicoides biting midges (lee 1979, st. george et  al. 1980, cybinski 1984, yanase et al. 2005, balenghien et al. 2014, kato et al. 2016a). akabane and aino viruses (akav and ainov) are the most studied among simbu viruses, and are known to cause outbreaks of abortion, stillbirth and congenitally deformed neonates, when susceptible pregnant ruminants are infected (markusfeld-nir and mayer 1971, kurogi et  al. 1975, inaba et  al. 1975, uchinuno et  al. 1988, noda et  al. 1998, brenner et  al. 2004a, tsuda et  al. 2004, brenner et  al. 2016). the recent emerging outbreaks of ruminant malformations in europe were clearly associated with the schmallenberg virus (sbv), a newly emerged simbu serogroup virus first identified in germany (hoffmann et  al. 2012, goller et al. 2012, wernike et al. 2014). other simbu serogroup viruses, including the peaton, shamonda, sathuperi and shuni viruses (peav, shav, satv and shuv), have also been implicated in the ruminant malformations (partsonson and mcphee 1985, yanase et  al. 2012, golender et  al. 2015). the malformations seen at birth, known as congenital arthrogryposis-hydranencephaly (a-h) syndrome, are correlated with the pregnancy stage at which the dam first contracts the infection (markusfeld-nir and mayer 1971, kurogi et al. 1975, inaba et al. 1975, brenner et  al. 2004a, tsuda et  al. 2004, bayrou et  al. 2014, brenner et  al. 2016). however, other clinical manifestations such as diarrhea, fever, reduced milk yield, and hypofertility (kono et  al. 2008, hoffmann et al. 2012, brenner et al. 2016) have also been reported in adult cattle. thus, the diseases arising from simbu serogroup virus infections have a significant adverse impact on animal health and welfare, the economy of livestock production, international trade, and movement of livestock worldwide (dominguez et  al. 2012, hoffmann et  al. 2012, wernike et al. 2014, martinelle et al. 2014). israel has been subjected to akav and ainov infections in the past. since 1969, there have been several outbreaks with clinical manifestations attributed to akav infections in ruminants (markusfeld and mayer 1971, shimshony 1980, brenner et al. 2004a, brenner et al. 2016). in november 2014, shuv (closely related to ainov) was isolated from the brains of congenitally malformed lambs with a-h syndrome (golender et al. 2015). 25 km sea of galilee dead sea gaza strip syria lebanon egypt west bank cluster #1 kfar rosh haniqra, beit haemeq, lochamei hagetaot cluster #2 heftzi ba, maoz haiim, tel-yoshep cluster #3 aielet hashachar, bei zera, ashdod yaakov cluster #4 zikim, karmia a�ected sheep farm bet dagan figure 1. locations of the serosurvey study on simbu serogroup viruses. 83 brenner et al. simbu serogroup viruses in israel veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 seroreactivity was assumed to have been positive in adult cattle before that date. accordingly, only heifers under the age of 10 months were tested. in addition, 5 serum samples from adult sheep were also included in this screening. the samples were collected from an infected sheep herd, where a-h syndrome had been observed, and shuv had been isolated (golender et al. 2015). serum samples from known exposed dams were collected to examine possible epidemiological links between the anticipated heifers’ seroconversion and the a-h  syndromes reported in this sheep flock and in cattle and goat herds in late 2014 and early 2015 (golender et al. 2015). screening samples for simbu serum‑reactivity by elisa all samples collected during the study were tested by elisa for the possible presence of simbu serogroup antibodies. the idexx schmallenberg ab test kit (3097 liebefeld-bern, switzerland), was used at the kvi in israel. the elisa procedure was carried out in accordance with the manufacturer’s instructions. since this elisa kit is based on the nucleocapsid protein of schmallenberg virus and detects antibodies against several simbu serogroup viruses, selected serum samples that tested positive by elisa were sent to kyushu research station, national institute of animal health, japan for specific virus neutralisation test (vnt) analysis. for this study, 13 dairy cattle farms in 5 regions in israel were selected. these farms are considered to be hotspots of culicoides activity, where akav (markusfeld and mayer 1971, brenner et  al. 2004a, brenner et al. 2016) and other culicoides-transmitted viruses have been isolated (brenner et  al. 2010, brenner et al. 2011, yadin et al. 2008, kedmi et al. 2011). the selected regions are three valleys in northern israel (zvulun valley cluster #1; jezreel valley cluster #2; sea of galilee cluster #3), each with 3 collective dairy farms. two farms were in the southern coastal plain (cluster #4), and one experimental dairy farm in bet dagan, in the central coastal plain where the kimron veterinary institute (kvi) is situated (figure 1). at each selected farm, 5 heifers and 10 milking cows were sampled (for a total of 75 and 140, respectively) at the beginning of the study. thereafter, only the 5  selected heifers were retested serologically every 2 to 3 weeks until seroconversion occurred. by analyzing the adult cow stock, we expected to gain anamnestic information about previous cumulative exposures prior to the sampling. conversely, the heifers, at an age of between 8 to 10 months, should have been serologically naïve in august/september 2014, and should serve as sentinels. therefore, we chose heifers which were born after october 2013. the main aim of the study was to find the etiology of the seroconversion in the selected heifers, if applicable. the bet dagan farm was tested because akav infection had been confirmed in it recently (2012) (brenner et  al. 2016). thus, simbu serogroup table i. total distribution of neutralizing antibody titers to each simbu serogroup virus for each sample that tested positive by elisa. — cont’d no. animal settlement (cluster #) antibody titer aino sathuperi akabane shamonda peaton 1 adult cows kfar rosh haniqra (cluster #1) 8 < 2 2 16 < 2 2 adult cows kfar rosh haniqra (cluster #1) 4 < 2 < 2 16 < 2 3 adult cows beit haemeq (cluster #1) 8 < 2 < 2 8 < 2 4 adult cows lochamei haghetaot (cluster #1) 8 < 2 < 2 8 < 2 5 adult cows lochamei haghetaot (cluster #1) 2 < 2 n/a n/a n/a 6 adult cows heftziba (cluster #2) 16 32 < 2 16 2 7 adult cows heftziba (cluster #2) 8 < 2 < 2 < 2 < 2 8 adult cows tel joseph (cluster #2) 2 16 < 2 4 < 2 9 adult cows tel joseph (cluster #2) < 2 < 2 < 2 8 < 2 10 adult cows maoz haim (cluster #2) 32 8 2 16 < 2 11 adult cows beit zera (cluster #3) 4 2 16 4 < 2 12 adult cows beit zera (cluster #3) 4 < 2 32 8 4 13 adult cows ashdot yaakov meuchad (cluster #3) 2 4 128 16 32 14 adult cows ayelet hashachar (cluster #3) 16 32 2 32 < 2 15 adult cows ayelet hashachar (cluster #3) 8 4 64 16 4 16 adult cows ziqim (cluster #4) > 256 128 64 32 n/a 17 adult cows ziqim (cluster #4) 64 16 32 8 < 2 18 adult cows karmia (cluster #4) 16 2 < 2 4 < 2 continued 84 simbu serogroup viruses in israel brenner et al. veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 table i. total distribution of neutralizing antibody titers to each simbu serogroup virus for each sample that tested positive by elisa. — cont’d no. animal settlement ( cluster #) antibody titer aino sathuperi akabane shamonda peaton 19 adult cows karmia (cluster #4) 64 16 16 4 32 20 heifer kfar rosh haniqra (cluster #1) 64 2 8 < 2 < 2 21 heifer kfar rosh haniqra (cluster #1) 32 8 < 2 < 2 < 2 22 heifer kfar rosh haniqra (cluster #1) 16 4 < 2 < 2 < 2 23 heifer lochamei haghetaot (cluster #1) 2 64 < 2 < 2 < 2 24 heifer lochamei haghetaot (cluster #1) 32 4 < 2 < 2 < 2 25 heifer lochamei haghetaot (cluster #1) 8 2 32 2 < 2 26 heifer beit haemeq (cluster #1) 16 4 < 2 < 2 < 2 27 heifer beit haemeq (cluster #1) 16 8 < 2 < 2 < 2 28 heifer beit haemeq (cluster #1) 128 < 2 < 2 < 2 < 2 29 heifer beit haemeq (cluster #1) 64 4 < 2 < 2 < 2 30 heifer tel joseph (cluster #2) < 2 < 2 < 2 < 2 < 2 31 heifer tel joseph (cluster #2) < 2 < 2 < 2 < 2 < 2 32 heifer heftziba (cluster #2) < 2 < 2 < 2 < 2 < 2 33 heifer heftziba (cluster #2) 32 2 < 2 < 2 < 2 34 heifer maoz haim (cluster #2) 16 2 < 2 < 2 < 2 35 heifer maoz haim (cluster #2) 64 2 < 2 < 2 < 2 36 heifer maoz haim (cluster #2) 32 4 < 2 < 2 < 2 37 heifer maoz haim (cluster #2) 64 2 < 2 < 2 < 2 38 heifer beit zera (cluster #3) 8 8 2 64 < 2 39 heifer beit zera (cluster #3) 64 16 2 32 < 2 40 heifer beit zera (cluster #3) 32 8 n/a n/a n/a 41 heifer ayelet hashachar (cluster #3) < 2 32 < 2 < 2 < 2 42 heifer ayelet hashachar (cluster #3) < 2 64 < 2 < 2 < 2 43 heifer ayelet hashachar (cluster #3) < 2 32 < 2 < 2 n/a 44 heifer ayelet hashachar (cluster #3) < 2 16 < 2 < 2 < 2 45 heifer ashdot yaakov meuchad (cluster #3) 2 < 2 < 2 32 < 2 46 heifer ashdot yaakov meuchad (cluster #3) 64 4 < 2 < 2 n/a 47 heifer ashdot yaakov meuchad (cluster #3) 16 2 < 2 < 2 < 2 48 heifer ashdot yaakov meuchad (cluster #3) 16 2 < 2 < 2 < 2 49 heifer ashdot yaakov meuchad (cluster #3) 2 < 2 < 2 32 < 2 50 heifer ziqim (cluster #4) 32 4 < 2 < 2 < 2 51 heifer ziqim (cluster #4) < 2 < 2 2 < 2 < 2 52 heifer ziqim (cluster #4) 32 16 16 4 < 2 53 heifer karmia (cluster #4) < 2 64 < 2 < 2 < 2 54 heifer karmia (cluster #4) < 2 64 < 2 < 2 < 2 55 heifer karmia (cluster #4) 32 4 < 2 < 2 < 2 56 heifer karmia (cluster #4) < 2 < 2 < 2 < 2 < 2 57 heifer beit dagan exp. farm < 2 < 2 < 2 < 2 < 2 58 heifer beit dagan exp. farm < 2 16 < 2 < 2 < 2 59 heifer beit dagan exp. farm 2 64 < 2 < 2 < 2 60 sheep yoqneam 16 2 16 32 < 2 61 sheep yoqneam 2 < 2 2 16 < 2 62 sheep yoqneam 8 8 < 2 4 < 2 63 sheep yoqneam 2 16 4 16 n/a 64 sheep yoqneam 2 32 64 < 2 < 2 85 brenner et al. simbu serogroup viruses in israel veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 responsible for serum reactivity found by elisa indicated that anti-ainov (8/10), anti-satv (1/8) and anti-akav (1/8) antibodies were detected in ruminants in this region during the seroconversion process (table 1). cluster #2 the 5 selected elisa-positive serum samples of adult animals (representing the jezreel valley region) suggested that ruminants in this region were previously exposed to ainov, satv and shav (table 1). in the 8 selected heifers only anti-ainov (5/8) antibodies were detected (table 1). three samples were negative by vnt despite being elisa-reactive. cluster #3 the 5 selected elisa-positive serum samples of adult animals (representing the regions around the sea of galilee) suggested that one or more ruminants in this region were previously exposed to all 5 tested viruses (e.i. ainov, satv, shav, akav, and peav) (table 1). in the 12 selected heifers anti-ainov (5/12), anti-satv(4/12) and anti-shav(3/12) antibodies were detected (table 1). cluster #4 the 4 selected elisa-positive serum samples of adult animals of cluster #4 representing the south coastal plain suggested that in this region ruminants were previously exposed to all 5 tested viruses (table 1). in the 7 selected heifers anti-ainov (3/7) and anti-satv (2/7) antibodies were detected. two samples were negative by vnt despite being elisa-reactive (table 1). the experimental farm at bet dagan in the 3 selected heifers only anti-satv (2/3) antibodies were detected (table 1). one sample was negative by vnt despite being elisa-reactive. infected sheep herd contrary to our expectations, in the 5 elisa-positive serum samples of the clinically affected sheep herd from which shuv had been isolated (golender et al. 2015), four of the five tested adult sheep reacted to at least 2 different simbu serogroup viruses (table 1, supplementary table 1). in one sheep only anti-shav antibodies were detected. specific virus neutralization tests a total of 64 serum samples, 59 cows (19 adults and 40 heifers) and 5 sheep were tested by specific vnts (table 1). the sera were subjected to vnts against 5 viruses from the simbu serogroup of the following respective strains: akav obe-1, ainov janar28, satv ksb-2/c/08, peav ksb-1/p/06, and shav ksb-6/c/02. after heat inactivation at 56°c for 30 min, the sera were serially diluted two-fold in serum-free eagle’s mem, containing 10 μg/ml gentamicin sulfate from 1:2-1:256 in the 96-well microplates. fifty microliters of serum dilution was mixed with an equal volume of virus inoculum containing 100 × the 50% tissue culture infective doses, and incubated at 37°c under 5% co 2 for 1 hour. then, 100 μl of the suspension of hmlu-1 cells in git medium (wako pure chemical industries, ltd., osaka, japan) was added to each well, and incubated at 37°c under 5% co 2 for 7 days. the antibody titer was calculated as the reciprocal of the highest serum dilution inhibiting the cytopathic effect. samples were deemed positive if they had neutralizing antibodies to the viruses at a dilution of at least 1:8 (kato et al. 2016b). results screening samples for simbu serum‑reactivity by elisa at time 0 (july 2014), all the milking cows tested positive by the elisa. in contrast, all the heifers from all tested sites tested negative by the elisa. subsequent elisa tests confirmed that seroconversion occurred in the heifers within less than 6 weeks of their first testing. simbu sero‑group virus neutralization tests antibodies against all of the five simbu serogroup viruses tested (ainov, akav, peav, satv and shav) were detected in one or more of the serum samples from adult cows, heifers, and sheep. their titer ranged from 1:8-1:256 (table 1, supplementary table 1). cluster #1 the 5 selected elisa-positive serum samples of adult animals (representing the anamnestic exposure to simbu serogroup viruses in the zvulun valley/ western galilee) suggested that one or more ruminants in this region were exposed to ainov and shav between 2008 and the sampling date (table 1). the 10 selected heifers’ serum samples which should have specified the simbu serogroup viruses 86 simbu serogroup viruses in israel brenner et al. veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 other viruses of the simbu serogroup (i.e. satv and shav) were probably also circulating in israel between 2008 and 2014. notably, antibodies to peav were detected in two adult cows. since cross reactivity between peav and sango virus (sanv) has been reported before (kinney and calisher 1981), our results might indicate for the first time, a remote circulation of either of these viruses in israel. while preparing this manuscript, peav genomic material was detected in a calf exhibiting micro-hydranencephaly, supporting the assumption that peav is currently (2017) circulating in israel (data not shown). similarly, the vnt results from the naïve heifers in the summer/autumn of 2014 also revealed concurrent seroconversion of mixed simbu serogroup viruses (mainly to ainov and satv, but also to shav and akav) in israel. shav has been shown to have cross reactivity with akav (kinney and calisher 1981). nonetheless, it is likely that each of these viruses was circulating in israel as there were both adult cows and heifers with high antibody titers to a single virus and very low or no antibodies to the other viruses (table 1). in addition, samples from known affected sheep of the flocks from which shuv had been isolated (golender et al. 2015) yielded vnt antibodies to sat, sha and aka viruses. consequently, this infected sheep herd provides indications that the israeli’ ruminants in 2014 were indeed exposed to more than one simbu serogroup viruses, concurrently or sequentially. the findings of this study are of epidemiological significance, not only because they shed new light on simbu serogroup virus infections in israel, but also because mixed viral infections may create the baseline conditions for viral re-assortment, as previously demonstrated by yanase and colleagues (yanase et  al. 2010, yanase et  al. 2012). the co-existence of several different simbu viruses in specific ‘pockets’ in israel may indicate that israel (and by extension the middle east) may have functioned as a hotspot for virus evolution and/ or re-assortment. similar re-assortment has been observed in the btv mixed infections. probably such processes were responsible to generate novel viruses leading to various clinical syndromes observed at infected sites in different geographical regions in israel (brenner et  al. 2010, brenner et  al. 2011). nevertheless, to further improve our understanding of the epidemiology of the simbu serogroup and arboviruses in general in israel, this work should be complemented by entomological studies, in parallel with sero-specific (namely, vnt) surveillance and genomic detection (pcr or virus isolation) in insect vectors and/or mammalian hosts. finally, from closer examination of the vnt results, it is fairly apparent that each individual cluster/location presented a distinct site-specific serum reactivity discussion the results of this study indicate that all tested ruminants had antibodies against one or more of the five simbu serogroup viruses tested by elisa and vnts. our results also show that the seroconversion detected in the naïve heifers by elisa during summer/autumn 2014 was the result of more than one simbu serogroup virus circulating simultaneously in israel. prior to 2012, only akav had been linked to a-h syndrome in israel (shimshony 1980, brenner et  al. 2016). our results reveal no evidence of akav in adult cows (2-8 years old) in two regions that have hitherto been considered akav ‘hotspots’. these results are in accordance with a previous study conducted in israel (kalmar et  al. 1975). moreover, there is little evidence of current infection with akav, as antibodies against akav rarely appeared in the seroconversion process of the selected heifers during summer/autumn 2014 (table 1). the unexpected absence of akav exposure at bet dagan since 2013 (table 1) also lends further support to this ‘anomalous’ finding. therefore, we postulate that akav is not the main simbu serogroup virus present in israel, as previously thought, but is only one of several etiological agents from this serogroup circulating in israel. even though vnts are considered to be the ‘gold standard’ for the assessment of other viruses, it is recognized that cross-reactivity between viruses belonging to the simbu serogroup should be expected and taken under consideration during the interpretation of such serological results. the differences between these viruses can be recognized to at least seven different species by specific serological tests such as cross-neutralization test and cross-heamagglutination-inhibition tests (plyusnin et  al. 2012). in addition, the close relationships between some of these viruses can also be revealed in their current taxonomic classification where the species sathuperi virus (satv) includes sathuperi, douglas and schmallenberg viruses; species shamonda (shav) virus includes shamonda, peaton (pea) and sango viruses; species akabane virus (akav) includes akabane, tinaroo and sabo viruses; species shuni virus (suhv) includes aino and shuni viruses and species simbu virus includes only simbu virus (goller et al. 2012). recognizing these limitations, five viruses representing different simbu species were chosen for this study. accordingly, several postulations are emerging from our data. the antibodies detected in the adult lactating cows indicate that besides akav, ainov and shuv, which have already been documented (brenner et  al. 2004a, golender et  al. 2015, brenner et  al. 2016), 87 brenner et al. simbu serogroup viruses in israel veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 conclusions this study shows for the first time, that israeli’ ruminants are exposed to a mixed infection of more than one simbu serogroup viruses, circulating in israel concurrently or sequentially. this is also the first serological indication of simbu serogroup viruses from the species shamonda and sathuperi in israel. due to potential cross reactivity, it is possible that viruses from the simbu serogroup, other than the ones included in the test, are also present. isolation and further genetic characterization of simbu serogroup viruses, including isolates from vectors and ruminants from different geographical regions, will be essential for understanding the molecular epidemiology and evolution of these viruses. acknowledgements we thank the teams of the veterinary services in the field, for sampling the cattle. profile (table 1). even locations that had been thought to belong to the same cluster, including those within a radius of only 5 to 10  kilometers, differed from each other. the mechanism behind the cyclical arbo-disease pattern, where a given simbu virus disappears from a certain location for relatively long periods, only to reappear at the same infected site several years later (brenner et al. 2004b), remains a mystery. it should be noted that mathew and colleagues (mathew et  al. 2015) published a similar study in tanzania, with similar methodology and conclusions. however, unlike tanzania, israel is small and crowded, with extensive agricultural areas and animal farms that create a very high density of ruminant population. therefore, the different distributions of simbu serogroup observed in this study, even within a radius of 5-10  km, may also indicate that ‘ecological pockets’ such as the ones chosen for our study indeed exist. investigating these ‘ecological pockets’ may improve our understanding of the arbo-spreading transboundary diseases. balenghien t., pagès n., goffredo m., carpenter s. et al., 2014. the emergence of schmallenberg virus across culicoides communities and ecosystems in europe. prev vet med, 116, 360-369. bayrou c., garigliany m.m., sarlet m., sartelet a., cassart d. & desmecht d. 2014. natural intrauterine infection with schmallenberg virus in malformed newborn calves. emerg infect dis, 20, 1327-1330. bravernam y. & chechik f. 1996. air streams and the introduction of animal disease born on culicoides (diptera, ceratopogonidae) into israel. rev sci tech oie, 15, 1037-1052. bumbarov v., brenner j., rotenberg, d., batten c., sharir b., gorohov a., golender n., shainin t., kanigswald g., asis i. & oura c. 2012. the presence and possible effects of bluetongue virus in goat flocks in israel. isr j vet med, 64, 237-244. brenner j., yadin h., chai d., stram y., kato t. & tsuda t. 2004a. serological and clinical evidence for reactivity of arboviral teratogenic simbu sero-group infection in israel: 2001/2003 episode(s). vet ital, 40, 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tsuda t. 2010. genetic characterization of aino and peaton virus field isolates reveals a genetic reassortment between these viruses in nature. virus res, 153, 1-7. yanase t., kato t., aizawa m., shuto y., shirafuji h., yamakawa m. & tsuda t. 2012. genetic reassortment between sathuperi and shamonda viruses of the genus orthobunyavirus in nature: implications for their genetic relationship to schmallenberg virus. arch viro, 157, 1611-1616. kalmar e., peleg b.a. & savir d. 1975. arthrogryposis hydranencephaly syndrome in newborn cattle, sheep and goats. serological survey for antibodies against the akabane virus. refuah vet, 32, 47-54. kato t., shirafuji h., tanaka s., sato m., yamakawa m., tsuda t. & yanase t. 2016a. bovine arboviruses in culicoides biting midges and sentinel cattle in southern japan from 2003 to 2013. transbound emerg dis, 63, 160-172. kato t., yanase t., suzuki m., katagiri y., ikemiyagi k., takayoshi k., shirafuji h., ohashi s., yoshida k., yamakawa m. & tsuda t. 2016b. monitoring for bovine arboviruses in the most southwestern islands in japan between 1994 and 2014. bmc vet res, 12, 25. kedmi m., galon n., herziger y., yadin h., bombarov v., batten c., shpigel n.y. & klement e. 2011. comparison of the epidemiology of epizootic hemorrhagic disease and bluetongue viruses in dairy cattle in israel. vet j, 190, 77-83. kinney r. m. & calisher c.h. 1981. antigenic relationships among simbu serogroup (bunyaviridae) viruses. am j trop med hyg, 30, 1307-1318. kono r., hirata m., kaji m., goto y., ikeda s., yanase t., kato t., tanaka s., tsutsui t., imada t. & yamakawa m. 2008. bovine epizootic encephalomyelitis caused by akabane virus in southern japan. bmc vet res, 13, 4-20. kurogi h., inaba y., goto y., miura y., takahashi h., sato k., omori t. & matumoto m. 1975. serologic evidence for etiologic role of akabane virus in epizootic abortion-arthrogryposis-hydranencephaly in cattle in japan, 1972-1974. arch virol, 47, 71-83. lee v.h. 1979. isolation of viruses from field populations of culicoides (diptera: ceratopogonidae) in nigeria. j med entomol, 16, 76-79. markusfeld-nir o & mayer e. 1971. an arthrogryposis/ hydranencephaly syndrome in calves in israel 1969/70; epidemiological and clinical aspects. refuah vet, 28, 144-151. mathew c., klevar s., elbers a.r., van der poel w.h., kirkland p.d., godfroid j., mdegela r.h., mwamengele g. & stokstad m. 2015. detection of serum neutralization antibodies to simbu sero-group viruses in cattle in tanzania. bmc vet res, 11, 208. martinelle l., dal pozzo f., gauthier b., kirschvink n. & saegerman c. 2014. field veterinary survey on clinical and economic impact of schmallenberg virus in belgium. transbound emerg dis, 61, 285-288. noda y., uchinuno y., shirakawa h., nagasue s., nagano n., ohe r. & narita m. 1998. aino virus antigen in brain lesions of a naturally aborted bovine fetus. vet pathol, 35, 409-411. partsonson i.m. & mcphee d.a. 1985. bunyavirus 89 brenner et al. simbu serogroup viruses in israel veterinaria italiana 2019, 55 (1), 81-89. doi: 10.12834/vetit.1397.7622.2 supplementary table i. distribution of neutralizing antibody titers to each simbu serogroup virus. annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: xx; issue: x; year: 2019; doi: 10.12834/vetit.1397.7622.2 animal virus antibody titers positive sera/ tested sera (%) adult cow ainov 7 5 3 1 2 0 1 12/19 (63.2) satv 12 1 3 2 0 1 0 7/19 (36.8) akav 11 0 2 2 2 1 0 7/18 (38.9) shav 5 5 6 2 0 0 0 13/18 (72.2) peav 15 0 0 2 0 0 0 2/17 (11.8) heifer ainov 17 2 6 8 6 1 0 23/40 (57.5) satv 25 4 4 2 5 0 0 15/40 (37.5) akav 36 1 1 1 0 0 0 3/39 (7.7) shav 35 0 0 3 1 0 0 4/39 (10.3) peav 37 0 0 0 0 0 0 0/37 (0.0) sheep ainov 3 1 1 0 0 0 0 2/5 (40.0) satv 2 1 1 1 0 0 0 3/5 (60.0) akav 3 0 1 0 1 0 0 2/5 (40.0) shav 2 0 2 1 0 0 0 3/5 (60.0) peav 4 0 0 0 0 0 0 0/4 (0.0) ainov = aino viruses; satv = sathuperi viruses; akav = akabane viruses; shav = shamonda viruses; peav = peaton viruses. 173 short communication parole chiave caratterizzazione molecolare, culicoides imicola, peste dei piccoli ruminanti, trasmissione. riassunto ad ottobre 2015 un focolaio di peste dei piccoli ruminanti (ppr) ha colpito antalya, una provincia della turchia caratterizzata da condizioni climatico ambientali ottimali per gli artropodi vettori. dal momento che non ci sono dati su un possibile ruolo svolto dalle specie di culicoides nel trasmettere il virus della ppr (pprv), questo studio si è posto come obiettivo quello di valutare se culicoides spp. possano effettivamente agire come vettori del virus. numerosi culicoides sono stati catturati da metà ottobre a metà dicembre 2015. dodici pool di femmine non ingorgate sono stati predisposti ed esaminati per rilevare la presenza del virus attraverso una rt-pcr real time specifica per il gene nucleocapside del pprv. dei 12  testati, 7 pool sono risultati positivi. l'analisi della sequenza del gene della subunità  i della citocromo ossidasi mitocondriale, effettuata su culicoides spp. per identificare la specie implicata, ha individuato c. imicola nei pool positivi. il confronto tra le sequenze genomiche dei ceppi di pprv responsabili dei focolai del 2015 e quelli rilevati in c. imicola suggerisce che c. imicola potrebbe aver avuto un ruolo nella trasmissione del pprv nei focolai di antalya. virus della peste dei piccoli ruminanti in culicoides imicola (diptera: ceratopogonidae) in turchia keywords peste des petits ruminants, transmission, culicoides imicola, molecular characterization. summary an outbreak of peste des petits ruminants (ppr) occurred in the antalya province in turkey during october 2015. the antalya province has suitable habitats for vectors. there is no information available on the role of culicoides spp. in the transmission of peste des petits ruminants virus (pprv). in this study we investigated the potential role of the culicoides spp. in the transmission of pprv. culicoides were trapped throughout middle of october and middle of december, 2015. a total of 12 pools of non-engorged females were analysed with real-time rt-pcr targeting the nucleocapsid (n) gene of the pprv. pprv rna was detected in 7 of 12 culicoides pools. these pools were negative for the bovine/ovine beta-actin mrna. culicoides spp. were identified to the species level by sequence analysis of the mitochondrial cytochrome oxidase subunit  i gene. the species of culicoides found pprv positive was culicoides imicola. molecular characterization of field isolates from recent outbreaks and pools of midges that tested positive for pprv suggests that pprv replication might occur in culicoides imicola, and it may have played a role in transmitting pprv. veterinaria italiana 2019, 55 (2), 173-177. doi: 10.12834/vetit.1069.5784.2 accepted: 08.08.2016 | available on line: 30.06.2019 1department of virology, veterinary faculty, hatay mustafa kemal university, hatay, turkey. 2department of molecular microbiology, konya veterinary control institute, turkey. *corresponding author at: department of virology, veterinary faculty, mustafa kemal university, hatay, turkey. tel.: +90 326 2455313 / 1561, fax: +90 326 2455704, e‑mail: dr_muratank@hotmail.com. murat şevik1* and mustafa emin oz2 detection of peste des petits ruminants virus rna in culicoides imicola (diptera: ceratopogonidae) in turkey 174 veterinaria italiana 2019, 55 (2), 173-177. doi: 10.12834/vetit.1069.5784.2 peste des petits ruminants in c. imicola şevik & emin oz germany). rna was extracted from the supernatant by using a qiaamp viral rna mini kit (qiagen) on a qiacube (qiagen). the real-time rt-pcr was performed to detect n gene of the pprv (batten et  al. 2011). furthermore, culicoides midges were tested for the recent intake of a blood meal by a quantitative real-time rt-pcr specific for beta-actin mrna (toussaint et  al. 2007). nuclease free water (qiagen, hilden, germany) was used as the negative control in all real-time rt-pcr assays. dna was extracted from the supernatant of the culicoides pools by using a qiaamp dna mini kit (qiagen). the species of culicoides spp. of the pprv positive pools was identified to the species level by sequence analysis of the mitochondrial cytochrome oxidase subunit i gene (coi) using the primers c1-j-1718 and c1-n-2191 (dallas et al. 2003). pprv rna was detected in 7 of the 12 pools assayed. the ct-values of the real-time rt-pcr ranged from 29.8 to 34.1. reported ct values generated by using the same assay from blood of experimental infected goats with a moroccan strain of pprv were 25.7-34.5 (hammouchi et  al. 2012). generally, 100-200  µl of sheep or goat blood are used for detection of pprv, whereas less than 1  μl of blood remains in a midge after a blood meal. therefore, ct values should have been 6-7 units higher when the biting midge is tested by real-time rt-pcr (hoffmann et al. 2009). additionally, bovine/ovine beta-actin mrna was not determined in positive pools. this result showed that the detection of pprv rna within the culicoides midges didn't result from recent blood meals from infected animals. it has been reported that sub-transmissible infections are common in culicoides and ct values can be used to define transmissible and sub-transmissible infections (mellor 2000, veronesi et  al. 2013). comparison of the ct-values (29.8 to 34.1) obtained from the pprv positive culicoides pools in this study with predicted quantities (25.7-34.5) of pprv in the experimental infection study (hammouchi et  al. 2012) demonstrated that pprv replication may occur in the biting midges. pprv-positive culicoides pools (n  =  7) and one positive sheep blood sample from each district (aksu, alanya, döşemealtı, korkuteli, manavgat and muratpaşa), where ppr outbreaks (n  =  6) have been recently reported, were selected for sequence analysis. the primers described by forsyth and barrett (forsyth and barrett 1995) were used to amplify the fusion (f) gene of the pprv, whereas n gene was amplified with the primer pairs n1/n2 (kerur et al. 2008). nucleotide sequences of the f and n genes were obtained from 2 of the 7 pprv positive culicoides pools (pool-c1 and pool-c4). the two positive nucleotide sequences obtained from f and n genes peste des petits ruminants (ppr) is a transboundary small ruminant disease characterised by high fever, nasal and ocular discharges, erosive stomatitis, conjunctivitis, pneumonia, and severe diarrhoea (gibbs et al. 1979, lefevre and diallo 1990). peste des petits ruminants virus (pprv), classified within the genus morbillivirus in the family paramyxoviridae, is the causative agent of the disease (gibbs et al. 1979). although pprv has also been reported in cattle and pigs, sheep and goats are considered the natural hosts of pprv (anderson and mckay 1994). vaccination of lambs and kids has been used to control ppr in turkey, however, sporadic outbreaks have been observed. in october 2015, an outbreak of ppr occurred in the antalya province, which is located in the mediterranean region of turkey. it is known that pprv needs close contact between infected and susceptible animals for transmission (lefevre and diallo 1990). the antalya province has suitable climatic circumstances for the growth and distribution of culicoides. several species of biting midges of the genus culicoides play a role in the transmission of viral diseases of ruminant livestock, including bluetongue (bt), epizootic hemorrhagic disease (ehd) and schmallenberg virus (sbv) (paweska et  al. 2002, ruder et  al. 2012, balenghien et  al. 2014). however, there are no evidence of vectorial transmission of ppr in affected (endemic) regions of africa, middle east and india. the purpose of this study therefore was to investigate the potential role of the culicoides spp. in the transmission of pprv. this study was conducted in the antalya province (29°20’-32°35’ e, 36°07’-37°29’ n) in south-west anatolia. the antalya province has mediterranean climate characterised by mild and rainy winters and hot, dry summers. during 1981-2014, the annual mean temperature was 18.6  °c and annual precipitation was 783.8 mm (turkish state meteorological service). its elevation is 30 metres, and the average temperature during the study was 17.8  °c. three sheep farms within a radius of 2  km from the pprv-infected flocks were selected for the collection of culicoides. two of the three farms were located in manavgat district and one in döşemealti district where the highest number of ppr cases occurred. culicoides were trapped at biweekly intervals between middle of october and middle of december 2015 by using onderstepoort-type blacklight traps. culicoides spp. were separated from other insects using a stereomicroscope, followed by species identification (dyce 1969). subsequently, they were grouped into males, non-engorged and blood-fed females (engorged). non-engorged females were used for detection of pprv rna. twelve pools of non-engorged females, 10 midges per pool, were homogenized in nuclease-free water (400  µl) by using a tissueruptor (qiagen, hilden, 175veterinaria italiana 2019, 55 (2), 173-177. doi: 10.12834/vetit.1069.5784.2 şevik & emin oz peste des petits ruminants in c. imicola the network analysis based on f gene sequences showed that isolates from this study and previously characterised turkish isolates clustered in the same taxon (figure 1). the isolates culicoides-pprv  f  01 and culicoides-pprv  f  02 differentiated into two nodes with field isolates of the present study and previously characterised turkish isolates. these two nodes were separated from each other with a single mutation. a previously characterized turkish isolate (kf478918) from the antalya province in 2012 was placed on the same node with the isolate culicoides-pprv f 02. the network analysis based on n gene sequences showed that eight isolates of the present study clustered in two taxa (figure 2). the isolates culicoides-pprv  n  01 and culicoides-pprv  n  02 formed a node with isolate of alanya district (ku325487). the field isolates of döşemealti district (ku325485 and ku325492) were the closest to this node with a single mutation. a turkish isolate characterised in 2009 (kf478928) was the most distant previously characterised turkish isolate from this node. a second node with six mutations was formed by three field isolates (ku325489, ku325491 and ku325493) of the present study. network analyses showed that the number of mutations, among the isolates of the present study, was higher in n genes compared with f genes of corresponding isolates. this is expected because pprv is more in culicoides were from the same samples (pool-c1 and pool-c4). furthermore, nucleotide sequences of the f and n genes were obtained from pprv positive field samples from the alanya, döşemealtı, manavgat and muratpaşa districts. nucleotide sequences of the f gene were not obtained from pprv positive field samples from the aksu and korkuteli districts, only nucleotide sequences of the n gene were obtained from these two field samples. the analysis of the f gene sequences revealed that the homology between the four field isolates in the present study ranged between 99.3% and 100%, whereas the similarity between two isolates from culicoides midges (ku175170 and ku1751702) and four field isolates ranged from 98.9-99.6%. the two sequences from culicoides midges showed 98.9% nucleotide homology with each other. the deduced amino acid homology of the isolates of the present study was 100%. the analysis of the n gene sequences revealed that the homology between the six field isolates in the present study ranged between 97.2% and 100%, whereas the similarity between two isolates from culicoides midges (ku175171 and ku325486) and six field isolates ranged from 97.6-99.4%. the two sequences from culicoides midges showed 99.8% nucleotide homology with each other. the deduced amino acid homology of the isolates of the present study ranged from 96.4-100%. 216 279 231 16 48 78 572145120144 273 60 81192021237 42 24 297 242 265 237 219 174 156 160 287 282 267 263 243 226 207 204 195 165 150 132 96 93 75 57 27 18 287 270 243 222 166 132 96 90 75 63 57 33 30 19 93 271 288 288 42 123 102 186 270 180 84 126 189 9 160 255 264 93 157 159 168 183 8 213 249 156 120 111 87 54 39 36 252 53 102 123 129 141 234 276 nigeria/75/1 (x74443) oman/ibri/83 (fr667553) qatar/a37/10 (fn995206) yemen/01b (fn995999) ethiopia/96 (fn995997) sudan/ksud71 (hq131956) iran/r22/10 (fn995204) china/tibet/07-1 (eu816772) iraq/02 (fn995440) pakistan/09 (fn996973) tr/konya/cihanbeyli/2009 (kf478920) tr/konya/guneysinir/2011 (kf478921) tr/konya/kulu/2011 (kf478922) tr/dosemealti/culicoides-pprv f 01 (ku175170) tr/antalya/2015/pprv f1464 (ku325484) tr/antalya/2015/pprv f1545 (ku325488) tr/antalya/2015/pprv f1578 (ku325490) tr/isparta/yalvac/2013 (kf478919) nepal/09 (fn996974) nepal/95 (fr667648) bangladesh/narayangonj/09 (jx094440) saudi arabia/94 (fr667645) india/sungri/96 (gq452015) bangladesh/00 (fr667556) pakistan/94 (fr667646) kuwait/99 (fr667644) tr/manavgat/culicoides-pprv-f 02 (ku175172) tr/antalya/2015/pprv f1531 (ku325486) tr/antalya/ibradi/2012 (kf478918) tr/konya/kulu/2013 (kf478923) tr/nigde/ulukisla/2011 (kf478924) tr/aksaray/2011 (kf478917) guinea/91 (fr667554) ivory coast/89 (fr667555) cote divoire/icv89 (eu267273) 179 66 lineage i lineage ii lineage iii lineage iv figure 1. phylogenetic network analysis based on the f gene of the field isolates with corresponding sequences published in the genbank. numbers along the branches represent the nucleotide changes separating the nodes. field isolates and isolates from culicoides midges in this study are marked with round black spot () and black star (), respectively. 176 veterinaria italiana 2019, 55 (2), 173-177. doi: 10.12834/vetit.1069.5784.2 peste des petits ruminants in c. imicola şevik & emin oz that c.  imicola plays a role in transmitting the schmallenberg, bluetongue and epizootic hemorrhagic disease viruses (paweska et  al. 2002, ruder et  al. 2012, balenghien et  al. 2014). however, to our knowledge, its role in the transmission of pprv has not been previously reported. in conclusion, the absence of bovine/ovine beta-actin mrna in pprv positive pools and the results of sequence and phylogenetic network analyses suggest that pprv might replicate in culicoides midges and c.  imicola may have played a role in the transmission of pprv. however, further experimental studies are needed to confirm pprv field isolates within c. imicola. prone to mutations on the n gene compared to the f gene (munir et al. 2015). sequence comparisons and network analyses (based on f and n genes) showed that circulating pprv isolates and isolates from culicoides midges are very closely related and pprv isolates from culicoides midges seem to have diverged from previously characterised turkish isolates (figure  1 and figure  2). these findings suggest that virus replication occurred in the biting midges. the sequence analysis of coi gene showed that the species of culicoides in pprv positive pools was c.  imicola. previous studies have reported 243 178 223 195 157 78 45 187 85 24 14 217 220 157 7 13 137 223 247 115 109 68 58 48 175 44 82 122 92 17 142 220 169 223 140 48 97 113 253 31 35 74 25 135 85 46 19 43 63 64 91 126 153 172 179 193 211 214 133 105 79 57 46 37 1456473102109139147177187190198214217244217 223 223 182 182 148 193 184 113 107 65 19 13 135 122 76 99 99 13 21 22 35 38 90 113 120 136 140 151 157 175 191 223 153 18 59 68 85 121 165 176 34 18 35 36 41 43 52 72 83 88 93 107 113 124 132 140 141 166 169 172 179 186 193 235 238 103 77 96 102 106 152 13 215 241 198 188 157 113 100 91 69 63 45 18 103 223 223 57 nigeria/76/1 (dq840164) nigeria/75/1 (dq840160) oman/ibri/83 (dq840168) united arab emirates/86 (dq840169) ethiopia/96 (dq840183) sudan/gedarif-ksud71 (hq131918) iran/98 (dq840185) china/tibet/0701 (eu360596) pakistan/faisalabad/2010 (jn009673) tr/konya/cihanbeyli/2009 (kf478928) tr/konya/guneysinir/2011 (kf478929) tr/konya/kulu/2011 (kf478930) guinea/88 (dq840170) cote divoire/89 (dq840199) cote divoire/icv89 (eu267273) lineage i lineage ii lineage iii lineage iv tr/antalya/2015 pprv n1464 (ku325485) tr/antalya/2015 pprv n1506 (ku325492) tr/antalya/2015/pprv n1545 (ku325489) tr/antalya/2015/pprv n1556 (ku325493) tr/antalya/2015/pprv n1578 (ku325491) tr/dosemealti/culicoides-pprv n 01 (ku175171) tr/manavgat/culicoides-pprv n 02 (ku325483) tr/antalya/2015/pprv n1531 (ku325487) india/sungri/96 (ay560591) tajikistan/04 (dq840198) saudi arabia/99/7 (dq840195) bangladesh/09 (hq131961) tr/isparta/yalvac/2013 (kf478932) tr/aksaray/2011 (kf478925) figure 2. phylogenetic network analysis based on the n gene of the field isolates with corresponding sequences published in the genbank. numbers along the branches represent the nucleotide changes separating the nodes. field isolates and isolates from culicoides midges in this study are marked with round black spot () and black star (), respectively. 177veterinaria italiana 2019, 55 (2), 173-177. doi: 10.12834/vetit.1069.5784.2 şevik & emin oz peste des petits ruminants in c. imicola anderson j. & mckay j.a. 1994. the detection of antibodies against peste des petits ruminants virus in cattle, sheep and goats and the possible implication to rinderpest control programme. epidemiol infect, 112, 225-231. balenghien t., pagès n., goffredo m., carpenter s., augot d., jacquier e., talavera s., monaco f., depaquit j., grillet c., pujols j., satta g., kasbari m., setier-rio m.l., izzo f., alkan c., delécolle j.c., quaglia m., charrel r., polci a., bréard e., federici v., cêtre-sossah c. & garros c. 2014. the emergence of schmallenberg virus across culicoides communities and ecosystems in europe. prev vet med, 116, 360-369. batten c.a., banyard a.c., king d.p., henstock m.r., edwards l., sanders a., buczkowski h., oura c.c. & barrett t. 2011. a real time rt-pcr assay for the specific detection of peste des petits ruminants virus. j virol methods, 171, 401-404. dallas j.f., cruickshank r.h., linton y.m., nolan d.v., patakakis m., braverman y., capela r., capela m., pena i., meiswinkel r., ortega m.d., baylis m., mellor p.s. & mordue luntz a.j. 2003. phylogenetic status and matrilineal structure of the biting midge, culicoides imicola, in portugal, rhodes and israel. med vet entomol, 17, 379-387. dyce a.l. 1969. the recognition of nulliparous and parous culicoides (diptera: ceratopogonidae) without dissection. j austr entomol soc, 1, 11-15. forsyth m.a. & barrett t. 1995. evaluation of polymerase chain reaction for the detection and characterisation of rinderpest and petse des petits ruminants viruses for epidemiological studies. virus res, 39, 151-163. gibbs e.p.j., taylor w.p., lawman m.p.j. & bryant j. 1979. classification of peste des petits ruminants virus as a fourth member of the genus morbillivirus. intervirology, 11, 268-274. hammouchi m., loutfi c., sebbar g., touil n., chaffai n., batten c., harif b., oura c. & el harrak m. 2012. experimental infection of alpine goats with a moroccan strain of peste des petits ruminants virus (pprv). vet microbiol, 160, 240-244. hoffmann b., bauer b., bauer c., bätza h.j., beer m., references clausen p.h., geier m., gethmann j.m., kiel e., liebisch g., liebisch a., mehlhorn h., schaub g.a., werner d. & conraths f.j. 2009. monitoring of putative vectors of bluetongue virus serotype 8, germany. emerg infect dis, 15, 1481-1484. kerur n., jhala m.k. & joshi c.g. 2008. genetic characterization of indian peste des petits ruminants virus (pprv) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments. res vet sci, 85, 176-183. lefevre p.c. & diallo a. 1990. peste des petits ruminants virus. rev sci tech, 9, 951-965. mellor p.s. 2000. replication of arboviruses in insect vectors. j comp pathol, 123, 231-247. munir m., saeed a., abubakar m., kanwal s. & berg m. 2015. molecular characterization of peste des petits ruminants viruses from outbreaks caused by unrestricted movements of small ruminants in pakistan. transbound emerg dis, 62, 108-114. paweska j.t., venter g.j. & mellor p.s. 2002. vector competence of south african culicoides species for bluetongue virus serotype 1 (btv-1) with special reference to the effect of temperature on the rate of virus replication in c. imicola and c. bolitinos. med vet entomol, 16, 10-21. ruder m.g., howerth e.w., stallknecht d.e., allison a.b., carter d.l., drolet b.s., klement e. & mead d.g. 2012. vector competence of culicoides sonorensis (diptera: ceratopogonidae) to epizootic hemorrhagic disease virus serotype 7. parasit vectors, 5, 236. toussaint j.f., sailleau c., breard e., zientara s. & de clercq k. 2007. bluetongue virus detection by two real-time rt-qpcrs targeting two different genomic segments. j virol methods, 140, 115-123. veronesi e., antony f., gubbins s., golding n., blackwell a., mertens p.p., brownlie j., darpel k.e., mellor p.s. & carpenter s. 2013. measurement of the infection and dissemination of bluetongue virus in culicoides biting midges using a semi-quantitative rt-pcr assay and isolation of infectious virus. plos one, 8, e70800. 169 introduction feline immunodeficiency virus (fiv) disease is one of the most important infectious diseases in domestic cats caused by a virus of the genus lentivirus within the family of retroviridae (pedersen et  al. 1987, miyazawa et  al. 1994). pathogenesis of fiv infection includes three different phases. the transient phase is the primary stage of the infection. during this phase the virus replicates rapidly (callanan et  al. 1992, beebe et  al. 1994, hartmann 2012). the asymptomatic phase is the second stage of fiv infection. in this phase the cat’s humoral immune response reduces the plasma virus load, but fails to clear the infection (addie et  al. 2000, mcdonnel et  al. 2013). as the disease progresses, a steady decline in cd4+ t helper cells occurs and antiviral immunity declines. within months to years, the plasma virus level increases throughout the infection and finally leads to an immune deficiency stage which is considered as the end stage of the disease (ackley et  al. 1990, hoffmann‑fezer et  al. 1992). diagnostic investigations are usually based on antibody detection by enzyme‑linked immunosorbent assay (elisa), and sometimes on virus detection by polymerase chain reaction (pcr) or on other tests detecting antigen (bienzle et  al. 2004, hartmann et  al. 2007). proviral dna quantification can provide useful information about disease staging, independent of the cd4+ count and can be detected by using the real‑time pcr method (shiramizu et  al. 2005, gueudin et  al. 2008, malnati et al. 2008). cycle threshold (ct) values are proportional to the amount of proviral dna 1department of veterinary clinical science, division of small animal internal medicine, faculty of veterinary medicine, university of tabriz, islamic republic of iran. 2department of veterinary pathobiology, division of molecular biology, faculty of veterinary medicine, university of tabriz, islamic republic of iran. *corresponding author at: faculty of veterinary science, university of tabriz, tabriz, iran. e‑mail: a.azadian90@ms.tabrizu.ac.ir. keywords feline immunodeficiency virus, cat, aggression, dna, pcr. summary a study was undertaken to determine the possible interaction between aggressive behavior and feline immunodeficiency virus (fiv) disease progression based on semi‑quantitative viral load levels and health status in naturally fiv‑infected cats. fiv status was determined in ninety‑six owned and stray cats, using nested polymerase chain reaction (pcr). aggressive tendencies were assessed based on observation and the cats’ demeanor as determined by the owners and shelter caretakers. results showed that forty‑seven cats (49%) were pcr‑positive for fiv infection and all aggressive cats were fiv‑positive (100%). fiv infection was significantly linked to extreme aggressive tendencies and the extremely aggressive fiv‑infected cats were more likely to have an unhealthy status compared to the non‑aggressive individuals (p = 0.022). there was also a significant difference (p = 0.012) in the mean cycle threshold (ct) values between the aggressive and non‑aggressive fiv‑infected cats and also between the unhealthy fiv‑infected cats with extreme aggressive tendencies and the healthy fiv‑infected individuals without aggression (p = 0.001). accordingly, results indicated that parameters associated with fiv disease progression are directly linked to aggression. the possible impact of fiv on the behavioral pattern of naturally infected cats should not be underestimated. however, there is an urgent need to conduct more experiments to support the assumptions about the possible exacerbation of aggression tendencies in naturally fiv‑infected cats following the direct effect of fiv through the course of the infection. amin azadian1*, mohsen hanifeh1 and masoumeh firouzamandi2 aggressive behavior in cats naturally infected with feline immunodeficiency virus (fiv) and its interaction with fiv disease progression veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 accepted: 18.06.2019 | available on line: 31.12.2020 170 fiv and aggressive behavior in cats azadian et al. veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 • they had conditions that could cause extreme pain like traumatic injuries. general health assessment was conducted after history taking by performing a routine physical and neurological examination and evaluating the complete blood count and biochemistry profile of the subjects. for determination of the health status, cats with at least one clinical abnormality related to common diseases and opportunistic infections following the terminal stage of fiv disease were considered as unhealthy (ishida and tomoda 1990, bęczkowski et  al. 2015). diseases considered to be associated with fiv disease progression to the terminal stage included: chronic stomatitis/ gingivitis with gingival score of 2/3 or 3/3 (signs: ptyalism, halitosis, and bleeding gingiva), chronic upper respiratory tract infections (signs: nasal discharge, stertorous breathing, ocular signs like conjunctivitis), chronic skin diseases e.g. parasitic, dermatophytosis or bacterial pyoderma (signs: pruritus, erythema, scaling, crusting, and alopecia). two to four ml of whole blood were collected from each cat after the clinical examination. blood collections were done via standard venipuncture from the jugular vein in sterile microtubes with edta. sedation with a dose of 0.05  mg  kg‑1 of acepromazine maleate 2% injected intramuscularly was done to sedate the cats with anxiety. blood samples were submitted for proviral dna extraction after isolation of the buffy coat layer via centrifugation (10,000 g, 6 minutes, 4 °c) and were stored at ‑ 20 ºc prior testing. after the clinical procedure, as stray cats spent a quarantine period as long as their fiv status was determined. they were handled by a carrier into the cat’s ward of the hospital and were kept in individually ventilated cages (1.5  x  2  meter in size). commercial dry cat food was given to the cats twice a day. wet cat diet was instead given to those cats which had gingivostomatitis. owned cats were sent back to their home and shelter cats were sent to the shelters after completing the sample collection and clinical procedure. during the quarantine period, the stray cats were taken out of the cage daily by a carrier and were brought to a room where they could interact with toys. dna extraction and pcr amplification of fiv dna was extracted from each blood sample with genomic dna purification kit (bioneer, korea) according to the instruction of the manufacturer. for the first stage reaction of fiv amplification, one pair of specific primers were used as described previously (matteucci  et  al.  1993). in samples and correlate to both free in fluid and cell‑associated proviral load. therefore, it can be used for interpretation of fiv proviral levels (désiré et al. 2001, luo et al. 2005, malnati et al. 2008). since biting and direct inoculation of infected blood and saliva are the main routes for transmission of fiv infection, intact male cats, which are more likely to display aggressive behavior following breeding and territorial fights, are usually more frequently infected (winkler et al. 1999, little 2005). fiv and hiv are both neurotropic viruses, and neurological impairments are believed to be a consequence of direct viral effect (pedersen and barlough 1991, meeker and hudson 2017). in experimentally infected cats, behavioral disturbances like extreme aggression following neurodegeneration are widely seen by using a sensitive behavioral recording. however, whether naturally fiv‑infected cats have the neurological disease it is still controversial. clinically relevant neurobehavioral impairments are rarely observed in these patients (english et al. 1994, phillips et al. 1994, meeker 2007). close monitoring of the aggressive behavior in naturally infected cats during the course of the infection can contribute to a better understanding of the neurobehavioral aspect of fiv disease. thus, the aim of the current study is to determine the possible interaction between aggressive behavior and fiv disease progression based on semi‑quantitative viral load levels and health status in naturally fiv‑infected cats. materials and methods subjects and sampling the study was conducted at the veterinary hospital of university of tabriz from september 2015 to august 2016. owned and stray cats older than 1‑year were recruited because it was believed that current aggression tendencies in cats younger than 1‑year may not be reflective of their aggression tendencies at adulthood since aggression in these cats could be related to their motivation of play in some situations. in the current study, most of the owned cats were recruited during the annual health check or during hospitalization for further workup of systemic illnesses. stray cats instead were rescued by animal welfare groups and brought to the hospital for a diagnostic checkup before entering shelters. all recruited cats were clinically examined by board‑certified small animal internal medicine specialists and each subject’s health status was determined afterward. cats were excluded from the study if: • they had a known fiv infection status (due to prior testing); 171 azadian et al. fiv and aggressive behavior in cats veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 status. the questionnaire included 3 questions concerning the cat’s aggression tendencies toward strangers, family members, and other cats (vapalahti et  al. 2016, ahola et  al. 2017). owners were asked to rate their cat’s aggressive behavior at five levels from “not at all” to “very often” by considering all related intolerant behaviors ranging from hissing, spitting and growling to inflicting physical injuries (bite and claws). the questionnaire had to reflect the range of aggression tendencies from “extremely aggressive with humans and other animals” to “extremely accustomed to interacting with humans and other animals and very comfortable with them”. those owned cats which were reported to show intolerant behaviors toward all three categories (family, strangers, and other cats) at “often” and “very often” level, were rated as aggressive cats (cats with extreme aggression tendencies). evaluation of the human‑directed aggressive behavior in the stray cats was also measured for hiss, bite, and slap/scratch during the clinical procedure before sedation, the quarantine period inside the ward while being fed and also during the time they were handled to the playroom by the small animal medicine intern. the long‑term behavioral history of the stray cats based on displaying inter‑cat aggression with related intolerant behaviors was also collected following observation by a caretaker for 90 days in the shelters while he or she was unaware of the cat’s fiv status. only those feral cats which showed aggressive behavior during the clinical procedures, feeding, and handling to the playroom area and which were also reported to often show aggressive behaviors toward other cats during 90 days of observation inside the shelter, were considered to be in the aggressive cats’ category. statistical analysis statistical analysis was performed in spss statistics version 24.0. chi‑square analysis was used to see whether fiv infection was more prevalent among the aggressive cats and to see if there is any significant association between health status of the fiv‑infected cats and their aggression tendencies. evaluating the difference in the mean ct values between the aggressive and non‑aggressive fiv‑infected cats was done, using student t‑test. a one‑way analysis of variance (anova) was then used to test statistically significant differences in the mean ct  values between the four groups of fiv‑infected cats including the healthy and unhealthy cats with and without extreme aggression tendencies, with ct values serving as the dependent variable and the health status and aggression serving as independent variables. data were presented as mean ± standard error of the mean (sem) and the statistical difference was considered significant at p < 0.05. the sequence of the forward primer was 5’‑ggcatatcctattcaaacag‑3’and the reverse primer was 5’‑aagagttgcattttatatcc‑3’. for the first stage reaction of the nested‑pcr, 5 µl of each extracted dna was used along with 1 µl of each primer, 12.5 µl of master mix (ampliqon, denmark) and 5.5 µl of nuclease‑free water to reach the total volume of the reaction mixture into 25 µl. thermal profile used for the first reaction included an initial denaturation at 94  °c for 3 min, followed by 40 cycles of 94  °c for 1 min for denaturation, 50  °c for 1 min for annealing, 72  °c for 120 sec for extension, and a final extension step at 72  °c for 5 minutes. amplification products were 1/10 diluted with nuclease‑free water in order to be submitted for the second reaction. real‑time pcr as the second stage reaction one pair of fiv specific primers were designed with primer3 software and checked with oligoanalyzer 3.1 software on fiv gene (ncbi access number: m25381.1) as following: the forward primer was 5'‑taataatggccgcaccaggg‑3', and the reverse primer was 5'‑tgcatcctagctggtgcaaa‑3'. five  µl of the diluted pcr product was submitted to a real‑time pcr along with 0.5 µl of each designed internal primer, 10 µl of syber green master mix for pcr (takara, usa), and 4 µl of nuclease‑free water to reach the total volume of the reaction mixture into 20 µl. thermal profile used for the second stage reaction performed at 95  °c for 10 min as initial denaturation, then followed by 40 cycles of 95  °c for 10 sec and 45 sec at 60  °c. all reactions were performed in triplicates. finally, 5 µl of real‑time pcr products were run for an electrophoresis with an 1% agarose gel containing dna safe stain with 100 bp dna ladder. evaluating the aggressive behavior in cats it is quite common to create and use a survey based on opinions of the cats’ guardians to evaluate the frequency and likelihood of aggressive behaviors in cats (stelow et  al. 2016). assessment of aggression was also done based on cats’ demeanor as determined by the owners or veterinarians (bande et al. 2012). thus, aggression tendencies in the current study were evaluated based on the subject’s behavioral background by considering a rating from a person familiar with the cat according to their knowledge of the cat’s typical behavior. a short questionnaire was given to the owners at the time of the history taking and before determination of the cats’ fiv 172 fiv and aggressive behavior in cats azadian et al. veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 category. all aggressive cats (100%) and twenty‑three of the non‑aggressive cats (31.9%) were fiv‑positive. results also showed that twenty cats out of twenty‑four aggressive fiv‑infected cats (83%) in the current study were unhealthy. the mean ct value which is semi‑quantitatively proportional to the amount of proviral dna in peripheral blood was 14.78 ± 0.91 (ranging from 9.91 to 29.27) for the aggressive and 21.39 ± 1.61 (ranging from 11.39 to 30.52) for the non‑aggressive fiv‑infected cats. statistical analysis revealed a significant association between fiv prevalence and aggressive behavior in cats, (p‑value = 0.000) (table ii). a significant association was also observed between health status and aggression in the fiv‑infected cats (p‑value = 0.022) (table iii). there was also a significant difference in the mean ct values between the aggressive and non‑aggressive fiv‑infected cats (p‑value = 0.012) (figure 1). one ‑way anova revealed a significant interaction in ct values between the four groups of fiv‑infected cats (the healthy aggressive vs. healthy non‑aggressive vs. unhealthy aggressive vs. unhealthy non‑aggressive cats), indicating that changes within the ct values between these results fifty‑two owned and forty‑four stray cats of different species and gender (32 females, 15 spayed females, 31 males, and 18 neutered males) were recruited for this study following inclusion and exclusion criteria. the median age of the whole study population was 3.48 ± 1.88 years, ranging from 1 to 12 years. overall, forty‑seven cats (49%) were considered to be clinically healthy in general and forty‑nine cats (51%) were grouped as unhealthy based on the health assessment records (considering those with diseases associated with the terminal stage of fiv disease). details of fiv‑positive and negative cats within each disease category were shown in table i. the first stage reaction amplified a 675  bp dna fragment and the second stage reaction amplified a 199  bp dna fragment for the positive samples. ct  values were also collected for the fiv‑positive samples following real‑time pcr amplifications. finally, forty‑seven cats (49%) out of ninety‑six subjects were diagnosed as fiv‑infected using the nested‑pcr method. after evaluating aggression tendencies in all subjects, twenty‑four cats were considered to be aggressive (25%) and seventy‑two cats (75%) were included in the non‑aggressive table i. clinical abnormalities detected during the health assessment in the unhealthy feline immunodeficiency virus (fiv) positive and negative cats (n = 49). the most frequent diseases detected in the unhealthy fiv-infected cats were feline chronic gingivostomatitis (fcgs) and urt diseases which were seen in 78% and 41% of the unhealthy fiv-positive cats, respectively. diseases associated with the terminal stage of fiv disease main signs/symptoms in patients the most common sign of the disease among patients (comments) number of unhealthy cats with clinical abnormalities (%) fiv-positive cats (n = 32) fiv-negative cats (n = 17) total unhealthy cats (n = 49) feline chronic gingivostomatitis (fcgs) ptyalism, oral pain, halitosis, bleeding gingiva bleeding gingiva (appeared in all patients with fcgs during the clinical examination) 25 (78%) 4 (24%) 29 (59%) chronic upper respiratory tract (urt) infections nasal discharge, stertorous breathing, conjunctivitis stertorous breathing (more frequent with rhinitis in the fiv-positive patients) 13 (41%) 4 (24%) 17 (34%) chronic skin diseases (including bacterial, fungal, and parasitic infections) pruritus, erythema, scaling, crusting and alopecia scaling and crusting 7 (22%) 1 (6%) 8 (16%) table ii. pattern of feline immunodeficiency virus (fiv) status and aggression within the whole study population. all aggressive cats were pcr-positive for fiv disease and there was a significant association between the prevalence of fiv infection and aggression tendencies in cats (p =0.000). fiv status with aggression without aggression total χ 2 p value positive 24 23 47 33.362 < 0.001 negative 0 49 49 total 24 72 96 table iii. pattern of aggression and health status within the feline immunodeficiency virus (fiv) positive cats. twenty cats (83%) out of the twenty-four aggressive fiv-positive cats were unhealthy. there was a significant association between the health status of fiv-infected cats and aggression tendencies (p = 0.022) which shows that the aggressive fiv-positive cats were more likely to have an unhealthy status compared to the non-aggressive cats. behavior status healthy unhealthy total χ 2 p value aggressive 4 20 24 non-aggressive 11 12 23 5.248 0.022 total 15 32 47 173 azadian et al. fiv and aggressive behavior in cats veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 1999, goldkamp et  al. 2008, lara et  al. 2008, gates and dale 2017). ct values which have been shown to be semi‑quantitative measures of the amount of virus in the clinical specimen can be obtained by real‑time pcr assays, and if associated with factors related to disease severity like health status, may provide a reliable way for determining the progression of fiv disease (bęczkowski et  al. 2015). to date, the level of fiv proviral dna among infected cats were evaluated in very few studies and all have revealed that as the disease progresses into the terminal stage, the amount of proviral dna will increase as well (klein et  al. 1999, pedersen et  al. 2001, ryan et al. 2003, pinches et al. 2007, leal et al. 2015). the fiv‑associated neurodegenerative disease is believed to cause a very gradual decline in the brain function with only a few overt signs until 5  to  8 years post‑infection and it is also believed that a progressive encephalopathy usually overlaps with the development of acquired immune dysfunction in infected cats (meeker and hudson 2017). however, neurobehavioral disturbances still remain a controversial consequence of fiv disease progression in naturally infected cats. to our knowledge, no study has been evaluated groups were significantly different [f (3.43) = 6.099, p‑value  =  0.001] (figure  2). the games‑howell post‑hoc test showed that the difference between the healthy group without extreme aggression tendencies (mean ct  value  =  25.210 ± 1.74) and the unhealthy group with extreme aggression tendencies (mean ct value  =  13.109 ± 1.97) was significant (p < 0.001). discussion results of the current study showed that fiv disease is more prevalent in the aggressive cats and the aggressive fiv‑infected cats were more likely to have an unhealthy status comparing to the non‑aggressive fiv‑ infected individuals. cats which displayed extreme aggression tendencies were all diagnosed as fiv‑positive and they had a significantly lower ct values and higher amount of proviral dna than the non‑aggressive fiv‑infected cats. the statistically significant difference in ct values between the four groups of healthy and unhealthy fiv‑ infected cats with and without extreme aggression tendencies (p < 0.001) also shows a direct link between the two parameters associated with fiv disease progression and aggressive behavior. gleich and colleagues (gleich et al. 2009) reported a fiv prevalence rate of 91% in the aggressive cats in their study. in another study, the prevalence of fiv disease was also reported as 20.7% in the aggressive cats while the prevalence in the non‑aggressive cats was much lower (9.6%) (bande et  al. 2012). other studies also demonstrated that cats with more aggression tendencies have a greater risk for fiv infection (yamamoto et al. 1989, bradshaw and hall m ea n c t va lu e aggressive fiv-positive cats 25 20 15 10 5 0 non-aggressive fiv-positive cats 14.78 21.39 figure 1. mean ct values for feline immunodeficiency virus (fiv) infected cats with and without aggressive behavior. there was a significant difference in the mean ct values between the aggressive and non-aggressive fiv-positive cats (p = 0.012) which shows that the aggressive fiv-positive cats significantly had a higher amount of proviral dna in their peripheral blood compared to the non-aggressive cats and might be in a more progressed stage of the disease. m ea n c t va lu e 25 15 healty aggressive fiv-positive cats 10 5 0 healty non-aggressive fiv-positive cats 30 20 unhealty aggressive fiv-positive cats unhealty non-aggressive fiv-positive cats 23.176 25.21 13.109 18.548 figure 2. the mean ct values of the feline immunodeficiency virus (fiv) positive cats grouped based on their aggression tendency and health status. the p-value for the mean ct values difference between the four groups was 0.001 showing that the difference between groups was significant. the post-hoc test revealed that the amount of proviral dna based on the ct values was significantly higher in the unhealthy aggressive cats compared to the healthy non-aggressive cats which shows a strong link between parameters associated with fiv disease progression and aggressive behavior. 174 fiv and aggressive behavior in cats azadian et al. veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 hypothesis. there are also other factors that may have impacts on aggression tendencies in cats. for instance, being an outdoor cat can cause stress in different conditions which not only can cause mood and behavioral changes but also can affect the immune system and subsequently, cause a poor immune response to the virus replication. current evidence also showed that dysregulation of the immune system may also be one of the mediators of social stress that causes exacerbation of aggression in both animals and humans (takahashi et al. 2018); thus, disarray of the immune function following fiv disease progression may cause behavioral disturbances per se and should be discussed in future studies. it is also important to know whether cats were also aggressive before and aggressive behaviors become more apparent and frequent recently or not. such a long‑term behavioral history can be collected from owned cats but in terms of having also free‑roaming or stray cats among the study population, this is actually a great challenge as the way these cats behave in shelters may vary with their behavioral characteristics in the outdoor environment. it is also possible that human‑directed aggression among the stray cats was the result of the ‘stress’ they experienced during the experimental procedure or because of the limited exposure to humans they had before. that is why we also tried to evaluate inter‑cat aggression tendencies in these cats via observation for 90 days inside the shelters. however, our measure of aggression tendencies may have some limitations; for example, the answers reported in each section of the questionnaire were the subjective opinions of the cats’ owners or caretakers. it would have been better to have objective observations in order to get more reliable answers. nonetheless, answers related to human‑directed aggression were strongly in accordance with the observed behaviors during the veterinary visits. overall, results of the current study showed that unhealthy status and high semiquantitative levels of fiv proviral load were significantly linked to extreme aggression tendencies. however, the subtlety of the results from the current study suggests the need for additional research on the topic. thus, findings should advisedly be confirmed in additional populations and further studies is also needed to support the assumption about the exacerbation of aggression tendencies in cats following the possible effect of fiv through the course of natural infection. acknowledgements we would like to thank all cat owners, clinicians and veterinary staff in university of tabriz veterinary hospital and central laboratory, animal shelters, the interaction between a behavioral trait like aggression and variables associated with fiv disease progression so far. while the aggressive behavior has been already reported as a risk factor for fiv infection, our results may provide the very first link between aggression tendencies and parameters associated with fiv disease progression into the terminal stage. aggression in cats is always considered as one of the important predisposing factors for fiv disease, and research studies have also reported a significant correlation between aggression and fiv infection (fromont et  al. 1997, goldkamp et  al. 2008, gleich et  al. 2009). lara and colleagues (lara et  al. 2008) suggested that being aggressive as a risk factor for fiv infection is probably more important than the way of life in cats. on the other hand, as it was previously reported in a study conducted on the experimentally infected cats, exacerbated forms of aggression which can be a manifestation of social dysfunction may also be a consequence of the neurodegenerative effect caused by fiv (dow et  al. 1990, meeker 2007). thus, fiv may play a critical role in disrupting social‑emotional behaviors like aggression in naturally infected cats similar to what has been documented in experimentally infected individuals and the progressive encephalopathy following fiv disease progression may exacerbate aggression tendencies in patients regardless of originally being a cat with extreme aggression tendencies. podell and colleagues (podell et al. 1997) also demonstrated that progressive encephalopathy parallels the reduction in the cd4/cd8 ratio among the experimentally infected fiv cats. therefore, in regard to the previous studies, it can be indicated that fiv disease progression could influence the neurobehavioral function of the experimentally infected cats. in this study fiv‑infected cats with aggression tendencies had higher amounts of proviral dna copies comparing to the healthy non‑aggressive cats; thus, an assumption can be raised about the possible occurrence of neurodegeneration following the direct effect of fiv in naturally fiv‑infected cats. it could be stated that the disease progression may possibly exacerbate aggression tendencies among the infected cats and consequently, increase the risk of fiv infection itself. however, the higher amount of proviral dna in cats displaying extreme aggression tendencies can also be due to the greater risk they have for fiv infection. they might be involved in inter‑cat fights when they were young and consequently, they have been infected earlier than non‑aggressive cats. hence, the aggressive fiv‑infected cats in the current study may be in a more progressive stage of the disease at the time of the diagnosis and have a higher amount of proviral dna than the non‑aggressive fiv positive cats. that is why further studies are needed to confirm this 175 azadian et al. fiv and aggressive behavior in cats veterinaria italiana 2020, 56 (3), 169‑176. doi: 10.12834/vetit.1795.9466.3 statement of animal rights ethical approval was discussed and obtained (approval code: fvm.rec.1396.940) by the ethics committee of faculty of veterinary medicine, university of tabriz in accordance with the national legislation. all cat owners and shelter groups were first informed about the objective of the study and those who agreed to participate in this study were then received a written informed consent to sign. and welfare groups for their participation in this study. this study was conducted as a part of a thesis approved for the degree of doctor of veterinary medicine (dvm) and partially funded by university of tabriz postgraduate research department. ackley c.d., yamamoto j.k., levy n., pedersen n.c. & cooper m.d. 1990. immunologic abnormalities in pathogen‑free cats experimentally infected with feline immunodeficiency 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m.l. & yamamoto j.k. 1987. isolation of a t‑lymphotropic virus from domestic cats 97 review veterinaria italiana 2021, 57 (2), 97-103. doi: 10.12834/vetit.1679.8914.5 accepted: 23.01.2020 | available on line: 31.12.2021 1universidade estadual paulista ‘júlio de mesquita filho’, brazil. 2universidade do estado de santa catarina, brazil. *corresponding author at: universidade estadual paulista ‘júlio de mesquita filho’, brazil. e‑mail: helenabaldini@gmail.com. maria helena mazzoni baldini1* and aury nunes de moraes2 keywords wild animals, epizootic haemorrhagic disease virus, bluetongue virus, clinical signs, pathology, orbivirus. summary bluetongue (bt) and epizootic haemorrhagic disease (ehd) are two oie-listed vector borne diseases of domestic and wild ruminants caused by two orbiviruses, bt virus (btv) and ehd virus, respectively. also wildlife can be infected by these two viruses. they can manifest a variable range of clinical signs and lesions. some species appear to be extremely susceptible showing, indeed, high mortality rates, others may act only as reservoirs, playing however, an important epidemiological role. the purpose of this review is to describe the clinical and pathological manifestations related to these diseases in wildlife species and to review available literature on btv/ehdv-infected wildlife species with emphasis for the south american scenario. bluetongue and epizootic haemorrhagic disease in wildlife with emphasis on the south american scenario isolated in 1955 from a white-tailed deer in new jersey; the animal was probably infected during an outbreak that led to the death of an estimated number of 500 to 700 deer (shope et  al. 1960). epizootic haemorrhagic disease has been reported causing clinical cases and seropositive animals in north america, australia, asia, africa and south america (alfieri et  al. 2007, werther and kawanami 2014, favero et  al. 2013). currently at least eight serotypes are recognized (oie 2009). vector abundance and distribution, among other factors, lead to the variation in geographic distribution of haemorrhagic diseases as bt and ehd. these factors, as well as the specific serotype present, pathogenicity, host immunity, and genetic variation contribute to the geographic distribution of ehdv and btv (stallknecht et al. 2002). among domestic species, sheep are notably susceptible to btv (erasmus 1975). clinical signs are not common in cattle and goats however, these animals may develop reproductive disorders as a result of infection (arida et  al. 2007). in many endemic countries, cattle rarely present clinical signs. they however can show a prolonged period of viraemia (katz et  al. 1994, singer et  al. 2001) and act as a reservoir of the virus (arida et  al. 2007). nevertheless, a higher occurrence of the disease bluetongue virus and epizootic haemorrhagic disease virus history and distribution bluetongue (bt) and epizootic haemorrhagic disease (ehd) are vector-borne infectious diseases caused by bluetongue virus (btv) and epizootic haemorrhagic disease virus (ehdv), respectively. both viruses belong to the genus orbivirus within the family reoviridae. these diseases affect domestic and wild ruminants with variable susceptibility depending on the species involved (duarte et  al. 2001). the first report of bt was published in the early 20th century in africa, but it was only in 1943, when an outbreak occurred in cyprus, that the disease was officially identified on other continents (gibbs and greiner 1994). currently, 1-24 classical and several novel atypical serotypes (which do not cause bt) are described (reviewed in cappai et al. 2019). besides the recent events in europe (reviewed in maclachlan et  al. 2019), bt has been found to circulate widely in africa, the middle east, australia, the south pacific, north and south america, and asia, and it may be present in some regions without causing clinical manifestations (werther and kawanami 2014). epizootic haemorrhagic disease virus was first 98 bt and ehd in wild ruminants mazzoni baldini & nunes de moraes veterinaria italiana 2021, 57 (2), 97-103. doi: 10.12834/vetit.1679.8914.5 susceptible to btv. at least 3,500 animals died during the two outbreaks of btv-17 in wyoming, usa, in 1972 and 1984 (thorne et  al. 1988). gross lesions included hemorrhages and oedema in the pericardial, subepicardial and subendocardial areas on the tunica adventitia of the dorsal aorta and pulmonary artery. hemorrhages were also present in the gastrointestinal tract, lymph nodes, urinary bladder and synovial surfaces of joint capsules (thorne et al. 1988). camelids appear to be resistant to the development of the clinical signs caused by both orbiviruses. batten and colleagues (batten et al. 2011) described an experimental infection with btv-1 in three adult camels. the animals were observed for 75 days. camels did not show clinical signs, however, they seroconverted in approximately 11 days. viraemia was detected at 7 dpi, and replication magnitude of the virus was lower in the experimentally infected camels than that observed in sheep. the same study revealed for the first time that btv could be isolated from the blood of infected camels with potential epidemiological consequences. several studies described btv seropositive camels in india, morocco and tunisia (chandel et  al. 2003, touil et  al. 2012, lorusso et  al. 2016, hassine et  al. 2017) and seropositive dromedaries were also found in the united arab emirates (wernery et  al. 2013). also llamas (lama glama) and alpacas (vicugna pacos) experimentally infected with btv did not show clinical signs. furthermore, viral rna levels in the blood of the infected animals were low and disappeared soon after seroconversion (schulz et al. 2011). despite the results of the previous study, btv was detected in the spleen of a 15-year-old female alpaca that died after weakness, recumbency and respiratory distress. the necropsy revealed hydrothorax, hydropericardium, marked pulmonary oedema, and acute superficial myocardial haemorrhage affecting the left ventricle. to the author’s knowledge this was the first report of lethal bt in a camelid in the americas (ortega et al. 2010). there is no report of ehdv causing clinical signs in south american camelids. wild sheep are susceptible to bt. in 2007, several european mouflons (ovis aries musimon) from a game reserve in spain developed clinical signs and died after a natural infection with btv-1. the pathological lesions were inflammation of the mucous membranes, congestion, swelling and haemorrhage, which suggests that, like domestic sheep, this species is highly susceptible to the virus (fernández-pacheco et  al. 2008). the susceptibility of bighorn sheep (ovis canadensis) was also suggested during an outbreak of btv-17 in 1991, when 13 sheep showed clinical signs of bt and died (singer et al. 1998). was reported in cattle than in sheep during the btv-8 outbreaks in northern europe. the clinical signs observed include loss of body condition, hyperthermia, nasal discharge and ulcers on the oral mucosa (thiry et al. 2006). information on the impact of btv and ehdv in wildlife in south america is rarely reported. while some studies indicate seroprevalence in wild populations and captive animals, information about circulating serotypes, distribution and species affected is extremely rare. therefore, the aim of this study is to review the information currently available on btv and ehdv in different species of wildlife in south america with the emphasis on clinical and pathological manifestations caused by these viruses. clinical and pathological manifestations in wildlife species vosdingh and colleagues (vosdingh et  al. 1968) 10  white-tailed deer (odocoileus virginianus) were experimentally expose to the california btv8 strain. all infected animals developed clinical signs. infection was fatal for all of the seven fawns and an adult female. the clinical signs observed in the fawns included: increase in temperature, anorexia, weakness, bloody diarrhoea and cyanotic tongues. gross lesions such as subendocardial haemorrhage, enteritis and haemorrhage in the tongue were present in most of the animals. histopathology revealed congestion haemorrhage, necrosis, thrombosis, and the most frequently affected organs were the tongue, heart, spleen, kidneys and lymph nodes. the histological lesions in deer resembled the lesions seen in infected sheep, and the major differences between these species appear to be the absence of extensive buccal erosions and foot lesions and a greater tendency for vascular thrombosis in deer (karstadt and trainert 1967). contrarily to the findings in white-tailed deer, experimental infections with btv and ehdv in black-tailed deer (odocoileus hemionus columbianus) were unable to cause clinical disease other than elevated temperatures (work et al. 1992). european red-deer (cervus elaphus) do not develop clinical signs after experimental infection with btv serotype 1 (btv-1) and btv serotype 8 (btv-8). the authors of this study suggested that red-deer may act as a carrier host for btv, maintaining the virus for long periods as rna was detected in the blood until the end of the experiment at 112 days post infection (dpi) (lópez-olivera et al. 2010). american bisons (bison bison) also do not show clinical signs after infection with btv (tessaro and clavijo 2001). pronghorn (antilocapra americana) is also 99 mazzoni baldini & nunes de moraes bt and ehd in wild ruminants veterinaria italiana 2021, 57 (2), 97-103. doi: 10.12834/vetit.1679.8914.5 several wildlife species. during an ehdv-2 outbreak in colorado-usa, which caused the death of two white-tailed deers, other species maintained in the same facility, including bisons, elks (cervus canadensis), domestic cattle, and domestic goats did not show clinical signs (nol et  al. 2010). however, disease and death were observed in yaks (bos grunniens) after natural infection by ehdv-2. clinical signs included anorexia, nasal discharge, conjunctivae and sores on the dental pad. necropsy revealed exudate from the nares, the conjunctivae were oedematous, were ulcerated areas on the dental pad and under the tongue. multiple petechiae were present on the serosal surface of the rumen, epicardial surface of the heart, papillary muscles, and the pulmonary artery, and serosanguineous fluid was found in the abdomen and thoracic cavities (campen et al. 2013). elk experimentally infected with ehdv did not manifest clinical signs of infection (hoff and trainer 1973). when experimentally infected with ehdv, white-tailed deer demonstrated petechial and ecchymotic haemorrhages, and the peritoneal cavity contained clear straw-coloured or slightly blood-tinged fluid. petechial and ecchymosis were also found on the serosal surfaces of the stomachs and intestines and haemorrhage and congestion were present in several organs such as liver, lungs and lymph nodes. histopathology demonstrated extravasated red blood cells in the parenchyma of the spleen, stomachs, heart musculature, and lymph nodes (shope et  al. 1960). given variable results on the susceptibility of white-tailed deer to ehdv, gaydos (gaydos 2002) measured the immune response and clinical signs in two subspecies, odocoileus virginianus borealis and odocoileus virginianus texanus experimentally infected with ehdv-2. the virus caused severe clinical disease with high mortality in o. virginianus borealis fawns, whereas in o. virginianus texanus fawns the disease was mild or non-detectable. despite the difference in manifestation of the disease, the viral titers and humoral immune response were similar in both subspecies. the results suggest that differences may be explained by innate disease resistance of o. virginianus texanus, which occurs in southern areas, such as texas, oklahoma, new mexico colorado and kansas, that are known to be endemic for the ehdv. bluetongue and epizootic haemorrhagic disease in south american wildlife free‑living wildlife animals the studies involving free-living animals in south despite the absence of clinical signs, several african carnivores were demonstrated to have antibodies to btv, including african wild dogs (lycaon pictus), jackals (canis spp.), cheetahs (acinonyx jubatus), lions (panthera leo), spotted hyenas (crocuta crocuta) and genets (genetta maculata) (alexander et  al. 1994). domestic cats and dogs also demonstrated to have detectable antibodies (alexander et  al. 1994, oura and harrak 2010). another study, conducted by jauniaux and colleagues (jauniaux et  al. 2008), reported a natural infection caused by btv-8 in lynx (lynx lynx) kept in a zoo in belgium. the outbreak led to the death of two animals. the lynx had been fed with ruminant fetuses from an area in which bt cases had been lately confirmed. necropsy findings were anemia, subcutaneous hematomas, petechial haemorrhage, pneumonia besides lung congestion and oedema. microscopic examination showed oedematous vascular walls, enlarged endothelial cells, and vasculitis in muscle, myocardium, peritoneum, and lung. the infection of carnivores with btv suggests that the number of natural btv hosts may be much larger than previously supposed. the possibility that the two lynx were infected by vectors cannot be ruled out, but this case strongly suggests that btv may also be transmitted by oral route to carnivores, while the development of clinical signs brings concern upon the extent of the disease in wildlife populations and questions upon the role of these species in the transmission and maintenance dynamics of btv. a study evaluated the presence of antibodies to btv in 187 domestic dogs from morocco finding a prevalence of 21%. as these dogs were consumed canned food only, and had no access to other meat products, it was suggested that the most likely source of infection was through infected culicoides midges (oura and harrak 2011). btv and ehdv are closely related to the african horse sickness virus which is known to infect carnivores, such as domestic dogs, hyenas, lions, jackals, cheetah and genets (alexander et  al. 1995). similar to what was discussed above about btv, a case of african horse sickness was reported in a domestic dog that died after a period of illness without apparent ingestion of horse meat (van sittert et  al. 2013). another study had reported that culicoides impunctatus from scotland had a blood meal on dogs, although less frequently that on other hosts, even when present in the same site as a rumimant host (blackwell, et  al. 1995). however, more studies are required to elucidate this question about possible btv transmission from carnivore species. in south america, the most abundant vector of btv is culicoides insignis although culicoides pusillus has been described as the main biological vector of orbiviruses in central america (mo et al. 1994). infections caused by ehdv are also reported in 100 bt and ehd in wild ruminants mazzoni baldini & nunes de moraes veterinaria italiana 2021, 57 (2), 97-103. doi: 10.12834/vetit.1679.8914.5 table i. bluetongue and epizootic haemorrhagic disease serological studies in free‑living wildlife in south america. country region specie number of animals btv ehdv reference brazil porto primavera ‑ sp blastocerus dichotomus 81 88% 74% pandolfi et al. (1998b) brazil pantanal ‑ ms ozotoceros bezoarticus 49 0% tomich et al. (2009) argentina buenos aires ozotoceros bezoarticus 7 0% 0% uhart et al. (2003) bolivia gran chaco mazama gouazoubira 15 0% 7% deem et al. (2004) argentina chubut lama guanicoe 20 0% karesh et al. (1998) argentina cieneguillas vicugna vicugna 128 0% marcoppido et al. (2010) brazil porto primavera ‑ sp pecari tajacu 49 *39 gerber et al. (2012) peru madre de dios pecari tajacu 106 7.5% **29% rivera et al. (2013) brazil pontal do paranapanema ‑ sp tapirus terrestris 35 14.29% may‑júnior (2011) *positive to orbivirus (btv/ ehdv); **orbivirus of the same serogroup, possibly ehdv another orbivirus of the same serogroup, possible ehdv (rivera et al. 2013). in buenos aires, argentina, seven pampas deer (ozotoceros bezoarticus) tested negative to btv and ehdv antibodies (uhart et  al. 2003). forty-nine animals of this species tested negative to btv in pantanal, brazil (tomich et al. 2009). the gray brocket deer (mazama gouazoubira), in gran chaco bolivia showed 0% and 7% of btv and ehdv seropositivity, respectively (deem et al. 2004). a serosurveillance study conducted on guanacos america are based on serological techniques. during the flooding of the porto primavera hydroelectric dam in brazil’s são paulo state, marsh-deer (blastocerus dichotomus) were captured and studied. a total of 81 individuals were tested for antibodies to btv and ehdv. a seroprevalence of 88% and 74% to btv and ehdv, respectively, was reported (pandolfi et  al. 1998a). in the same area, another study was carried out on peccaries (pecari tajacu) reporting a seroprevalence of 39% to btv (gerber et  al. 2012). the seroprevalence of peccaries in madre de dios, peru, was reported to be 7.5% for btv and 29.2% for figure 1. studies involving bluetongue virus (btv) and epizootic hemorrhagic disease virus (ehdv) in wildlife in south america. n 0 1,000 2,000 km ehdv isolation btv isolation btv positive rt‑pcr / btv and ehdv positive serology btv negative serology btv and ehdv negative serology btv positive serology btv and ehdv positive serology ehdv positive serology 101 mazzoni baldini & nunes de moraes bt and ehd in wild ruminants veterinaria italiana 2021, 57 (2), 97-103. doi: 10.12834/vetit.1679.8914.5 gaps and considerations for the future the role of wildlife species in the epidemiology of bt and ehd in south america appears to be variable. some species are extremely susceptible while others are less and some others may act only as maintenance hosts. it is of great importance to understand how wildlife species respond to btv and ehdv since this may have a considerable economic impact if wildlife species are serving as spillover source for domestic species. these viruses also impact the conservation of wildlife such as some species of deer and other ruminants like pronghorns (antilocapra americana). as already written, btv and ehdv are closely related to the african horse sickness virus, which is known to affect carnivores (lubroth 1992). reports of seropositive canids and felids in addition to the isolation of btv from a lynx suggest that the range of hosts could be much wider than previously suggested. only few studies on ehd and bt epidemiology and clinical outcomes in free-living deer and other species are available from south america. however, evidence suggests that endangered species such as the brazilian dwarf brocket deer may show high mortality related to the haemorrhagic manifestations of the disease. unfortunately, the dense forest present in some south america biomes, and the shy behavior of most deer, hamper the visualization of clinical signs and the evaluation of diseases. indeed, these animals are rarely captured to evaluate their health status. south american camelids do not appear to play an important role in the epidemiology of btv, possibly due to their innate resistance and the adverse climate conditions of their habitat for the survival of vector species. south america has a colossal biodiversity, unique biomes and in most parts of its territory btv and ehdv are endemic. more studies are necessary to identify vectors responsible for the transmission of btv and ehdv in this continent and to better comprehend the additional potential host species and the epidemiological role they play in the maintenance and spread of these viruses. acknowledgements we are very thankful to m.sc pedro henrique de farias peres for his help with the elaboration of the map. (lama guanicoe) and vicugnas (vicugna vicugna), from argentina, demonstrated that these animals were negative for btv antibodies (karesh et al.1998, marcoppido et al. 2010). in pontal do paranapanema, in são paulo brazil, 35 tapires (tapirus terrestres) were tested for btv and 14.29% of the animals showed antibodies to the virus (may-júnior 2011). studies involving free-living wildlife in south america are summarized in figure 1 and in table i. wildlife in captivity the seroprevalence of ehdv and btv in brazilian deer belonging to a scientific breeding center in são paulo state, brazil, was investigated. out of 22  animals, 23% presented antibodies to btv and 9% to ehdv (pandolfi 1998b). a retrospective study performed in the same institution by kawanami and colleagues (kawanami et  al. 2018) analyzed paraffin samples of organs from 42 animals that died showing clinical signs of haemorrhagic disease. these animals were marsh-deer (blastocerus dichotomus) and brocket-deer (genus mazama). seven of the 42  animals tested positive to btv rna presented clinical signs that included loss of appetite, lethargy, lesions on the tongue or mouth, diarrhoea or soft faeces, emaciation, drooling, and oedema of the head. the most relevant macroscopic findings observed were haemorrhagic intestinal contents, petechiae in organs such as the heart, tongue and stomachs, reddish gastrointestinal mucosa and necrosis/ulceration in the mouth or tongue. in 2008 ehdv was isolated in cell culture from a brazilian dwarf brocket deer (mazama nana) kept in a zoo in south brazil. this animal died and necropsy revealed hemorrhages in several organs. this was the first isolation of ehdv ever recorded in brazil (favero et al. 2013). another study identified btv as most likely cause of death of deer from a conservation center in south brazil. this shelter has been suffering from outbreaks of haemorrhagic disease leading to the death of several deer for a period of 15 years (baldini et  al. 2018). five btv serotypes (btv-3, btv-14, btv-18, btv-19 and btv-22) were isolated from samples collected from five brazilian dwarf brocket deer that had died in recent years (oie 2018). none of these serotypes had previously been recorded in brazil. interestingly, from only one out of 32 animals was possible to evidence btv antibodies. this aspect suggests that this species is extremely susceptible to this disease (baldini et al. 2018). 102 bt and ehd in wild ruminants mazzoni baldini & nunes de moraes veterinaria italiana 2021, 57 (2), 97-103. doi: 10.12834/vetit.1679.8914.5 alexander k.a., maclachlan n.j., kat p.w., house c., o'brien s.j., lerche, n.w., sawyer m., frank l.g., holekamp k., smale l., mcnutt j.w., laurenson m.k., mills m.g.l. & osburn a.i. 1994. evidence of natural bluetongue virus infection among african carnivores. am j trop med hyg, 51 (5), 568-576. alfieri a.a., alfieri a.f., takiuchi e. & lobato z.i.p. 2007. reoviridae. in virologia veterinária (e.f. flores, ed) 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veterinary medicine vienna, 1210 vienna, austria. 2institute of microbiology, department for pathobiology, university of veterinary medicine vienna, veterinärplatz 1, 1210 vienna, austria. 3bioinformatics and biostatistics platform, department for biomedical sciences, university of veterinary medicine vienna, veterinärplatz 1, 1210 vienna, austria. 4veterinary health service tyrol, wilhelm-greil-straße 17, 6020 innsbruck, austria. 5regional veterinary office tyrol, wilhelm-greil-straße 17, 6020 innsbruck, austria. *corresponding author at: university clinic for ruminants, department for farm animals and veterinary public health, university of veterinary medicine vienna, 1210 vienna, austria. e-mail: johannes.khol@vetmeduni.ac.at. sophie gschaider1, judith köchler1, joachim spergser2, alexander tichy3, christian mader4, matthias vill5, paul ortner5, josef kössler5 and johannes lorenz khol1* keywords boot swab, cattle, faecal shedding, johne’s disease, mycobacterium avium subsp. paratuberculosis. summary individual faecal samples were collected from adult animals in 275 cattle farms previously positive for mycobacterium avium subsp. paratuberculosis (map). in addition, boot swab samples were collected in 30 randomly chosen farms. faecal samples were tested for map by a combination of bacterial culture and pcr. a logistic regression and the pearson correlation were used to calculate the relation between the number of map-positive cows and boot swab results. in 66.9% of all previously tested herds, no positive individual faecal sample was detected, indicating possible fadeout of the infection. in 9 (30.0%) of the 30 selected farms, at least one map-shedding animal was detected in faecal samples individually collected, while only 5 (16.7%) of these farms were found positive when the boot sampling method was used. the sensitivity of the boot swab sampling increased up to 92% (95% ci: 41%-99%), if at least 12 animals were faecal map-shedders in a herd. the current study shows possible fadeout of jd in a substantial percentage of previously infected herds. furthermore, in small herds, a relatively high within-herd prevalence of map-shedding animals is needed to assure reliable positive boot swab results. individual faecal and boot swab sampling to determine john's disease status in small cattle herds signs of jd, such as watery diarrhoea and weight loss despite normal appetite, is quite variable, they usually do not become evident in cattle younger than 2 years of age (sweeney 1996). the disease is incurable (fecteau and whitlock 2011). vaccines, which are available in some countries, contribute to reduce production losses. map infections in cattle are emerging in most parts of the world. in europe, up to 68% of cattle herds were found map positive, based on the available data (nielsen and toft 2009). in austria, the herd prevalence was 19.1% (baumgartner et al. 2005). the elisa is the most commonly indirect detection method used for jd today (gilardoni et  al. 2012). considering its low sensitivity, this method is not appropriate for either detecting the early stages of introduction johne’s disease (jd), which is also called paratuberculosis, is caused by mycobacterium avium ssp. paratuberculosis (map; sweeney 1996). jd is a chronic infection in cattle, usually transmitted via the faecal-oral route within the first months of life. a period of at least two years of latency usually follows the early exposure (sweeney 2011). in this early stage of the infection, neither faecal shedding of map nor specific antibodies (ab) occurs, thereby hampering the detection of infected individuals  (sweeney  1996). as the infection progresses, the faecal shedding of map and the production of specific ab commences, but clinical signs of jd are still absent at this time (sweeney 2011). even though the onset of characteristic clinical 20 map-status in small cattle herds gschaider et al. veterinaria italiana 2021, 57 (1), 19-27. doi: 10.12834/vetit.1389.7584.2 materials and methods background between 2013 and 2014, the veterinary animal health service tyrol and the regional veterinary office conducted an investigation to evaluate the map herd status of cattle farms in the austrian province of tyrol. the purpose of this investigation was to establish a program for the control and eradication of jd in cattle. in the course of this voluntary survey, boot swab samples were collected as previously described (donat et  al. 2016) on 4,679  farms and tested for the presence of map by bacterial culture and pcr. map was detected in 349 (7.5%) boot swab samples (köchler et al. 2017). study population of the map-positive farms detected in the previous survey (2013-2014), 275 farms, voluntarily joined the individual animal testing-program launched by the veterinary health service in autumn 2015, were enrolled in the present study. resampling, as part of the present study, was performed approximately 18  months (min. 13, max. 26 months) after the first boot swab collection. on these farms, individual faecal samples were taken from all animals with a minimum age of 2 years. furthermore, another boot swab sample was collected at the same time from 30 randomly selected farms. these farms were chosen by simple randomisation from the 275 positive farms, but selection of farms had to be modified due to willingness of the owners to participate in the study. this resulted in a total of 3,758 individual faecal samples from 275 farms. an overview of the study population and its origin is given in figure 1. the size of the farms enrolled in the study ranged from one to 82 tested individuals with a minimum age of 2 years. the 275 farms are referred to as “all farms” and had a mean of 14 individuals tested per farm (median 11). the 30 randomly selected herds, called “selected farms”, held a mean of 24  (median  19.5) animals with a minimum age of 2  years. animals tested throughout the study were kept in tight stalls or loose housing systems and pastured throughout the summer. details for the 30 selected farms are given in table ii. collection and testing of boot swab samples boot swab samples, for determining the map-herd status, were collected as previously described (donat et al. 2016). samples for the 2013-2014 study were collected by the local veterinarians, except for the re-evaluation of the 30 randomly chosen farms, map infection in individual animals (diéguez et  al. 2009) or defining a map herd status on cattle farms (donat et al. 2012). bacterial culture and identification is considered the “gold standard” in map diagnostics. it requires from 8 to 20 weeks and can be applied to faecal and tissue samples (gilardoni et al. 2012, whittington 2010). because of the long incubation time of bacterial culture, pcr is increasingly used for map-detection in faecal samples with comparable results concerning sensitivity and specificity. the pcr sensitivity is even improved if applied on faecal samples following enrichment culture (fawzy et  al. 2015). sample pooling with pool size of 10 samples collected either from the animals or from the areas of the stable with high animal density may reduce the costs of map-diagnosis. the sensitivity of pooled samples were reported to range from 48% to 69% (van shaik et  al. 2007, tavorpanich et  al. 2004). a more recent study stated that pool sizes of five or ten faecal samples may reduce costs with an acceptable reduction of the sensitivity (mckenna et  al. 2018). environmental sampling was able to correctly identify 70% of map positive herds, if six samples were collected from different places throughout the farm (wolf et al. 2017). in 2013, boot swab sampling was suggested for the first time as a technique to establish the map-herd status in cattle, with 90.6% of map-infected herds being detected (eisenberg et  al. 2013). disposable cover boots, equipped with an absorbent material on the sole and worn while walking around the animals inside the barn and the milking parlour are just what this sampling method required (eisenberg et  al. 2013). after walking through the herd, the absorbent material soaked with manure is removed and used for map detection by pcr or culture (eisenberg et al. 2013). in a recent study, the sensitivity of boot swabs for map-detection was calculated to be only 43.5% (wolf et al. 2016). another paper showed that the probability for the detection of map-positive farms by boot swabs ranged between 50% and 90%, depending on the intra-herd-prevalence of map (donat et al. 2016). the aim of the present study was to evaluate the development of the map-herd status determined by boot swab samples in small structured cattle herds. furthermore, the association of the boot swab results with the number of animals shedding map with faeces within a herd should be investigated. therefore, individual faecal samples were collected in previously map-boot swab positive cattle farms. in some of these farms, boot swabs were taken for a second time simultaneously in order to reassess the map-herd status. 21 gschaider et al. map-status in small cattle herds veterinaria italiana 2021, 57 (1), 19-27. doi: 10.12834/vetit.1389.7584.2 ludwigsburg, germany) per litre, were inoculated with each sample. all heym media used for the study were prepared as single batch and underwent quality control including control for sterility and prove of map growth by inoculation of 10 media with 100 cfu/ml of map at 37 °c. the heym tubes were then incubated at 37 °c and checked for growth once a week. after 4 weeks of incubation, one of the 4 tubes per sample was rinsed with 200 µl pbs and the fluid was used for the subsequent map detection by real-time pcr. the qiamp dna stool mini kit (qiagen n.v., venlo, netherlands) was used for the extraction of the dna following the user’s manual. subsequently, the map specific sequence element was amplified using the vetmaxtm map real-time pcr screening kit (fisher scientific austria gmbh; vienna, austria), again following the manufacturer’s instructions using a c1000 touch thermal cycler (bio-rad laboratories gmbh, vienna, austria) for the amplification. the 3 remaining tubes were incubated at 37  °c for another 8 weeks and checked for growth of map weekly. if growth of map occurred colonies were sampled by pcr as described above for confirmation and the culture rated as map-positive. culture tubes not showing any map colonies were rated as negative after 12 weeks of incubation. more samples were positive in the combination of culture and pcr than in the culture alone, which is in accordance with the study of köchler and colleagues (köchler et  al. 2017). therefore, results of the described combination of culture and pcr are presented here and were used for statistical evaluation only. collection and testing of individual faecal samples individual faecal samples were taken directly from the rectum, using a new single use plastic glove for each animal. the faeces were put into a sterile plastic container, stored cooled, and sent to the institute of microbiology at the university for veterinary medicine in vienna. for the detection of map, bacterial culture on heym and real-time pcr were performed following the procedure previously used for boot swab samples. statistical analysis statistical analysis of the data was performed using the ibm spss statistics v19 software (ibm österreich internationale büromaschinen gesmbh, vienna, austria). a logistic regression to calculate the relation of the number and percentage of map-positive cows to a negative or positive boot swab result in the 30 randomly selected farms was performed. the boot swab result (positive/negative) was used as dependent and the amount of animals wherein the boot swab samples were collected by the authors of this paper (gschaider and köchler). to avoid contamination, sample takers had to put on single use plastic overshoes first. then, the actual boot swabs (sodibox, névez, france) made out of knitted jersey, were pulled over the bottom of the overshoes. in tie-stalls with cows tethered in rows next to each other, sample takers were instructed to walk through the manure channel while, in free stall barns, samples were collected by walking along the alleyways, including the spaces around feeding or watering devices and the waiting area in front of the milking parlour (eisenberg et al. 2013). to collect a sufficient amount of sampling material, the boot swabs had to be soaked with at least 50 g of manure. after collection, the boot swabs were put into sterile plastic twirl type bags, stored cooled and sent to the institute of microbiology at the university for veterinary medicine in vienna for map-detection. for the detection of map, the boot swab samples were transferred to stomacher® bags (seward ltd., worthing, uk) and then homogenized in a lb 400 circulator (vwr international llc, vienna, austria) for 60 seconds after adding 50 ml of phosphate buffered saline (pbs). following centrifugation at 3,000 x g for 15 minutes, the supernatant was discharged and 3 g of the remaining manure were resuspended with 30 ml of 0.75% hexadecylpyridinium chloride (hpc, sigma aldrich handels gmbh, vienna, austria). thereafter, samples were shaken for 60 minutes and left for 5 minutes for the sedimentation of the large particles afterwards. following sedimentation, 15 ml of the supernatant were filled into a sterile tube and incubated in the dark for 48 hours at room temperature. subsequently, the samples were centrifuged again at 3,000 x g for 15 minutes, the supernatant was discharged and the pellets remixed with 1 ml of 0.75% hpc. four tubes of herrolds egg yolk medium (heym), prepared in house and containing 2 mg of mycobactin j (idexx gmbh, 349 (7.5%) positive boot swab samples 1st sampling 2013/2014 boot swab sampling in 4,679 cattle farms 245 farms individual faecal sampling 3,038 animals > 2 years 30 farms individual faecal sampling 720 animals > 2 years additional boot swab sample 2nd sampling 2015 275 boot swab positive farms figure 1. study population and sampling scheme. 22 map-status in small cattle herds gschaider et al. veterinaria italiana 2021, 57 (1), 19-27. doi: 10.12834/vetit.1389.7584.2 intra-herd-prevalence of cattle shedding the bacterium with their faces in farms with at least one positive animal turned out to be 21.2% (min. 1.2%, max. 75.0%), with a mean samples size of 17 (median 14) tested animals in farms with at least one positive result. the intra-herd-prevalence of map-shedding animals in relation to the herd size is shown in figure 2. four cattle premises turned out to have 50% or more animals shedding map with faeces, but the size of those 4 farms was small with a maximum of 12 animals sampled (figure 2). the highest intra-herd-prevalence of map-shedders found was 75%, but this farm was holding 4 animals above 2 years of age only. altogether, map was detected in individual faecal samples of less than 20% of the individuals tested in more than half of the map-positive cattle premises. the pearson correlation showed a weak negative correlation between the herd size and amount of map-shedding individuals with a correlation coefficient of 0.36. relation of map‑shedding individuals and boot swab result in 9 (30.0%) of the 30 selected farms, in which individual faecal testing as well as boot swab sampling was performed simultaneously, at least one map-shedding individual was found (table  i). in total, 36 (5.0%) animals turned out to be map-shedders, and boot swab samples were shedding map with faeces as independent variable (probability of entry 0.5, removal of 0.10, iterate 20 and cut 0.5). because of the small sample size, leading to a possible bias when referring to the quantity and the percentage of samples, the probability related to the total number of positive individuals was calculated, combining the quantity of map-positive animals with the predicted probability for a positive boot swab result from the logistic regression. additionally, the pearson correlation was calculated to define the relation between the number of map-shedders and the herd size. results detection of map in individual faecal samples no map-positive individual faecal sample was detected in 184 (66.9%) of the previously boot swab positive farms, together holding 3,417 animals with a minimum age of 2 years (table i). altogether, 248 animals (6.6%) originating from 83  (30.2%) different farms were tested positive for map in the individual faecal samples (table  i). relating to map-positive cattle premises only, 17.3% (248 out of 1,430) of cows or heifers tested were shedding map with their faces. the mean table i. detection of mycobacterium avium subsp. paratuberculosis (map) in boot swab samples and individual faecal samples, absolute numbers with the percentage in brackets. positive negative missing total farm breed housing system animals tested1 map positive2 map boot swab 16 rf3 loose housing 17 2 (11.8%) negative 17 as4 tight stall 2 1 (50.0%) negative 18 bs5 tight stall 4 0 (0.0%) negative 24 tg6 loose housing 15 0 (0.0%) negative 53 as tight stall 14 3 (21.4%) positive 55 as loose housing 20 4 (20.0%) positive 56 as tight stall 18 6 (33.3%) positive 57 bs tight stall 17 3 (17.6%) negative 58 bs loose housing 34 12 (35.3%) positive 88 fv loose housing 29 4 (13.8%) positive 89 fv tight stall 19 0 (0.0%) negative 109 as tight stall 14 0 (0.0%) negative all farms farms 83 (30.2) 184 (66.9) 8 (2.9) 275 (100) individuals 248 (6.6) 3,417 (90.9) 93 (2.5) 3,758 (100) selected farms farms 9 (30.0) 21 (70.0) 0 (0) 30 (100) individuals 36 (5.0) 684 (95.0) 0 (0) 720 (100) boot swab samples 5 (16.7) 25 (83.3) 0(0) 30 (100) 1number of cattle with a minimum age of 2 years, included in the study; 2number of animals with mycobacterium avium subsp. paratuberculosis-positive faecal samples with percentage in brackets; 3red frisian; 4austrian simmental; 5brown swiss; 6tyrolean grey cattle. 23 gschaider et al. map-status in small cattle herds veterinaria italiana 2021, 57 (1), 19-27. doi: 10.12834/vetit.1389.7584.2 table ii. results of mycobacterium avium subsp. paratuberculosis (map) detection in individual and boot swab samples in selected farms. farm breed housing system animals tested5 map positive6 map boot swab7 16 rf1 loose housing 17 2 (11.8%) negative 17 as2 tight stall 2 1 (50.0%) negative 18 bs3 tight stall 4 0 (0.0%) negative 24 tg4 loose housing 15 0 (0.0%) negative 53 as tight stall 14 3 (21.4%) positive 55 as loose housing 20 4 (20.0%) positive 56 as tight stall 18 6 (33.3%) positive 57 bs tight stall 17 3 (17.6%) negative 58 bs loose housing 34 12 (35.3%) positive 88 fv loose housing 29 4 (13.8%) positive 89 fv tight stall 19 0 (0.0%) negative 109 as tight stall 14 0 (0.0%) negative 111 as loose housing 29 0 (0.0%) negative 112 as tight stall 30 0 (0.0%) negative 114 as loose housing 31 0 (0.0%) negative 115 as loose housing 82 1 (1.2%) negative 123 as tight stall 27 0 (0.0%) negative 126 as tight stall 23 0 (0.0%) negative 130 as tight stall 32 0 (0.0%) negative 131 as tight stall 37 0 (0.0%) negative 137 bs loose housing 17 0 (0.0%) negative 138 bs loose housing 30 0 (0.0%) negative 163 bs tight stall 15 0 (0.0%) negative 165 tg tight stall 7 0 (0.0%) negative 166 tg tight stall 4 0 (0.0%) negative 167 as tight stall 3 0 (0.0%) negative 168 tg tight stall 3 0 (0.0%) negative 170 bs loose housing 22 0 (0.0%) negative 207 as loose housing 44 0 (0.0%) negative 239 rfxas loose housing 81 0 (0.0%) negative 1red friesian; 2austrian simmental; 3brown swiss; 4tyrolese gray cattle; 5number of cattle with a minimum age of 2 years, included in the study; 6number of animals with mycobacterium avium subsp. paratuberculosis-positive faecal samples with percentage in brackets; 7result of boot swab testing for mycobacterium avium subsp. paratuberculosis. these farms. marcé and colleagues (marcé et  al. 2011) observed spontaneous fadeout of jd in 43% of cattle herds within 2 years after map introduction. altogether, the infection was not detected in 66% of herds within several years in this model. in this study, it was furthermore shown, that more farms remained infected, if animals with clinical jd stayed in the herd for a prolonged time and map removal from the environment was reduced (marcé et al. 2011). most of the tyrolean cattle herds spend at least two months on alpine pastures during the summer. cows in weak condition might be slaughtered before positive in 5 (16.7%) of these farms (table ii). there was no farm without a map shedding animal giving a positive boot swab result, but 4 premises, with at least one individual animal shedding the bacterium, showed a map-negative boot swab sample. the detailed results of the selected farms are shown in table ii. the calculation of the logistic regression revealed that the probability of obtaining a map-positive result in the boot swab sample depends on the within-herd prevalence of animals shedding map with their faeces (figure 3a). as sample size, which was small in this study, can produce a bias referring to the quantity and the percentage of positive samples, the probability related to the total number of positive individuals is shown in figure  3b. when the number of positive animals was combined with the predicted probability for a boot swab to be positive, (figure 3a), the sensitivity of the map-detection methods was 48% if there are 6 map shedding animals in a herd and 92% if the map shedding animals are 12 (figure 3b). as seen in figure 3b, the probability of a positive boot swab sample drops to 28% (95% ci: 4%-79%) if there are 4 map-shedding animals in a herd. discussion all of the farms included in the present study had shown a positive boot swab result for  map  before  (2013  or  2014). about 18 months later, map could not be detected in individual faecal samples of any of the adult cattle in two third of these farms. although it has to be considered that map is not shed continuously in the faeces of animals with subclinical  jd (mitchell et  al. 2015), this result may point to a possible change in the map-status of number of animals in the herd > 2 years of age 0 0 10 20 30 40 50 60 70 80 20 40 60 80 100m a p ‑p o si ti ve in d iv id u al fa ec al s am p le s (% ) figure 2. relation between the within-herd prevalence of mapshedding individuals and the herd size. 24 map-status in small cattle herds gschaider et al. veterinaria italiana 2021, 57 (1), 19-27. doi: 10.12834/vetit.1389.7584.2 contributes to the reduction of map contamination of the environment and, thereby, to the fadeout of the infection. further studies, including a follow up of the farms enrolled in the present study, are needed to elucidate possible spontaneous fadeout of map in cattle herds. a negative correlation exists between the herd size and the number of map-shedding individuals. the mean within herd prevalence of map-shedding animals in the present study turned out to be 21.2%. farms with a maximum of 15 animals showed a mean within herd prevalence of map-shedding of 24.6%, whereas it was 16.8% in herds with 16 to 82 animals only. this finding is in contradiction to the literature, where an increasing herd size has been reported to correlate with a higher map within-herd prevalence in infected cattle farms (hirst et  al. 2004, muskens et al. 2003). one possible explanation for this could be the close relationships of the individuals in small structured cattle herds. the farmers often keep the female offspring of a cow over several years for breeding, if it is believed to be of high genetic value. therefore, if this cow is map positive, there is an increased chance that its offspring is infected with map as well, as map infection can occur in utero, via colostrum or via manure-contaminated teats (sweeney 1996). the size of the cattle herds in the part of austria where the study was performed is quite small compared to dairy cow premises worldwide. the mean herd size in our study was 14 sampled animals with a minimum age of 2 years per herd only. in the german federal states thuringia, hesse, and saxony, boot swab sampling was performed in 77  cattle herds with a known jd status and an average sample size of 272  animals per herd (donat  et  al.  2016). in that study, it was shown that the map within-herd prevalence had to be at least 2.39% to obtain a positive boot swab result with a probability of 50% (donat et  al. 2016). for the probability of positive boot swab results to be raised to 90%, the within-herd prevalence of animals shedding map with their faeces had to be at least 5.85%, when the samples were tested by faecal culture and pcr simultaneously (donat et al. 2016). our study on the tyrolean cattle premises showed that the within-herd prevalence of animals shedding map had to exceed 25% to obtain a probability of at least 50% for a positive boot swab result. this is a markedly higher percentage than found in the aforementioned study (donat et  al. 2016), but interpretation of the results of the present study is hampered by the small sample size, questioning the relevance of the results. importantly, only 25 of the 83 positive farms held more than 25% of the animals shedding map with their faeces in our study population. in relation to the total number of map positive individuals in a herd, statistical calculations in our study revealed pasturing, and map-positive animals likely removed from the herd. during this time, barns are also usually thoroughly cleaned with a high pressure cleaner and left empty until the animals return in autumn. this management practice may lead to a significant reduction of map in the stable and could, therefore, contribute to possible fadeout of the infection, as evidenced in the present study. however, map-dna was detected in the environmental samples after the complete destocking of a herd with a known history of clinical paratuberculosis as well as 24 months after cleaning and disinfection (moravkova et  al. 2012). clinical jd is a notifiable disease in austria and affected cattle have to be culled within 3 days after confirmation of a map-infection (khol et  al. 2007). this timely removal of clinically ill animals p ro b ab ili ty o f m a p ‑p o si ti ve b o o t sw ab r es u lt p ro b ab ili ty o f m a p ‑p o si ti ve b o o t sw ab r es u lt fr o m f ig . 3 a map intra herd prevalence in % map‑positive animals within the herd 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 5040 tested logistic 3020100 121086420 a b figure 3. logistic regressions for mycobacterium avium subsp. paratuberculosis (map) positive boot swab results depending on the within-herd prevalence of map-shedding individuals. a. probability of obtaining a map-positive result in the boot swab sample depending on the within-herd prevalence. b. probability of obtaining a map-positive boot swab sample as calculated in figure 3a, related to the total number of map-positive animals (test results and logistic regression). 25 gschaider et al. map-status in small cattle herds veterinaria italiana 2021, 57 (1), 19-27. doi: 10.12834/vetit.1389.7584.2 sting et al. 2014). unfortunately, no data concerning the overall test performances for the combination of protocols for dna extraction and pcr used in the present study are available. although this should be considered when interpreting the results, it can be assumed to be of minor importance, as in this and in the 2013-2014 surveys, samples were tested in the same laboratory using the same protocols. the results of the present study indicate that examination of individual faecal samples is more sensitive than boot swabs for detecting map positive herds, as 4 out of 9 farms showed positive individual faecal results and were boot swab negative (table ii). individual faecal sampling is not an option to establish the map-herd status in large herds because of financial and management issues, although it has been shown that may reduce costs with an acceptable reduction of sensitivity (mckenna et  al. 2018). nevertheless, in small premises, holding a few adult cattle only, individual faecal samplings might give better results compared to boot swab sampling because of a possible low sensitivity of the latter, with a reasonable increase of costs and workload. boot swabs are a suitable sampling method for defining the map-herd status in cattle, as they are quick and easy to perform as well as cost effective (eisenberg et al. 2013, wolf et al. 2016). the sensitivity of boot swab samples for map assessement depends on the within-herd prevalence (donat et  al. 2016) and the total amount of animals shedding map with their faeces. nevertheless, results from the present study indicate that boot swab sampling have to be used with caution in smaller herds, as a relatively high within-herd prevalence of map-shedding animals is needed to assure reliable results. to avoid false negative results, repeated sampling should be applied. due to the long incubation period and chronic nature of jd in cattle with a late onset of faecal map-shedding, as well as intermittent shedding of the bacteria, repeated faecal sampling increases the probability to detect infected individuals (gilardoni et  al. 2012). consequently, repeated sampling also increases the chance to detect map-positive herds, by both environmental and boot swab sampling (eisenberg et al. 2013, khol et al. 2009). furthermore, combining boot swab with the slurry tank sampling for map as suggested before (donat et  al. 2016) might increase the sensitivity. according to the results of this study, on very small premises, holding a few adult cattle only, individual faecal samplings should be considered instead of boot swab sampling. further studies, including larger sample sizes and long-term investigations, are needed to elucidate the use of boot swab samples for map-detection in small cattle herds and possible spontaneous fadeout of the disease. that the boot swab sample will be map positive with a probability near 90% when there are at least 12 map-shedding animals in a herd, irrespective of the herd size. if there are at least 6 map-shedding individuals, the calculated probability of a positive boot swab result drops to 50%. because of the small number of positive farms detected in the present study 95% confidence intervals are rather wide, which has to be considered when interpreting the results. nevertheless, the calculated within-herd prevalence of cattle shedding map in their faeces must be 10 times higher in our study than in the aforementioned one (donat et  al. 2016) in order to achieve a positive boot swab sample with a probability of 50%. the reason for this marked difference remains unknown, but the small size of the herds included in our study may contribute to this finding. the housing conditions could possibly also influence the results of the boot swab samples. map-shedding cows in tethered stalls may lead to a smaller contamination of the environment than animals in a free stall which are able to move around. because of the relatively low number of boot swab positive farms in the present study, a comparison of the two housing systems was not performed and warrants further investigations. furthermore, it has been shown, that bedding material, such a straw as well as low outside temperatures are able to hamper the detection of map in environmental samples (wolf et al. 2017). overall, due to the small sample size and corresponding to the fact that only 16.7% of the farms turned out to be map positive by boot swab sampling, the conclusions of the present study must be interpreted with caution. a prior study reported a rather low sensitivity of boot swap samples to detect map-positive cattle herds of 43.5% only (wolf et  al. 2016), thus questioning their application for assessment of the map-herd status. possible methodological deficiencies of the sampling procedure applied in our study, related to either the procedure itself or the sampling person should be considered. comparison of the results of the present study to the literature also is hampered by the different detection methods applied. a test protocol combining solid culture and pcr was used in our study, while map was detected by 3 different pcr protocols by donat and colleagues (donat et al. 2016) and by liquid culture in the investigation of wolf and colleagues (wolf et  al. 2016). it has been shown, that the combination of bacterial culture for the enrichment of map, followed by pcr has a high sensitivity for the detection of map (fawzy et  al. 2015). then again previous studies showed that the method of dna extraction is crucial for the success of map detection in faecal samples and differs between different protocols (fernando et  al. 2013, 26 map-status in small cattle herds gschaider et al. veterinaria italiana 2021, 57 (1), 19-27. doi: 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p. 2017. detection of mycobacterium avium subspecies paratuberculosis infected cattle herds using environmental samples: a review. berl münch tierärztl wochenschr, 130, 4-12. whittington r.j. cultivation of mycobacterium avium subsp. paratuberculosis. 2010. in paratuberculosis: organism, disease, control (d.m. collins & m.a. behr, eds). cab international, wallingford, oxfordshire, cambridge, 244-266. wolf r., orsel k., de buck j., kanevets u. & barkema h.w. 2016. short communication: evaluation of sampling socks for 179 short communication riassunto il morbillivirus felino (femv) è un nuovo paramyxovirus rilevato nei gatti. femv è sospettato di essere associato a nefrite tubulointerstiziale, ma il suo ruolo patogenetico non è stato ancora ben compreso. in questa breve comunicazione sono riportate le sequenze dell'intero genoma dei primi due ceppi femv isolati in italia. isolamento e sequenziamento del genoma di due ceppi di feline morbillivirus genotipo 1 parole chiave feline morbillivirus, isolamento, sequenziamento. keywords feline morbillivirus, isolation, sequencing. the genus morbillivirus includes several enveloped negative‑sense single‑stranded rna viruses infecting humans and animals. feline morbillivirus (femv) is a novel morbillivirus infecting cats and first described in stray cats from hong kong nearly ten years ago (woo et  al. 2012). soon after, femv circulation was detected worldwide (furuya et  al. 2014, park et al. 2014, sakaguchi et al. 2014, lorusso et al. 2015, sieg et al. 2015, sharp et al. 2016, yilmaz et al. 2017, darold et al. 2017). femv isolation on cell culture has been described to be difficult and time consuming (sakaguchi et  al. 2014) and a limited number of viral isolates and related whole genome sequences are, indeed, veterinaria italiana 2019, 55 (2), 179‑182. doi: 10.12834/vetit.1847.9883.1 accepted: 06.06.2019 | available on line: 30.06.2019 summary feline morbillivirus (femv) is a novel viral paramyxovirus detected in cats. femv is suspected to be associated to tubulointerstitial nephritis, but its pathogenic role is far to be clearly understood. in this short communication, we report the whole genome coding sequences of the first two femv strains isolated in italy. #the authors equally contributed to this manuscript 1dipartimento di scienze veterinarie, università di messina, italy. 2istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo italy. 3faculty of veterinary medicine, veterinary teaching hospital, university of teramo, teramo, italy. 4national reference center for whole genome sequencing of microbial pathogens: database and bioinformatic analysis, istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo italy. * corresponding author at: national reference center for whole genome sequencing of microbial pathogens: database and bioinformatic analysis, istituto zooprofilattico sperimentale dell'abruzzo e del molise 'g. caporale', campo boario, 64100, teramo italy. tel.: +39 0861 332440, fax: +39 0861 332251, e-mail: a.lorusso@izs.it. giulia donato1#, eliana de luca2#, paolo emidio crisi3, federica pizzurro2, maria masucci1, maurilia marcacci4, francesca cito2, daria di sabatino2, andrea boari3, nicola d’alterio2, maria grazia pennisi1 and alessio lorusso4* isolation and genome sequences of two feline morbillivirus genotype 1 strains from italy publicly available. here, we describe the complete genome coding sequences of two femv isolates from italy. urine samples were taken in march 2018 from two male cats (tremedino and pepito, 1 and 7 year old, respectively), living in reggio calabria (calabria region, southern italy). the two cats did not show clinical and laboratory signs of renal damage (donato, manuscript in preparation). briefly, the first cat (tremedino) showed a good body condition (bcs  3/5), stomatitis and an enlargement of popliteal and submandibular lymph nodes, whereas the second cat (pepito) was overweight (bcs  4/5) with signs compatible with stomatitis and bilateral otitis. in both cats, no abnormalities were recorded during kidneys 180 veterinaria italiana 2019, 55 (2), 179‑182. doi: 10.12834/vetit.1847.9883.1 isolation and characterization of femv donato et al. cells were incubated at 37  °c in a humidified atmosphere with 5% of co 2 and observed daily for cytophatic effect by microscopy. at the 1st cell passage, syncytia were evident at day 8. cells were stained by may grunwald‑giemsa (figure  1a). rna was purified (qiaamp® viral rna) from 140 µl of cell culture supernatants and tested by real time rt‑pcr for femv (c q 26 and 23 for tremedino and pepito, respectively). fea cells that tested positive by real time rt‑pcr were also fixed in chilled acetone at ‑ 20 °c for 20 min. fixed cells were incubated with 1:100 dilution of rabbit polyclonal antibody against the n protein of femv (kindly provided by dr shigeru morikawa, national institute of infectious diseases, tokyo), followed by incubation with a fitc‑goat anti‑rabbit igg (sigma aldrich) 1:32 diluted. cells were then examined under a fluorescence microscope and imaged using the leica tcs sp5 ii confocal laser scanning microscope. uninfected fea cells were used as negative control. infected cells tested positive for femv (figure 1b). isolates were named femv tremedino/2018 italy and femv pepito 2018/italy, further passaged and stored at ‑  80°. total rna was purified from 300  µl of supernatant of the first passage by using the qiaamp viral rna minikit (qiagen). sequencing was performed by using a combination of sequence‑independent/single‑primer amplification (sispa) and next generation sequencing (ngs) as previously described (marcacci et  al. 2015). library preparation was carried out by using the nextera xt library prep kit (illumina inc.) according to the manufacturer’s protocol. sequencing was performed on the nextseq 500 (illumina inc., san diego,ca) using the nextseq 500/550 mid output reagent cartridge v2, 300 cycles and standard palpation. in the first cat, the haemato‑biochemical profile indicated mild eosinophilia [2.85 k/µl, reference interval  (ri)  =  0.17‑1.57 k/µl], and severe thrombocytopenia (platelet count 20 k/ µl, ri  =  300‑700  k/µl; low platelet estimate). creatinine was 1.1  mg/dl (ri  =  0.8‑2.4 mg/dl) with serum symmetric dimethylarginin (sdma) within normal limits (10  μg/dl, ri  =  ≤ 14 μg/dl). urine specific gravity (usg) was 1,056 (ri  =  >  1035), with a urine protein to creatinine ratio (upcr) of 0.09 (reference range > 0.4); struvite crystals were also observed. in the second cat, haemato‑biochemical profile and urinalysis were unremarkable with serum creatinine (1.0 mg/dl, ri  = 0.8‑2.4 mg/dl) sdma (8  μg/dl, ri  = ≤  14  μg/dl) and usg (1,038, ri = > 1,035) within normal limits. when urine were collected, an aliquot was immediately 1:8 diluted with mem for virus isolation. rna was purified from 280 µl of undiluted urine samples (biosprint 96 one‑for‑all‑vet kit) and tested by a femv specific real time rt‑pcr (de  luca et  al. 2018). resulting c q values were 35 and 32, for tremedino and pepito, respectively. as for virus isolation, five hundred μl of diluted femv rna‑positive urine were centrifuged at 3,000 rpm for 5 min to remove debris and filtered through 450 nm disc filters (millipore). tpck trypsin (sigma‑aldrich, zwijndrecht, the netherlands) was then added to a final concentration of 0.1 µg ml‑1. samples were incubated at 37  °c for 15 minutes. the mixture was then inoculated into feline embryonic fibroblast (fea) cells in 24‑well plates serum‑free minimum essential medium eagle (mem) (sigma‑aldrich) supplemented with penicillin (100 units ml‑1) and streptomycin (100 µg ml‑1) (invitrogen). after 8  hours, inocula were replaced with mem (total volume 1 ml) supplemented by 3% heat inactivated fetal calf serum and antibiotics. figure 1. fea cells infected by femv pepito2018/italy. multinucleated syncytium was observed by may grünwald-giemsa staining (a); strong and specific cytoplasmic fluorescence (green color), nuclei are stained with dapi (blue) (b). scale bar = 100 µm (a), 75 µm (b). a b 181veterinaria italiana 2019, 55 (2), 179‑182. doi: 10.12834/vetit.1847.9883.1 donato et al. isolation and characterization of femv of nt identity with femv gt2 sequences. overall, our results confirm the viral heterogeneity existing between femv circulating strains and that the strains described in this study belong to the femv genotype 1. further molecular analysis of femv strains circulating in southern italy is currently underway (donato, manuscript in preparation) as well as the assessment of a serum‑neutralization assay to quantify specific femv antibodies in cat serum. nucleotide sequence accession numbers nucleotide sequences of tremedino2018/italy and pepito2018/italy have been deposited in genbank with accession numbers mk088516 and mk088517, respectively. acknowledgements funding were provided by the italian ministry of health (msrcte06/17, ricerca corrente 2017 “nuovi flussi diagnostici in sanità animale: dalla ngs alla banca antigeni”, recipient alessio lorusso). mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo italy. 150  bp paired‑end reads. the resulting 3,539,382 and 1,421,904 reads for tremedino and pepito, respectively, were de  novo assembled by spades v3.8.0. a total number of 266,976 and 388,432 reads mapped on a reference femv sequence (genbank accession number ab924120, strain otjp001). the length of the final de  novo assemblies were of 16,027 and 15,946  bp for femv tremedino2018/ italy and femv pepito2018/italy, respectively. the obtained nucleotide (nt) genome sequences were compared to those of extant femvs available online and genetic distances were calculated by using megalign (lasergene 15.0, madison‑wi, usa). the genome sequences of tremedino2018/ italy and pepito2018/italy were found to be nearly identical as they share the 99.2% of nt identity. nt identity between sequences obtained in this study and extant whole femv genome sequences ranges from 98.7% to 78.1%. tremedino2018/italy and pepito2018/italy showed the highest % of nt sequence identity with the japanese strains ss1 (98.7%, ab910309) and otjp001 (98.5%, ab924120); nt identity was lower with the early femv strains 761u and 776u (87.8%, jq411014 and jq411015) isolated in hong kong (woo et  al. 2012) and with strain us1 from usa (87.9%‑87.8%, kr014147). tremedino2018/italy and pepito2018/italy strains share the 88.1% of nt identity with piuma/2015, the first femv strain described in italy in 2015 (lorusso et al. 2015). very recently, a new genotype of femv, tentatively named femv genotype 2 (femv gt2), was described in germany (sieg et  al. 2019). femv sequences obtained in this study share the 78.1% 182 isolation and characterization of femv donato et al. veterinaria italiana 2019, 55 (2), 179‑182. doi: 10.12834/vetit.1847.9883.1 darold g.m., alfieri a.a,. muraro l.s., amude a.m., zanatta r., yamauchi k.c., alfieri a.f. & lunardi m. 2017. first report of feline morbillivirus in south america. arch virol, 162 (2), 469‑475. de luca e., crisi p.e., di domenico m., malatesta d., vincifori g., di tommaso m., di guardo g., di francesco g., petrini a., savini g., boari a. & lorusso a. 2018. a real‑time rt‑pcr assay for molecular identification and quantitation of feline morbillivirus rna from biological specimens. j virol methods, 258, 24‑28. furuya t., sassa y., omatsu t., nagai m., fukushima r., shibutani m., yamaguchi t., uematsu y., shirota k. & mizutani t. 2014. existence of feline morbillivirus infection in japanese cat populations. arch virol, 159, 371‑373. lorusso a., di tommaso m., di felice e., zaccaria g., luciani a., marcacci m., aste g., boari a. & savini g. 2015. first report of feline morbillivirus in europe. vet ital, 51 (3), 235‑237. marcacci m., de luca e., zaccaria g., di tommaso m., mangone i., aste g., savini g., boari a. & lorusso a. 2015. genome characterization of feline morbillivirus from italy. j virol methods, 234, 160‑163. park e‑s., suzuki m., kimura m., maruyama k., mizutani h., saito r., kubota n., furuya t., mizutani t., imaoka k. & morikawa s. 2014. identification of a natural recombination in the f and h genes of feline morbillivirus. virology, 468‑470, 524‑531. sakaguchi s., nakagawa s., yoshikawa r., kuwahara references c., hagiwara h., asai k., kawakami k., yamamoto y., ogawa m. & miyazawa t. 2014. genetic diversity of feline morbilliviruses isolated in japan. j gen virol, 95, 1464‑1468. sharp c.r., nambulli s., acciardo a.s., rennick l.j., drexler j.f., rima b.k., williams t. & duprex w.p. 2016. chronic infection of domestic cats with feline morbillivirus, united states. emerg infect dis, 22 (4), 760‑762. sieg m., heenemann k., ruckner a., burgener i., oechtering g. & vahlenkamp t.w. 2015. discovery of new feline paramyxoviruses in domestic cats with chronic kidney disease. virus genes, 51 (2), 294‑297. sieg m., busch j., eschke m., böttcher d., heenemann k., vahlenkamp a., reinert a., seeger j., heilmann r., scheffler k. & vahlenkamp t.w. 2019. a new genotype of feline morbillivirus infects primary cells of the lung, kidney, brain and peripheral blood. viruses, 11, 146. woo p.c.y., lau s.k.p., wong b.h.l., fan r.y.y., wong a.y.p., zhang a.j.x., wu j., choi g.k.y., li k.s.m., hui j., wang m., zeng b‑j., chan k.h. & yuen k‑y. 2012. feline morbillivirus, a previously undescribed paramyxovirus associated with tubulo‑interstitial nephritis in domestic cats. proc natl acad sci usa, 109, 5435‑5440. yilmaz h., tekelioglu b.k., gurel a., bamac o.e., ozturk g.y., cizmecigil u.y., tarakci e.a., aydin o., yilmaz a., berriatua e., helps c.r., richt j.a. & turan n. 2017. frequency, clinicopathological features and phylogenetic analysis of feline morbillivirus in cats in istanbul, turkey. j feline med surg, 19 (12), 1206‑1214. 265 veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 accepted: 17.07.2019 | available on line: 31.12.2021 1faculty of veterinary medicine, university of belgrade, serbia. 2veterinary institute of the republic of srpska ‘dr. vaso butozan’, bosnia and herzegovina. *corresponding author at: faculty of veterinary medicine, university of belgrade, serbia. e‑mail: andrea.zoric@vet.bg.ac.rs. andrea radalj1*, nenad milic1, oliver stevanovic2 and jakov nisavic1 keywords asymptomatic horses, ehv-1, ehv-4, ehv-5, phylogenetic analysis. summary nasal swabs originating from 112 apparently clinically healthy and unvaccinated horses of different age, breed and from diverse rearing conditions from serbia and bosnia and herzegovina were examined for the presence of equine herpesviruses 1, 4 and 5 using multiplex nested pcr (mn-pcr) and virus isolation. the detected viruses were subsequently characterised by gb gene nucleotide sequencing and their phylogenetic analysis was performed. the infections with ehv-1, ehv-4, and ehv-5 in the examined horse populations are apparently chronic, subclinical and persistent, whilst the shedding of ehv-1 and ehv-5 was confirmed by their successful isolation. a connection was established between the finding of ehvs and rearing conditions since horses kept together in stables were positive for at least one ehv in contrast to animals held free grazing or individually. ehv-5 was found most often in younger horses, however descending in frequency in animals up to 10 years of age. the phylogenetic analysis showed that the identified ehv strains group mostly with turkish and german strains of respective viruses. a certain degree of genetic heterogeneity was determined regarding the identified ehv-5 strains in contrast to ehv-1 and ehv-4. the detection and phylogenetic analysis of equine herpesviruses 1, 4 and 5 identified in nasal swab samples of asymptomatic horses from serbia and bosnia and herzegovina mostly in host lymphoid tissues (welch et  al. 1992, edington et  al. 1994, wang et  al. 2007, diallo et  al. 2008). equine herpesviruses 1 and 4 are categorized as economically important viruses of horses, both leading to respiratory disease, whilst ehv-1 is often found as the causative agent of abortion as well as of severe cases of myeloencephalopathy (van  maanen 2002, patel and heldens 2005, wang et  al. 2007, slater 2014, ataseven et  al. 2016). the clinical impact of equine gammaherpesviruses is still unclear; these viruses are commonly detected in various samples from both diseased and clinically healthy horses (dunowska et  al. 2002, wang et  al. 2007, negussie et  al. 2017, radalj et  al. 2018, stasiak et  al. 2018). equine herpesviruses 2 and 5 are believed to cause immunosuppression and are often mentioned as predisposing factors for the appearance of other respiratory diseases in horses as well as activation of latently present equine alphaherpesviruses (welch et  al. 1992, edington et al. 1994, dunowska et al. 2002, hartley et al. 2013, introduction the family herpesviridae consists of a large number of diverse and important viruses of both humans and animals and is divided into three subfamilies based on their cellular tropism, nature of latent infection as well as genomic sequences (van regenmortel et al. 2000, davison et  al. 2009). equine herpesviruses  1 and 4 (ehv-1 and ehv-4) are members of the genus varicellovirus within the alphaherpesvirinae subfamily, whilst equine herpesvirus es 2 and 5 (ehv-2 and ehv-5) have recently been included in the percavirus genus of the gammaherpesvirinae subfamily (davison et al. 2009). alphaherpesviruses establish latency in both sensory ganglia and lymphoid tissues and are characterized by short replication cycles which is best demonstrated in vitro since they rapidly produce a cytopathic effect (cpe) in cell culture (slater et  al. 1994, oie 2018, radalj et al. 2018). differently, gammaherpesviruses replicate more slowly and establish latency 266 veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina radalj et al. symptoms, the reactivation of latently present equine herpesviruses causing their consequential spread between animals (slater et  al. 1994, foote et  al. 2004, patel and heldens 2005, slater 2014). considering that often clinically normal horses can excrete ehvs, it is important to investigate their occurrence and genetic diversity in the equine population. the aim of this study was to examine nasal swab samples originating from apparently clinically healthy horses of different breeds and from different rearing conditions for the presence of equine herpesviruses 1, 4 and 5. furthermore, another objective of this investigation was to perform sequence analysis of the herpesviruses identified in horses from serbia and the republic of srpska (bosnia and herzegovina) in order to support the basic diagnostic tools used in the study and establish the phylogenetic similarities and differences with other representative ehv strains from other geographical regions of the world. materials and methods samples a total of 112 non-vaccinated, clinically healthy horses from the territories of the republic of serbia (50 animals) and republic of srpska, bosnia and herzegovina (62 animals) were included in this study. nasal swabs collected in serbia were taken from each horse available on a single visit in the period between august 2015 and september 2017, whilst samples from the republic of srpska were gathered in march 2016. all samples collected in the republic of serbia originated from horses of different breeds reared by individual owners from zlatibor (zlatibor district), gornji krivodol (pirot district), tutin and progorelica (raska district) aging from 3 to 27 years. all horses from zlatibor district (9 animals) and some from raska district (15 animals) were kept together in a stable. in contrast, horses from pirot district (14 animals) and some from raska district (12 animals) that roamed freely on mountain pastures most of the time or reared individually. nasal swab samples collected in the republic of srpska originated from 20 horses aging from 2 months to 14 years reared by an individual owner in mrkonjic grad municipality and 42 horses from ‘vucijak’ lipizzaner stable in prnjavor municipality. lipizzaner horses aged from 1 to 18 years. the breed age and origin of the sampled horses are presented in table i, while the locations where the sampling was performed are shown in figure 1. immediately after sampling, the nasal swab specimens were immersed into 2 ml of minimum negussie et al. 2017). moreover, ehv-5 has recently been proved to induce fibrotic changes in the lungs, a disease called equine multinodular pulmonary fibrosis (williams et  al. 2013). equine herpesviruses are widespread in equine populations around the world and most animals are infected in the first months of life (gilkerson et  al. 1999, dunowska et  al. 2002, nordengrahn et  al. 2002, marenzoni et  al. 2010, laabassi et  al. 2017, negussie et  al. 2017, stasiak et  al. 2018). latently infected horses are considered to be the most important reservoir of equine herpesviral infections which are most commonly spread in the horse population by direct or indirect contact between animals (welch et  al. 1992, edington et  al. 1994, gilkerson et  al. 1999, foote et  al. 2004, patel and heldens 2005, hartley et  al. 2013, slater 2014). reactivation episodes, during which the virus is shed, are rarely followed by marked clinical symptoms, thus representing an important maintenance mechanism of endemic infection cycles within the horse population (edington et  al. 1994, slater et  al. 1994, gilkerson et  al. 1999, van maanen 2002, foote et  al. 2004, patel and heldens 2005, hartley et  al. 2013, slater 2014). equine gammaherpesviruses are genetically and antigenically distinct from ehv-1 and ehv-4 and no cross-reactivity between neutralization antibodies against these groups of viruses has been observed. however, due to existing antigenic similarities within the viral subfamilies, it is difficult to distinguish the representative herpesviruses using neutralization tests (hartley et al. 2013, slater 2014, milic et  al. 2018, oie 2018). the utilization of genome sequencing made possible the analysis of equine herpesvirus genomes and showed significant differences that are used in molecular techniques such as polymerase chain reaction (pcr) as reliable markers for their distinction in both clinical samples and inoculated cells (wang et  al. 2007, negussie et  al. 2017, bilge dagalp et  al. 2018, milic et  al. 2018, radalj et  al. 2018, stasiak et  al. 2018). the reasons for the widespread use of pcr are its numerous advantages over standard virological methods in terms of speed, specificity, sensitivity, especially in cases of small amounts of samples and when to know whether the virus, in the examined sample, is still alive is not important. furthermore, genome sequencing data enable the tracking of different viral strains on a global level (turan et  al. 2012, ataseven et  al. 2016, negussie et  al. 2017, bilge dagalp et  al. 2018, milic et  al. 2018, radalj et  al. 2018, stasiak et  al. 2018). when possible, it is best to combine virus isolation (vi), which permits the discovery of viable virus particles in the examined samples with the fast and specific molecular methods. horses are subjected to numerous stressful situations that may induce, often without any clinical 267veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 radalj et al. equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina was grown in roux flasks for further testing. dna was extracted from the cells when the monolayer was 60% destroyed using genejet genomic dna purification kit (thermo scientific, usa) according to the manufacturer's instructions. the virus isolates were subsequently identified by multiplex nested pcr (wang et  al. 2007). the ehv-1 strain with a titre of 6.25  log10 50% tissue culture infective dose (tcid50) which was kindly provided by the scientific veterinary institute of serbia was used as positive control. internal laboratory reference strains of ehv-4 and ehv-5, titre 3.5 log10 tcid50 and 3.9  log10 tcid50, respectively, were also used as positive controls. furthermore, an archived ehv-1 strain marked as d1 (titre 6.9 log10 tcid50) isolated during the abortion storms that occurred in  ‘ljubicevo’ horse stable (former yugoslavia) during the eighties, was also used as a positive control. multiplex nested pcr detection (mn‑pcr) the primers for the first and second round of mn-pcr (metabion international, germany) used for the amplification of glycoprotein b (gb) genes of ehv-1, ehv-4, and ehv-5 were described by wang and colleagues (wang et  al. 2007). the final essential medium (mem, capricorn scientific, germany) with 2% foetal calf serum (fbs-12a, capricorn scientific, germany) supplemented with antibiotics and chilled on ice during transport to the laboratory. in the laboratory, samples were homogenized using a vortex mixer and centrifuged for 10 min at 1,677 × g. the supernatants were filtered using sterile 0.22 μm millex syringe filter units (merck, usa) and frozen at 20 °c pending inoculation on cell culture, whilst the deoxyribonucleic acid (dna) was extracted from the cell debris using genejet genomic dna purification kit (thermo scientific, usa) according to the manufacturer’s instructions. all nasal swab samples were collected in order to examine the presence of equine herpesviruses 1, 4 and 5. the obtained results were further analyzed in relation to the breed of sampled horses, age and rearing conditions. all horses were divided into age groups, namely: animals under 1 year (foals), yearlings, horses aging from 2 to 5, 6 to 10, 11 to 15, 16 to 20 and over 20 years of age. virus isolation rabbit kidney-13 cell line (rk-13, atcc ccl-37, izsler, brescia, italy) was used for virus isolation. the examined samples were individually inoculated onto 24-h old and 80% confluent monolayer of rk-13 cell line in 24-well microtitre plates. each well was inoculated with 100 μl of sample and incubated for 1 h at 37 °c in an atmosphere with 5% co 2 . subsequently, 500 μl of mem (capricorn scientific, germany) supplemented with 2% foetal calf serum (fbs-12a, capricorn scientific, germany) were added. the plates were incubated in the previously mentioned conditions and observed each day for the appearance of cytopathic effect (cpe). if cpe was not visible after 7 days, the plates were frozen and thawed three times and passaged onto new rk-13 confluent monolayers for two more times at 7-day intervals. the sample was considered negative when no cpe was visible after the third passage. uninoculated cell monolayers were used as cell controls (figure 2a). if cpe appeared, the virus table i. description of horse breeds, age, origin, and number of positive and negative animals determined by multiplex nested pcr. number of sampled horses breed age district ehv-1 ehv-4 ehv-5 negative 2 english thoroughbred 3-4 years raska 2 / / / 44 lipizzaner 1-18 years zlatibor, raska, prnjavor 30 / 10 11 3 croatian posavac 5-27 years zlatibor 3 1 / / 23 bosnian mountain horse 2 months-14 years zlatibor, mrkonjic grad 17 / 6 / 38 domestic mountain horse 3-16 years pirot, raska 13 / / 25 1 arabian 12 years zlatibor 1 / / / 1 haflinger 4 years zlatibor 1 / / / figure 1. map of sampling locations in serbia (zlatibor, zlatibor district; gornji krivodol, pirot district; tutin and progorelica, raska district) and the republic of srpska, bosnia and herzegovina (municipalities of prnjavor and mrkonjic grad) created with qgis software. 268 veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina radalj et al. multiplex nested pcr detection (mn‑pcr) multiplex nested pcr method was applied in order to investigate the presence of equine herpesviruses directly in the nasal swabs. from 112  collected samples 76 were found to be positive for equine herpesvirus (67.86%). in nasal swabs from horses originating from serbia, equine herpesviruses 1 and 4 were detected in 24 samples. ehv-4 was found in one sample, while in another a mixed infection with ehv-1 was detected. positive samples were all collected from horses in zlatibor and raska whilst all positive animals originated from individual households and were kept in small groups. other samples from serbia that were found to be negative (in total 26 nasal swabs) originated from horses which were kept individually (raska district) or roamed freely on mountain pastures for most of the time like the domestic mountain horses in pirot district. single and mixed infections of ehv-1 and ehv-5 were detected in the horse nasal swab samples from the republic of srpska. all bosnian mountain horses reared by an individual owner in mrkonjic grad were positive. of these, 6 animals from 2 to 5 year old were positive for ehv-5, whilst the other 14 horses from 2 months to 14 years of age were positive for ehv-1. of the 42 nasal swab samples from the lippizaner stable ‘vucijak’ in prnjavor, 32  were positive for  ehv-1 and/or ehv-5. in the first group consisting of 16 animals from one stable, ehv-1 was detected in 15 clinically healthy mares from 7 to 18 years of age, whilst ehv-5 was found in the nasal swab from one yearling animal. in a second group of 16 mares held together in a different stable, ehv-1 was detected in 14 animals, whilst ehv-5 was found in 9. of these, 7 were also positive for ehv-1. ehv-5 was detected in horses from 6 to 8 years of age. in a third group of 10 stallions 8 to 17 year old which were held separately from the mares, the mn-pcr was negative. specific pcr products were visualized using 1.5% agarose gel electrophoresis. sterile nuclease-free water was used as negative control, whilst the dna extracts of the above-mentioned strains used as positive controls during vi were also used as positive pcr controls. ehv‑1, ehv‑4, and ehv‑5 gb gene sequencing and phylogenetic analysis the ehv-1 specific primers from the first round of pcr amplifying the fragment of 1,180 bp along with ehv-4 and ehv-5 second round pcr primers used for the amplification of 677 bp and 410 bp products, respectively, were used for sequence analysis. the samples were sent to the macrogen europe laboratory, amsterdam, netherlands, for sequencing. the obtained nucleotide sequences from both directions were assembled together to obtain consensus sequence, aligned and compared with documented virus sequences available in the genbank database using blast software1. evolutionary analyses were conducted with mega x software. the phylogenetic trees for ehv-1, ehv-4, and ehv-5 strains were constructed using maximum likelihood algorithm with 1,000 bootstrap replicates. the evolutionary distance was computed using maximum composite likelihood method. results virus isolation from the total of 112 inoculated samples, cpe was noted in 28 samples (25%), i.e. in 8 samples after the first, while in 20 after the second passage in rk-13 cell line (figure 2b, 2c). all positive samples came from horses originating from the republic of srpska. the mn-pcr confirmed 25 isolates as ehv-1, and the remaining 3 as ehv-5. ehv-4 was not isolated. 1 http://www.ncbi.nlm.nih.gov/blast/. figure 2. cytopathic effect (cpe) in rk-13 cell line. a = negative control uninoculated cells; b = equine herpesvirus 1 cpe 48 h after first inoculation; c = equine herpesvirus 5 cpe at day 5 of the second passage. 269veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 radalj et al. equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina republic of srpska. however, the ehv-1_z_1 strain from zlatibor standed out and was 98% similar to all analyzed domestic and international strains and clustered with german ehv-1 strain cp4b. there is noticeable clustering between serbian, ehv-1 strains from the republic of srpska and those from turkey (tr-02, tr-04, tr-05) and away from certain strains from sweden, usa and germany (figure 4). partial gb gene nucleotide sequence of the ehv-4_z identified in this study, was 98% homogenous to the ehv-4 sequences found in genbank databases. the phylogenetic analysis evidenced a cluster with ehv-4 strains from turkey, japan and australia (figure 5). the partial gb gene nucleotide sequences of representative ehv-5 strains from the republic of srpska (ehv-5_mg_13, ehv-5_v_4 and ehv-5_v_5) were 98 to 99% homologous to each other. however, the strain ehv-5_mg_13 from mrkonjic grad and ehv-5_v_5 from prnjavor shared more similarities than ehv-5_v_5 and ehv-5_v_4 which originated from horses kept in the same stable. these three strains clustered with ehv-5 strains from turkey (rd-ehv5-tr2011, m-ehv5-tr2011, is-ehv5-tr2011) and south korea (krehv5/71) sharing nucleotide similarities from 96 to 99%. the strains originating from iceland and usa formed a different cluster and were 95-96% homologous to representative ehv-5 strains from the republic of srpska (figure 6). results relative to breed, rearing conditions and age the analysis of the likelihood of a certain horse breed to be positive for the presence of different equine herpesviruses was impaired by the fact that some breeds were more represented than others in this study. from the available results, it seems that the serbian indigenous domestic mountain horse is the breed least likely to be positive for infection with ehvs (table i). however, when reviewed from the perspective of rearing conditions, the results showed that this parameter might be more trustworthy since all positive horses in this investigation originated from smaller or larger groups held by individual owners or in stables. differently, all horses held individually in households as well as animals that were free grazing for most of the time were negative for equine herpesviruses. this very fact may have affected the negative result obtained in the case of the nasal swab samples from domestic mountain horses since this indigenous breed is most often held free grazing. furthermore, the distribution of different ehvs in several age groups appointed before is shown in figure 3. yearlings and horses up to 10 years of age were most frequently infected with ehv-5, however, the number of positive animals decreased with age. equine herpesvirus 1 was in most cases evenly distributed within the given age groups. comparison between the results of virus isolation and multiplex nested pcr out of 112 examined nasal swab samples, one or more ehvs were detcted in 76 samples (67.86%) using mn-pcr, whilst vi was successful in 28 nasal swabs (25%) (table ii). ehv‑1, ehv‑4, and ehv‑5 gb gene sequencing and phylogenetic analysis the nucleotide homology of the selected gb sequences of ehv-1 strains from serbia and republic of srpska to each other as well as with ehv-1 strains from genbank was from 98 to 100%. all serbian strains were 100% homogenous to each other, with the archived isolate marked as ehv-1_d1, with strains from other countries as well as with strains from the 0 20 40 60 80 100 < 1 1 2 to 5 6 to 10 11 to 15 16 to 20 > 20 ehv-1/ehv-4 ehv-1/ehv-5 ehv-5 ehv-1 % o f p o si ti ve a n im al s in a g e g ro u p age groups (years) figure 3. the distribution of different equine herpesviruses (ehv-1, ehv-4 and ehv-5) alone and in mixed infections in appointed age groups of sampled horses showing their frequency of detection in nasal swabs from 112 horses. table ii. comparison of the results of diagnostic methods used for the identification of equine herpesviruses 1, 4, 2 and 5 (ehv-1, ehv-4, ehv-2 and ehv-5) in nasal swabs of horses from serbia and the republic of srpska, bosnia and herzegovina. multiplex nested pcr virus isolation ehv-1 ehv-4 ehv-2 ehv-5 ehv-1 + ehv-4 ehv-1 + ehv-5 ehv-1 ehv-4 ehv-2 ehv-5 serbia 24 1 / / 1 / / / / / bosnia and herzegovina 43 / / 16 / 7 25 / / 3 total 67 1 / 16 1 7 25 / / 3 270 veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina radalj et al. these authors, similarly to heldens and colleagues (heldens et  al. 2001) consider that the sensitivity of isolation of ehv-4 could be augmented by the application of equine-derived cell lines. besides, one of the important factors that ensure the detection and isolation of ehv-4 is the sampling, i.e. the length of transport can affect the results (diallo et al. 2008, ploszay et  al. 2013). this coud have influenced the results of this study especially when sampling was carried out in the warmer period of the year. the isolation of equine herpesviruses from nasal swabs often fails given the sensitivity of the virus, the presence of virus-neutralizing antibodies in nasal secretions, the time of sampling, as well as the amount of viable virions necessary for the appearance of cpe in cell culture. polymerase chain reaction is therefore the method of choice since it can be equally, if not more sensitive than standard virus detection techniques (nordengrahn et  al. discussion the isolation of herpesviruses in rk-13 cell line followed by their identification using molecular methods represents a standard procedure in the diagnostics of equine herpesviral diseases (slater et  al. 1994, dunowska et  al. 2002, diallo et  al. 2008, ataseven et al. 2010, hartley et al. 2013, williams et al. 2013, oie 2018). the mentioned cell line was used to isolate equine herpesviruses from nasal swab samples of horses originating from serbia and the republic of srpska. virus isolation was able to isolate ehv-1 and ehv-5. many studies have confirmed the suitability of rk-13 cells for the isolation of ehv-1 but not of ehv-4 since it is considered that ehv-4 readily grows mostly in equine-derived cell lines (welch et al. 1992, edington et al. 1994, heldens et al. 2001, diallo et al. 2008, oie 2018). the cytopathic effect of 3 ehv-5 isolates was observed after the second passage which is in accordance with the results of other authors stating that equine gammaherpesviruses often fail to show cpe or do so slowly and after a few blind passages (dunowska et  al. 2002, wang et  al. 2007, diallo et  al. 2008, williams et  al. 2013, radalj et al. 2018). ehv-4 was not isolated in our study. on the other hand, mn-pcr used directly on nasal swab samples detected ehv-4 with ehv-1 in one sample. ploszay and colleagues (ploszay et al. 2013) describe the first successful isolation of ehv-4 in poland using vero cell line, nevertheless vi proved to be less sensitive when compared to pcr and real-time pcr. figure 4. maximum likelihood phylogenetic tree based on 1,180 bp gb gene fragment of equine herpesvirus 1 (ehv-1) strains from serbia (ehv-1_d1, ehv-1_t1, ehv-1_t2, ehv-1_z1), the republic of srpska (ehv-1_v1, ehv-1_v2, ehv-1_mg3) and gb genes of international ehv-1 strains. evolutionary analyses were conducted in mega x. figure 5. maximum likelihood phylogenetic tree based on 677 bp gb gene fragment of equine herpesvirus 4 (ehv-4) strain from serbia (ehv-4_zl) and gb genes of international ehv-4 strains. evolutionary analyses were conducted in mega x. figure 6. maximum likelihood phylogenetic tree based on 410 bp gb gene fragment of equine herpesvirus 5 (ehv-5) strains from the republic of srpska (ehv-5_v4, ehv-5_v5, ehv-5_mg13) and gb genes of international ehv-5 strains. evolutionary analyses were conducted in mega x. 271veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 radalj et al. equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina in the first group of 16 mares kept together in close confinement, ehv-1 was identified in 15 clinically healthy animals, whilst ehv-5 was found in a yearling animal. from the second group of 16 mares, ehv-1 was detected in 14 and ehv-5 in 9 samples from animals between 6 and 8 years, in 7 these ehv-5 was detected together with ehv-1. the third group of 10 stallions between 8 and 17 years which were kept separately from the above-mentioned mares was negative for equine herpesviruses. these results are in accordance with other authors claiming that the population of mares and foals represents the primary reservoir of ehv-1 in large groups of horses (gilkerson et al. 1999, foote et al. 2004). in this study, the presence of ehv-5 was correlated with age, since all positive horses ranged from 1 to 8 year old. a number of studies conducted by other authors showed similar results associating the presence of equine gammaherpesviruses with the age of infected animals (dunowska et  al. 2002, nordengrahn et  al. 2002, bell et  al. 2006). marenzoni and colleagues (marenzoni et  al. 2010) showed that the occurrence of ehv-5 in nasal swabs and leucocytes of younger horses was 73.3% and 80%, respectively, which was significantly higher compared to older categories of animals. moreover, negussie and colleagues (negussie et  al. 2017) established a high prevalence of ehv-5 in horses younger than 3 years. laabassi and colleagues (laabassi et  al. 2017) examined the presence of equine herpesviruses in 100 nasal swabs of horses aging from 1 to 27 years using real-time pcr and established that 97.3% horses aging up to 3 years are infected with ehv-5, whilst its prevalence and the amount of excreted virus decrease with age. stasiak and colleagues (stasiak et  al. 2018) also reported that yearlings represent the category most often positive for ehv-5. since the nucleotide sequence coding the synthesis of gb is a highly conserved region of the ehv_1 and ehv_4 genomes, the genome sequence similarities between the viral strains from this study and the ehv-1 and ehv-4 strains from other parts of the world were expected. however, the phylogenetic analysis showed that ehv-1 and ehv-4 strains from serbia and the republic of srpska clustered with the homologous strains from turkey. the analysis of the phylogenetic tree constructed in this study showed that ehv-1 strains from serbia and republic of srpska mostly clustered with strains from turkey and germany. bilge dagalp and colleagues (bilge dagalp et  al. 2018) determined that gb gene nucleotide sequences of turkish ehv-4 strains are very similar and create a distinct group along with sequences reported by turan and colleagues (turan et al. 2012), whilst one strain separated with european and japanese strains. the nucleotide homology of gb gene of the ehv-4 strain examined in this study was 2002, van maanen 2002, wang et al. 2007). all nasal swabs in this study were examined by mn-pcr that enabled the detection of more positive samples than vi (67.86% compared to 25%), as well as the presence of mixed infections with equine alphaand gammaherpesviruses. standard virological methods are more expensive and last much longer than mnpcr, making it inappropriate in clinical practice (milic et al. 2018). moreover, the appearance of cpe in equine gammaherpesviruses is much slower prolonging the time necessary to obtain a proper diagnosis (dunowska et  al. 2002, diallo et  al. 2008, williams et  al. 2013, radalj et  al. 2018). multiplex nested pcr represents a sensitive and specific test designed for rapid and simultaneous identification of various different equine herpesviruses in the examined samples (wang et al. 2007, negussie et al. 2017, bilge dagalp et al. 2018). the mn-pcr set up by wang and colleagues (wang et al. 2007) was created in order to simultaneously identify multiple equine herpesviruses in equine clinical samples. as in this study, the authors used mn-pcr to analyze nasal swab samples from horses with and without clinical symptoms of respiratory disease and showed that it was more specific and sensitive than vi. the absence of clinical symptoms in animals whose nasal swabs were positive for ehv-1 and/or ehv-5 can be explained in various ways. equine herpesviruses are often present in nasal secretions of horses more than 4 weeks after infection, a period during which clinical symptoms of respiratory disease are often missing. moreover, latently infected horses can not show symptoms in the period of virus reactivation (slater et  al. 1994, gilkerson et  al. 1999, foote et  al. 2004, patel and heldens 2005, slater 2014). in the nasal swabs from horses originating from individual breeders from serbia, ehv-1 and ehv-4 were detected in 24 samples by mn-pcr, however, ehv-4 was found in a mixed infection with ehv-1 in only one sample. all positive animals were kept in small groups, negative animals, instead, were kept individually or roamed freely. these results agree with well-known facts about the epizootiological features of equine herpesviral infections. equine herpesviruses 1 and 4 spread quickly in the population by both direct and indirect contact. the role of aerosol in their transmission depends on the quantity of infectious virions, climate, aeration of horse stables and the distance between animals (van maanen 2002, patel and heldens 2005, slater 2014). similar results were recorded in the republic of srpska. in the group of 20 bosnian mountain horses reared by an individual owner, ehv-5 was detected in nasal swabs from 6  animals between 2 and 5  years, whilst the other 14 horses between 2 months and 14 years were all positive for ehv-1. of the 42 nasal swab samples from lipizzaner horses of ‘vucijak’ stable, 32 were positive for ehv-1 and/or ehv-5 by mn-pcr. 272 veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 equine herpesviruses in asymptomatic horses from serbia and bosnia and herzegovina radalj et al. the republic of serbia and bosnia and herzegovina are apparently chronic, subclinical and persistent. the higher number of positive samples identified using the mentioned molecular method was to be expected since its results are independent of the amount and viability of virions, thus making it a suitable procedure for rapid screening of large horse populations. virus isolation was successful in a smaller number of samples and the only viruses isolated were ehv-1 and ehv-5, however, this finding is significant since it confirms the fact that not only horses with marked clinical symptoms are herpesvirus shedders in the equine population. during this examination, a correlation was found concerning the finding of equine herpesviruses and rearing conditions of sampled animals which showed that horses kept together in stables were mostly positive for at least one ehv in contrast to animals kept free grazing on pastures or individually in households. furthermore, a connection could be established between the age of horses and ehv-5 infection which was found most often in young horses and, descending in frequency, in animals up to 10 years of age. the results of the phylogenetic analysis performed in this study showed that the strains of ehv-1, ehv-4, and ehv-5 identified in serbia and bosnia and herzegovina group mostly with turkish strains of respective viruses which is probably due to the geographical location of the balkan region and active trade occurring over this territory. furthermore, a certain degree of genetic heterogeneity was determined regarding the identified ehv-5 strains in contrast to ehv-1 and ehv-4 which were mostly homologous. given the data presented here, we encourage field veterinarians who should take samples from horses with clinical symptoms and from larger horse agglomerations more often in order to confirm or exclude the role of herpesviruses in potential disease outbreaks. acknowledgments we thank damjan radoja and drago nedić for technical support. grant support the study was supported by the ministry of education, science and technological development of the republic of serbia (contract number 451-0368/2022-14/200143). 98% when compared to other strains from genbank, however the results of the phylogenetic analysis showed its clustering with turkish, japanese and australian ehv-4 strains, comparable to the results obtained by bilge dagalp and colleagues (bilge dagalp et  al. 2018). the similarity of the strains of equine herpesviruses from this region with turkish strains can be explained by the active trade and transport of animals occurring between europe and asia over the balkans. the phylogenetic analysis of turkish gammaherpesviruses performed by bilge dagalp and colleagues (bilge dagalp et  al. 2018) showed a high degree of genetic heterogeneity between their gb gene nucleotide sequences, in contrast to the above-mentioned alphaherpesviruses. the phylogenetic analysis of ethiopian gammaherpesvirus strains showed their vast genetic heterogeneity with similarities of the identified ehv-5 strains ranging from od 95.1 to 100% (negussie et  al. 2017). the nucleotide homology between gb gene sequences of ehv-5 strains in our study as well as their similarities with other ehv-5 strains from genbank ranged from 95 to 99%. the phylogenetic analysis of partial gb gene nucleotide sequences of ehv-5 strains in our study showed significant similarities and grouping with ehv-5 strains from turkey. turkish ehv-5 strains analyzed in the study of ataseven and colleagues (ataseven et al. 2010) were separated from european strains on a different branch, and when compared, their nucleotide sequences were 77.3-90.2% similar, showing the genetic heterogeneity amongst gb gene sequences of different ehv-5 strains. bilge dagalp and colleagues (bilge dagalp et  al. 2018) obtained similar results to ours regarding the overall similarities of identified ehv-5 strains which were from 98.8 to 99.7%. comparable results were also reported by stasiak and colleagues (stasiak et  al. 2018) regarding the similarities between gb gene nucleotide sequences of ehv-5 from poland and other countries which ranged from 90.8 to 99.6%. from the obtained results it can be concluded that equine herpesvirus infections are common amongst asymptomatic horses from both serbia and bosnia and herzegovina. to the authors' knowledge, this is the first evidence of ehv-1 and ehv-5 presence in bosnia and herzegovina. equine herpesviruses 1, 4, and 5 were detected using mn-pcr in nasal swab samples of horses belonging to different breeds and age groups. infections with ehv-1, ehv-4, and ehv-5 in horse populations from 273veterinaria italiana 2021, 57 (4), 265-274. doi: 10.12834/vetit.1767.9329.3 radalj et al. equine herpesviruses in asymptomatic horses from serbia and bosnia and 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microbiol, 121, 18-28. 35 introduction bovine brucellosis is an economically important zoonotic disease of cattle and buffaloes caused by brucella abortus, less frequently by b.  melitensis and rarely by b.  suis. it has been recognized as a major cause of contagious abortion in cattle and may account for 80% incidence in naive herds (radostits et al. 2007, selim et al. 2014). the disease is characterized by abortion in last trimester of gestation, retained placenta and impaired fertility in the cow (oie 2016a). the disease is usually asymptomatic after the first abortion. infected animals often become chronic carriers and continue to shed bacteria in milk and uterine discharges (cfsph 2009). infectious bovine rhinotracheitis (ibr)/ infectious pustular vulvovaginitis (ipv) is caused by bovine alphaherpesvirus‑1 (bohv‑1) belonging to the genus varicellovirus, subfamily alphaherpesvirinae, family herpesviridae and order herpesvirales (murphy et  al. 1999, muylekns et  al. 2007). the disease is characterized by rhinotracheitis, pustular vulvovaginitis, balanoposthitis, conjunctivitis, enteritis, encephalitis, decreased milk production, weight loss and abortion (muylekns et  al. 2007). ibr/ipv is the major cause of viral abortion in bovines and it may account for 5‑60% of abortions in non‑vaccinated herds (tibary 2016). following primary infection, the virus becomes latent in the sensory neurons of trigeminal ganglia. during stress/ immune suppression, the virus reactivation occurs and the virions are secreted through nasal secretion, lachrymal secretion, genital secretion, semen, etc. (kutish et al. 1990, jones et al. 2006). both bovine brucellosis and ibr/ipv are endemic #these authors contribute equally to this work. 1nddb r&d laboratory, hyderabad, iil campus, gachibowli, hyderabad, telengana 500 032, india. 2jawaharlal nehru technical university, kukatpally, hyderabad 500 085, telangana, india. 3national dairy development board, anand, gujarat 388 001, india. *corresponding author at: national dairy development board, anand, gujarat 388 001, india. e-mail: skrana@nddb.coop. keywords bovine abortion, bovine alphaherpesvirus, brucella, diagnosis, infectious bovine rhinotracheitis, multiplex, real‑time pcr. summary a duplex real‑time pcr was developed and validated for the simultaneous detection of brucella and bovine alphaherpesvirus‑1 (bohv‑1) from bovine clinical specimens. the bcsp31 gene of brucella and gb gene of bohv‑1 were used as targets in the assay. the limit of detection for bohv‑1 was 0.03 tcid 50 of virus and 10 plasmid copies containing the target gene while for brucella it was 4.1 × 101 cfus. intra‑assay and inter‑assay values showed high repeatability and reproducibility of the assay. the diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the duplex assay were determined by screening 443 clinical specimens and comparing the results with the respective individual assays. the dsn and dsp for detection of brucella were found to be 95.24% and 95.65%, respectively whereas for bohv‑1, the dsn (100%) and dsp (99.47%) were slightly higher. the duplex assay had a very good degree of agreement with the respective individual real‑time pcr test {kappa value 0.97 for brucella and 0.95 for bohv‑1}. the results of the current study suggest that the duplex assay would be a cost‑effective and time‑saving alternative for the individual real‑time pcr assay for the detection of brucella and bohv‑1. laxmi narayan sarangi1#, supriya polapally2#, samir kumar rana3*, vijay shriram bahekar1, kota sri naga leela surendra1, rachamreddy venkata chandrasekhar reddy1, aparna sri raichur2, nadikerianda muthappa ponnanna1, and girish kumar sharma3 development and validation of duplex real-time pcr for simultaneous detection of brucella and bovine alphaherpesvirus from clinical specimens veterinaria italiana 2020, 56 (1), 35‑41. doi: 10.12834/vetit.1728.9123.2 accepted: 24.05.2019 | available on line: 24.04.2020 36 veterinaria italiana 2020, 56 (1), 35‑41. doi: 10.12834/vetit.1728.9123.2 duplex real‑time pcr for detection of brucella and bovine alphaherpesvirus‑1 sarangi et al. cloning vector (rana et al. 2011) was used as positive control in the real‑time pcr assay for bohv‑1 detection. the dna extracted from the brucella abortus strain 19 was used as positive control for brucella real‑time pcr reaction. preparation of mock specimens nasal and vaginal swab samples collected from ibr and brucella sero‑negative animals and tested negative in real‑time pcr were used as known negative samples. aliquots of these known negative samples were pooled to obtain a large volume of uniform sample matrix. the pooled sample matrix was further tested by real‑time pcr to confirm the absence of bohv‑1 and brucella. an aliquot of this sample matrix was spiked with a fresh culture of brucella abortus strain19 (4.1  ×  107 cfu/ml) and serial ten‑fold dilutions were prepared. similarly, serial ten‑fold dilutions of bohv‑1 virus (2  ×  105.0 tcid 50 /ml) were prepared in sample matrix. extraction of dna from samples the qiamp® blood dna mini kit (qiagen, germany) was used for extraction of dna from the nasal and vaginal swabs. the manufacturer’s protocol was followed albeit addition of 2 µl of carrier rna to the samples before extraction for increasing the dna yield. a sample volume of 200 µl was used for dna extraction and elution was made in 100 µl of buffer ae. the eluted dna was stored at ‑ 20 °c till further use. the dna from abortive tissues was extracted by qiamp® cador® pathogen mini kit (qiagen, germany) as per the manufacturer’s protocol. real‑time pcr for detection of bohv‑1 and brucella dna extracted from swabs and abortive tissues were processed by real‑time pcr for detection of bohv‑1 and brucella. the uniplex assay for detection of bohv‑1 used primers and probes targeting gb gene of bohv‑1 (wang et  al. 2007) and the test was performed as per the protocol described earlier (rana et  al. 2011, sarangi et  al. 2018). the uniplex real‑time pcr for brucella targeting bcsp31 gene was performed as per the established protocol (mukherjee et  al. 2015). the duplex real‑time pcr assay used the same primers and probes combination, targeting bcsp31 and gb gene, as in the respective individual (uniplex) assays with a change in the reporter dye for bcsp31 probe. the premix ex taq (takara bio) was used for reaction set‑up and real‑time pcr was carried out in 7,300 real‑time pcr system (applied biosystems) or rotor‑gene q (qiagen). the duplex real‑time in india and cause huge economic loss to the dairy industry (trangadia et  al. 2010, mukherjee et  al. 2015, krishnamoorthy et  al. 2016). quick, accurate and comprehensive diagnosis of pathogenic agents in a herd is of utmost importance to initiate control and preventive actions. the conventional diagnostic methods consist of serological tests and isolation of the causative agent. the bacteriological isolation of brucella is still considered the gold standard but is poorly sensitive, labour intensive, with long turnaround time and requires skilled personnel. isolation of brucella is bio hazardous and requires biosafety level iii facility. serological tests that detect antibodies cannot identifty the early phase of infection and require paired sera for confirmation. hence, the development and combination of fast, quantitative and accurate molecular tests based on pcr are strongly reccommended. both brucella and bohv‑1 cause reproductive disorders in dairy animals including abortion, establish latent/ chronic carrier status in the animals and are excreted in nasal, vaginal and lacrimal secretions and also in semen of infected bulls. a duplex assay targeting both the pathogens will be an effective tool in abortion investigations and also in determining the infection status of the animals in the herd aiding implementation of effective disease control. in this study, a duplex real‑time pcr for simultaneous detection of brucella and bohv‑1 from clinical samples was standardized. further, attempts were made to validate the assay as per the guidelines of the oie (oie 2016b). materials and methods reference sample virus guk57/2007 strain (bohv‑1.1 genotype) stored at the repository of national diary development board research and development (nddb r&d) laboratory, hyderabad, was used as reference virus. the titer of the virus was 2  ×  105.0 tcid 50 /ml (reed and muench 1938). bacteria brucella abortus strain 19 (vaccine strain) was used as reference strain. the bacterial count was determined by pour plate method and the bacterial load was found to be 4.1 × 107 cfus/ml. plasmid and dna a plasmid construct containing the target 97bp of bohv‑1 glycoprotein b (gb) gene cloned into pdrive 37veterinaria italiana 2020, 56 (1), 35‑41. doi: 10.12834/vetit.1728.9123.2 sarangi et al. duplex real‑time pcr for detection of brucella and bovine alphaherpesvirus‑1 determination of analytical specificity of the assay the analytical specificity of the assay was determined by using dna extracted from pathogens that might be present in the nasal swabs/ vaginal swabs or aborted tissues of bovine which may cause similar clinical signs to brucellosis and ibr/ipv. the microorganisms included in the analytical specificity study are listed in table i. repeatability and interference assays reference test samples for the determination of repeatability and interference assay were prepared by mixing dna extracted from the mock specimens with various concentrations of brucella and bohv‑1. inter‑assay and intra‑assay variations were also evaluated. the combinations of the two pathogens were tested in both duplex as well as with the uniplex real‑time pcr assays, in triplicates (intra‑assay variation) and for three consecutive days (inter‑assay variation). positive controls and negative controls were included in the tests. determination of diagnostic sensitivity and specificity the diagnostic sensitivity (dsn) and diagnostic specificity (dsp) were determined for diagnosis of pcr reaction contained 4.5 µm of primers, 4 µm of probes of both the targets and rox as passive reference dye. the thermal profile used in the uniplex as well as in the duplex real‑time pcr was 95 °c for 30 seconds (s), followed by 45 cycles of 5 s at 95 °c and 35 s of 60 °c. the threshold level was usually set manually at the starting of exponential phase of the positive controls which were higher than that of background. the cut‑off used in the respective uniplex assays were also retained for the duplex assay. any sample showing a cycle threshold (ct) value less than 40 in fam fluorophore detection channel was regarded as positive for bohv‑1. similarly, any sample showing a ct value less than 38 in hex fluorophore detection channel was regarded as positive for brucella dna. determination of limit of dilution (lod) /analytical sensitivity of the assay the lod of the assays was determined by spiking experiment. dna was extracted from the mock specimens (serially diluted spiked sample) and was tested in quadruplicate by both uniplex and duplex real‑time pcr assay. the highest dilution of bohv‑1 and brucella, which was showing positive amplification in all the replicates, was considered as the lod (probability point 100%). the lod was confirmed by testing 10 replicates of this highest dilution sample. table i. bacteria and viruses used to evaluate the specificity of the duplex real-time pcr . name the of the species source /origin amplification in fam channel (bohv-1) amplification in hex channel (brucella) brucella abortus 544 atcc (23448) negative positive brucella abortus s19 vaccine strain (usda/mar. 98) negative positive brucella isolate 1 from placenta field strain (nddb) negative positive brucella isolate 1 from milk field strain (nddb) negative positive brucella isolate 2 from milk field strain (nddb) negative positive brucella isolate 3 from milk field strain (nddb) negative positive brucella isolate 4 from milk field strain (nddb) negative positive positive plasmid harbouring gb gene of bohv-1 nddb positive negative bovine alphaherpes virus isolate 1 field strain (nddb) positive negative bovine alphaherpes virus isolate 2 field strain (nddb) positive negative bovine alphaherpes virus isolate 3 field strain (nddb) positive negative escherichia coli atcc (51299) negative negative staphylococcus aureus atcc (25923) negative negative klebsiella pneumonia atcc (700603) negative negative listeria monocytogenes atcc (19111) negative negative pseudomonas aeruginosa atcc (27853) negative negative campylobactor foetus atcc (27374) negative negative trichomonas foetus atcc (30003) negative negative 38 veterinaria italiana 2020, 56 (1), 35‑41. doi: 10.12834/vetit.1728.9123.2 duplex real‑time pcr for detection of brucella and bovine alphaherpesvirus‑1 sarangi et al. for the detection of brucella and bohv‑1 by using the same primers and probe combination, targeting bcsp31 and gb gene respectively, as in the respective uniplex assays with a change in the reporter dye for bcsp31 probe. the duplex real‑time pcr was validated by comparing its results with the respective uniplex real‑time pcr. initially, 6‑fam and hex fluorophores were evaluated for their suitability in the individual real‑time pcr reactions for both bohv‑1 and brucella. the correlation coefficient between the results obtained in 6‑fam and hex for detection of brucella was 0.997 (95% ci: 0.990 to 0.999), whereas for bohv‑1 it was 0.97 (95% ci: 0.927 to 0.989). however, the ct value obtained with hex fluorophore for bohv‑1 was comparatively higher brucella and bohv‑1 by both uniplex and duplex real‑time pcrs from 443 clinical specimens. the samples were tested in duplicate and any replicate showing positive amplification (ct values < 40 for bohv‑1 and < 38 for brucella) was considered positive. the degree of agreement between these two tests was calculated by using kappa statistics (altman 1991, fleiss et al. 2003). results and discussion individual real‑time pcr assay have been validated and routinely used in our laboratory for detection of bohv‑1 and brucella from clinical samples (rana et al. 2011, mukherjee et al. 2015). in this study, a duplex real‑time pcr was optimized uniplex bohv-1 ct value 20 15 20 25 30 35 40 25 30 35 40 d u p le x b o h v -1 c t va lu e uniplex brucella ct value 15 15 20 25 30 35 40 20 25 30 4035 d u p le x b ru ce lla c t va lu e a b figure 1. correlation of ct values of uniplex and duplex real-time pcrs for detection of bohv-1 (a) and brucella (b). serial dilution of bohv-1 virus (300 tcid 50 to 0.03 tcid 50 /reaction) 15 10 5 0 20 25 30 35 40 10-5 10-4 uniplex ibr real-time pcr (mean ct) duplex ibr real-time pcr (mean ct) lineare [uniplex ibr real-time pcr (mean ct)] lineare [duplex ibr real-time pcr (mean ct)] 10-3 10-2 10-1 m ea n c t va lu e y = -3.9145x + 40.493 r2 = 0.9931 y = -3.7736x + 39.916 r2 = 0.9971 figure 2. comparison of standard curve of uniplex and duplex real-time pcr assay for detection of bohv-1 using serial dilution of bohv-1 [guk57/2007 (bohv-1.1 genotype)] strain infected cell culture supernatant 39veterinaria italiana 2020, 56 (1), 35‑41. doi: 10.12834/vetit.1728.9123.2 sarangi et al. duplex real‑time pcr for detection of brucella and bovine alphaherpesvirus‑1 any of other micro flora included in the study viz., e.  coli, staphylococcus spp., listeria monocytogenes, pseudomonas aeruginosa, klebsiella pneumonia, campylobacter foetus and trichomonas foetus. the rna viruses were not included in the specificity test as no reverse transcriptase step was involved. further, primers and probes used in the current study were taken from published works which already investigated the specificity (wang et  al. 2007, rana et  al. 2011, mukherjee et  al. 2015). the primers and probe used for amplification of gb gene of bohv‑1 could successfully amplify bohv‑1.1, bohv‑1.2 and bohv‑5 and no cross‑reaction was reported with bvdv, pi3, bsrv and human herpesvirus 1‑5 (wang et al. 2007). one major drawback encountered in the multiplex pcr is the reduced efficiency at detecting the less abundant pathogen in mixed infections. although there were minor differences in the ct values obtained in the mixed sample versus the samples from bohv‑1 alone, there was no noticeable difference in amplification efficiency of bohv‑1 even with the than the 6‑fam fluorophore. therefore, 6‑fam labelled probe was retained for bohv‑1 and hex labelled probe was used for brucella. limit of detection (lod) was carried out to measure the analytical sensitivity of the assay. serial ten‑fold dilutions of the mock specimens spiked with the respective organism were used. the highest dilution at which amplification was observed in all four replicates was considered as the lod (100% probability point). the lod for detection of bohv‑1 was found to be 0.03 tcid 50 per reaction in both uniplex and duplex real‑time pcr (figure 1a and 2). similarly, the lod for brucella was determined to be 41 cfus/reaction in both uniplex as well as duplex real‑time pcr (figure 1b and 3). the lods for the duplex assay were further confirmed by testing 10 replicates of the dilution series containing 0.03 tcid 50 virus per reaction of bohv‑1 and 41 cfus of brucella per reaction. the analytical specificity study suggested that the duplex assay was able to detect only brucella and bohv‑1 isolates but did not amplify dna from serial dilution of brucella s19 strain (410,000 to 41 brucella cfus/reaction) 15 10 5 0 20 25 30 35 40 10-5 10-4 10-3 10-2 10-1 m ea n c t va lu e y = -4.434x + 40.395 r2 = 0.9945 y = -4.3568x + 40.855 r2 = 0.9866 uniplex brucella real-time pcr (mean ct) duplex brucella real-time pcr (mean ct) lineare [uniplex brucella real-time pcr (mean ct)] lineare [duplex brucella real-time pcr (mean ct)] figure 3. comparison of standard curve of uniplex and duplex real-time pcr assay for detection of brucella using serial dilution of s19 reference strain. table ii. intra-assay and inter-assay variations observed in individual (uniplex) and duplex real-time pcr for detection of bohv-1 and brucella. data presented in co-efficient of variation in mean ct values obtained in real-time pcr; samples of each combination were tested in triplicate. brucella and bohv-1 combination inter-assay intra-assay brucella concentration ibr concentration uniplex bohv-1 duplex bohv-1 uniplex brucella duplex brucella uniplex bohv-1 duplex bohv-1 uniplex brucella duplex brucella 10-2 10-2 0.45-1.89 0.57-0.92 0.10-2.33 0.57-0.92 1.98 2.09 2.84 4.76 10-2 10-5 0.55-1.52 1.79-3.57 0.19-1.25 1.79-2.59 2.26 5.61 5.56 10.24 10-5 10-2 0.12-0.89 0.15-0.41 0.53-2.75 0.15-0.41 0.88 1.94 1.35 10.37 10-5 10-5 0.59-2.07 0.57-3.14 0.47-3.08 0.57-3.24 4.44 3.78 2.90 7.07 brucella pc ibr pc 1.64-2.33 0.40-2.11 0.40-2.11 0.0-1.45 1.27 2.49 1.34 1.01 40 veterinaria italiana 2020, 56 (1), 35‑41. doi: 10.12834/vetit.1728.9123.2 duplex real‑time pcr for detection of brucella and bovine alphaherpesvirus‑1 sarangi et al. standard deviations of 1.64, 1.76, 2.0, and 1.34 were observed for the uniplex brucella, uniplex bohv‑1, duplex brucella and duplex bohv‑1 real‑time pcr, respectively, which is within an acceptable range. bland‑altman plot was used to simultaneously display and analyze the results obtained in uniplex and duplex pcr reactions carried out on each sample. the ct values obtained in the analytical sensitivity and repeatability study were included in the analysis. results suggest that, on average, the ct values obtained in duplex real‑time pcr for bohv‑1 is 0.15 more than the uniplex bohv‑1 real‑time pcr method. similarly, on an average, the ct values obtained in duplex real‑time pcr for brucella is 0.07 less than the uniplex brucella real‑time pcr method. the presence of inhibitors in the sample can result in false‑negative results in pcr based assays. however, the majority of the samples tested in this study were nasal and vaginal swabs collected in virus transport media which are not usually known to contain pcr inhibitors (buckwalter et  al. 2014). hence, the need for inclusion of an inhibition control in this qualitative duplex real‑time pcr assay was not considered but yet suggested for testing samples containing pcr inhibitors. the dsn and dsp were calculated by screening dna extracted from 443 samples. the results of the uniplex and duplex real‑time pcr for brucella and bohv‑1 are presented in table iv. the dsn and dsp of the duplex real‑time pcr for the detection of brucella were recorded as 95.24% (95% ci: 76.18% to 99.88%) and 100.00 % (95% ci: 99.13% to 100.00%), respectively. the dsn and dsp for the detection of bohv‑1 were 95.65% (95% ci: 87.82% to 99.09%) and 99.47% (95% ci: 98.08% to 99.94%), respectively. the degree of agreement between the duplex and the respective uniplex assays was 0.974 (95% ci: 0.92 to 1.00) and 0.957 (95% ci: 0.92 to 0.99) for brucella and bohv‑1, respectively. the disagreement between the test methods occurred at very low analyte concentrations (ct value > 38 for bohv‑1 and > 37.5 for brucella) resulting in amplification in either duplex and the respective uniplex. the duplex real‑time pcr developed and validated in the present study could be a cost effective and time saving alternative for routine diagnostic use over the individual real‑time pcr assays. acknowledgement the authors are grateful to the management of the national dairy development board (nddb), anand for providing necessary facilities and funding to carry out this work. presence of a high concentration of brucella dna in the reaction (table iii). although, the amplification efficiency of brucella was reduced in presence of high concentration of bohv‑1 (>  2.3  tcid 50 per reaction), brucella dna with concentration higher than 410  cfus could be successfully amplified (table iii). in actual field scenario, the possibility of obtaining such high concentration of bohv‑1 in swabs is remote, and hence the interference in the detection of brucella is negligible. further, during screening of 443 field samples, the presence of dna of both the pathogens were detected in 7 samples by the respective uniplex assays. the duplex real‑time pcr could also detect all the 7 cases suggesting in none of the cases, the dominant amplification of one analyte competitively inhibited amplification of the another analyte. inter‑assay and intra‑assay analyses were performed. all the combination of dna mixture were tested in triplicates for three consecutive days. the coefficient of variation (cv) in terms of ct values were found to be similar for the duplex and the respective uniplex assays (table ii) and were within the acceptable range suggesting that the tests are repeatable. the inter‑run and intra‑run cv of ibr uniplex real‑time pcr was earlier reported to be 0.81‑1.02 and 0.51‑1.37, respectively (wang et al. 2007). the correlation between uniplex and duplex real‑time pcr for detection of brucella and bohv‑1 was evaluated by scatter diagram using the data obtained from the lod experiment. there was high correlation between the duplex assay and the uniplex assays [correlation coefficient (r) ≥  0.99 between the assays]. to evaluate the day to day variation, a histogram was plotted for ct values obtained in the uniplex and the duplex real‑time pcr for positive controls tested on 16 different days. table iii. effect of different amounts of target pathogen on the sensitivity of the duplex real-time pcr. all the combinations were tested in triplicates and the average ct value is presented. dna concentration ct value in uniplex bohv-1 ct value in duplex bohv-1 ct value in uniplex brucella ct value in duplex brucella high high 24.38 25.23 22.44 26.69 high medium 25.73 23.36 high low 26.09 22.60 medium high 31.38 32.14 29.94 37.10 medium medium 31.72 31.23 medium low 31.89 30.03 low high 35.17 36.58 35.55 41.45 low medium 35.02 38.89 low low 35.38 37.08 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animal health (oie). 2017. oie manual of diagnostic tests and vaccines for terrestrial animals 2017. chapter 2.4.12. infectious bovine rhinotracheitis/ infectious pustular vulvovaginitis (version adopted in may 2017). 73 parole chiave cavalli, risposta immunologica, vaccino, virus della west nile. riassunto il virus della west nile (wnv) è un virus a rna del genere flavivirus, famiglia flaviviridae che, trasmesso da zanzare, può causare morbilità e mortalità significative in uccelli, umani e cavalli. questi ultimi sono ospiti a fondo cieco. ad oggi non esiste una cura per la malattia e le misure preventive sono fondamentali; per i cavalli, occorre ridurre al minimo l'esposizione ai vettori o vaccinare. in europa sono in uso tre vaccini per cavalli che, quando sono stati utilizzati, hanno impedito la manifestazione dei segni clinici della wnd. per valutare la risposta immunologica alla vaccinazione sono stati scelti quaranta cavalli sierologicamente negativi al wnv e divisi in due gruppi da venti unità. un gruppo è stato vaccinato (richiamo dopo 28 giorni) con un intero ceppo virale inattivato e il secondo gruppo con uno vivo ricombinante per il canarypox virus che esprime i geni codificanti per le proteine virali prem / e del virus della west nile. per 360 giorni igm, igg e anticorpi neutralizzanti sono stati risposta immunologica indotta da due vaccini per il virus della west nile: inattivato e ricombinante keywords west nile virus, vaccine, horses, immunological response. summary west nile virus (wnv) is an rna virus belonging to flavivirus genus, family flaviviridae. transmitted by mosquitoes, the virus may cause significant morbidity and mortality in birds, humans and horses. humans and horses are dead‑end hosts. as no specific treatment for the disease is currently available, preventive measures are critical. in horses the prevention can be achieved by minimizing the exposure to the vectors or through vaccination. in europe three products have been registered for horses. when used, they are capable of preventing wnd clinical signs. to evaluate the immunological response following vaccination, 40 wnv serologically negative horses were selected and divided in two groups of 20 animals. one group was vaccinated (booster after 28 days) with a whole inactivated viral strain and the second group with a live recombinant canarypox virus expressing the genes coding for the wnv prem/e viral proteins. igm, igg and neutralizing antibodies were monitored by class specific elisas and serum neutralization assay for 360 days. in both groups, igm antibodies were first detected 7 days post vaccination lasting up to 42 dpv and 52 dpv in the animals vaccinated with the inactivated and the recombinant product respectively. a similar (p < 0.05%) number of horses [30%; 95% confidence interval (ci):14.59%‑52.18%] showed igm antibodies after vaccination with the recombinant vaccine (rec‑wnv) compared to the group (32%; 95% ci: 15.39‑54.28%) vaccinated with the inactivated vaccine (k‑wnv). both vaccines induced a detectable igg antibody response as early as 7 days following the vaccinations till the end of the trial. both vaccines induced the rise in neutralizing antibodies even though neutralizing antibody responses induced by the live canarypox virus‑vectored vaccine were higher (mean titres 1:298 vs 1:18.9) and lasted longer than did those induced by the killed‑virus vaccines. in fact, one year after the vaccination, neutralizing antibodies were still detectable in the horses which received the canarypox virus‑based vaccine but not the group vaccinated with the killed product. the use of vaccines is a valuable tool to prevent wnv disease in horses and the availability of different products facilitate the control of the disease in endemic areas. veterinaria italiana 2019, 55 (1), 73‑79. doi: 10.12834/vetit.1820.9611.1 accepted: 24.10.2018 | available on line: 31.03.2019 1istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. 2istituto zooprofilattico sperimentale della sicilia ‘a. mirri’, via gino marinuzzi 3, 90129 palermo, italy. *corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861 332446, e‑mail: f.monaco@izs.it. federica monaco1*, giuseppa purpari2, annapia di gennaro1, francesco mira2, patrizia di marco2, annalisa guercio2 and giovanni savini1 immunological response in horses following west nile virus vaccination with inactivated or recombinant vaccines 74 veterinaria italiana 2019, 55 (1), 73‑79. doi: 10.12834/vetit.1820.9611.1 west nile virus vaccines in horses monaco et al. previously duvaxyn® wnv, pfizer, us) (emea 2008), the recombinant canarypox virus vcp2017 strain, that expresses the wnv prm/pre genes (recombitek equine wnv vaccine, merial) (emea 2011) and the inactivated chimaeric flavivirus strain of yellow fever virus presenting the genes for the structural proteins e and prm of wnv (equilis® west nile, intervet international bv, netherlands) (emea 2013). these products are capable of protecting horses against possible development of clinical manifestations of the infection which can lead to a severe and long lasting neurological impairment of the animal. aim of this study was to look into the dynamic of the serological response in horses vaccinated with two different products widely used in eu member countries to protect horse population, the inactivated vaccine (equip® wnv) and the recombinant canarypox virus expressing the wnv prm/pre genes (recombitek equine wnv). materials and methods forty healthy horses, serologically negative to wnv, were randomly selected and divided into two groups of twenty animals. one group was vaccinated with the inactivate wnv strain, the other with the recombinant canarypox expressing the wnv prm/pre genes. both groups received a booster shot 28 days after the first dose. following the vaccination, all horses were regularly bled up to 1 year to monitor the immune response. to this purpose, blood samples were collected by jugular venipuncture in dry tubes at the indicated time points (figure 1). sera were prepared and stored at ‑20  °c until analysis. a commercial elisa kit (id screen west nile competition multi‑species ‑ id‑vet, montpellier, france) was used to detect igg immunoglobulins while the west nile virus igm introduction west nile virus (wnv) is a member of the flavivirus genus family flaviviridae. included in the japanese encephalitis group, it is one of the most widespread arbovirus in the world. in europe, wnv has been reporting since the mid 1960s (filipe et  al. 1969, joubert et  al. 1970) but the virus circulation has increased dramatically in the last decades (calistri et al. 2010, savini et al. 2018). in italy, wnv epidemics caused by genetically divergent isolates have been recorded since 2008 (savini et al. 2008, monaco et al. 2010, savini et  al. 2012) and most of the territory is nowadays endemic. the infection in nature in a mosquito‑bird cycle involving culex species of ornitophilic mosquitoes as main vectors and an extensive variety of birds as reservoir hosts (komar et  al. 2001, mancini et  al. 2017). mammals including humans and equidae are susceptible to the infection and can show clinical symptoms ranging from a flu‑like syndrome to a fatal meningoencephalitis (komar 2000, debiasi and tyler 2006). because of the low grade of viraemia and the lack of viral shedding, the virus cannot be transferred further and an infected horse acts as a dead‑end host (bunning et  al. 2002). nevertheless, the development of the severe clinical symptoms might raise devastating emotional effects and significant financial burden to owners. there is no specific anti‑viral treatment for the disease and prevention can be achieved by minimizing the exposure to the vector or, in equines, through vaccination (amanna and slifka 2014). to date, three vaccines have obtained the marketing authorization in european union (eu) member countries: the inactivated vaccine, produced from the vm‑2 strain (equip® wnv, zoetis, belgium monitorati con elisa specifici per classe e test di sieroneutralizzazione. in entrambi i gruppi, gli anticorpi igm sono stati rilevati per la prima volta dopo 7 giorni di vaccinazione con una durata fino a 42 dpv e 52 dpv negli animali vaccinati rispettivamente con il prodotto inattivato e il prodotto ricombinante. dopo la vaccinazione, sia il gruppo sottoposto al ricombinante (rec‑wnv) sia quello trattato con l’inattivato (k‑wnv) ha mostrato anticorpi igm in un numero simile (p < 0,05%) di cavalli con risultati rispettivamente pari a 30%; 95% intervallo di confidenza (ci): 14,59%‑52,18% e a 32%; ic 95%: 15,39‑54,28%. entrambi i vaccini hanno indotto una risposta anticorpale igg rilevabile già 7 giorni dopo le vaccinazioni fino alla fine dello studio. entrambi i vaccini hanno indotto l'aumento degli anticorpi neutralizzanti, anche se le risposte al vaccino vivo ricombinante sono state più elevate (titoli medi 1: 298 vs 1: 18,9) e sono durate più a lungo di quelle indotte dal vaccino con il virus inattivato. a un anno dalla vaccinazione infatti, non erano rilevabili nei cavalli di quest’ultimo gruppo si riscontravano in quelli del primo gruppo. i vaccini sono uno strumento prezioso per prevenire la malattia del wnv nei cavalli e la disponibilità di diversi prodotti facilita il controllo della malattia nelle aree endemiche. 75veterinaria italiana 2019, 55 (1), 73‑79. doi: 10.12834/vetit.1820.9611.1 monaco et al. west nile virus vaccines in horses after vaccination with the recombinant vaccine (rec‑wnv ) compared to the group (32%; 95% ci: 15.39‑54.28%) vaccinated with the inactivated vaccine (k‑wnv ). in both groups, igm antibodies were first detected after 7 days. however, in the group vaccinated with the inactivated product, the igm antibodies were detected up to 42 dpv (in 1 animal) while in the group immunized with the recombinant product igm antibodies were detected up to 52 dpv (in 5 animals) (figure 1). in the k‑wnv group, although the idexx elisa was capable of detecting a higher percentage of positive animals (32%; 95% ci:  15.39‑54.28%) than the id‑vet kit (21%; 95% ci: 8.66‑43.66%), this difference was not statistically significant (p > 0.05%) (figure 1). both vaccines induced a detectable igg response as early as 7 days following the vaccinations whatever the vaccine used. in the k‑wnv group, all animals seroconverted on day 18 pv while in the rec‑wnv group, all the animals become igg positive following the booster injection (on day 35 pv). igg antibodies were detected in the animals of both groups until the end of the trial (one year after vaccination). in the group vaccinated with the modified canarypox strain, neutralizing antibodies were first detected on day 18 pv in 1 horse. from day 42 pv till the end of the trial, neutralizing titers were detected in all vaccinated animals. the peak was observed on day 56 pv (mean antibody titer  =  1:298) (figure  2). in contrast, not all the animals vaccinated with the killed wnv strain developed a neutralizing response following vaccination. in fact, neutralizing titers were first detected on day 21 pv and 3 weeks later (day 42  pv) all except one animal seroconverted. after peaking on day 42 (mean antibody titer  =  1:18.9), the neutralizing titers decreased and, at the end of the trial, only 4 animals showed detectable titers (1:5) (figure 2). none of the animals in the trial developed antibody elisa kit (idexx laboratories, inc., maine, us) to evaluate the igm response in both groups. igm response in the group of animals receiving the recombinant product was also assessed by the id screen west nile igm capture (idvet, grabels, france). the tests were performed according to the manufacturer instructions. to confirm the presence of wnv antibodies, to define their titers and to exclude any cross reaction with other co‑circulating related flaviviruses such as usutu (savini et al. 2011), serum samples were also examined by serum neutralization (sn) assay in microtitre format (di gennaro et  al. 2014). for each proportion, 95% confidence intervals (ci) were calculated through the bayesian approach using the beta (s+1, n‑s+1) distribution where s is the total number of positives and n is the total number of tested animals. any wnv circulation in the area where the animals were kept was investigated according to the procedure defined in the wnv national surveillance plan (italian ministry of health, 20161). results no adverse effects were observed in the animals following vaccination. igm antibodies were detected in both groups of animals following vaccination (figure  1). a similar (p  <  0.05%) proportion of horses [30%; 95% confidence interval (ci):  14.59%‑52.18%] developed igm antibodies 1 italian ministry of health. 2016. piano nazionale integrato di sorveglianza e risposta al virus della west nile. circolare 10/08/2016, n. 23689. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 50 100 150 200 250 300 350 0 7 11 14 18 21 28 days post vaccination m ea n n eu tr al iz in g t it re p er ce n ta g e o f h o rs es w it h n eu tr al iz in g a n ti b o d ie s 35 42 49 56 90 360 % positive horses k-wnv % positive horses rec-wnv mean titre k-wnv mean tire rec-wnv figure 2. mean titres and percentage of animals with detectable neutralizing antibodies in two groups of horses following vaccination against the west nile virus (wnv) with an inactivated (k-wnv) or a recombinant canaripox wnv vaccine (rec-wnv). 0 7 11 14 18 21 28 35 42 52 56 k-wnv (1) 0 0.1 0.32 0.16 0.11 0.11 0.11 0 0 0 0 k-wnv (2) 0 0.1 0.21 0.21 0.16 0.11 0.11 0.11 0.05 0 0 rec-wnv 0 0.05 0 0.25 0.3 0.25 0.1 0.2 0.25 0.25 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 % p o si ti ve h o rs es days post vaccination figure 1. detection of igm antibodies in 2 groups of horses (n = 20) vaccinated either with an inactivated west nile virus vaccine (k-wnv) or with a recombinant canarypox wnv vaccine (rec-wnv). k-wnv (1) and rec-wnv = igm response detected by ‘west nile virus igm antibody’ test (idexx laboratories, inc., maine, us). k-wnv (2) = igm response detected by id screen west nile igm capture (idvet, grabels, france). 76 veterinaria italiana 2019, 55 (1), 73‑79. doi: 10.12834/vetit.1820.9611.1 west nile virus vaccines in horses monaco et al. this study confirmed, once more, how difficult is to predict the igm kinetic following vaccination suggesting that, in endemic areas or within a wnv surveillance plans, it is not possible to differentiate infected horses from recently vaccinated horses based on the presence/absence of igm antibodies. as a consequence, positive igm results should be carefully interpreted by verifying the vaccination history of the horses especially when vaccination against wnv is commonly practiced. when dealing with wnv igm antibodies, particular attention should also be paid to the igm elisas used since variations may exist in the igm elisa performances which often demonstrate different diagnostic sensitivity and potential for false positive results (davidson et  al. 2005, beck et  al. 2017). in this study, although in the number of positive animals detected by the two elisas after the vaccination with the inactivated product was different, this difference was not statistically significant. both vaccines were capable of evoking an igg  response in all vaccinated animals even if differences between groups were observed in the timeframe elapsed to stimulate the complete seroconversion. all horses immunized with the inactivated product developed a detectable igg  response starting from the 18th dpv while 35 days were necessary to achieve the complete seroconversion in the group which received the recombinant vaccine. such difference may rely on the different nature of the two vaccines. the viral antigens are ‘ready to be used’ in the inactivated vaccines, while with the recombinant product the same antigens need to be expressed by the vaccinated host vaccinated. differences were also noted in the duration and intensity of the neutralizing antibodies response elicited by the two vaccines. both vaccines induced the rise in neutralizing antibodies as observed by other authors (davies et al. 2008, joò et al. 2017, seino et al. 2007) even though neutralizing antibody responses induced by the live canarypox virus‑vectored vaccine were higher and lasted longer than did those induced by the killed‑virus vaccines. one year after the vaccination, neutralizing antibodies were still detectable in the horses which received the canarypox virus‑based vaccine but not in the group vaccinated with the killed product. long‑term immunity is not a characteristic of inactivated vaccines, and field studies demonstrated the drop of neutralizing titers 5 to 7 months after vaccination (davidson et  al. 2005). in our k‑wnv group of horses, the presence of neutralizing antibodies was detected only in four animals with titers below the threshold (< 1:10). it is known that the protective immune response to wnv requires both innate and adaptive immunity (de filette et al. 2012) and there is strong evidence neutralizing antibodies against usutu virus nor wnv circulation was detected in the area during the study period. discussion in accordance with the directive 2003/99/ec on monitoring of zoonoses and zoonotic agents, european member states collect data in order to define hazards, to evaluate exposures and to assess risks related to zoonoses and zoonotic agents. as a consequence, member states have implemented surveillance programs and, since 2012, they have been collecting data and reporting wnd cases. in austria, france, greece, italy, and united kingdom, an integrated animal‑human‑vector approach is already in place (gossner et  al. 2017). veterinary surveillance usually relies on the combination of passive measures, based on reporting clinical wnd cases in horses, and active surveillance, based on detecting seroconversion in sentinel horses (humblet et  al. 2016). the serological tests most commonly used are elisas (for detection of igg and igm antibodies) for screening and vnt as confirmatory test. the humoral response following wnv infection includes the production of igm antibodies, which are detectable 4‑7  days after wnv infection, and of igg, detectable 5  to  7  days after the infection (bunning et al. 2002). as the igm lifespan is considered to be less than three months in horses, the presence of igm antibodies in this species is regarded as a valuable indicator of recent infections (castillo‑olivares and wood 2004) and, as a consequence, the wnv igm elisa the test of choice for diagnosis of recent infection. it is also claimed that vaccination only occasionally elicits an igm response making it possible to differentiate acutely‑infected from recently vaccinated horses by using an igm‑based elisa (emea 2008). the possibility to differentiate a recently infected horse from one that has been (recently) vaccinated is particularly useful to early identify viral circulation either in wnv‑free or endemic areas. in this study, wnv recombinant and killed vaccines were examined and even if the igm response of the inactivated vaccine was lower, shorter and involving fewer animals, both products were capable of stimulating the production of igm  antibodies in vaccinated horses. these results were in line with what found in similar trials by other authors (porter et al. 2004, jonquiere et al. 2011, joò et  al. 2017). surprisingly, in few animals vaccinated with the recombinant vaccine, the igm antibodies persisted for 52 days after vaccination showing a kinetic similar to that observed in naturally infected horses in which igm antibodies can be detected 3  months after infection (ostlund et  al. 2000). 77veterinaria italiana 2019, 55 (1), 73‑79. doi: 10.12834/vetit.1820.9611.1 monaco et al. west nile virus vaccines in horses reported, the cell‑mediated response is still poorly explored (nelson et al. 2010). the use of vaccines has been demonstrated as a valuable preventative strategy against wnv disease in horses (grosenbaugh et al. 2004, siger et al. 2004, joò et  al. 2017). the 2018 has been regarded as an exceptional year referring to wnv circulation in eu countries (haussig et  al. 2018, riccardo et  al. 2018) with the infection of a high number of horses even in endemic areas (https://westnile.izs.it/j6_wnd/ docbolletmeditperiodico?annodocumento=2018). thus, the availability of different products provides a valuable tool to reduce the impact of the severe clinical symptoms in this species funding this work was supported by the italian ministry of health (msrcsi 01, 2012). mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the oie reference laboratory for west nile fever of the istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. that neutralizing antibodies provide long‑term protection from clinical signs of the disease (pierson and diamond 2015). different neutralizing responses however do not necessarily reflect dissimilarities in the protective capacity induced by the two vaccines. many trials demonstrated that the level of neutralizing antibody titer is not predictive of protective immunity in horses or hamsters (tesh et  al. 2002, seino et  al. 2007) since protection may occur in the absence of detectable antibodies. the requirements of significant levels of neutralizing ab at the time of exposure may not be critical as long as vaccination properly primes the immune system and response is rapid (minke et al. 2004). the absence or scarce level of humoral response does not preclude the efficacy of the cellular response. in fact, the role of cell mediated immunity in protecting against wnv and other related flaviviruses has been demonstrated in experimental murine studies (diamond et  al. 2003, shrestha and diamond 2004). the capacity of flavivirus infection to induce both innate and adaptive immune response is crucial to prevent the dissemination of these viruses in the organism (diamond 2003). effective vaccine‑based protection requires both responses and if the humoral has been frequently amanna i.j. & slifka m.k. 2014. current trends in west nile virus vaccine development. expert rev vaccines, 13 (5), 589‑608. beck c., lowenski s., durand b., bahuon c., zientara s. & lecollinet s. 2017. improved reliability of serological tools for the diagnosis of west nile fever in horses within europe. plos negl trop dis, 11 (9):e0005936. doi: 10.1371/journal.pntd.0005936. bunning m.l., bowen r.a., cropp c.b., sullivan k.g., davis b.s., komar n., godsey m.s., bake, d., hettler d.l., holmes d.a., biggerstaff b.j. & mitchell c.j. 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239‑247. 79 monaco et al. west nile virus vaccines in horses veterinaria italiana 2019, 55 (1), 73‑79. doi: 10.12834/vetit.1820.9611.1 siger l., bowen r.a., karaca k., murray m.j., gordy p.w., loosmore s.m., audonnet j.c., nordgren r.m. & minke j.m. 2004. assessment of the efficacy of a single dose of a recombinant vaccine against west nile virus in response to natural challenge with west nile virus‑infected mosquitoes in horses. am j vet res, 65, 1459‑1462. tesh r.b., arroyo j., travassos da rosa a.p., guzman h., xiao s.y. & monath t.p. 2002. efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of west nile virus encephalitis in hamster model. emerg infect dis, 8, 1392‑1397. the mediterranean basin. plos one, 13 (6), e0196429. doi: 10.1371/journal.pone.0196429. seino k.k., long m.t., gibbs e.p.j., bowen r.a., beachboard s.e., humphrey p.p., dixon m.a. & bourgeois m.a. 2007. comparative efficacies of three commercially available vaccines against west nile virus (wnv ) in a short‑duration challenge trial involving an equine wnv encephalitis model. clinical and vaccine immunol, 14 (11), 1465‑1471. shrestha b. & diamond m.s. 2004. role of cd8 t cells in control of west nile virus infection. j virol, 78, 8312‑8321. 281 department of veterinary medical sciences, alma mater studiorum, university of bologna, bologna, italy * corresponding author at: european college of animal welfare and behavioural medicine, via tolara di sopra 50, 40064 ozzano dell’emilia (bo), italy. department of veterinary medical sciences, alma mater studiorum, university of bologna, bologna, italy. tel.: +39 051 2097594, mob.: 335 8169721, e‑mail: angelo.peli@unibo.it. parole chiave benessere, cavallo da corsa, circonferenza dello stinco, lesione muscoloscheletrica, scienza forense, morfometria. riassunto nel cavallo sportivo le lesioni interessano con maggiore frequenza la regione del metacarpo. numerosi studi si sono occupati delle fratture a carico degli arti del cavallo ma pochi hanno indagato le possibili relazioni tra l’insorgenza di fratture e la morfometria dei segmenti ossei interessati. tuttavia, nei regolamenti di alcune corse tradizionali italiane, come il palio di siena, la misurazione della circonferenza dello stinco rappresenta un requisito imprescindibile affinché il cavallo possa essere ammesso alla corsa. scopo dello studio è stato verificare quanto la misurazione della circonferenza dello stinco eseguita nell’ambito dell’esame post‑mortem possa differire da quella rilevata sull’animale in vita, per valutare la validità del suo impiego in ambito forense. a tal fine sono stati esaminati gli arti anteriori di undici cavalli appartenenti a cinque differenti razze. la circonferenza dello stinco è stata misurata in tre momenti: nell’animale in vita subito prima della macellazione o dell’eutanasia, cinque ore dopo la morte e, infine, dopo un periodo di conservazione a ‑20°c per 14 giorni. gli arti sezionati nel corso delle due misurazioni post‑mortem sono stati inoltre pesati. la circonferenza media degli stinchi degli animali in vita è risultata di 24,0 ± 2,4 cm, di 22,9 ± 2,5 cm cinque ore dopo la morte e di 22,4 ± 2,3 cm dopo conservazione a ‑20°c per 14 giorni. la differenza tra queste misure si è rivelata statisticamente significativa (p  <  0,001). tra la misurazione effettuata nell’immediato post‑mortem e quella successiva allo scongelamento è stato inoltre registrato un significativo calo del peso medio degli arti sezionati, pari al 9,3% (p  <  0,001). secondo questo studio, la validità della misurazione della circonferenza dello stinco in ambito forense risulta essere limitata dato che la rilevazione post‑mortem, anche a distanza di poche ore dal decesso dell’animale, sottostima la circonferenza misurata nell’animale in vita. misurazione della circonferenza dello stinco nei cavalli in vita e nell'esame post mortem: attendibilità in ambito forense keywords forensic science, musculoskeletal injury, morphometrics, racehorse, shin circumference, welfare. summary catastrophic injuries in racehorses mostly involve the metacarpal region. although many studies describe fractures of equine limbs, few examine the relationship between bone morphometrics and musculoskeletal problems in racing horses. and yet, according to the regulation of some italian traditional races, the shin circumference represents a qualifying prerequisite for horses to be admitted to races. this study aims to evaluate the conformity of the shin circumference measurement in living animals and in post‑mortem examinations, in order to evaluate the forensic reliability of these measurements. the right and left distal forelimbs from 11 horses of 5 different breeds were examined. the shin circumference was measured at 3 time points: in the living animal before slaughter/euthanasia, 5 hours post‑mortem, and after 14‑days of cold storage. the isolated limbs were also weighed in both of the post‑mortem examinations. in the examined sample, the mean shin circumference was 24.0 ± 2.4 cm in living animals, 22.9 ± 2.5 cm 5 hours post‑mortem, and 22.4 ± 2.3 cm after 14‑days of cold storage, with a highly significant difference between these measurements (p < 0.001). there was also a significant decrease in the limbs’ weight between the 2 post‑mortem examinations (p < 0.001). according to our findings, the post‑mortem measurement significantly underestimates the in vivo dimensions of the shin circumference, even when performed a few hours after death; the forensic soundness of this parameter is therefore limited. angelo peli* and mariana roccaro the reliability and forensic soundness of the equine shin circumference measurement in living animals versus post‑mortem examination veterinaria italiana 2018, 54 (4), 281‑286. doi: 10.12834/vetit.1497.8069.2 accepted: 22.02.2018 | available on line: 31.12.2018 282 veterinaria italiana 2018, 54 (4), 281‑286. doi: 10.12834/vetit.1497.8069.2 forensic soundness of horse shin circumference peli & roccaro age, pre‑existing pathology and past traumas, biomechanics (conformation), but also race‑related factors such as race surface and training schedules (kobluk et al. 1990, magnusson and thafvelin 1990, mohammed et al. 1991, dolvik and klemetsdal 1996). there are very few reports on the relationship of overall body conformation to musculoskeletal problems in racing thoroughbred (anderson et  al. 2004). morphometrical data of equine limb bones are scant, perhaps because of the difficulty involved in making consistent and meaningful measurements of complex shapes, or because of the lack of standard field‑measurement procedures. in order to prevent cmis, some italian traditional races like the palio of siena have introduced regulations around the measurement of the ‘shin circumference’. measurements are made with a measuring tape in the thinnest part of the metacarpal region of the racing horse. in order to be admitted to the race, the shin circumference must not be below than a given value. the value is different from race to race, but usually around 18‑19 cm. although there are many reports describing fractures in the bony elements of equine limbs, very few of them provide information about specific morphological details and morphometrical measures of the affected bones. nevertheless, according to current regulations, a designated veterinarian must measure the shin circumference of any horse prior to being admitted to race. if a competing horse sustains a career‑ending injury or euthanasia and the veterinarian is sued for malpractice, a second examination of the shin circumference may be demanded during the legal proceeding. the aim of this study is to evaluate the conformity between the measurement of the shin circumference in the living animal and in post‑mortem examination in order to assess its forensic soundness. introduction catastrophic musculoskeletal injuries (cmis) of thoroughbred racehorses have been reported as the main reason for wastage (jeffcott et  al. 1982, rossdale et  al. 1985, robinson et  al. 1988, lindner and dingerkus 1993, bailey et  al. 1997). these injuries occur either during racing or training and are not only noxious to the welfare of the horses (evans 2002), but can also adversely affect public perceptions of racing. minimising and managing risk factors for this type of injury are therefore important considerations for those who are involved in this industry. in 97% of cases of injury to horses, the limbs are involved, and in particular the forelimbs, with the distal part being more susceptible to injuries such as fractures than other structures (jeffcott et  al. 1982, williams et al. 2001, perkins et al. 2005). in a study carried out in canada by cruz and colleagues (cruz et  al. 2007) on 76 thoroughbred horses with catastrophic musculoskeletal injuries, the 3 most affected regions were found to be the metacarpal‑metatarsal region (29%), followed by carpus (19.7%), and proximal sesamoid bones (18.4%). in a retrospective cohort study of thoroughbred racing in the national hunt in great britain from 2000 to 2013, more than 75% of fatalities resulted from catastrophic fracture, with most involving the third metacarpal (mciii) or third metatarsal (mtiii) (allen et al. 2017). race injuries in horses are considered to have a multifactorial aetiology, including genetics and figure 1. shin circumference measurement in the living horse. the measurement was performed right above the fetlock joint. sample 1b. figure 2. shin circumference measurement of the isolated limb five hours after death. sample 1b. 283veterinaria italiana 2018, 54 (4), 281‑286. doi: 10.12834/vetit.1497.8069.2 peli & roccaro forensic soundness of horse shin circumference examination was preceded by 24 hours of defrosting. for some limbs, immersion in water was necessary to hasten this process. statistical analysis was performed using the repeated measures anova in order to investigate changes in mean scores over the 3 time points. the bonferroni correction was used for post‑hoc analysis. a paired t‑test was used to compare the limbs’ weight at the 2 post‑mortem time points. a p‑value < 0.05 was regarded as statistically significant. results table i lists the subjects included in the study and their respective shin circumference measurements and distal limb weights. in the examined sample, the mean shin circumference was 24.0 ± 2.4 cm in the living animals, 22.9 ± 2.5 cm 5 hours post‑mortem, and 22.4  ±  2.3  cm after 14 days of storage in the refrigerator. the mean shin circumference measured 5 hours after death was therefore 1.1 cm shorter than in the living animal, whereas the mean circumference after 14 days of refrigeration was 1.6 cm shorter. this materials and methods in this study we examined the right and left distal forelimbs from 11 horses (10 sent to slaughter and 1 euthanised for tetanus). the animals were selected randomly and the sample included 5 breeds: 4 saddlebreds, 3 italian trotters, 2 thoroughbreds, 1 haflinger, and 1 arabian horse. the shin circumference of each horse was measured at 3 time points: in the living animal before slaughter/ euthanasia (t1), 5 hours post‑mortem (t2), and after 14 days of storage in sealed plastic bags in a refrigerator set at ‑20°c (t3). figure 1 shows the shin circumference measurement of the living animal taken by placing the measuring tape right above the fetlock joint. in post‑mortem examinations, we transected the distal limbs at the level of the radiocarpal joint or at the intercarpal joint. figure 2 illustrates the measurements taken on the isolated distal limb, 5 hours post‑mortem. the isolated limbs were also weighed in both of the post‑mortem examinations. the last post‑mortem table i. shin circumference measurements in the living animal (t1), 5 hours after death (t2) and after 14-days cold storage (t3). distal limb weight at t2 and t3. sample id breed right/left forelimb shin circumference (cm) distal limb weight (kg) t1 t2 t3 t2 t3 1a it right 23.0 23.0 21.5 2.0 1.5 1b it left 23.0 21.0 21.0 2.0 2.0 2a sb right 26.0 25.5 23.0 3.0 2.0 2b sb left 27.0 24.0 24.0 3.0 2.5 3a sb right 26.0 25.5 25.0 3.0 3.0 3b sb left 25.5 25.5 24.5 3.0 3.0 4a th right 21.3 19.5 18.5 1.5 1.0 4b th left 21.3 20.0 20.0 1.5 1.5 5a th right 23.0 21.0 21.0 2.0 2.0 5b th left 22.5 21.0 21.0 2.0 2.0 6a it right 24.5 24.5 23.5 2.3 2.0 6b it left 24.0 23.0 22.5 2.3 2.0 7a it right 22.0 21.5 20.5 2.0 2.0 7b it left 25.0* 23.5 22.5 2.0 2.0 8a sb right 29.0 28.0 28.0 4.0 3.0 8b sb left 29.0 28.0 26.0 4.0 3.0 9a sb right 23.0 23.0 23.0 2.0 2.0 9b sb left 23.0 22.5 22.5 2.0 2.0 10a ha right 24.0 23.0 23.0 3.0 3.0 10b ha left 24.0 23.0 23.0 3.0 3.0 11a ar right 20.0 19.5 19.5 2.5 2.5 11b ar left 21.0 19.0 19.0 2.5 2.5 the horse breeds are abbreviated as follows: it = italian trotter; sb = saddlebred; th = thoroughbred; ha = haflinger; ar = arabian horse. * the limb was swollen. 0 1 2 3 4 5 t2 t3 d is ta l l im b w ei g h t (k g ) figure 4. box and whiskers plot of the distal limbs weight at the two post-mortem examinations. t1 15 20 25 30 35 t2 t3 sh in c ir cu m fe re n ce (c m ) figure 3. box and whiskers plot of the shin circumference measurements at the three time points. 284 veterinaria italiana 2018, 54 (4), 281‑286. doi: 10.12834/vetit.1497.8069.2 forensic soundness of horse shin circumference peli & roccaro underlying synovial bursa. all of these events critically contribute to the decrease in volume of the studied structure and thence to the reduction of the shin circumference. moreover, storage conditions of the carcass such as the temperature and elapsed time prior to refrigeration, the unfreezing process, the beginning of putrefactive phenomena, the potential blood loss that follows sectioning, and the prolonged compression caused by the riding bandage or tendon boots left in place even during storage can contribute to an underestimation of the actual shin circumference in the living animal. given the results of this study, we recommend that forensic pathologists consider all factors, including normal post‑mortem phenomena, which are likely to cause a reduction of the shin circumference. they should moreover be aware that any measure acquired during the necropsy is an underestimation of the actual shin circumference in the living animal. according to our study, this underestimation is quantifiable as 4.6% 5 hours after death and 6.7% after 14‑day storage in a refrigerator set at ‑20°c. the forensic soundness of this dimensional parameter is limited. the correlation between shin circumference and the incidence of catastrophic musculoskeletal injuries has not yet been established. in order to objectively evaluate the relationship between conformation and the horse soundness, 2  requirements must be met: conformation has to be quantified in an accurate and repeatable way and reliable epidemiological data relating to type and incidence of injury should be available. however, only a small amount of data concerning the morphometrics of the mciii is available. a 2006 cohort study carried out on 108 national hunt racehorses, aimed to provide a set of baseline standard conformational traits within the thoroughbred population. the study took into account 98 conformational parameters consisting of segment lengths, joint angles, inclinations, deviations, and circumference measurements, including the mid‑metacarpus circumference. the mean circumference was 20.15 cm, with a minimum size of 18.00 cm and a maximum of 22.00 cm. significantly different circumference measurements were found between left and right limbs (weller et al. 2006). a more recent study aimed at identifying morphometrical variations of equine metacarpal, proximal phalangeal, and proximal sesamoid bones recorded the proximodistal length and mid‑shaft width and depth of the mciii after boiling, cleaning, and drying the bone. in thoroughbred horses, the mean mid‑shaft widths of the right and left mciii were respectively 4.09 ± 0.04 cm and 4.02 ± 0.04 cm, reduction corresponds respectively to the 95.4% and 93.3% of intra‑vitam values, and was found to be highly significant (p  <  0.001). figure 3 is a box‑and‑whisker plot illustrating the distribution of the shin circumference measurements at the 3 different time points. in the living animal (t1) the median circumference value was 23.5 cm, with a minimum circumference of 20.0 cm and a maximum of 29.0 cm. five hours post‑mortem (t1) the median circumference was 23.0 cm, with a minimum value of 19.0 cm and a maximum of 28.0 cm. after 14 days of refrigeration (t3), the median value decreased to 22.5 cm, with a minimum circumference of 18.5 cm and a maximum of 28.0 cm. the limbs’ weight was also found to be significantly different between the 2 post‑mortem examinations (p  <  0.001). a 9.3% decrease in the mean weight between t2 (2.48 kg) and t3 (2.23 kg) was observed. figure 4 is a box‑and‑whisker plot displaying the distribution of the distal limb weights 5 hours post‑mortem and after 14 days of cold storage. at t2, the median was 2.3 kg, with a minimum value of 1.4 kg and a maximum of 4.0 kg. at t3, the median was 0.3 kg less, with a minimum value of 1.0 kg and a maximum of 3.0 kg. discussion competitive activity in horses involves a unique challenge in terms of muscular and athletic abilities, which predispose them to particular types of injury or disease. concern about the welfare of horses involved in the racing industry together with the popularity of the more well‑known races, such as the palio of siena in italy, raises an intense public debate. regulated examinations should help to ensure that the welfare of racing horses is not compromised, as they prevent horses that are unfit from competing. the shin circumference is considered a formal requirement for any horse to be admitted to various traditional races in italy. therewith, if a racing horse sustains a career‑ending injury or euthanasia, the designated veterinarian’s conduct could be questioned and a second measurement of the shin circumference required during the post‑mortem investigation, most likely on a cold‑stored carcass or limb after unfreezing. this study has identified significant morphometric variations between the shin circumference in living horses that are then examined post‑mortem, especially after cold storage time. this difference can be explained by taking into consideration post‑mortem phenomena that normally occur after death, including the arrest of blood flow and consequent dehydration, the loss of muscle tone and tissue turgidity, the autolytic processes, and the decrease in volume of the 285veterinaria italiana 2018, 54 (4), 281‑286. doi: 10.12834/vetit.1497.8069.2 peli & roccaro forensic soundness of horse shin circumference affect musculoskeletal disease, while conformation variables associated with metacarpophalangeal joint problems were long pasterns, offset ratio, carpal angle, and radio‑metacarpal angle. contrary to this, according to davies and watson (davies & watson 2005), longer mciii bones are associated with ticker dorsal cortices at the mid‑shaft in racehorses that were exercising at racing speed, suggesting that the longer bones do bend more and therefore might be expected to fracture more easily. in light of these considerations, further studies are needed to assess the effects of bone morphology on fracture incidences. such data will enable veterinarians to better estimate the relative importance of conformational variables, such as the shin circumference, for future soundness in racehorses. whereas the mean depths were 3.23 ± 0.07 cm for the right mciii and 3.26 ± 0.05 cm for the left mciii (alrtib et al. 2013). the purpose of arltib’s study was to address the lack of information on the normal range in size and shape of these bones, and to identify reliable techniques for measuring them that are not 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racehorse injuries, clinical problems and fatalities recorded on british racecourses from flat racing and national hunt racing during 1996, 1997 and 1998. equine vet j, 33 (5), 478‑486. and tibial stress fractures in thoroughbred racehorses in training in the uk. prev vet med, 74, 21‑35. weller r., pfau t., may s.a. & wilson a.m. 2006. variation in conformation in a cohort of national hunt racehorses. equine vet j, 38, 616‑621. 287 veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 accepted: 19.07.2019 | available on line: 31.12.2021 1department of pathology, institute of veterinary medicine of serbia, belgrade, serbia. 2department of pathology, faculty of veterinary medicine, university of belgrade, serbia. 3department of ruminants and swine diseases, faculty of veterinary medicine, university of belgrade, serbia. 4department of virology, institute of veterinary medicine of serbia, belgrade, serbia. 5department of food hygiene, institute of veterinary medicine of serbia, belgrade, serbia. 6department of immunology, institute of veterinary medicine of serbia, belgrade, serbia. 7university clinical center of serbia, clinic for digestive surgery, belgrade, serbia. *corresponding author at: department of pathology, institute of veterinary medicine of serbia, belgrade, serbia. tel.: +381112851096, e-mail: branislavkureljusic@yahoo.com. branislav kureljušić1*, sanja aleksić kovačević2, božidar savić1, radiša prodanović3, nemanja jezdimirović1, vesna milićević4, jelena maksimović zorić4, jasna kureljušić5, jadranka žutić6, đorđe knežević7, ljiljana spalević6 and vladimir kukolj2 keywords swine, liver, hev, microscopic changes, immunohistochemistry. summary hepatitis e virus (hev), the zoonotic agent of infectious hepatitis, is present in swine farms in different geographical areas. little is known about the mechanism of liver damage and type of local immune response by hev in swine. therefore, the aim of this study was to determine the morphological and immunophenotypic characteristics of hepatic lesions caused by hepatitis e virus in naturally infected swine. in this study, liver samples of 12 slaughtered 10 weeks old pigs which were rt-pcr positive for hev rna in rectal swab samples have been used. livers were macroscopically examined and samples were taken for histopathological, immunohistochemical (cd3, cd79α and tgf-β1), semiquantitative, morphometric analysis, rt-nested-pcr, pcr and bacteriological analysis. microscopically, mild and moderate multifocal lymphoplasmacytic hepatitis was observed. apoptotic bodies were observed as areas of focal eosinophilic condensation in the cytoplasm of 33.33% liver samples, while in 16.67% liver samples portal fibrosis was detected. immunohistochemically, portal and lobular lymphocytes in the mononuclear liver infiltrate were predominantly cd3+ t cells (234.80 ± 79.98). an intense tgf-β1 positive reaction was observed within the mononuclear cell infiltrate as well as polymorphonuclear cells in liver samples with apoptosis of hepatocytes. in all 12 tested liver samples hev rna was detected by rt-nested-pcr. hev is noncytopathic, and this finding provides further evidence for an immune mediated pathogenesis in hepatitis e virus infection in swine. also, the role of cd3+ cells in hepatocyte damage is clearly demonstrated. morphological and immunophenotypic characteristics of the liver of swine naturally infected with hepatitis e virus of proves have demonstrated the zoonotic nature of hepatitis e virus. swine and human hev strains are genetically related suggesting both a zoonotic and a possible foodborne transmission (meng 2003, di  bartolo et  al. 2008, caruso et  al. 2017, bansal et al. 2017). usually, there are no gross lesions in the liver of hev infected pigs. occasionally mild hepatic enlargement and scattered yellowish discoloration foci can be found in some samples (lee et al. 2007). introduction hepatitis e virus, the zoonotic agent of infectious hepatitis, is present in swine farms in a number of geographical areas (aprea et  al. 2018). different species of domestic and wild animals have been reported to have anti-hev antibodies (okamoto 2007, roić et  al. 2018), and/or to be infected with viruses closely related to hev strains infecting humans (tei et  al. 2003, banks et  al. 2004). swine infected with hev are asymptomatic. a number 288 veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 hepatitis e virus in swine kureljušić et al. injury may be attributed to immune-mediated damage by cytotoxic t cells and natural killer cells (prabhu et al. 2011). although many studies have described histological alterations of the swine liver, little is known about the mechanism of cell damage or the type of local immune response by the host cells to hev. therefore, the aim of this study was to determine the immunophenotypic characteristic of immune cells in swine liver naturally infected with hev. materials and methods sampling rectal swabs were taken from 153 weaned pigs from the same farm and tested for the presence of hev rna by rt-nested-pcr. thereafter, 12 positive pigs were slaughtered in the slaughterhouse and livers were macroscopically examined and liver samples were taken for histopathological, immunohistochemical, semiquantitative, morphometric analysis, rt-nested-pcr and bacteriological analysis. additionally, spleen samples were taken for pcr analysis for swine torque teno virus (ttv) and porcine circovirus 2 (pcv2), whereas lung samples were used for rt-pcr for porcine reproductive and respiratory syndrome virus (prrsv) detection. the research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals (ethics committee of faculty of veterinary medicine, university of belgrade, 01.06.2011, no. 01-624). liver samples from 10 pigs which were negative for hev rna were used as negative controls. previously, swine sera from both groups were tested negative for pcv-2 and prrsv antibodies. histopathological and immunohistochemical studies liver samples for histopathological and immunohistochemical studies were fixed in 10% buffered formalin and after standard processing in an automated tissue processor (dehydration, illumination and impregnation), cast in paraffin blocks. the paraffin sections 4 μm thick were stained with hematoxylin and eosin (he) and masson trichrome method (mth) for light microscopic examination. previously, fresh liver samples were fixed in fixative consisting of 9 parts by volume of absolute ethyl alcohol to one part of 40% formaldehyde neutralized with mgco 3 and paraffin sections were stained by best’s carmine technic for excluding accumulation of glycogen in hepatocytes. additionaly, cryostat sections of the liver samples microscopically, naturally and experimentally hev-infected pigs showed evidence of acute hepatitis characterized by mild to moderate multifocal and periportal lymphoplasmacytic hepatitis, with mild focal hepatocellular necrosis (lee et  al. 2007, halbur et  al. 2001, de deus et  al. 2008, lee et al. 2008). viral infection causes host cell injury either directly, through the action of an infectious agent, indirectly, as a consequence of the antiviral host immune response or through the cumulative effects of both direct and indirect damage (srivastava et  al. 2007). while hev is effectively cleared by a strong immune response, hepatic damage of a variable severity is a common consequence of cytotoxicity. conversely, an inadequate immune response results in viral persistence, although with little liver damage (srivastava et al. 2007). studying the direct cytopathic effect of hev has proven difficult, due to the inability to efficiently culture the virus in  vitro (srivastava et  al. 2007). an aggressive immune response leads to effective viral clearance that is accompanied by a variable degree of hepatic damage (srivastava et al. 2007). on the other hand, an inadequate cytotoxic response results in viral persistence, albeit with little liver damage (srivastava et  al. 2007). in any case, the mechanisms which regulate the intensity of the immune response have a key role in the viral infection defense. it has been hypothesized that liver damage induced by hev infection in humans may be due to the immune response to the invading virus and may not be a direct cause of viral replication in the hepatocytes (purcell 1996). nevertheless, some authors emphasize that the immune-mediated liver injury by lymphocytes might be mainly involved in the pathogenesis of hepatitis e, but liver injury induced directly by hev could not be excluded (zhao et al. 2001). in cases of acute fulminant human hepatitis e, the lymphocyte infiltrate consisted predominantly of cd3+ t cells. these cells contained a predominant cytotoxic (cd8+) cell subpopulation in 81.8% of cases with hepatitis e infection (agrawal et al. 2012). the cellular composition of the liver inflammatory infiltrate was different in patients with b and c hepatitis where t helper lymphocytes comprised 50-60% of the inflammatory infiltrate. approximately 25% were t cytotoxic lymphocytes; b lymphocytes comprised 15% of the inflammatory infiltrate and other cells, including natural killer cells (nk), 10% in total (waleska-zielecka et al. 2008). immunohistochemical findings of the liver biopsy samples of hev infected patients clearly demonstrate the role of cd8+ t cells and natural killer cells in hepatocyte damage and disease pathogenesis. the virus is noncytopathic, and therefore, liver 289veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 kureljušić et al. hepatitis e virus in swine cell b), and the cell count per mm2 was calculated. this was compared between the observed cases and controls. digital images were taken by microscope olympus bx51 with digital camera olympus color view iii. rna extraction and hev rt‑nested‑pcr and pcr rectal swabs were prepared by immersion into sterile pbs and vigorous vortexing. organ samples were homogenized in sterile pbs in final dilution 1:10. after homogenization, the suspensions were centrifuged for 10 min at 2000 rpm. the supernatant was used for nucleic acid extractions. dna was isolated from liver samples by using qiaamp dna mini kit (qiagen, hilden, germany), following the recommended tissue protocol. viral rna from rectal swabs and liver samples was extracted by using qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer’s instructions. rt-pcr and nested-pcr were carried out in 50  µl reactions using onestep rt-pcr kit and hotstar taq mastermix kit (qiagen, hilden, germany), respectively. two degenerate primer sets were selected for the amplification of orf2 region. primers used for the first round were hevorf2con-s1 5’gacagaattratttcgtcggctgg 3’ and hevorf2con-a1 5’cttgttcrtgytggttrtcataatc 3’. for the nested-pcr, primers hevorf2con-s2 5’gtygtctcrgccaatggcgagc 3’ and were stained by sudan iii for excluding potentially fatty liver degeneration, as well. immunohistochemical procedure a three-step indirect immunohistochemical technique was performed on 4  µm formalin-fixed and paraffin embedded sections. after antigen retrieval (table i) the sections were then treated with methanol containing 0.3% hydrogen peroxide for 15 minutes at 22 ± 3 °c in order to inactivate the endogenous peroxidase. non-specific binding of secondary antibodies was minimized by incubating with 50% normal goat serum in pbs for 20 minutes. sections were incubated with appropriate primary antibodies (table i) diluted in pbs. three primary antibodies were applied: rabbit-anti-human cd3 (pan t-cell marker), mouse-anti-human cd79α (b-cell marker) and rabbit polyclonal antibody tgfβ1  (v). all rinsing procedures and serum dilutions were done in pbs (ph 7.2-7.4). the detection kit was dako cytomation lsab2 system-hrp, rabbit/mouse (dako, k0675). positive reactions were visualized by applying dab+ (dako, k3468) for 5 to 10 minutes. counterstaining was performed using mayer haematoxylin for 2 seconds. aqueous medium glycergel (dako, c563) was used on stained sections for mounting. liver sections not treated with the primary antibody were used as negative controls. as positive controls for immunohistochemistry (ihc) reactive lymph nodes of swine were used. semiquantitative analysis semiquantitative analysis was conducted according to criteria which were used for assessment of distribution and density of lymphoplasmacytic infiltrates in swine livers experimentally infected with hev (halbur et  al. 2001). score for severity of lymphoplasmacytic hepatic lesions are shown in table ii. morphometric analysis immunopositivity was quantified by counting the total number of lobular and portal cd3 and cd79α immunopositive cells in 25 random selected fields at 40× magnification. the area of the liver specimen was calculated using image analysis software (olympus table i. primary antibodies used for immunohistochemistry. antibodies source dilution antigen retrieval incubation cd3 dako a0452 1/50 microwave, 560w, 21 minutes in citrate buffer 1 h, in humid chamber at 22 ± 3 °c cd79 dako m7051 1/50 microwave, 560w, 21 minutes in citrate buffer 1 h, in humid chamber at 22 ± 3 °c tgf-β1 (v) santa cruz biotechnology, sc-146 1/10 proteinase k treatment for 40 minutes at 22 ± 3 °c overnight, in humid chamber at 4 °c table ii. criteria for semiquantitative analysis. score for severity of lymphoplasmacytic hepatic lesions description 0 no inflammation 1 1 to 2 focal lymphoplasmacytic infiltrates/ 10 hepatic lobules 2 2 to 5 focal lymphoplasmacytic infiltrates/ 10 hepatic lobules 3 6 to 10 focal lymphoplasmacytic infiltrates/ 10 hepatic lobules 4 > 10 focal lymphoplasmacytic infiltrates/ 10 hepatic lobules 290 veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 hepatitis e virus in swine kureljušić et al. demonstrated by macroscopic examination. additionally, mildly to moderately enlarged hepatic lymph nodes in 6 (50%) out of 12 examined livers were also observed. in control group of pigs those findings were not noted. a markedly dilated gallbladder was observed in four cases (33.33%) among infected pigs and in two cases among pigs from control group (20%). the gallbladder was enlarged, protruding over the edge of the liver and filled with a large amount of bile. histopathological findings mild to moderate multifocal lymphoplasmacytic hepatitis in the portal tracts and/or irregularly distributed in the liver parenchyma was observed (figures 1, a and b). the infiltrate comprised predominantly mononuclear cells, usually observed close to the area of necrosis of the hepatocytes. in all of the 12 examined samples a mild and moderate mononuclear infiltrate was localized in the portal tract. in 10 out of the 12 examined cases (83.33%) a multifocal mononuclear infiltrate and hydropic degeneration were observed in the liver parenchyma. intracellular edema was present randomly, but with a higher prevalence in the centrilobular area. activated kupffer cells were detected in 7 out of 12 cases (58.33%). apoptotic bodies which represent areas of focal eosinophilic condensation in the cytoplasm were observed in 4  out of 12 (33.33%) samples (figure  1c). apoptotic bodies were frequently observed in all three lobule areas, but they were more frequently found in the centrilobular and midzonal area. in 2 out of 12 cases, severe hepatocyte necrosis was observed mainly in the centrilobular area. in the remaining 10 cases, focal points of necrosis of the hepatocytes were observed mainly in the centrilobular and midzonal area and less frequently in the periportal areas. these focal points of necrosis were visualized as areas of parenchyma showing a complete destruction of cells and its transformation in cellular debris with lymphocytic and occasionally neutrophilic infiltrate. the degree of inflammatory cell infiltrate was significantly increased in the necrotic and degenerated liver parenchyma. borders between the portal tracts and lobuli were intact, and the lobular liver architecture was intact. in uninfected control livers of pigs, microscopically there were no lesions in four samples. in six samples, very mild focal lymphoplasmacytic infiltrates were observed. in this group there were no signs of hepatocyte degeneration, apoptosis and/or necrosis. in addition to the accumulation of large amounts of connective tissue in the portal tracts, around the dilated bile ducts, proliferative epithelium surrounded by multiple smaller bile ductules were observed (figure 1d). the blood vessels in the portal hevorf2con-a2 5’gttcrtgytggttrtcataatcctg -  3’ were used, yielding a final product of 145 bp (erker et al. 1999). both reactions were accomplished following the thermal profiles previously described (di bartolo et al. 2008). for ttv, pcv2 and prrs detection, we used the previously described primers and protocols (oleksiewicz et al. 1998, lukač et al. 2016, kekarainen et al. 2006). bacteriological analysis for isolation of pathogenic bacteria (staphylococcus aureus, trueperella pyogenes, escherichia coli, klebsiella  spp., proteus spp., salmonella spp., clostridium novyi, listeria spp.) from the liver samples, pre-enrichment was performed in various selective broths followed by plating on selective media as per the method described by cruickshank and colleagues (cruickshank et  al. 1975). identification of the bacteria and biochemical tests were done according to the standard procedures given by cowan and steel (1993). statistical analysis after counting the total number of cd3 and cd79α immunopositive cells, the average number ( χ ), standard deviation (sd), minimal and maximal value (x min , x max ) were calculated for each examined group. the cell counts among the groups were compared using anova test. the significance of differences was determined by the level of significance of 5% and 1%. statistical comparison of score for severity of lymphoplasmacytic hepatic lesions between infected and control groups has been done with mann-whitney test. statistical analysis was carried out using graphpad prism (ver. 6.01). results hev rt‑nested‑pcr and pcr in all 12 liver samples tested, hev rna was detected by rt-nested-pcr. all tested samples were negative for the presence of nucleic acid of pcv2, prrs and ttv. bacteriological results all liver samples were tested negative for the presence of pathogenic aerobic and anaerobic bacteria. macroscopic findings mild liver enlargement with blunt edges was 291veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 kureljušić et al. hepatitis e virus in swine were predominantly cd3+ t cells (figure 2a). immunopositivity was clearly demonstrated in the area of the plasma cell membrane. a smaller number of cd79α+ cells was detected in swine liver (figure  2b). these cells were individually located within the regions of t-cell infiltration. tgf-β1 positive cells were detected within mononuclear cells infiltrate, as well as polymorphonuclear cells located in the liver parenchyma (figure 2c). the intense positive reaction to tgf-β1 showed inflammatory cells in liver samples with hepatocyte apoptosis. morphometric analysis number of cd3 and cd79α positive cells was higher in infected pigs than in control pigs (p < 0.05, p < 0.01) (table iii). discussion there is a lot of literature data on the prevalence of hev infection in pigs in different countries throughout europe. in addition, numerous studies tracts had a thickened tunica media with clearly visible amplified smooth-muscle cells. this finding is suggesting a chronic liver injury and fits into the portal liver fibrosis pattern. semiquantitative analysis results liver samples of infected pigs had the most frequent lymphoplasmacytic hepatic lesions severity score  1 (41.67%), following by score 2 in 3 (25%) tested samples. the most severe score 4 was as prevailing as score 3, both found in 2 cases (16.67%), respectively. in the tested liver samples of control pigs, the most common score was 0 (40%), and score 1, as well as score 2 were found in 3 cases (30%), respectively. a mann-whitney test indicated that the infected pigs' livers (mean rank = 14.25) were rated more severe than the control group (mean rank = 8.20), z = -2.261, p = 0.014. the difference between tested groups is statistically significant (p < 0.05). immunohistochemistry immunohistochemically, portal and lobular lymphocytes in the mononuclear liver infiltrate figure 1. microscopic changes in the liver of pigs under study. a. severe portal mononuclear infiltrate. b. mononuclear inflammatory cells observed closed to the area of necrosis of the hepatocytes. c. councilman apoptotic bodies, arrow shows councilman apoptotic body. d. portal fibrosis, he. a c b d 292 veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 hepatitis e virus in swine kureljušić et al. hev-infected pigs show evidence of acute hepatitis characterized by mild to moderate multifocal and periportal lymphoplasmacytic hepatitis, with mild focal hepatocellular necrosis (lee et al. 2007, halbur et al. 2001, lee et al. 2008, de deus et al. 2007), which was confirmed in our investigation with naturally infected pigs. furthermore, in all 12 samples (100%) the infiltrate was localized in the portal area of the liver, and in 10 tested samples (83.33%) a multifocal infiltrate was observed in the liver parenchyma. hepatocyte damage ranged from mild intracellular edema and vacuolar degeneration to hepatocyte necrosis. a similar finding was established in people with hepatitis e (zhao et al. 2001). the dominant cell population in the mononuclear cellular infiltrate were cd3 positive lymphocytes. the population of cd79α positive lymphocytes was less frequent. liver injury can be attributed to immune-mediated damage by cytotoxic t cells which are within the cd3+ cell subpopulation. similar findings by agrawal and colleagues and prabhu and colleagues (agrawal et al. 2012, prabhu et  al. 2010) were proven in cases of hepatitis e in humans. generally, damage to liver cells by viruses may occur as a result of the direct effects of the virus or indirectly as a result of the antiviral immune response of the host, or a combination of both. it is possible that the powerful immune response leads to the efficient removal of viruses and liver damage of various degree, a weaker immune response leads to the persistence of the virus and liver damage of lower intensity (srivastava et al. 2007). the mechanisms that regulate the intensity of the immune response are probably crucial in defending the body from the virus. although many studies have described histological alterations of the swine liver, little is known about the mechanism of cell damage or the type of local immune response by the host cells to swine hev. demonstration of the immunophenotype of infiltrating lymphocytes in swine liver tissue infected with hev, as performed in this study, has not been reported previously. are based on the molecular epizootiology and genotyping of hepatitis e virus (di bartolo et  al. 2008, vasickova et  al. 2009, forgach et  al. 2010). some reports were based on pathomorphological investigations in hepatitis e (lee et al. 2007, lee et al. 2008, de deus et  al. 2007), whereas studies on the local immune response in hev infected animals have not been reported previously. due to these reasons, the current investigations were conducted. macroscopic examination of the liver of pigs naturally infected with hepatitis e revealed a slight increase in volume which is consistent with the descriptions of other authors (lee et  al. 2007). in assessing this finding, one should be very cautious with regard to the enlargement of the liver, as it may be the result of increased amounts of blood and other infectious agents (pcv2, ttv, prrs and bacteria such as clostridium novyi, listeria spp., trueperella pyogenes, leptospira spp., salmonella spp.). however, in the current study, these agents were excluded by molecular methods and bacteriological analysis. the markedly dilated gallbladder cannot be linked to the hev infection, due to the fact that such a pathological change usually occurs in the case of bile duct obstruction and in cases where the animals do not consume food and there are no stimuli for the secretion of bile. in this case it can be cosequence of feed restriction before slaughtering, which is usual practice. the observed increase in volume in hepatic lymph nodes in 50% cases might be due to reactive hyperplasia which is also described by bouwknegt and colleagues (bouwknegt et al. 2009). microscopically, naturally and experimentally table iii. number of cells expressed per mm2 (n/mm2). all values are given as mean values ± standard deviation. cell marker infected group χ ± sd ( x min x max ) (n = 12) control group χ ± sd ( x min x max ) (n = 10) p value cd3 234.80 ± 79.98 (166-350) 183 ± 8.49 (177-189) p < 0.05 cd79 26.00 ± 17.71 (10-49) 3.00 ± 1.41 (2-4) p < 0.01 p < 0.01 = statistically significant at the level of 99%; p < 0.05 = statistically significant at the level of 95%. figure 2. immunohistochemical characteristics of the liver of swine. a. cd3 positive cells. b. cd79α positive cells. c. tgf-β1 positive cells, lsab2. a b c 293veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 kureljušić et al. hepatitis e virus in swine yellow fever, researchers revealed cd4+ t cells as the dominant cell population present in the mononuclear cell infiltrate, as well as hepatocytes which express tgf-β. it is believed that tgf-β is responsible for apoptosis of hepatocytes which in this disease was the dominant finding, as well as a weaker inflammatory response (quaresma et  al. 2006). a source of tgf-β1 in the liver are kupffer cells, endothelial cells, hepatocytes, epithelial cells of the biliary duct and perisinusoidal stellate cells (hinz et  al. 2007). when hepatocytes undergo apoptosis they actively secrete cytokines and chemokines that activate the transmission signal to adjacent cells and release inflammatory cytokines and chemokines including tgf-β1. in our study, portal liver fibrosis was confirmed in two cases. according to some authors the release of tgf-β1 is the key event in the pathogenesis of fibrosis (takiya et al. 1995, kisseleva and brenner 2008). tgf-β1 has multiple functions during organogenesis, tissue damage, and recovery. in the development of liver fibrosis not only increased synthesis of tgf-β1 is present, but also activation of latent to biologically active form is more intense (friedman et al. 1999). conclusions our finding provides further evidence for an immune mediated pathogenesis in swine naturally infected with hepatitis e virus. detected liver lesions, as well as the local immunological response, are similar to those detected in human hev infection. both morphological features, i.e. the dominant cd3+ t cell population in the mononuclear infiltrate, as well as the finding of apoptotic bodies in the liver of pigs naturally infected with hev have not been reported previously. acknowledgements this work was supported by the projects iii46009 and tr31062 from the ministry of education, science and technological development of the republic of serbia. the observed presence of apoptotic bodies in liver of infected pigs was considered as a morphological manifestation of apoptosis. apoptosis is established in experimental infections of pigs with porcine and human strains of hepatitis e virus (halbur et al. 2001). it is known that apoptosis is also a physiologically active process in the regulation of the population of different cells, which is characterized by specific biochemical and morphological changes. various signals within a cell or signals outside the cell can activate biochemical reactions in the cell which can result in apoptosis. in this complex process, cysteine proteinases are included. these are enzymes of the family of caspases, which play a key role in the process of apoptosis (salvesen and dixit 1997, roulston et al. 1999). massive necrosis of hepatocytes in the centrilobular and midzonal area was accompanied by a dense mononuclear infiltrate. it can be assumed that the number of lymphocytes is in direct relationship with the resultant necrotic process. this finding suggests that hepatocyte damage occurs as a result of immunoreactivity and highlights the importance of present lymphocytes in the pathogenesis of damage (lee et al. 2007, halbur et al. 2001, de deus et al. 2008, lee et al. 2008, de deus et  al. 2007, meng et  al. 1997). studies on hepatitis e in humans have shown that immune-mediated liver damage is likely to arise as a result of the action of cytotoxic t lymphocytes and natural killer cells (nk cells) (prabhu et  al. 2011). the presence of activated kupffer cells is due to increased synthesis of proinflammatory cytokines. an increased number of kupffer cells was found in the liver of people with hepatitis e (zhao et al. 2001). the presence of tgf-β1 positive cells located in the mononuclear cell infiltrate in the liver parenchyma can be associated with apoptosis of hepatocytes, which was present in a third of tested pigs, and a reduced inflammatory response. this hypothesis is based on the fact that tgf-β1 induced apoptosis in the liver and bile ducts in a number of liver pathologies (takiya et al. 1995, quaresma et al. 2006). upon examination of pathological changes occurring in the liver of humans suffering from 294 veterinaria italiana 2021, 57 (4), 287-295. doi: 10.12834/vetit.1813.9553.3 hepatitis e virus in swine kureljušić et al. agrawal v., goel a., rawat a., naik s. & aggarwal r. 2012. histological and immunohistochemical features in fatal acute fulminant 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kasorndorkbua c., gilbert c., guenette d., potters m.b., purcell r.h., emerson s.u., toth m.e. & meng x.j. 2001. comparative pathogenesis of infection pigs with hepatitis e viruses recovered from a pig and a human. j clin microbiol, 39 (3), 918-923. hinz b., phan s.h., thannickal v.j., galli a., bochaton-piallat m.l. & gabbiani g. 2007. the myofibroblast – one function, multiple origins. am j pathol, 170 (6), 1807-1816. kekarainen t., sibila m. & segales j. 2006. prevalence of swine torque teno virus in post-weaning multisystemic 236 wasting syndrome (pmws)-affected and non-pmws-affected pigs in spain. j gen virol, 87, 833-837. kisseleva t. & brenner d.a. 2008. mechanisms of fibrogenesis. exp biol med (maywood), 233 (2), 109-122. doi: 10.3181/0707-mr-190. lee s.h., kang s.c., kim d.y., bae j.h. & kim j.h. 2007. detection of swine hepatitis e virus in the porcine hepatic lesion in jeju island. j vet sci, 8 (1), 51-55. lee y.h., ha y., ahn k.k. & chae c. 2008. localisation of swine 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[pathological and virological studies of the liver tissues from the patients with sporadic hepatitis  e]. zhonghua shi yan he lin chuang bing du xue za zhi, 15 (3),205-207. quaresma j.a., barros v.l., pagliari c., fernandes e.r., guedes f., takakura c.f., andrade h.f. jr., vasconcelos p.f. & duarte m.i. 2006. revisiting the liver in human yellow fever: virus induced apoptosis in hepatocytes associated with tgf-β, tnf-α and nk cells activity. virology, 345 (1), 22-30. roić b., terzić s., florijančić t., prpić j., ozimec s., jemeršić l., bošković i., jungić a. & keros t. 2018. preliminary serological and molecular investigation of selected viral pathogens in croatian cervid species. acta veterinaria-beograd, 68 (1), 65-79. roulston a., marcellus r.c. & branton p.e. 1999. virus and apoptosis. annu rev microbiol, 53, 577-628. salvesen g.s. & dixit m.v. 1997. caspase: intracellular signaling by proteolysis. cell, 91 (4), 443-446. srivastava r., aggarwal r., jameel s., puri p., gupta v.k., ramesh v.s., bhatia s. & naik s. 2007. cellular immune responses in acute hepatitis e virus infection to the viral open reading frame 2 protein. viral immunol, 20 (1), 56-65. takiya s., tagaya t., takahashi k., kawashima h., kamiya 141 short communication squamous cell carcinoma (scc) is a common malignant tumor of the skin in both humans (zur  hausen 2009) and animals (ahmed and hassanein 2012). it derives from the more superficial epidermal layers (keratinocytes) and mucocutaneous borders; it is noted for its invasive behavior and metastatic abilities. classification of scc is based on the histological pattern (acantholytic, spindle cell, verrucous, pseudovascular, adenosquamous) and on the tumor, node, metastasis (tnm) staging system (weedon et  al. 2006). squamous cell carcinoma affects both pets and farm animals, with a reported peak age per species and breed susceptibility (ladds and entwistle 1977), though its detection in meat animals is frequently inaccurate because of their short life expectancy (valentine 2004, goldschmidt and hendrick 2002, ahmed and hassanein 2012). its prevalence is variable but is widespread in sheep, with most cases reported from arid and sunny regions such as western australia (daniels and johnson 1987). several studies have highlighted a greater susceptibility in breed‑selected species characterized by a lack of skin pigmentation e.g., merinos (daniels and johnson 1987) or milk‑producing sheep such as sarda breed (vitiello et al. 2017). the most “common localization” in sheep and goat is dorsally exposed areas and/or areas with poor skin pigmentation and hair, including the head, base of the horn, eyelids, conjunctiva, hind limbs, shoulder, back, abdomen, flank region, vulva, and inner part of the tail (banadiam et  al. 2010, ahmed and hassanein 2012, tmumen et  al. 2016). other sites (e.g., nasal or intracranial) are considered rare (zeman and cho 1986, banadiam et  al. 2010, pace et  al. 2010).the etiology seems to be multifactorial, although ultraviolet (uv) radiation, especially uvb ray exposure, appears to be the main cause of tumor development due to activation of proto‑oncogenes or inactivation of tumor suppression factors expressed in uv irradiated body areas (kubo et  al. 2002). tissues exposed to greater solar radiation produce, as result of tissue damage, a greater amount of reactive oxygen species (ros) that induce cell damage and possible dna mutations, 1istituto zooprofilattico del piemonte, liguria e valle d'aosta, torino, italy. 2università degli studi di perugia, perugia, italy. *corresponding author at: istituto zooprofilattico sperimentale del piemonte liguria e valle d'aosta via bologna 148, 10154 torino, italy. tel.: +39 06 59948749, e-mail: fabrizio.lazzara@izsto.it. keywords intracranial, ovis aries, papillomavirus, squamous cell carcinoma. summary squamous cell carcinoma (scc) is a malignant mucoepithelial tumor that affects pets and farm animals. common sites are dorsal areas and/or areas of poor skin pigmentation exposed to mutagenic ultraviolet (uv) radiation. novel ovine papillomavirus (oapv3) was recently described in scc lesions in sardinia breed ovines. in 2017, a 7‑year‑old half‑breed aries was presented with symptoms compatible with a vestibular syndrome. the animal was euthanized 1 month after the onset of clinical signs due to a lack of response to treatment and poor prognosis. a complete postmortem examination was performed. necropsy revealed only a loss of incisors, associated with alveolar necrotic osteomyelitis, and left unilateral purulent nasal discharge. no other thoracic or abdominal lesions were observed. opening of the skull revealed a cauliflower‑like space‑occupying mass. histological examination showed trabecules and islands of squamous, neoplastic epithelial cells with the formation of concentric keratin layers. this raised the suspicion of scc, which was confirmed with cytokeratin‑positive immunostaining. simplex pcr on the frozen tissue mass was negative for oapv1, oapv2, and oapv3. this case report suggests that scc, although rare, should be included in the differential diagnosis of cases of vestibular disorder. fabrizio lazzara1*, federica giorda1, katia varello1, maria teresa mandara2, simona zoppi1, mariella goria1, milena monnier1, tiziana avanzato1, cristina casalone1 and barbara iulini1 intracranial squamous cell carcinoma in an ovis aries veterinaria italiana 2020, 56 (2), 141‑144. doi: 10.12834/vetit.1911.10405.2 accepted: 31.01.2020 | available on line: 31.12.2020 142 veterinaria italiana 2020, 56 (2), 141‑144. doi: 10.12834/vetit.1911.10405.2 intracranial squamous cell carcinoma in an ovis aries lazzara et al. et  al. 2017). briefly, dna was extracted using a commercial kit (qiamp dna mini kit, qiagen, milan, it), according to the manufacturer’s instructions, and then amplified with sets of primers specific for oapv type  1, type  2, and type 3. pcr master mix compositions and thermal profiles were applied as reported in table  і. after separation by electrophoresis in agarose gel (2% w/v containing gel red nucleic acid stain, biotium) in tbe buffer 1x at 5 mv/cm for 90 min, the amplification products were visualized by exposure to uv in a gel doc apparatus (biorad, segrate, milan, it). postmortem examination revealed rupture and loss of incisors, associated with alveolar necrotic osteomyelitis, and left unilateral purulent nasal discharge; bacteriological and virological examinations isolated no specific pathogens. no thoracic or abdominal lesions were observed. opening the skull revealed a cauliflower‑like space‑occupying mass, white‑grayish in color, firm to hard, walnut in size, with a broad basis. the mass rested under the left temporal bone, laterally in contact with the petrous portion that showed evident hyperostosis. the pedunculated part of the mass extended into the left cerebral hemisphere, creating a voluminous deep fovea and compression of the underlying cerebral tissue (figure 1). there was no gross evidence of enlargement of the trigeminal ganglion and no gross evidence of masses in the retrobulbar space, corneal process, frontal sinus, regional lymph nodes or in other organs. microscopical examination revealed trabeculae and islands of squamous neoplastic epithelial cells with the formation of concentric keratin layers (keratin pearls). the cells showed moderate pleomorphism with abundant eosinophilic cytoplasm, large and hyperchromatic nuclei with prominent nucleoli, usually single (figure 2). rare mitotic figures and resulting in the development of pre‑cancerous lesions (solar keratosis). in italy, recent studies of cases of ovine scc have signaled the involvement of a novel genus of papillomavirus oapv3 in these lesions (alberti et al. 2010). papillomavirus can infect the basal layer or skin lesions and cause cutaneous fibropapillomas (oapv 1 and 2) or rarely progressive skin cancer lesions in humans and animals (tilbrook et al. 1992, bocaneti et  al. 2016), suggesting a synergic action with other pro‑cancer factors (vitiello et  al. 2017). the present report describes a rare and “uncommon localization” of intracranial scc in a domestic sheep (ovis aries) in italy. in 2017, a 7‑year‑old half‑breed aries, bred in gassino in turin province, was presented with symptoms suggestive of vestibular syndrome. detailed clinical findings were head pressing and tilt, circling and loss of sense of balance, pain and difficulty in chewing and walking, and fever. the animal was euthanized one month after the onset of clinical signs due to lack of treatment response and poor prognosis. the carcass was promptly delivered to the laboratory and submitted to complete necropsy. brain tissue was partly frozen for bacteriological and molecular investigations and partly fixed in 10% buffered formalin solution. fixed tissue was routinely processed for neuropathological examination; 4  ±  2  μ sections were cut and stained with hematoxylin and eosin (he) for light microscopy evaluation. immunohistochemistry investigation for vimentin (clone v9, dako, glostrup, dk, dilution 1:150) and cytokeratin (clone ae1/ae3, dako, glostrup, dk, dilution 1:50) were performed on selected sections. tissue samples from the frozen mass were investigated for ovine papillomavirus type 1, 2, and 3 by simplex pcr methods targeted to the l1 gene, as described previously (vitiello table i. set up of pcr detection for oapv1, 2, and 3 investigations. primer pairs, amplicon length, master mix composition, and thermal profile. target primer pairs amplicon length (bp) master mix composition (final volume of 50 µl) thermal profile l1 gene opv-1 f: cgcccgtctccctacggtgc 177 pcr buffer 1x; mgcl2 1.5 mm; primers concentration: 0.5 μm each; dntps: 0.2 mm each; dna hot-start taq polymerase 1.25 u/reaction (platinum taq, invitrogen); dna template: 50÷300 ng in 5 µl. initial denaturation: 95°c x 5 min 40 cycles: 95 °c x 30 s; 56 °c x 30 s; 72 °c x 30 s final elongation step: 72 °c x 5 min r: ctgcaacgcctccggacccc l1 gene opv-2 f: cgcaccacagcccaaggcac 147 r: tccagcgtccacacggtctga l1 gene opv-3 f: aactggacttgtcttccatg 127 initial denaturation: 95°c x 5 min 40 cycles: 95 °c x 30 s; 57 °c x 30 s; 72 °c x 30 s final elongation step: 72 °c x 5 min r: aaagactcggtattgggagg bp = base pair; f= forward; r = reverse. 143veterinaria italiana 2020, 56 (2), 141‑144. doi: 10.12834/vetit.1911.10405.2 lazzara et al. intracranial squamous cell carcinoma in an ovis aries the neoplastic cells (figure 5). histological findings classified the neoplasm as a well differentiated scc. the simplex pcr analysis for oapv1, oapv2 and oapv3 detected no gene fragments referable to these oapvs types. scc is a common tumor of animals and intracranial localization is rarely described in the literature (zeman and cho 1986, pace et al. 1997, raheja et al. 2016). “the uncommon localizations” of scc have been demonstrated in cows, in which metastasis spreads from a specific primary site of the skin or muco‑cutaneous zone, through a perineural pathway (mendenhall et al. 2007) or from the foramen orbitorotundum, with or without alteration of nearby bone structures (hinkley 1951, pace et  al. 1997, zeman and cho 1986). epidermoid cysts, derived from rathke’s pouch in humans, due to incarceration of vestigial remains of the oropharyngeal ectoderm, are another possible origin for the development of intracranial scc, involving the sellar region in particular (pace et al. 1997). multifocal peritumoral lymphoplasmacytic infiltrates were also detected (figure 3). immunohistochemical staining for vimentin was negative (figure 4); cytokeratin staining showed specific positivity in figure 1. gross appearance. the cauliflower-like mass appears pedunculated and with a broad basis, laterally in contact with the petrous left temporal bone. figure 4. immunohistochemical stain negative for vimentin clone v9, ihc (original objective10x). figure 3. microscopical examination. peritumoral multifocal lymphoplasmacytic infiltrates. h&e. (original objective10x). figure 2. microscopical examination. trabeculae and islands of squamous, neoplastic epithelial cells associated in concentric keratin layers “keratin pearls”. h&e. (original objective10x). figure 5. immunohistochemical stain positive for cytokeratins ae1/ ae3, ihc (original objective10x). 144 veterinaria italiana 2020, 56 (2), 141‑144. doi: 10.12834/vetit.1911.10405.2 intracranial squamous cell carcinoma in an ovis aries lazzara et al. the frozen tissue did not confirm the presence of papillomavirus nucleic acid. histological pattern and immunohistochemical staining unequivocally confirmed an epithelial origin of the tumor, thus allowing us to discern between other neoplasms such as meningothelial meningioma or psammomatous meningioma (pace et al. 1997). this case report strongly suggests that scc, although rare, should be included in the differential diagnosis of cases in which there is a vestibular disorder. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not‑for‑profit sectors. the possibility of an intracranial primary origin of scc in the present case cannot be ruled out, considering the absence of macroscopically detectable neoplasms in the oculonasal region, the thoracic and the abdominal cavity, and the large size of the intracranial mass. the clinical signs and the evidence of hyperostosis of the petrosal bone, contiguous laterally to the mass, point to a possible primary source from the medium acoustic meatus. alternatively, the neoplasm may have originated from the bone labyrinth and induced a vestibular syndrome. no oculofacial abnormality referable to horner's syndrome could be determined in this animal, unlike previous cases of intracranial scc (pace et al. 1997). in contrast to previous studies (alberti et  al. 2010, vitiello et  al. 2017), our virologic investigation on ahmed a.f. & hassanein k.m.a. 2012. ovine and caprine cutaneous and ocular neoplasms. small rumin res, 106, 189‑200. alberti a., pirino s., pintore f., addis m.f., chessa b., cacciotto c., cubeddu t., anfossi a., benenati g., coradduzza e., lecis r., antuofermo e., carcangiu l. & pittau m. 2010. ovis aries papillomavirus 3: a prototype of a novel genus in the family papillomaviridae associated with ovine squamous cell carcinoma. virology, 407, 352‑359. baniadam a., moezzi n. & mohammadian b. 2010. nasal squamous cell carcinoma in a cow. turk j vet anim sci, 34, 303‑305. bocaneti f., altamura g., corteggio a., velescu e., roperto f. & borzachiello g. 2016. bovine papillomavirus: new insights into an old disease. transbound emerg dis, 63, 14‑23. daniels p.w. & johnson r.h. 1987. ovine squamous cell carcinoma. vet bull, 57, 153‑167. goldschmidt m.h. & hendrick m.j. 2002. tumors of the skin and soft tissues. in tumors in domestic animals, 4th ed. 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(p.e. leboit, g. burg & d. weedon, eds). iarc press, lyon, 20‑25. zeman d.h. & cho d.y. 1986. intracranial squamous cell carcinoma in a cow. cornell vet, 76, 236‑240. zur hausen h. 2009. papillomaviruses in the causation of human cancers – a brief historical account. virology, 384, 260‑265. 15 parole chiave gestione degli alveari, inquinamento ambientale, mortalità delle api, monitoraggio, aree naturali protette. riassunto il presente studio riporta i risultati del monitoraggio dello stato di salute delle api effettuato da ottobre 2009 a dicembre 2010 in cinque aree naturali protette italiane, scelte per rappresentare le aree biogeografiche alpina, continentale e mediterranea. all'interno di ciascuna è stato posizionato un apiario di 20 alveari vicino a potenziali fonti di inquinamento (ad esempio aree agricole, aree industriali o insediamenti urbani) e un altro apiario di 20  alveari lontano da possibili fonti di inquinamento. per monitorare lo stato di salute delle api, è stata messa in relazione la mortalità degli alveari con la presenza di malattie delle api, con l'ambiente (naturality index, presenza di prodotti fitosanitari ed esposizione a metalli pesanti) e la gestione dell’apiario. non sono stati osservati effetti significativi degli inquinanti di origine antropica e dei patogeni sulla mortalità degli alveari, mentre la capacità di gestione degli alveari da parte degli apicoltori è risultata strettamente correlata alla mortalità delle colonie. monitoraggio dello stato di salute delle api in cinque aree naturali protette italiane keywords apiary management, environmental pollution, honey bee mortality, monitoring, natural protected area. summary the health status of the honey bee populations has attracted a great amount of interest in recent years. we investigated honey bee health in five natural protected areas in italy from october 2009 to december 2010. areas were selected to represent a wide range of bio‑geographical zones including alpine, continental, and mediterranean. within each of these natural protected areas, one apiary of 20 colonies near potential pollution sources (e.g., agricultural areas, industrial areas, or urban settlements) and another apiary of 20  colonies far from possible sources of pollutants have been placed. to monitor honey bee health, colony mortality was related to: honey bee pathologies, environment (naturality index, plant protection products and heavy metal exposure), and apiary management. anthropogenic pollutants and pathogens did not have significant effects on colony mortality while environment and the poor colony management skills of the beekeepers did. veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 accepted: 11.09.2017 | available on line: 06.03.2019 1istituto superiore per la protezione e la ricerca ambientale, via vitaliano brancati 60, 00144 rome, italy. 2istituto zooprofilattico sperimentale del lazio e della toscana ‘m. aleandri’, via appia nuova 1411, 00178 rome, italy. 3dipartimento di scienze veterinarie, università degli studi di pisa, viale delle piagge 2, 56124 pisa, italy. 4dipartimento di scienze e tecnologie agro-alimentari (distal), alma mater studiorum, università di bologna, via zamboni 33, 40126 bologna, italy. 5istituto zooprofilattico sperimentale delle venezie, national reference laboratory for beekeeping, viale dell’università 10, 35020 legnaro (pd), italy. *corresponding author at: istituto zooprofilattico sperimentale del lazio e della toscana ’m. aleandri’, via appia nuova 1411, 00178 rome, italy. tel.: +39 06 79099328, e-mail: giovanni.formato@izslt.it. valter bellucci1, stefano lucci1, pietro bianco1, alessandro ubaldi2, antonio felicioli3, claudio porrini4, franco mutinelli5, sabrina battisti2, valentina spallucci2, antonella cersini2, marco pietropaoli2 and giovanni formato2* monitoring honey bee health in five natural protected areas in italy 16 veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 honey bee health in italy bellucci et al. materials and methods the monitoring project was conducted from october 2009 to december 2010 using 200 honey bee colonies within five italian npas (figure 1) as representative of a wide range of the bio‑geographical regions in italy including alpine, continental, and mediterranean areas. these npas were all located near agricultural, industrial, or urban settlements and are listed as follows: • parco nazionale delle dolomiti bellunesi (dolomiti), alpine bioregion, northeastern italy; • parco dei gessi bolognesi e dei calanchi dell’abbadessa (calanchi), continental bioregion, northern italy; • parco di migliarino san rossore massaciuccoli (san rossore), mediterranean bioregion, central italy; • parco dei monti simbruini (simbruini), mediterranean bioregion influenced by sub‑continental conditions, central italy; • riserva naturale statale litorale romano (litorale), mediterranean bioregion, central italy. for the present study, two apiaries of 20 healthy colonies were established in each npa (40 colonies/ npa). one apiary was classified as a ‘non‑exposed’ and called ‘apiary a’ (combined with site names as dolomiti a, calanchi a, san rossore a, litorale a, simbruini a) and located in ecosystems with a modest level of anthropogenic pressure. the other apiary was classified as ‘exposed’ and called introduction the decline of honey bee (apis mellifera l.) colonies, observed in the last few decades in both europe and in the united states (ellis et al. 2010, potts et al. 2010), have a multi‑factorial origin (neumann and carreck 2010) including plant protection products (ppps), beekeeping practices, pest and pathogens, queen failure, genetic weakness, nutrition, and weather patterns. farming techniques and crop protection procedures play a pivotal role in the proper management of honey bee colonies and their possible exposure to ppps (alaux et  al. 2010, brodschneider and crailsheim 2010, johnson et al. 2010). environment pollution, especially pesticides, can negatively affect the health of honey bee colonies. numerous studies have reported on the negative effects of exposure to ppps on honey bees (koch and wisser 2001, forster 2009, anne and gavin 2010, efsa 2012, henry et  al. 2012, whitehorn et  al. 2012). in addition, honey bee pests and pathogens probably play a crucial role in honey bee colony losses, especially losses caused by varroa destructor (v.  destructor) combined with viruses and nosema ceranae (vanengelsdorp et  al. 2009, neumann and carreck 2010, martin et  al. 2013). the interaction of these pathogens with pesticides also causes losses of hives (pettis et al. 2012, pettis et al. 2013). aside from ppps, honey bee pests and pathogens, even beekeeping management contributes to endangering the status of honey bee health (oldroyd 2007, vanengelsdorp et al. 2008). about 75,000 beekeepers in italy manage more than 1,317,000 colonies [commission implementing regulation (eu) n. 768/2013]. the responses from 874 surveys conducted in 2007‑2009 by the organization named prevention of honeybee colony losses, using the cost action fa0803 framework, showed winter mortality ranged from 11% (abruzzo) to 38% (emilia romagna), with an average of 23.5% (mutinelli et  al. 2010, van der zee et  al. 2012). a pan‑european epidemiological study on honeybee colony losses called epilobee (laurent et  al. 2015), set up from 2012 to 2014 in 17  european member states, showed a overwintering colony mortality in italy ranging from 5.5% (winter 2012‑2013) and 4.8% (winter 2013‑2014). this study, coordinated by the italian national institute for environmental protection and research was promoted in 2009‑2010 by the italian ministry of environment, territory and sea to evaluate honey bee mortality within five natural protected areas (npas) in italy, in order to verify the effects of chemical pollution, apiary management, and bee pathogens on honey bee’s health. figure 1. locations of the five natural protected areas included in the survey. dolomiti bellunesi simbruini calanchi bolognesi litorale romano san rossore 17veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 bellucci et al. honey bee health in italy used 1 week as a unit of time; this corresponds to the weekly beekeeper check of hive status. the differences between the survival curves of a and b colonies of the five apiaries were evaluated through a log‑rank test (thrusfield 1995). the association of both the cumulative and winter mortality indices with honey bee diseases and the weekly count of the under‑basket mortality (>  200 bees/colony) was measured by the spearman’s rank correlation coefficient (r s ); p  <  0.05 was selected as the level of significance. stata 12.0 software was used for statistical analysis. the honey bee diseases investigated were: varroasis, seven main honey bee viruses (acute bee paralysis virus, abpv; black queen cell virus, bqcv; chronic bee paralysis virus, cpbv; deformed wings ‘apiary b’ (dolomiti b, calanchi b, san rossore b, litorale b, simbruini b)and located close to potential anthropogenic pollutant sources with agricultural, industrial, and/or urban contaminants. apiaries a and b were established in similar climatic conditions. data sheets were prepared to standardize data collection related to colony inspections (health status and strength), honey bee mortality, samplings, and the environment. supplementary inspections and samplings of honey bees and pollen were performed in case of variations in beehive health status (e.g., colony depopulation, disease, death, higher mortality of adult honey bees) detected by the beekeepers. laboratory analyses were carried out in an accredited laboratory in conformity with uni cei en iso/iec 17025 (international organization for standardization and the international electrotechnical commission international standards). to assess the health status and the strength of the colonies for each apiary, four ad hoc trained inspectors conducted quarterly clinical inspections. moreover, bee mortality was assessed weekly. colony mortality was measured as ‘cumulative mortality’, ‘winter mortality’, and ‘mortality rate’. a dead colony was assessed if no honey bees of the colony were found alive. ‘cumulative mortality’ indicates the ratio of the number of dead colonies in each apiary found throughout the entire observational period (1 year) and the number of colonies (20) monitored in each apiary at the beginning of the project. ‘winter mortality’ indicates the ratio of the number of dead colonies in each apiary found throughout the winter season (from 1 october to 1 april) and the number of colonies (20) monitored in each apiary at the beginning of the project (1 october). the ‘mortality rate’ (colony‑month at risk) indicates the mortality rate calculated on a monthly basis during the entire follow‑up period (thrusfield 1995). under‑basket cages were used as a supplementary tool to monitor bee mortality within each colony (human et al. 2013). these cages were placed in front of each colony for a weekly count of the number of dead adult honey bees in each colony. whenever the number of dead honey bees exceeded the threshold of 200 honey bees/week in the same colony (porrini et  al. 2003), an additional inspection of the colony was combined with samplings for pathogens and pollutants (ppps and heavy metals) to find the cause of the increased mortality. to compare data related to cumulative mortality, survival curves in exposed and non‑exposed colonies were drawn using the non‑parametric method of kaplan‑meier (kaplan and meier 1958). this model represents the survival function as the probability that a colony will survive over a given period. the present study table i. pathogens, contaminants, methods and matrices used to monitor the honey bee health of the apiaries located in the natural protected areas. pathogen/ contaminant diagnostic methods matrix varroasis (varroa destructor) visual identification of the parasite adult honey bees with symptoms of disease; honey bee brood of hives with symptoms of disease main honey bee viruses: dwv, abpv, cpbv, bqcv, sbv, kbv, iapv reverse transcriptase polymerase chain reaction (rt-pcr) (singh et al. 2010) adult honey bees (10 adult honey bees) american foulbrood afb (paenibacillus larvae) cultural method (oie 2008) honey bee brood (3-5 affected larvae) european foulbrood efb (melissococcus plutonius) cultural method (oie 2008) honey bee brood with symptoms of the disease nosemosis (n. apis and n. ceranae) polymerase chain reaction (pcr) and microscopic examination (oie 2008) adult honey bees (10 adult honey bees and 30 honey bees, respectively) ascosphaera apis cultural method honey bee brood (affected larvae) main plant protection products (ppps): organochlorine, organophosphorous, pyrethroids, neonicotinoids and carbamates high resolution gas chromatography separation analyses method for neonicotinoids adult honey bees (200 bees/hive) high resolution gas chromatography honey (500 g/apiary) high resolution gas chromatography (for neonicotinoids only) pollen (10 cc) heavy metals: pb, cd, cr and cu atomic absorption spectrophotometry honey (500 g/apiary) palynological analysis optical microscopy pollen (10 g/colony) 18 veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 honey bee health in italy bellucci et al. adopted for analyzing ppps. because neonicotinoid compounds are thermally unstable, high‑resolution gas chromatography separation analysis was preferred instead of high‑resolution liquid chromatography. palynological analyses were used to identify pollen grains using optical microscopy (model b‑500tph, optika srl, ponteranica, bg, italy) on the beebread samples from dead or depopulated colonies, so as to relate the poisonings of honey bees by ppps to the treated plants. to evaluate the apiary management, a score from 1 (weak) to 5 (excellent) was given to the beekeeper skills, considering both their commitment to beekeeping and their ability to adopt good beekeeping management practices. the grades were assigned according to an evaluation grid designed and approved by the working group (table ii). in four npas, the two apiaries were managed by different beekeepers according to their customary practices. for the san rossore npa only, the same beekeeper managed both apiaries. all the beekeepers had to follow the same measurement protocols. the relationship between the skill of beekeepers (beekeepers score) and the mortality indices (cumulative mortality, winter mortality) was measured by the spearman’s rank correlation coefficient. then, linear regression was used to measure their reciprocal influence. results a total of 826 clinical inspections of beehives and 733 laboratory analyses were conducted to check the health status of honey bees and their exposure to pollutants. table iii shows detailed data about the analytical activities conducted for the project. table iv reports the results of hive mortality for the ten apiaries as cumulative mortality, mortality rate, and winter mortality. the colonies of the b apiaries of litorale and san rossore parks showed higher mortality compared with the corresponding a apiaries. the log‑rank test applied on kaplan‑meier curves representing aggregated data of colony mortality rates according to apiary exposure to pollution virus, dwv; israeli acute paralysis virus, iapv; kashmir bee virus, kbv; sac brood virus, sbv), american foulbrood (paenibacillus larvae), ascosphaera apis, european foulbrood (melissococcus plutonius), and nosemosis (nosema apis and nosema ceranae). table i provides the laboratory methods adopted for the above‑mentioned analyses. to compare the frequency of each infectious and parasitic disease within exposed and non‑exposed apiaries, a series of 2  ×  2 tables for each disease and npas was developed and the fisher’s exact test was conducted. when appropriate, risk measure was expressed as a risk ratio (rr). when the colonies experienced depopulation, death, relatively high mortality of adult bees or honey bee pathologies, extra‑inspections and extra‑samplings were conducted to assess chemical and/or biological causes of the related problems. to investigate the relationships between honey bee mortality and environment, data were collected related to a naturality index, including land use, wild vegetation, and crops. farming techniques were also recorded. around each apiary, a 1.5‑km radius buffer area (honey bee flight area) was evaluated; maps of land use and vegetation coverage were produced using a scale of 1:10,000. land use and vegetation polygons were delineated using both photo interpretation and field surveys. the identified polygons were referred to the european university information systems, co‑ordination of information on the environment biotopes, and natura 2000 (council directive 92/43/eec) categories according to the european environment agency (eunis 2007). the current agricultural use (presence of vegetable crops, vineyards, and corn) was extrapolated in detail from these buffer maps. the number of vegetation categories and the surface area for each of them were calculated with the naturality index (expressed as natural + natural like/urban + agricultural surfaces), shannon diversity index, and the simpson dominance index. naturality index was associated with cumulative mortality through pearson chi square test with yates correction, furthermore a linear regression was used to analyze correlation between cumulative and agricultural land coverage. honey samples were collected monthly from each colony to analyze any heavy metal residues of pb, cd, cr, and cu with atomic absorption spectrophotometry (table i). to test for differences in the average concentration of heavy metals in honey taken from the a and b apiaries, the student’s t‑test was performed for each npa and each individual metal. to investigate the residues of ppps, adult bees and honey were sampled monthly from each apiary. moreover, the same types of samples were collected from each colony in all cases of abnormally high colony mortality to monitor the exposure to ppps and heavy metals. table i provides the methods table ii. scoring criteria used to evaluate the management commitment and the success in adopting good beekeeping practices. scores range from 1 (weak) to 5 (excellent). commitment excellent sufficient weak management excellent 5 4 2 sufficient 4 3 2 weak 2 2 1 19veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 bellucci et al. honey bee health in italy the comparison of the cumulative mortality between colonies placed in buffers at different naturality index values showed higher values for cumulative mortality in areas with a lower naturality index (figure 4). this difference resulted significant (p = 0.047) table vii shows the amounts of several heavy metals found in the honey samples; the statistical analysis showed no significant differences between exposed (area b) and non‑exposed (a) apiaries. in addition, no residues of ppps were found in the monthly honey samples with the exception of one case where the active principle imidacloprid was detected in a dead honey bee sample, at a concentration of 0.0096  mg/kg [equivalent to about ¼ of the lethal dose 50% (ld 50 ) for honey bees], in march 2010, in the non‑exposed apiary a of calanchi. varroosis was detected at different levels in all the apiaries (table viii). the spearman’s rank correlation coefficient used to measure the relationship between heavy varroa infestation and the mortality (figure 2) showed that the mortality rate detected in the exposed (b) apiaries was significant higher compared to the mortality rate detected in the non‑exposed (a) apiaries (p = 0.0002). colony survival on the 30th week (210 days) was 87% for non‑exposed and 75% for exposed apiaries. table  v reports the excesses of adult honey bee weekly mortality (> 200 dead honey bee) found in the under‑basket cages, for each individual hive. this parameter was not observed to be related to colony mortality (both cumulative and winter mortality) in our study. the vegetation surveys conducted in the honey bee flight area provided a critical contribution to the study. table vi shows the synthetic characteristics of land use, vegetation coverage, naturalness, and the diversity of the above‑mentioned buffer zones. in all five npas, linear regression showed only a weak correlation (r 2  = 0.0373) between increased mortality of the colonies with an increase in agricultural land coverage (cultivation of carrots, forage crops, mixed crops, horticultural crops, alfalfa, corn, melons, olive plantations, potatoes, rape, savoy cabbage, sorghum, vineyards, and watermelon; figure 3) index of the possible use of ppp. table iii. activities performed to verify the health status of honey bees and their exposure to pollutants. activities numbers clinical inspections of the hives 826 samples for honey bee viroses 117 samples for nosemosis 108 samples for american foul brood (afb) 24 samples for european foul brood (efb) 1 samples for ascosphaera apis 3 samples for neonicotinoids 109 samples for other ppps 123 samples for heavy metals 96 palynological analyses 27 table iv. mortality rates (cumulative and winter mortalities) observed in non exposed and exposed apiaries located in natural protected areas. apiary do lo m iti ca la nc hi li to ra le sa n ro ss or e si m br ui ni a b a b a b a b a b cumulative mortality 15% 15% 0 0 20% 70% 25% 70% 5% 45% winter mortality 5% 15% 0 0 15% 45% 25% 70% 5% 45% a = non exposed apiaries; b = exposed apiaries. 0.00 0 100 200 time (days) su rv iv al p ro b ab ili ty (% ) oct nov dec jan feb mar apr may jun jul aug sep oct 300 400 0.25 0.50 0.75 1.00 non exposed apiaries exposed apiaries figure 2. kaplan-meier survival estimate curves for apiaries that were exposed and not exposed to high levels of pollutants. table v. weekly mortality threshold excess found in the under basket cages of the apiaries located in selected natural protected areas (npa). npa excesses of weekly mortality threshold (> 200 honey bees/ hive/week) month simbruini a 2 may (1st week) simbruini b 0 litorale a 9 may (1st and 2nd week) litorale b 10 november (2nd week) december (4th week) august (1st week) dolomiti a 0 dolomiti b 2 may (4th week) june (4th week) calanchi a 0 calanchi b 1 october (1st week) san rossore a 1 may (3rd week) san rossore b 1 october (3rd week) 20 veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 honey bee health in italy bellucci et al. 65 samples of honey bees (table viii). the frequency of the seven viruses did not significantly differ between a and b apiaries, with the exception of the following five cases: abpv in calanchi, cbpv in dolomiti and simbruini, kbv in litorale, and sbv in calanchi. however, the high variability of the rr did not allow any statement on a presumed major risk of viral diseases in the b apiary areas compared with the a ones. the spearman’s rank correlation coefficients (r s ) used to measure correlation among the prevalence of the seven main honey bee viruses listed above and the cumulative and winter mortality indices showed that the variables are unrelated for most of the cases with the exception of abpv and kbv. acute bee paralysis virus was positively and significantly related with both cumulative and winter mortality (r s   =  0.6862 and 0.6790, p  =  0.028 and 0.031, respectively). kashmir indices (cumulative mortality, winter mortality) showed no correlation. american foulbrood (afb) was detected in two colonies of the non‑exposed dolomiti apiary a, in three colonies of the litorale apiary a, in four colonies of the litorale apiary b, and in nine colonies of the simbruini apiary b (table viii). the prevalence of afb ranged from 0 to 45% between the apiaries, with an average of 5% in the a apiaries and of 13% in b apiaries. the spearman’s rank correlation coefficient calculated between the frequency of afb and both cumulative and winter mortality showed no correlation between them. however, no case of european foulbrood (efb) was detected during the study. seven main honey bee viruses (abpv, bqcv, cbpv, dwv, iapv, kbv, and sbv) were investigated in table vi. characteristics of the honey bee flight areas used for statistical analyses. dolomiti calanchi litorale san rossore simbruini a b a b a b a b a b n. polygons 491 1,806 610 789 273 333 120 298 748 1,381 average size of polygons (ha) 1.44 0.39 1.16 0.90 2.61 2.12 5.94 2.37 0.94 0.53 n. corine/eunis categories 30 30 40 39 32 39 29 34 33 42 n. natural categories 16 7 18 16 15 20 21 14 17 16 shannon diversity index* 2.73 2.1 1.80 2.23 2.64 2.24 2.39 2.23 2.30 1.80 simpson dominance index** 0.03 0.12 0.04 0.06 0.06 0.15 0.05 0.01 0.06 0.08 rate naturalness (natural+ semi-natural/agricultural+urban) 6.32 0.74 1.38 0.29 0.53 0.98 8.68 0.34 6.79 1.04 % forests 73.93 17.54 20.90 13.89 6.24 18.24 37.51 10.27 56.27 45.16 % meadows and pastures 8.73 21.41 19.92 7.12 36.53 26.23 4.75 11.91 25.19 4.18 % built 5.08 23.86 2.53 13.52 6.83 11.56 1.19 9.67 5.79 19.08 % agricultural 7.99 29.09 27.04 58.86 73.35 33.94 3.68 55.38 6.20 28.41 % vegetable crops 1.87 5.94 2.52 18.22 0.60 7.03 0 0.05 1.87 5.94 % vineyards 0.06 3.30 0.40 0.45 % corn 1.8 2.12 a = non-exposed apiaries; b = exposed apiaries; * calculated on the percentage of coverage; ** calculated on the number of polygons. y = 0.2155x + 19.519 r² = 0.0374 0 10 20 30 40 50 60 70 80 0 20 40 60 80 agricultural land coverage (ha) c u m u la ti ve m o rt al it y (% ) figure 3. relationship between the cumulative beehive mortality and the agricultural land coverage. 0 5 10 15 20 25 30 35 40 c u m u la ti ve m o rt al it y (% ) beehives with prevalence of natural habitats in the bu�ers natural index > 1 beehives with prevalence of anthropic habitats in the bu�ers natural index < 1 figure 4. cumulative mortality in colonies placed in buffers at different naturality index values. 21veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 bellucci et al. honey bee health in italy a as well as apiary b within both the dolomiti and calanchi sites (score 5). the relationship between the beekeeper score and the winter and cumulative mortality indices, showed that the beekeepers’ skill was significantly related to both indices. the spearman’s rank correlation coefficients showed a negative correlation between the beekeeper score and those two parameters, with values of ‑  0.7730 and ‑  0.7722, respectively (p  =  0.009 in both cases). two linear regressions showed that a one‑point increase in the beekeeper score corresponded to a 14% and 16% decrease in winter (figure 5) and cumulative mortality (figure 6), respectively. discussion the weekly counts of dead adult honey bees in the under‑basket cages in our study were not related to bee virus was positively and significantly related with cumulative mortality (r s  = 0.6351, p = 0.048). the diagnosis of nosemosis was performed on 64 honey bee samples. while n. ceranae was present in the samples from both a (average of 78.1%) and b (average of 65.6%) apiaries of the five npas, n. apis was never found (table viii). statistical analysis showed that the frequency of n. ceranae in the a and b apiaries was not significantly different. the spearman’s rank correlation coefficient used to measure correlation between the prevalence of n.  ceranae and the cumulative and winter mortality indices showed no significant correlation between them. table ix provides the results of the beekeeping skill assessment. the worst score for beekeepers’ management skill was given to litorale b and simbruini b (score 2) and the best was given to apiary table vii. average and standard deviation of heavy metal concentration detected (mg/kg) in each apiary of selected natural protected areas. he av y m et al dolomiti calanchi litorale san rossore simbruini a b a b a b a b a b pb 0.042±0.021 0.045±0.032 0.042±0.027 0.045±00.041 0.033±0.015 0.035±0.020 0.038±0.028 0.053±0.019 0.033±0.036 0.053±0.042 cu 0.369±0.402 0.551±0.436 0.178±0.035 0.165±0.075 0.567±0.461 0.304±0.288 0.204±0.082 0.150±0.058 0.183±0.075 0.299±0.218 cd 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 cr 0.038±0.029 0.047±0.020 0.101±0.082 0.104±0.096 0.091±0.080 0.054±0.030 0.066±0.052 0.076±0.095 0.063±0.030 0.084±0.055 a = non-exposed apiaries; b = exposed apiaries. table viii. pathogens detected in apiaries located in selected natural protected areas for the non-exposed and the exposed apiaries. dolomiti calanchi litorale san rossore simbruini mean a b a b a b a b a b a b varroa observations on adult honey bees 33% 33% 8% 13% 10% 5% 62% 47% 25% 20% 27.6% 23.6% american foul brood 2/20 (10%) 0/20 (0%) 0/20 (0%) 0/20 (0%) 3/20 (15%) 4/20 (20%) 0/20 (0%) 0/20 (0%) 0/20 (0%) 9/20 (45%) 5/100 (5%) 13/100 (13%) acute bee paralysis virus 0/7 (0%) 2/7 (28.6%) 4/7 (57.1%) 0/7 (0%) 7/7 (100%) 7/7 (100%) 3/5 (60%) 4/5 (80%) 2/6 (33.3%) 3/7 (42.8%) 16/32 (50%) 16/33 (48.5%) black queen cell virus 0/7 (0%) 0/7 (0%) 1/7 (14.3%) 0/7 (0%) 5/7 (71.4%) 5/7 (71,4%) 3/5 (60%) 0/5 (0%) 0/6 (0%) 0/7 (0%) 9/32 (28.1%) 5/33 (15.2%) chronic bee paralysis virus 6/7 (85.7%) 0/7 (0%) 5/7 (71.4%) 4/7 (57.1%) 7/7 (100%) 7/7 (100%) 2/5 (40%) 0/5 (0%) 1/6 (16.7%) 6/7 (85.7%) 21/32 (65.6%) 17/33 (51.5%) deformed wings virus 3/7 (42.8%) 0/7 (0%) 4/7 (57.1%) 5/7 (71.4%) 7/7 (100%) 6/7 (85.7%) 4/5 (80%) 4/5 (80%) 6/6 (100%) 6/7 (85.7%) 24/32 (75%) 21/33 (63.6%) israeli acute paralysis virus 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/5 (0%) 0/5 (0%) 0/6 (0%) 0/7 (0%) 0/32 (0%) 0/33 (0%) kashmir bee virus 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 2*/7 (28.6%) 0/7 (0%) 0/5 (0%) 4*/5 (80%) 0/6 (0%) 2*/7 (28.6%) 2/32 (6.3%) 6/33 (18.2%) sac brood virus 5/7 (71.4%) 5/7 (71.4%) 6/7 (85.7%) 1/7 (14.3) 6/7 (85.7%) 6/7 (85.7%) 4/5 (80%) 2/5 (40%) 1/6 (16.7%) 1/7 (14.3%) 22/32 (68.7%) 15/33 (45.5%) nosema ceranae 7/7 (100%) 7/7 (100%) 6/7 (85.7%) 3/7 (42.9%) 7/7 (100%) 5/7 (71.4%) 1/5 (20%) 2/5 (40%) 4/6 (66.7%) 4/6 (66.7%) 25/32 (78.1%) 21/32 (65.6%) nosema apis 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/7 (0%) 0/5 (0%) 0/5 (0%) 0/6 (0%) 0/6 (0%) 0/32 (0%) 0/32 (0%) a = non-exposed apiaries; b = exposed apiaries. 22 veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 honey bee health in italy bellucci et al. et  al. 2013). a more accurate study is needed to substantiate the relationship between abpv and kbv and colony mortality in combination with the varroa infestation level. the prevalence of n. ceranae did not significantly differ in a and b apiaries. no ppp residues were found in this 1‑year study, excluding one case in which, during spring 2010, a low level of imidacloprid (0.0096 mg/kg) was found in a dead honey bee sample of apiary b at calanchi. the active amount of imidacloprid found corresponded to about ¼ of the ld 50 for honey bees. however, in that apiary, bee mortality did not exceed the mortality threshold in the under‑basket cages, and no abnormal mortality was found. it should be stressed that, in italy, a rule1 (oj of italian republic n. 221 of 20 september 2008) established an immediate precautionary suspension of the use of ppps for seed dressing (but not other formulations, e.g., spray applications) when those ppps contain any of several active substances such as clothianidin, the colony mortality observed in the five npas; this was probably due to an absence of strong acute toxic effects caused by ppps during the year of monitoring activity. the kaplan‑meier survival estimates (figure  2) expressed mortality events more evident in exposed apiaries respect to non‑exposed apiaries during october/november, january/february, august. in these months, hive mortalities are usually related to v.  destructor. the spearman’s rank correlation coefficient used to measure the relationship between severe v.  destructor infestations and the mortality indices showed no correlation between them. however, in acquiring this information, we should also consider that a more accurate method may be used to assess the level of varroa infestation; this would have to be applied to achieve a robust conclusion, avoiding the different interpretations of the four inspectors that evaluated the hives in the five npas. indeed, the on‑field evaluation methods to detect the level of varroa infestation in adult honey bees, such as the use of icing sugar (lee et al. 2010) or detergent solutions (rinderer et  al. 2004), were still not in use at the time of our protocol definition. while efb was not found in the present study, afb was found with a higher prevalence in apiary b (13%) respect to apiary a (5%) (table viii). the spearman’s rank correlation coefficient did not highlight any correlation between the mean afb prevalence and the mortality indices. at simbruini b apiary, afb caused the highest mortality with nine colonies affected (45%); this was caused by the poor awareness of the beekeeper. with regard to the honey bee viruses, abpv was positively related to both winter and cumulative mortality, while kbv was positively related only to cumulative mortality. this could be explained by the prevalence of kbv, which appears to be higher in summer than in winter (formato et  al. 2012, cersini table ix. beekeepers’ honey bee management skill score in the investigated apiaries. scores range from 1 (weak) to 5 (excellent). npa apiary beekeepers score dolomiti a 5 b 5 calanchi a 5 b 5 san rossore a 3 b 3 litorale a 4 b 2 simbruini a 3 b 2 a = non-exposed apiaries; b = exposed apiaries. 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 beekepers score c u m u la ti ve m o rt al it y (% ) y = -15.638x + 84.362 r² = 0.5471 figure 5. relationship between beekeeper management skill score and cumulative mortality (%). 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 beekepers score w in te r m o rt al it y (% ) y = -13.652x + 73.014 r² = 0.5243 figure 6. relationship between beekeeper management skill score and winter mortality (%). 1 decreto dirigenziale. 2008. sospensione cautelativa dell'autorizzazione di impiego per la concia di sementi, dei prodotti fitosanitari contenenti le sostanze attive clothianidin, thiamethoxam, imidacloprid e fipronil, ai sensi dell'articolo 13, comma 1, del decreto del presidente della repubblica 23 aprile 2001, n. 290. oj, 221 of the 20.09.2008. 23veterinaria italiana 2019, 55 (1), 15‑25. doi: 10.12834/vetit.1209.6739.4 bellucci et al. honey bee health in italy the a and b apiaries. in fact, heavy metals do not decompose and are easily transported at considerable distances by air currents, being spread out in an area independently from their natural, rural, urban, or industrial characteristics (devillers et al. 2002). finally, the managerial skills of beekeepers were significantly related both to the winter and cumulative mortality, confirming that beekeeping management, like honey bee pathogens and environmental pollution, can contribute to threaten the health of honey bee colonies (oldroyd 2007; vanengelsdorp et al. 2008). conclusions our monitoring activity did not reveal a significant effect on colony mortality caused by either anthropogenic pollutants or honey bee pathogens that were observed in all the monitored apiaries in different proportions, even if there was a significant increase in risk in exposed areas (b apiaries) compared to unexposed ones (a apiaries). the results demonstrate that the application of the under‑basket cage criterion used to evaluate colony mortality is not effective in apiaries that are not involved in acute toxic effects (e.g., acute toxic ppp effects). the relation between honey bee mortality and naturality index is interestingly observed even in the context in which the present study was conducted, i.e. natural protected areas. in the areas most heavily affected by the colony mortality (litorale, san rossore, and simbruini), the poor colony management skills of the beekeepers played the most important role in colony losses. this study confirms previous studies showing that colony collapse is the consequence of the interaction of multiple factors, both natural and anthropogenic, that affect honey bee health. fipronil, imidacloprid, and thiamethoxam. as a consequence, since 2009 during corn sowing season, no neonicotinoid‑dressed seeds were allowed in italy and only two honey bee mortality outbreaks related to neonicotinoid‑dressed seeds were recorded in that year in italy. for this reason, the ban was then extended until june 2013, and no further cases of this type of mortality have been reported. it could have somehow reduced the possibility of detecting neonicotinoid residues in the investigated matrices, despite the permitted use of neonicotinoids on fruit trees, vineyards, and other shrubs. in npas, the vegetation coverage resulted related with the health status of colonies, even if it is not clear the influence of intensive agricultural techniques. however, it has to be considered that, within the npas, large areas of industrial or agricultural lands were not usually present. the failure to find a direct link between colony mortality and the crop coverage may be caused by the heterogeneity of the farming procedures used in the study area (e.g., the diversity of the treatments and cropping systems employed). despite the presence of large areas of farmland, vegetable crops, and vineyards, the low colony mortality recorded in both types of apiaries of calanchi npa, should be attributed to the spreading of sustainable and organic agricultural production promoted by the park authorities (naylor and ehrlich 1997), as well as to the apiary management skills of highly professional beekeepers.. the colonies of the apiaries b in litorale and san rossore parks, showed 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rinderer t., de guzman l. & sylvester h.a. 2004. re‑examination of the accuracy of a detergent solution for varroa mite detection. am bee j, 144 (7), 560‑562. singh r., levitt a.l., rajotte e.g., holmes e.c., ostiguy n., vanengelsdorp d., lipkin w.l. depamphilis c.w., toth a.l. & cox‑foster d.l. 2010. rna viruses in hymenopteran pollinators: evidence of inter‑taxa virus transmission via pollen and potential impact on non‑apis hymenopteran species. plos one, 5, e14357. spearman c. 1904. the proof and measurement of association between two things. am j psychol, 15, 72‑101. 13 introduction paratuberculosis (ptb), also known as johne’s disease, is a chronic intestinal infection caused by mycobacterium avium subsp. paratuberculosis (map) which primarily affects domestic and wild ruminants. however, many species were described as susceptible to the disease and transmission between wildlife and domestic animals has been reported (stevenson et  al. 2009). particularly, deer and wild rabbits may play a significant role in map epidemiology (carta et  al. 2013). wildlife may pose a risk for domestic animals, potentially introducing map into free herds (corn et al. 2005). the infection is frequently subclinical and the clinical signs, including weight loss and unresponsive watery diarrhoea, develop at late‑stage of the infection, thus map can persist undetected for many years at herd level. in domestic ruminants, infection leads to economic losses due to milk production decrease and reduced slaughter value (ott et al. 1999). at post‑mortem,the pathogen might be responsible for terminal ileal mucosa thickening due to lymphocitic infiltrates (granulomatous enteritis) (behr and collins 2010). in cattle, the risk of infection decreases after six months of age. juvenile animals are mostly infected via the fecal‑oral route or by the consumption of milk and colostrum from infected cows (rathnaiah et  al. 2017). concerns on the zoonotic potential of the disease have been raised because of the similarity with crohn’s disease, however, evidence linking map and crohn’s disease is far from conclusive (griffiths 2002). due to the economic importance and potential public health threat, most of the european countries have established control programmes, mainly in cattle. these programmes are based on testing and culling strategy. in cattle, the use of vaccines may interfere with both intradermal and serological tests for the diagnosis of tuberculosis, and with tuberculosis control programmes (garcia and shalloo 2015). nevertheless, the use of vaccines in small ruminants has been useful to reduce losses 1department of veterinary sciences, university of turin, grugliasco (to), italy. 2department of veterinary medicine, university of bari, valenzano (ba), italy 3department of animal health, regional campus of international excellence ‘campus mare nostrum’, university of murcia, murcia, spain. *corresponding author at: department of veterinary sciences, university of turin, largo paolo braccini 2, 10090 grugliasco (to), italy. e-mail: angela.fanelli@unito.it. keywords paratuberculosis, europe, epidemiological status, spatial distribution, multilevel model, wahis. summary mycobacterium avium subsp. paratuberculosis (map) is the etiological agent of paratuberculosis (ptb), a disease affecting domestic and wild ruminants. map may also play a zoonotic role in crohn’s disease. although both governments and industries are carrying out programmes to prevent and control the infection, there is a lack of harmonization across europe. moreover, the success of these programmes is influenced by the current lack of sensitivity of the diagnostic tests used. for these reasons, it is complex to evaluate the overall epidemiological situation of this disease. this study describes the european distribution of ptb from 2010 to 2017 using the information reported by member countries to the oie. countries were classified in three categories (‘absent’, ‘epizootic’, ‘enzootic’) depending on the disease epidemiology, and the trend of countries reporting the disease presence was computed throughout the study period. a multilevel model with random slope was built for twelve countries, with complete reporting history. most of the countries (57.44%) were classified as ‘enzootic’. the percentage of countries reporting the disease presence slightly increased along the study period, probably due to the improvement of ptb monitoring, rather than to a deterioration of the epidemiological situation of the disease in europe. results of the model account for different dynamics in the number of outbreaks reported by ‘enzootic’ and ‘epizootic’ countries. angela fanelli1*, domenico buonavoglia2, carlos martinez carrasco pleite3 and paolo tizzani1 paratuberculosis at european scale: an overview from 2010 to 2017 veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 accepted: 02.01.2020 | available on line: 24.04.2020 14 parabtubercolosis in europe fanelli et al. veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 countries that regularly provided data for this eight‑year period were considered in the analysis (forty‑seven member countries). quantitative data were grouped by semester, and member countries were classified in three categories: a. ‘absent’ countries: member countries where the disease was reported as absent throughout the whole study period b. ‘epizootic’ countries: member countries where the disease was reported as present but for which there was at least a two‑years period with no report of cases c. ‘enzootic’ countries: countries where the disease was present and for which all periods of absence were shorter than two years. the two‑year period of absence, to identify epizootic and enzootic countries was used, as it is the most frequent time range used in the oie terrestrial animal health code, to consider countries free from a disease (even if no specific chapter is available for ptb). however, it is important to notice that for countries in which in the previous two years the clinical disease was not reported, this does not mean that paratuberculosis is not present, if a surveillance program is not implemented, because the incubation period of ptb is 2‑15 years. the disease status in the different countries was mapped using quantum gis version 3.2.0 (qgis development team 2017), for both domestic animals and wildlife. the trend in percentage of infected countries per semester was computed throughout the study period to evaluate the dynamics of the epidemiological situation of the disease. average yearly figures on number of veterinarians engaged in animal health activities as well as national bovine, sheep and goat populations, were obtained from the annual reports submitted to the oie by the national veterinary authorities of the member countries3. the ratio between the average number of veterinarians and selected animal populations was used as a proxy to evaluate countries capacity for disease monitoring. pairwise one‑tailed wilcoxon tests were used to compare the number of veterinarians (normalized to susceptible animals) in the three disease categories. boxplots were drawn for graphical evaluation. all statistical analyses were performed using r software version 3.5.0 (r core team 2018). significant differences were considered at p < 0.05. a multilevel model was built to assess the effects (fridriksdottir et al. 2000). control programmes find some constraints in being accepted by farmers and veterinarians because of the cost, the effort required and the duration (khol and baumgartner 2012). the european surveillance framework is heterogeneous, with countries such as sweden performing a rigorous mandatory control programme with a stamping out policy (sfs 1999:657)1, other countries such as spain with voluntary regional programmes and countries with no control programmes at all. the success of disease control is influenced by the lack in sensitivity of diagnostic tests. tests reliability is low at early stages of infection, representing one of the major limits for ptb control (maroudam et  al. 2015). ante mortem diagnostic tests include direct and indirect methods, however, none of the available tests is recommended to be used alone, by the oie manual of diagnostic tests and vaccines for terrestrial animals [world organisation for animal health (oie) 2018]. for these reasons, a negative test result is not enough to prove that the animal is map free (manning and collins 2001). the complexity of map infection and the different conditions across european countries represent the major constraint to set up an epidemiological framework at regional scale. moreover, there is no disease case definition available in the oie terrestrial animal health code. to assess the heterogeneity of ptb situation in europe; member countries were classified into three disease categories depending on ptb occurrence throughout the study period (‘absent’, ‘epizootic’, ‘enzootic’), b) maps on countries disease status and presence of ptb in wildlife were built to spatially describe the disease, c) the percentage of the affected member countries per semester was computed during the period 2010‑2017, d) a hierarchical model was built to examine the impact of individual‑level (country) and group‑level (disease status) on the number of map outbreaks reported per semester, and e) the level of disease surveillance was evaluated comparing the number of veterinarians (normalized by susceptible animal populations) at country level. materials and methods a database containing data on the occurrence of ptb and disease outbreaks for the period 2010‑2017 was built for the european countries. data were retrieved from the oie world animal health information system (wahis)2. wahis is a dynamic database constantly updated, and data included in this study refers up to 1 february 2019. only member 1 swedish ministry of agriculture.2018. epizootic act (sfs1999:657). http://rkrattsbaser.gov.se/sfst?bet=1999:657 (accessed on 5 january 2019). 2 https://www.oie.int/animal‑health‑in‑the‑world/the‑world‑animal‑health‑information‑system/the‑world‑animal‑health‑information‑system/. 3 http://www.oie.int/ wahis_2/wah/health_v7_en.php. 15 fanelli et al. parabtubercolosis in europe veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 countries (italy, spain, and belgium) report details at administrative division level. table i summarizes wild species reported by countries throughout the study period. information on species affected is provided in the database since 2012. trend of the disease along the period of study the trend in percentage of countries reporting the disease ‘present’ per semester is depicted by figure  3. a slight and constant increase in the number of countries reporting the disease presence was observed along the period of study (mean increase of 0.6% per semester). hierarchical model for number of outbreaks reported the best model is a random slope model with the relationship between number of outbreaks and disease status varying across countries. table  ii presents model results, with all the variables that resulted significant (fixed and random effects). the percentage of variance explained by the selected model is high (adjusted r2  =  0.88). country and disease status contribute equally to the explained random effect variance (47%). the mean number of outbreaks per year reported across countries is  3.2. outbreak number shows a significant decrease along the period of study, with significant differences among status: the average reduction in number of outbreaks in ‘epizootic’ countries is 0.04 vs 3.1 in ‘enzootic’ countries. in of group‑level (disease status) and individual‑level (countries) on the number of outbreaks reported per semester. countries that didn’t report quantitative data along with countries reporting ptb absent during the study period were excluded from the analysis (twelve countries selected). data normalization was applied to the number of outbreaks (log transformation). hierarchical analysis was performed with r version 3.5.0 (r core team 2018). the lme4 v1.1‑20 (bates et al. 2015) and the lmertest v3.1‑0 (kuznetsova et al. 2017) packages were used. the akaike’s information criterion (aic) (akaike 1973) and the likelihood ratio test were used to select the best model. homogeneity of variance was assessed with diagnostic plots, from lattice v0.20‑35 (sarkar 2008). results geographic distribution of the disease and species affected figure 1 shows european countries based on their animal health status. the majority of member countries were classified as ‘enzootic’ (57.4%). ‘epizootic’ member countries are concentrated in central europe (12.7%). ‘absent’ member countries (29.7%) are located both in eastern and north europe. figure 2 shows wildlife status. data accuracy for wildlife varies among the countries: four member countries (germany, hungary, netherlands, and united kingdom) provide the information at national scale, three member figure 1. status of ptb in european countries based on information reported by countries in the period 2010‑2017 (data from world animal health information system, wahis). 1000 0 1000 2000 3000 4000 km ‘enzootic’ ‘epizootic’ ‘absent’ excluded 16 parabtubercolosis in europe fanelli et al. veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 veterinary surveillance at country level the number of veterinarians engaged in animal health activities (normalized to susceptible animals) was significantly lower for ‘enzootic’ countries compared to ‘epizootic’ (p value = 0.02) and to ‘absent’ countries (p value = 0.02) (figure 5). no statistically significant differences were observed between ‘absent’ and ‘enzootic’ countries (p value = 0.2). discussion this paper reports the status of ptb at european level, summarizing the official information reported particular, the divergence between disease status is 0.06 for time unit. the selected model fits very well with the heterogeneous data on reported outbreaks (figure 4). table i. reported wild species for the selected countries for ptb from 2012 to 2017 [data from world animal health information system (wahis)]. country reported wild species affected by ptb belgium capreolus capreolus cervus elaphus cabra ibex germany cervidae hungary cervus elaphus sus scrofa italy cervus elaphus capreolus capreolus rupicapra rupicapra cabra ibex argali sheep muflon ovis ammon ovis musimon dama dama netherlands cervidae (unidentified) spain capra pyrenaica cervus elaphus dama dama switzerland wildlife (species unkown) united kingdom of great britain & n. ireland camelidae cervidae 0.6 0.4 0.2 0.0 2010 2012 2014 2016 figure 3. percentage of countries reporting ptb presence from 2010 to 2017. black line shows the real values and red line shows the trend. [data from world animal health information system (wahis)]. figure 2. ptb presence in wild species reported by european countries in the period 2010‑2017 (data from world animal health information system (wahis). 1000 0 1000 2000 3000 4000 km present excluded 17 fanelli et al. parabtubercolosis in europe veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 european countries, and that a significant increase of the percentage of countries reporting the disease as present was observed during the study period. this finding may reflect the improvement of countries efforts for the early identification of map, through ad  hoc programmes for active surveillance, rather than a deterioration of the epidemiological situation of the disease in the region. this is especially true if we considering the characteristics of the disease, that easily becomes endemic in affected countries, along with the difficulties in diagnosis and in performing proper monitoring. the success of targeted surveillance programmes requires a long term commitment from both government and agricultural sector (benedictus and kalis 2003). levels of control vary over europe being regional or national programmes, compulsory or voluntary based. for instance, in italy, guidelines for the control of bovine ptb were developed to meet the request of china, india and russia which are the leading importer of italian milk and dairy‑products (luini et al. 2013). although reporting cases is compulsory, certification of farms is on voluntary basis and, therefore, disease monitoring and reporting is not homogeneous. in spain a national surveillance programme is not implemented, and most of disease surveillance is based on regional initiatives. among by veterinary services to the world organisation for animal health. few papers have described a global overview of the epidemiological situation of the disease, being most of them more focused on national or subnational aspects. in this context the main value of the present work is to provide information on the current status of the disease under a european perspective, and its dynamics during the last eight years. data show that ptb is present in a large majority of table ii. multilevel model results. details for random effects and fixed effects are displayed. random effects variance std. dev. country (intercept) 2.8 1.7 status 2.8 1.7 residuals 0.4 0.6 fixed effect estimate std. error p value intercept 3.23 0.6 0.002 semester 0.04 0.013 0.001 ds epidemic 3.1 0.6 0.002 semester* ds epidemic 0.06 0.02 0.003 o u tb re ak s (l o g s ca le ) 6 4 2 0 austria ‘enzootic’ semester 6 4 2 0 greece ‘enzootic’ 4 8 12 6 4 2 0 poland ‘epizootic’ czech republic ‘epizootic’ hrvatska ‘enzootic’ 4 8 12 slovenia ‘epizootic’ estonia ‘enzootic’ italy ‘enzootic’ 4 8 12 spain ‘enzootic’ germany ‘enzootic’ norway ‘epizootic’ 4 8 12 switzerland ‘enzootic’ figure 4. model fitting per country. 18 parabtubercolosis in europe fanelli et al. veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 can be either conducted as general surveillance (passive surveillance) or targeted surveillance (active surveillance) (kuiken et  al. 2011). in case of ptb, specific targeted surveillance is declared by the following countries: belgium, bulgaria, netherlands, norway, switzerland and united kingdom (data reported on wahis interface ‑ www.oie.int/wahid.), while general surveillance programmes on wildlife are present also in other countries. considering the difficulties in monitoring wildlife, the countries able to conduct active surveillance in wild population can be considered as having a high level of monitoring of the disease. concerning the affected species, ptb was reported in europe mainly in cervids (table i), however transmission of map between rabbits and cattle has been reported (stevenson et al. 2009) and high prevalence of ptb in wild rabbit population may be associated with high prevalence in domestic animals (shaughnessy et  al. 2013). despite these facts, no case in lagomorphs has been reported by countries during the study period. some reports in unusual species (i.e. in camelidae from uk), are due to the fact that monitoring of the disease concerns not only native wildlife, but also zoo and captive wild animals. the multilevel model presented in this study indicates that the number of outbreaks in ‘epizootic’ countries significantly decreased in time compared with the ‘enzootic’ countries. considering that, the analysis focused only on countries with constant level and quality of information provided, this reduction may be considered as a real improvement of disease situation in the concerned countries, probably due to a successful implementation of control programmes. multilevel models are routinely used in veterinary epidemiology, but most commonly in their simplest form using the random intercept approach (stryhn et  al. 2006). the model selected in this study is a random slope model, which accounts of both group (disease status) and individual (country) variability. this model was able to properly describe the variance in the number of outbreaks reported along the study period, and so to provide a better insight of the disease dynamics. finally, our work shows that limited veterinary workforce in animal public health field may be a big constraint for successful control of diseases. a strong relationship was in fact observed between the number of veterinarians, normalized for the susceptible animals, and disease situation of countries. in particular, ‘enzootic’ countries were found to have less veterinarians (normalized to susceptible animals) engaged in animal health activities than ‘epizootic’ or ‘absent’ countries. nevertheless, the positive result of surveillance programme doesn’t depend only on the number of veterinarians but also on the quality of veterinary services as well as the extent to which farmers are them, a specific programme to reduce economic losses of cattle infection is implemented in the basque autonomous community (nielsen 2009). other countries like sweden included the disease control in the swedish epizootic act (sfs 1999:657), and currently declare the absence of ptb at national level (frössling et al. 2013). according to the criteria used, twenty‑seven countries were classified as ‘enzootic’, fourteen as ‘epizootic’, and six as ‘absent’ . the number of ‘absent’ countries is probably overestimated, considering the epidemiological aspects of the disease [few clinical cases expressed, disease difficult to detect at herd level, long incubation period (2‑15 years)] along with the poor sensitivity of diagnostic tests during the latent period (maroudam et al. 2015). the geographic distribution of the disease, as it appears from the information officially reported by veterinary services, has to be evaluated critically, in the light of the difficulties in disease surveillance and detection. it is important to remind that the effectiveness of any surveillance strategies is influenced by a clear understanding of the advantages of ptb control. although ptb is notifiable in most countries, there is a lack of awareness on the economic impact of the disease and, therefore, countries tend to allocate resources for other animal diseases. moreover, countries that undertake a control program have difficulties in measuring its impact (whittington et al. 2019). a correct assessment of a control programme and the understanding of ptb economic burden are fundamental for long‑term control activities. official data reported by countries concerned also wildlife but surveillance programmes are not homogeneous across countries, so the absence of reporting may not be considered as a true absence. although surveillance of the disease in wildlife may be difficult, it must be considered as important as surveillance in domestic animals (vallat 2008). in fact, wild animals can transmit map to domestic species, either by direct or indirect contact (chiodini and hermon‑taylor 1993). as highlighted above, wildlife surveillance varies across europe and it v et er in ar ia n s/ su sc ep ti b le a n im al s 20*10-4 15*10-4 10*10-4 5*10-4 0 enzootic epizootic absent figure 5. number of veterinarians (normalized to susceptible animals) in the three disease status categories [data from world animal health information system (wahis)]. 19 fanelli et al. parabtubercolosis in europe veterinaria italiana 2020, 56 (1), 13‑21. doi: 10.12834/vetit.1829.9692.3 describing the epidemiological situation of ptb at regional scale using data of the oie reporting system (wahis). the different levels of reporting of the epidemiological situation of the disease, mainly for what concerns quantitative information (no constant quality of information provided by all the countries along the period of study), may bias some of our results. despite this, the main strength of the study is that it takes into consideration only information reported by veterinary services at the oie, and represents so the most complete officially reported situation of the disease in europe. this is also one of the few epidemiological studies implementing a multilevel model to describe heterogeneous data on the number of outbreaks reported. the results presented must be carefully interpreted in the light of the disease epidemiology and different level of surveillance. for a better control of the disease, countries should improve their monitoring systems, in order to increase surveillance and probability of outbreaks detection in both domestic animals and wild species. this study will serve as a basis for further studies on the epidemiological status of ptb at regional scale. willing to participate into surveillance programmes. for instance, in 2006 denmark, which have reported map presence throughout the whole study period, has initiated a programme called ‘operation paratuberculosis’ characterized by standard education of farmers and local health advisors (nielsen et  al. 2007). also in this case, these results must be considered cautiously as global indicator of the importance of veterinary services, and not as an index of each member countries efficiency in disease detection and control. conclusions map infection leads to economic losses in farms. the bacteria may also have a role in the development of crohn’s disease in humans. for these reasons, ptb control has arisen interest of countries over time. the restriction of livestock and dairy marketing in case of infection imposed by some countries has globally led to develop more efficient surveillance programmes. despite these attempts, there is still a wide variation both in map reporting and monitoring among countries. this is the first study akaike h. 1973. information theory and an extension of the maximum likelihood principle. in proc. the 2nd international symposium on information theory (petrov b.n. & csaki f., eds). akadémiai kiadó, 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caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 1; year: 2020; doi: 10.12834/vetit.1829.9692.3 supplementary material. model selection procedure. table a. anova table: likelihood ratio test to find the best fixed structure. model adjusted r2 aic loglik pr (> chisq)* linear semester and status interaction (single level model) as explanatory variable 0.41 690 340.12 random intercept semester and disease status interaction as explanatory variable 0.86 431 209.84 < 2.2e-16*** random slope semester and disease status interaction as explanatory variable 0.88 420 202.11 0.0004415*** *p value of the likelihood ratio chi-squared statistic. signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 1. first step: finding the optimal structure of random component (table a). table b. anova table: likelihood ratio test to find the best fixed structure. random slope model adjusted r2 aic loglik pr (> chisq)* null multilevel model 0.86 437 213.39 semester as explanatory variable 0.86 435 211.69 0.065077 disease status as explanatory variable 0.87 428 208.20 < 2.2e-16*** semester and disease status as explanatory variable 0.87 427 206.50 0.065052 interaction of disease status and semester as explanatory variable 0.88 420 202.11 0.003066** *p value of the likelihood ratio chi-squared statistic. signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 2. second step: finding the optimal fixed structure (table b). 3. third step: the best model is presented (please see the results part in the manuscript). 117 veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 accepted: 18.05.2020 | available on line: 16.11.2022 1department of veterinary medical sciences, university of bologna, via tolara di sopra 50,40064, ozzano dell’emilia (bo), italy. 2division of avian diseases, kimron veterinary institute, bet dagan p.o. box 12 5025001, israel. 3department of animal medicine, production and health, university of padua, viale dell’università 16, 35020, legnaro (pd), italy. *corresponding author at: department of veterinary medical sciences, university of bologna, via tolara di sopra, 50,40064, ozzano dell’emilia (bo), italy. e‑mail: giulia.mescolini3@unibo.it. giulia mescolini1*, caterina lupini1, antonietta di francesco1, irit davidson2, viviana felice1, laura bellinati1, mattia cecchinato3 and elena catelli1 keywords b19 haplotype, cochin chicken, marek’s disease virus, meq gene, mhc, paralysis. summary the present study investigates an outbreak of classical marek’s disease (md) in backyard cochin chickens reared for hobby in italy. examined chickens showed spastic paralysis of the legs and at necropsy, enlargement and discoloration of the peripheral nerves and plexuses that matched microscopic aand btype md lesions. molecular analysis of the meq gene of the detected gallid alphaherpesvirus 2 (gahv-2) strain, showed typical markers of low virulence and the strain shared the entire meq gene sequence with strains circulating in italian backyard chickens. furthermore, the haplotype b19 of the major histocompatibility complex (mhc) was defined in the affected chickens, showing that the birds possessed a genetic profile of high susceptibility to md, allowing the appearance of a classical nervous clinical form after infection with an apparently low pathogenicity gahv-2 strain. trade of live ornamental purebred chickens occurs frequently between hobby farmers and biosecurity practices, such as quarantine periods, should be applied to avoid the introduction of infected animals. veterinarians should raise awareness of this issue and promote the use of vaccines against md. marek’s disease in genetically susceptible cochin chickens in italy: a case report within the gahv-2 genome, the marek’s eco ri-q (meq) gene has been recognized as playing a crucial role in gahv-2-induced neoplastic transformation, in association with other auxiliary genes. the meq gene is generally 1,020 base pairs (bp)-long and encodes for meq, a 339-amino-acid basic leucine zipper (bzip) protein (chang et  al. 2002). the meq gene sequence analysis is frequently used for the prediction of gahv-2 strain virulence thanks to the polymorphic nature of the gene and to the presence of molecular markers of virulence (shamblin et  al. 2004, renz et  al. 2012, padhi and parcells 2016), such as the number of stretches of four proline molecules (pppp) within the transactivation domain of the meq protein. the well-known genetic resistance to md in chickens is associated with several chicken genes, in particular, the b locus of the chicken major histocompatibility complex (mhc) seems to play a significant role in conferring resistance to md introduction marek’s disease (md) is a disease of chickens caused by a lymphotropic and ubiquitous oncogenic virus, gallid alphaherpesvirus 2 (gahv-2), belonging to the genus mardivirus of the alphaherpesvirinae subfamily. four gahv-2 pathotypes are currently recognized: mild (m), virulent (v), very virulent (vv) and very virulent plus (vv+) (witter 1997). gahv-2 infection may induce neoplastic transformation of t cells resulting in development of lymphomas in the visceral organs, peripheral nerves or muscles and skin (schat and nair 2013). birds may experience different clinical signs according to the virulence of the strains. weakly virulent strains are responsible for the classical form of the disease characterized by spastic paralysis of the extremities, while infection with highly virulent strains results in the development of multiple visceral lymphomas or paralysis (witter 1997). case report 118 veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 outbreak of classical marek’s disease in susceptible cochin chickens mescolini et al. 10% neutral buffered formalin and embedded in paraffin blocks. sections (4 μm thick) from paraffin embedded tissues were stained with haematoxylin and eosin following standard histological techniques (dalle zotte et al. 2017). dna extraction and pcr amplification of the meq gene of gahv‑2 dna was extracted from pools of five feather tips per bird using the commercial kit high pure pcr template preparation kit (roche diagnostics gmbh, mannheim, germany), with a subtle adjustment to the manufacturer’s instructions as previously described (mescolini et  al. 2019). total dna was then subjected to the pcr protocol described by mescolini and colleagues (mescolini et  al. 2019), capable of amplifying the full-length meq gene. sequencing and phylogenetic analysis the amplification products of the meq gene were purified using exosap-it™ express pcr product cleanup (thermo fisher scientific, massachusetts, usa) and sequenced using pcr primers ecor-q for 5’-ggt gat ata aag acg ata gtc atg-3’ and ecor-q rev 5’-ctc ata ctt cgg aac tcc tgg ag-3’ (shamblin et  al. 2004) plus an additional internal primer, meq-f, 5’-atg tct cag gag cca gag ccg-3’ (hassanin et  al. 2013), by a commercial sequencing service (macrogen europe, amsterdam). the obtained nucleotide and deduced amino acid (aa) sequences were edited with bioedit sequence alignment editor version 7.0.5.3 (tom hall, ibis therapeutics, carlsbad, california, usa), aligned and compared using clustal w software with the meq gene sequences of reference gahv-2 strains, available in the genbank database, and with 33  gahv-2 italian strains recently detected during md outbreaks in backyard and commercial flocks (mescolini et  al. 2019, mescolini et  al. 2020a) and in a turkey flock (mescolini et  al. 2020b). the number of stretches of four consecutive proline molecules (pppp), contained in the transactivation domain of the meq protein, was counted. phylogenetic analysis based on the amino acid sequences of the meq gene of reference strains and of selected italian gahv-2 strains was performed with the maximum likelihood method under the jones-taylor-thornton model using megax (kumar et  al. 2018). bootstrap values, obtained with 1,000 replicates, were considered significant when equal or greater than 70. identification of the chicken’s haplotype by pcr‑ssp the chicken’s haplotype was molecularly determined, according to zheng and colleagues (zheng et  al. (bumstead and kaufman 2004). the existence of a hierarchy of resistance determined by b haplotypes has been revealed, with b21 haplotype conferring the strongest resistance to md and b19 conferring the greatest susceptibility (briles et  al. 1977, blankert et al. 1990). rearing backyard chickens is a common practice worldwide, especially in rural areas. although infectious disease surveillance in backyard flocks is limited (felice et al. 2019) and mostly undocumented, md is recognized as one of the leading causes of mortality in these birds in several countries. md in backyard flocks has been reported in mexico (rodriguez et  al. 1997), the united kingdom (whitehead and roberts 2014), finland (pohjola et al. 2015), ethiopia (demeke et al. 2017), the united states (mete et  al. 2013, crespo and senties-cue 2015, mete et al. 2016, bell et al. 2019, cadmus et al. 2019, vaught et al. 2019), canada (brochu et al. 2019), italy (mescolini et al. 2019), brazil (chacón et al. 2019) and israel (davidson, unpublished data). the present study investigates an outbreak of md in backyard cochin chickens reared for hobby. clinical signs as well as macroscopic and microscopic findings are described and molecular analysis of the meq gene of the detected gahv-2 is performed. finally, the mhc haplotype of the affected chickens is molecularly established. materials and methods birds and sample collection in 2014, two md unvaccinated frizzle cochin bantam chickens from a multi-species backyard farm in italy (sarsina, fc) were presented to the avian pathology service of the department of medical veterinary sciences of the university of bologna for diagnostic investigation. the flock consisted of approximately thirty chickens of various breeds (brahma, cochin, silkie, leghorn). peacocks and pigeons were raised on the same farm but in different fences. three birds showed clinical signs suggestive of classical md at two months of age, two of them were presented for examination. in addition, another young bird died but was not conferred for post-mortem examination. both birds were clinically examined and necropsied after euthanasia, and liver, lung, kidney, proventriculus, bursa of fabricius, brachial plexus, sciatic plexus and sciatic nerve were collected for microscopic examination. feather tips were also collected for pcr analysis. histopathology portions of the organs listed above were fixed in 119veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 mescolini et al. outbreak of classical marek’s disease in susceptible cochin chickens results clinical signs and post‑ mortem lesions both examined chickens showed an inability to stand upright and walk, and showed spastic paralysis of legs (figure 1). at necropsy, enlargement of the peripheral nerves and nervous plexuses was observed (figure 2). in particular, brachial and sciatic plexuses, and brachial, sciatic, vagus and intercostal nerves were involved. the nerves had lost their typical cross-striation and showed a yellow discoloration and oedematous appearance. no lymphomas were grossly visible in the visceral organs. at microscopic examination of the nerves, aand b-type lesions, typical of md according to payne and biggs (payne and biggs 1967), were detected. a-type lesions, considered of a neoplastic nature, consisted of masses of pleomorphic infiltration of small, medium and large lymphocytes (figure 3a). b-type lesions, considered of an inflammatory nature, were characterized by interneuritic oedema and diffuse infiltration of small lymphocytes and plasma cells (figure 3b). 1999). b‑lβii family genes were first amplified from genomic dnas extracted from pulpy feathers, using a pair of b‑lβii family specific degenerated primers (upstream primer: 5’ cg ttc ttc ttc trc ggt rbg at 3’ and downstream primer: 5’ ta gtt gtg ccg gca gam csy g 3’) amplifying a 235 bp fragment conserved in all known b‑lβii family alleles. b‑lβii family specific pcr was carried out by adding 3 μl dna to a 22 μl reaction mixture containing 0.125 μl gotaq® g2 flexi dna polymerase (promega, madison, wi), 5 μl 5x colorless go-taq flexi buffer, 1.75 μl mgcl2 solution, 0.5 μl dntps, 13 μl h 2 o for molecular biology, 1 μl upstream primer and 1  μl downstream primer. cycling conditions were as follows: 5 min of denaturation at 95°c followed by 35 cycles, each consisting of denaturation at 95  °c for 30 s, annealing at 50°c for 30 s, and extension at 72 °c for 30 s. a final elongation step at 72 °c for 5 min completed the reaction. for the differentiation of the more commonly encountered b haplotypes, the pcr products were then subjected to a further pcr using sequence-specific primers (pcr-ssp) internal to the b‑lβii family specific primers. five different pairs of specific primers 4up8/1dn69, 1up32/1dn65, 3up8/2dn69, 2up8/1dn66 and 1up8/2dn66 (zheng et  al. 1999) designed on the basis of published sequences of standard haplotypes were used in pcr-ssp to amplify b‑lβii family alleles associated with five of the more frequent haplotypes known b2, b13, b15, b19 and b21, respectively. the reaction mixture used was the same described above, changing the primer sets, and cycling conditions were the following: 94 °c for 5 min followed by 25 cycles each consisting of denaturation at 94 °c for 30 s, annealing at 50, 55 or 60 °c for 30 s, and extension at 72 °c for 30 s. final elongation phase was conducted at 72 °c for 5 min. the pcr products were separated on agarose gel (2%), stained with ethidium bromide, and visualized under ultraviolet light after an electrophoretic run at 110 v and 400 ma for 35 min. the amplicons obtained were purified, sequenced in both directions and submitted to the basic local alignment search tool (blast) to confirm pcr results with a similarity search. accession numbers nucleotide sequences of the meq gene of the detected gahv-2 strains were submitted to the genbank database and are available under the following accession numbers: mn518896 and mn518897. b‑lβii family allelic nucleotide sequences were submitted to the genbank database under accession numbers mn518898 and mn518899. figure 1. clinical examination. a. bird in lateral decubitus due to spastic paralysis of the limbs. b. bird showing one leg stretched forward and the other back. 120 veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 outbreak of classical marek’s disease in susceptible cochin chickens mescolini et al. gahv‑2 detection and molecular characterization of the meq gene feathers from both examined chickens were pcr positive for gahv-2. the two detected viruses, named gahv-2/italy/ck/419/14 and gahv-2/italy/ ck/420/14, showed 100% identity. the meq gene sequence had an overall length of 1,257 base pairs (bp), and, when compared with the meq sequence of the vaccine strain cvi988/rispens (genbank accession number dq534538), showed an insertion of 57 bp, encoding 19 aa. meq protein sequence had nine pppp motifs and a proline content of 23.6%. moreover, the detected virus shared the entire meq gene sequence with three strains detected in italian backyard chickens exhibiting neurologic signs (genbank accession numbers: mk13966, mk139664 and mk139665) (mescolini et al. 2019). the phylogenetic tree, based on meq amino acid sequences (figure 4), confirmed this latter finding as the investigated viral strain formed a cluster including other strains detected in italian backyard flocks (mescolini et al. 2019). identification of mhc haplotypes genomic dnas from the examined chickens were amplified only when the primer pair targeting b19 haplotype was used. amplicons of 213 bp were obtained and sequenced. both sequences showed 100% identity with sequences of the mhc class ii beta chain gene corresponding to the chicken haplotype figure 2. macroscopic lesions. the following nerves and plexuses showed a yellowish discoloration, are oedematous and enlarged, and have lost their typical cross‑striation: brachial plexuses and nerves (a); intercostal nerves (b); sciatic plexuses (c); sciatic nerve (d); vagus nerve (e). figure 3. histopathological lesions. a. sciatic nerve. pleomorphic infiltration of small, medium and large lymphocytes, compatible with a‑type lesions (40×). haematoxylin and eosin (he). b. sciatic nerve. interneuritic oedema with diffuse infiltration of small lymphocytes and plasma cells, compatible with b‑type lesions (40×). he. 121veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 mescolini et al. outbreak of classical marek’s disease in susceptible cochin chickens strain whose meq gene sequence showed typical markers of low virulence. in contrast to previous md reports in backyard chickens, which focused on clinical and post-mortem findings (rodriguez et al. 1997, mete et al. 2013, crespo and senties-cue 2015, pohjola et al. 2015, brochu et al. 2019, cadmus et  al. 2019, vaught et  al. 2019, chacón et  al. 2019), our study provides molecular data on the causative agent and genetic susceptibility of the affected birds. the low oncogenic potential of the gahv-2 strain was confirmed by clinical, macroscopic and microscopic findings suggestive of classical nervous form of md. shamblin and colleagues (shamblin et al. 2004) and renz and colleagues (renz et al. 2012) demonstrated the correlation between meq gene molecular features and the degree of virulence of gahv-2 strains, and proposed this as the basis to scrutinize virulence relying on meq gene sequencing. viral strains with meq containing a higher number of pppp repeats exhibit a lower virulence, while virulent gahv-2 strains show the lowest number of proline repeats. in this study, the gahv-2 strain detected showed a meq protein sequence with the highest number of pppp repeats (n = 9) when compared with other gahv-2 strains. our results, coupling sequence analysis of the meq gene with the observation of clinical and pathological outcomes of the infection, confirm the validity of the molecular method for gahv-2 classification. we believe that providing data confirming the correlation between strain sequence and virulence will reinforce the notion of a pathotyping that can avoid the use of in vivo studies. however, at the moment, in vivo studies conducted as described by witter and colleagues (witter et al. 2005) remain mandatory for gahv-2 pathotyping. the phylogenetic analysis showed that the strain detected in the present study belong to the same cluster as other italian low pathogenicity viruses, which are exclusively detected in backyard flocks (mescolini et  al. 2019) and not in the commercial sector (mescolini et al. 2020a). trade of live ornamental purebred chickens occurs frequently between hobby farmers while prophylactic measures like biosecurity, quarantine periods or vaccination are not regularly applied. while commercial chickens routinely receive vaccines against md, vaccination of backyard birds is quite unusual because owners are unaware of the disease and the prophylactic measures, or because the vaccine commercial formulations are too expensive for such small flocks (elkhoraibi et  al. 2014). given the circulation in the italian rural sector of gahv-2 strains capable of causing clinical outbreaks, veterinarians should raise awareness of this issue and promote the use of vaccines against b19 published in the genbank database (accession numbers ab426151.1; dq008584.2; aj248583.1). discussion the present study reports a md outbreak in cochin chickens following their infection with a gahv-2 figure 4. phylogenetic tree based on complete meq amino acid sequence of gahv‑2/italy/ck/419/14 and gahv‑2/italy/ck/420/14 (underlined in the tree) and italian and reference strains retrieved from genbank. accession numbers are located before the name of each strain along with a black dot (), indicating strains detected in backyard flocks, or a black triangle (), indicating strains detected in commercial flocks (when available). abbreviations were used for gahv‑2 pathotypes (m = mild, v = virulent, vv = very virulent, vv+ = very virulent plus) and for gahv‑2 host species (ck = chicken, ty = turkey). 122 veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 outbreak of classical marek’s disease in susceptible cochin chickens mescolini et al. in terms of vaccinal immunity and md incidence after vaccination (bacon and witter 1992, bacon and witter 1993, bacon and witter 1994a, bacon and witter 1994b). more recently it has been shown that also non-mhc host genes are able to affect the vaccine-induced protection against md (chang et  al. 2010, chang et  al. 2011, chang et  al. 2012, xie et  al. 2017). in conclusion, ad hoc vaccination programs could be developed when the host genetic background and the presumptive virulence of gahv-2 circulating strains are known. statement of animal rights the case report object of the present study, conducted on private pet birds with the consent of the owner, is to be considered as a veterinary clinical practice for non-experimental purposes pursuant to article 2, paragraph 1, letter b of the italian legislative decree no. 26/2014 (approval issued by the animal welfare committee of the university of bologna protocol no. 254249). md. in recent years, some italian hobby farmers have begun to vaccinate their flocks with cell-free lyophilized turkey herpesvirus (hvt) vaccine, which is cheaper and easier to handle than cell-associated formulations, with good results on md control in flocks where weakly virulent strains were circulating. results of mhc haplotype analysis showed that both affected chickens possessed the b19 haplotype, which is typical for susceptibility to md, which explains the severity of the clinical and pathological effects observed in non-vaccinated young chickens infected with a low-virulence gahv-2 strain. this finding demonstrates the importance of virus-host interaction in complicating the scenario. it has been reported that the chicken mhc haplotype b19 is correlated with a high susceptibility to md, since b19 chickens develop more severe gross and histological lesions and show a higher level of viral replication after challenge with a very virulent gahv-2 strain (gao et  al. 2015). mhc haplotype seems also to influence vaccine protective efficacy, as chickens expressing certain mhc b haplotypes seems to respond better to different vaccine strains 123veterinaria italiana 2022, 58 (1), 117-124. doi: 10.12834/vetit.2120.12221.1 mescolini et al. outbreak of classical marek’s disease in susceptible cochin chickens bacon l.d., witter r.l. & silva r.f. 2001. characterization and experimental reproduction of peripheral neuropathy in white leghorn chickens. avian pathol, 30, 487-499. bell a.s., kennedy d.a., jones m.j., cairns c.l., pandey u., dunn p.a., szpara m.l. & read a.f. 2019. molecular epidemiology of marek's disease virus in central pennsylvania, usa. virus evol, 5, vey042. biggs p.m., 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(d.e. swayne, j.r. glisson, l.r. mcdougald, l.k. nolan, d.l. suarez, & v. nair, eds). wiley-blackwell publishing, ames, 515-552. shamblin c.e., greene n., arumugaswami v., dienglewicz r.l. & parcells m.s. 2004. comparative analysis of marek’s disease virus (mdv) glycoprotein-, lytic antigen pp38and transformation antigen meq-encoding genes: association of meq mutations with mdvs of high virulence. vet microbiol, 102, 147-167. vaught m.e., gladden j.n., rozanski e.a. & graham j. 2019. reasons for evaluation on an emergency basis of and 71 veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 accepted: 27.07.2020 | available on line: 27.07.2021 1department of veterinary public health and preventive medicine, university of ibadan, nigeria. 2department of veterinary pathology, university of ibadan, nigeria. *corresponding author at: pmb 001, department of veterinary public health and preventive medicine, university of ibadan, nigeria. phone: +2348036976193, e-mail: olayinkaishola@yahoo.com. olayinka olabisi ishola1*, obokparo godspower ohore2 and oladimeji david adigun1 keywords antibodies, awareness, dogs, enzyme linked immunosorbent assay, rabies. summary this study was conducted to profile the antibody levels to rabies in dogs presented at veterinary clinics and determine rabies awareness among dog owners in abeokuta, nigeria. records of dogs’ rabies vaccination were obtained to determine their vaccination status and number of times they had been vaccinated. sera from 138 dogs of consenting owners were analysed using indirect elisa technique to detect rabies antibodies. structured questionnaire was administered to 138 dog owners to determine their awareness on rabies. data were analysed using descriptive statistics, chi-square, anova and t-test at p  ≤  0.05. of 138 dogs screened, 114 (82.6%) had history of vaccination against rabies. of these 114, 87  (76.3%) were seronegative; however, 5 (3.6%) of the 24 unvaccinated dogs were seropositive. overall, 32 (23.2%) comprising 15 (10.8%) males and 17 (12.3%) females had positive rabies antibodies level. five (3.6%), 3 (12.1%) and 24 (17.4%) were seropositive among dogs of <  6 months, 6-12 months and > 1 year of age, respectively. dogs > 1 year had significantly higher antibodies than < 6 months (p < 0.05). most (86.9%) of the dog owners were aware of rabies. the low seroconversion in vaccinated dogs and prevalence of rabies antibodies in unvaccinated dogs are of public health concern. there is need for regular sero-profiling of vaccinated and unvaccinated dogs. rabies awareness among dog owners and detection of antibody levels against rabies in dogs presented for treatment at selected veterinary clinics in abeokuta, ogun state, nigeria are susceptible to rabies, although canine rabies presents the greatest threat to humans, as exposure to rabid dogs contributes up to 99% of all rabies transmissions to humans worldwide (who 2019). otolorin and colleagues (otolorin et  al. 2015) reported that 99% of human rabies in nigeria were due to dog bite. domestic dogs are the most common reservoir of the virus, with more than 99% of human deaths caused by dog-mediated rabies (who 2019). the vast majority of humans, who develop rabies, die because of the infection (tekki et  al. 2013, hurisa et  al.  2015). about 60,000 people die of rabies every year in over 150 countries, with 95% of cases occurring in africa and asia (who  2019). due to widespread underreporting and uncertain estimates, the true burden of disease could even be more; as in the case of nigeria with only a total of 78 human deaths recorded in health care institutions in various states, spanning from introduction rabies is a zoonotic disease caused by the rabies virus (rabv) of the lyssavirus genus within the family rhabdoviridae (who 2019). it is a typical zoonosis that is considered as the oldest communicable disease of human, existing for more than 4,300 years (fooks et al. 2012, yibrah and damtie 2015, barecha et  al. 2017). the rabies virus genome consists of a single stranded, non-segmented, negative sense rna of approximately 12 kb (saito et  al. 2013). rabies transmission is usually via virus-laden saliva of an infected animal through bite or scratch from animal to animal or animal to man. rabies virus is highly neurotropic; it has high affinity for the central nervous system (barecha et al. 2017). rabies remains a zoonotic viral disease that affects human, domestic and wild animals. all mammals 72 rabies awareness among dog owners and detection of antibody levels against rabies in dogs ishola et al. veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 2011 (nvri  2012). the voluntary routine vaccination of dogs against rabies at the veterinary clinics in nigeria places responsibility on the individual dog owners to bring their dogs to the clinic or to book for ambulatory services at a fee with registration of the dogs and issuance of rabies vaccination license and certificate (adeyemi et al. 2005). alternatively, there is the mass vaccination campaigns usually sponsored by government and non-government agencies, which are commonly administered free of charge or at subsidised cost; although not carried out regularly (adeyemi et al. 2005). enzyme linked immunosorbent assay (elisa) has been useful as an acceptable method which is fast and practical in the quantitative detection of antibody levels to rabies (olugasa et al. 2010, moore et  al. 2017). several studies have been carried out to detect rabies antibody levels in dogs in nigeria (ohore et  al. 2007, olugasa et  al. 2011, oluwayelu et al. 2015, awoyomi and ogundipe 2019). antibody response to rabies vaccine greater than or equal to 0.5  iu/ml serum is considered adequate to protect against the development of the infection (hurisa et  al. 2015). according to olayemi and colleagues (olayemi et al. 2017), 88.6% of the dogs sampled had sufficient antibody levels in abuja, nigeria. however, some reports have shown low antibody responses in dogs vaccinated in nigeria with commercial rabies vaccines, both local and imported (ohore et al. 2007, awoyomi and ogundipe 2019, ishola et  al. 2019). majority of such studies have focussed on the general dog population with less emphasis on those presented at veterinary clinics. furthermore, there is the general believe that all vaccinated dogs are protected; and have adequate antibody levels. rabies vaccination failures in dogs, although rare, have been documented while falsification of records has resulted in the entry of canine rabies virus variant infected dogs into rabies-free countries (rota nodari et  al. 2017, wallace et  al. 2017). therefore, in addition to veterinary records, many countries require that dogs seeking entry into rabies-free jurisdictions also provide serologic evidence of vaccination (wallace et al. 2017). this study therefore aimed at determining the antibody levels against rabies in dogs routinely presented for treatments at selected veterinary clinics as well as the dog owners’ awareness of rabies in abeokuta, ogun state nigeria. materials and methods study site, sampling and sample collection a cross sectional study was conducted involving collection of blood for serum from confined dogs 1980 to 2014 (richard et  al. 2015). as much as 99% of rabies cases in developing countries are dog-mediated and the burden of disease is largely borne by the rural poor communities (who 2019). while majority of human rabies associated with dogs has reduced in the last two decades in mexico, south america and the carribean, dog-associated human rabies has increased in parts of sub-saharan africa in the same period (olugasa et al. 2011). rabies is a 100% vaccine-preventable zoonotic disease and marked reductions, often progressing to the elimination of rabies, have been achieved in countries embarking on rabies elimination programmes (who 2019). prevention or elimination programmes often revolve around mass dog vaccination campaigns using rabies vaccines, where at least 70% of the dog population should be covered in order to prevent the disease or break the cycle of transmission in dogs, and to humans (fitzpatrick et  al. 2012, who 2019). according to the world health organization (who), at least 500,000 people are given post-exposure vaccinations every year and mass vaccination of domestic dogs has been the most effective measure in reducing human rabies (who 2005, yang et al. 2011). in order to achieve the global target of “zero human rabies deaths by 2030” set by the who, world organisation for animal health (oie), food and agriculture organization of the united nations (fao) and global alliance for rabies control (who 2019), there is a need to intensify efforts at vaccinating dog populations with effective vaccines. dog owners and policy makers should be well informed of vaccination schedule in order to ensure effective vaccination programmes (olugasa et  al. 2011). types of rabies vaccines used for immunisation include modified live vaccine, inactivated rabies vaccine and oral modified live vaccine. although these have largely proven to be safe and efficacious worldwide, new-generation rabies vaccines including recombinant rabies virus-based vaccines, vectored vaccines, dna-based vaccines, and plant vaccines have recently been explored to overcome the limitations of the conventional vaccines (yang et al. 2013). in nigeria, pre-exposure vaccination of dogs usually involves a primary dose of low egg passage (lep) flury strain vaccine produced by the national veterinary research institute, vom, nigeria (nvri), given at 3 months of age. yearly booster doses are administered to sustain threshold immunity against rabies since rabies is enzootic in the country (adeyemi et al. 2005, ohore et  al. 2007, olugasa et  al.  2011). in addition to the rabies vaccines produced by nvri, imported rabies vaccines from other countries of the world are used since the local production by the nvri cannot meet up with the demand for the vaccine in nigeria (awoyomi and ogundipe 2019). annual rabies vaccine production for dog was less than 30,000 doses in 73 ishola et al. rabies awareness among dog owners and detection of antibody levels against rabies in dogs veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 spss (version  20), anova and student t-test. the significance was put at p < 0.05. results from the total of 138 dogs sampled, 51.4% (71/138) were male and 48.6% (67/138) female. proportions of 20.3% (n  =  28) were less than six months of age, 9.4% (n  =  13) were between 6-12 months of age while 70.3% (n  =  97) were greater than one year of age. the breed distribution showed that 45.7% (n  =  63) were alsatian, 13.8% (n  =  19) boerboel, 10.1% (n = 14) caucasian, 9.4% (n = 13) mastiff, 7.2% (n = 10) rottweiler and 13.8% (n = 19) other breeds (table i). vaccination records showed that 82.6% (114/138) had been vaccinated; 18.8% (26/138) of the sampled dogs had received only a single dose (primer dose), 63.8% (88/138) had received multiple doses of rabies vaccines while the remaining dogs (n = 24) were unvaccinated (table i). at the established spr cut-off of 0.25 (equivalent of 0.5 iu/ml), only 23.2% (32/138) of the screened dogs had adequate rabies antibodies levels. absence of detectable seroconversion was observed in 87 (76.3%) of the 114 vaccinated dogs screened. a proportion of 14.5% (20/138) of the seronegative dogs had received single vaccination dose and 48.6% (67/138) multiple vaccination doses. however, 3.6% (5/138) among the unvaccinated dogs were seropositive (table i). the study showed that 10.9% (15/138) males and 12.3% (17/138) females were seropositive. although, more females 17/32 (53.1%) were among the seropositive than males, there was no significant difference between the two sexes (p  >  0.05). there was an increasing seroconversion rate with age (table 1). the number of positive dogs >  1 year is significantly higher than those < 6 months (p < 0.05) (table i). based on breed of dogs, the number of seropositive alsatian breed dogs were significantly higher than rottweiler breed (p  <  0.05;  table  i). seroconversion rate among the dogs that had received multiple doses of rabies vaccines was higher 15.2% (21/138) than in those that received only a single dose 4.4% (6/138); but the difference was not statistically significant (p > 0.05, table i). the data from questionnaire respondents revealed that 121 (87.7%) of the screened 138 dogs were kept for guard duties while 17 (12.3%) were owned as pets. a total of 113 (81.9%) of the dogs were acquired through purchases and 18 (13.0%) as gifts. majority of the vaccines used were imported from usa (n = 103; 90.3%) followed by korea (n  =  6; 5.3%), france (n = 3; 2.6%), czech (n = 1; 0.9%) with only (n  =  1; 0.9%) produced in nigeria. there was no significant difference in the seroconversion rate based on the types of vaccines used. among the dog owner presented for various veterinary treatments at three major veterinary clinics in abeokuta, ogun state, nigeria, being the largest city in the state. consent of the dog owners was obtained after explaining the purpose and benefits of the study to them. only consenting dog owners thereafter presented their dogs for sampling. a total of 138 dogs were sampled (thrusfield 2007, oluwayelu et  al. 2015); sampling was done weekly for a period of six weeks. the age, breed and sex of each dogs were documented. records of vaccination history of the dogs were obtained to determine the vaccination status and how many times they had been vaccinated. a volume of 5 ml of blood was collected using sterile needle and syringe via cephalic venipuncture of sampled dogs and poured into respective glass tubes without anticoagulant. the blood samples were allowed to clot at room temperature for about 5-6 hours and the sera were separated into appropriately labeled eppendorf tubes and stored at 20 °c until serological test was performed. rabies antibody detection rabies antibody was assayed using the quantitative indirect elisa (i-elisa, department of pathology, university ibadan, ibadan, nigeria) technique as described previously (ohore et  al. 2007, oluwayelu et  al. 2015). the cut-off sample to positive ratio (spr) was 0.25 (equivalent of 0.5 iu/ml), which corresponded to twice the optical density (o.d.) value of the negative control serum. the o.d. was read at 450  nm. results were valid when the difference in the mean o.d. of the positive and negative controls was greater than 0.25, and the mean o.d. negative control was less than or equal to 0.25. vaccinated dogs under this study with spr greater than 0.25 were considered to have adequate antibody level antibodies against rabies. questionnaire a structured questionnaire (n = 138) was administered to the dog owners to obtain information about the demography of the dogs (age, breed, sex, purpose of keeping the dogs, source of the dogs and management type). information about the number of rabies vaccinations received, type and source of given vaccine and the last vaccination date as well as the awareness of the owners on rabies was also assessed. data analysis data obtained from the questionnaire were analysed using descriptive statistics and chi square. the mean optimal density was analysed with 74 veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 rabies awareness among dog owners and detection of antibody levels against rabies in dogs ishola et al. to inadequate immune response despite high (82.6%) level of vaccination. this phenomenon could be responsible for the significant occurrence of rabies infection in dogs and subsequently in humans despite the vaccination coverage of over 70% as recommended by who for the control of rabies transmission (who  2019). the low antibody level in the vaccinated dogs may be due to vaccine failure arising from poor cold-chain storage that leads to poor vaccine quality (dairo and osizimete 2016). in a study of factors influencing the antibody response of dogs vaccinated against rabies in the uk, kennedy and colleagues (kennedy et  al. 2007) observed considerably high failure rates for different vaccines tested. furthermore, vaccination of diseased or infected dogs coupled with non administration of booster doses, as at when recommended, could be contributory factors to the low seroconversion in vaccinated animals (olugasa et  al. 2011). the low level of antibody response could be associated with age, route of vaccination, breed of the dog, haplotype of specific breeds of dogs and sex respondents, 120 (86.9%) were aware of how rabies could be contracted (table ii). discussion although, vaccination of dogs has been shown to be the most effective method of prevention of rabies in humans, cases of human rabies remain a significant problem in many developing countries including nigeria. in this study, only 23.2% of the sampled dog population had the expected protective level of antibodies against rabies. this percentage is higher than that found in confined, hunting and roaming dogs in ogun and oyo states, nigeria (oluwayelu et al. 2015) but lower than that found in non-hunting confined dogs in ogun state, nigeria (awoyomi and ogundipe 2019). the result of this study also agrees with the report of seroconversion in only 32.6% of dogs presented for rabies vaccination at veterinary clinics in lagos state, nigeria, while majority (68.4%) of the dogs had low rabies seroconversion despite the fact that > 90% of the dogs had been vaccinated (ishola et  al. 2019). these indicate a consistently low population of protected animals’ consequent table i. prevalence of rabies antibodies based on sex, age, breed and vaccination status of dogs in abeokuta. variable total sample collected positive (%) negative (%) p value sex male 71 15 (10.9) 56 (40.6) female 67 17 (12.3 ) 50 (36.2) 0.15 total 138 32 (23.2) 106 (76.8) age < 6 months 28 5(3.6) 23(16.7) 6-12 months 13 3(2.2) 10(7.3) 0.80 > 1 year 97 24(17.4) 73(52.8) 0.04* total 138 32 (23.2) 106 (76.8) breed alsatian 63 14 (10.1) 49 (35.5) 0.02* boerboel 19 3 (2.2) 16 (11.6) 0.14 mastiff 13 4 (2.9) 9 (6.5) 0.27 caucasian 14 3 (2.2) 11 (7.9) 0.21 rottweiler 10 3 (2.2) 7 (5.07) others 19 5(3.6) 14(10.1) 0.07 total 138 32 (23.2) 106 (76.8) dose of rabies vaccine single dose 26 6 (4.4) 20 (14.5) 0.89 multiple dose 88 21 (15.2) 67 (48.6) 0.07 unvaccinated 24 5 (3.6) 19 (13.7) total 138 32 (23.2) 106 (76.8) * significant at p <0.05 table ii. demographics and awareness of dog owners on rabies and related epidemiological data on dog vaccination at selected veterinary clinics in abeokuta, ogun state, nigeria. variable/category frequency (n) percentage (%) purpose of keeping dogs pets 17 12.3 guards 121 87.7 total 138 100.0 source of dogs local 7 5.1 bought 113 81.9 gift 18 13.0 total 138 100.0 vaccination status vaccinated 114 82.6 unvaccinated 24 17.4 total 138 100.0 source of vaccine used usa 103 90.3 korea 6 5.3 france 3 2.6 czech 1 0.9 nigeria 1 0.9 total 114 100.0 awareness of dog owners on rabies transmission yes 120 86.9 no 18 13.1 total 138 100.0 75 ishola et al. rabies awareness among dog owners and detection of antibody levels against rabies in dogs veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 and awoyomi and ogundipe (gunatilake et al. 2003, awoyomi and ogundipe 2019); it may be due to poor immunological development in the young dogs. mansfield and colleagues (mansfield et  al. 2004) reported that dogs less than a year old had a slightly higher probability of responding poorly than those more than one year old. kennedy and colleagues (kennedy et  al. 2007) similarly observed that dogs less than one year of age elicited lower antibody response than adults. interference of early postnatal vaccination before the recommended period in dogs possessing maternal immunity may be partly responsible for the poor seroconversion in the vaccinated puppies (ohore et al. 2007). the mean antibody titre of dogs over one year of age which was significantly higher than those of less than six months of age could therefore be as a result of immature immune system and/or maternal antibodies interference in the young dogs. this may be evidence of impaired serocoversion to rabies vaccine among puppies less than 3 months of age, as earlier documented (morters et  al. 2015). the higher prevalence of seropositivity observed in the female dogs may be due to better care received by females kept for breeding purpose in order to confer a greater maternal immunologic response on the puppies (ohore et  al. 2007). anecdotal evidence shows that dog owners who are aware of rabies, more often ensure rabies vaccination of their dogs prior to breeding for the protection of the puppies. the significantly higher (p < 0.05) number of alsatian breed seropositive dogs compared to the rottweiler may be due to acquired innate properties that confer a higher immune response and a more lasting immune-protection than in rottweiler. this finding supports the observations of kennedy and colleagues (kennedy et  al. 2007) that larger breeds of dog show lower immune response than smaller breeds. while the precise factors responsible for this appears unclear, the phenomenon confers a shorter immune-protection in the larger breed rottweiler compared to other smaller dog breeds (kennedy et  al. 2007). dogs which had multiple (booster) doses of the vaccine however, had higher antibody levels and prevalence 21 (15.2%) compared to dogs that had received a single (primer) dose. this is expected as most dogs achieve adequate amnestic seroconversion after booster vaccination especially when the vaccines are highly immunogenic. it is supposed that immunization of over 70% of dog population in rabies endemic regions prevent transmission of the disease (who 2019). it is worrisome however that the low seroconversion rate of only 23.2% found in 76.3% of the 114 vaccinated dogs investigated (which represented 82.6% of the total population sampled) would portend failure in arresting rabies transmission within the dog population and consequently, human/public health hormones (coyne et  al. 2001, mansfield et  al. 2004, kennedy et  al. 2007). this low level of antibody might thus be due to the type of vaccine used and mode of vaccine storage which is mostly challenging in the country due to electric power fluctuations (ohore et  al. 2007) since most of the rabies vaccines given to these dogs were imported. this study showed that majority of the vaccines were imported from usa and korea while few were from france and czech. only 0.9% of the vaccines used was made in nigeria. the findings of this study agree with awoyomi and ogundipe (awoyomi and ogundipe 2019) who reported that apart from the rabies vaccines produced by nvri in nigeria, rabies vaccines are imported from other countries of the world since the local production by the nvri could not meet up with the demand of the country. ishola and colleagues (ishola et  al. 2019) reported that more than two-thirds (77.6%) of dogs vaccinated against rabies at veterinary clinics in lagos state, nigeria were vaccinated with imported rabies vaccines, only few (13.2%) were vaccinated with the local nvri rabies vaccine; majority (51.3%) of these imported rabies vaccines originated from asia. the absence of significant difference in seroconversion with the types of vaccines used found in this study remains consistent with earlier report of ohore and colleagues (ohore et  al. 2007) who observed that there was no significant difference between the various types of rabies vaccines given. they concluded that there was a relative uniform but low potency among the commonly available rabies vaccines in use in nigeria (ohore et al. 2007). incorrect or sub-optimal doses of vaccine used, inability of some dogs to seroconvert even with repeated doses of vaccine and poor immunogenicity of the vaccine, probably due to poor storage conditions or break in the cold chain could cause low immunogenicity of the vaccines (ohore et  al. 2007, awoyomi and ogundipe 2019). the success of efforts against vaccine-preventable diseases is attributable in part to proper storage and handling of vaccines; vaccines exposed to temperatures outside the recommended ranges could have reduced potency and protection (cdc 2019). vaccines have to be protected from heat and light and should be stored as recommended by manufacturers and national immunization program. irreversible and permanent loss of vaccine potency may occur by exposure to heat and/or light. nearly all the inactivated and live attenuated vaccines require refrigerator storage temperatures between 35  °f to 46  °f (2  °c to 8  °c), with a desired average temperature of 40  °f (5  °c) (arsalan 2014). vaccine quality is maintained using a cold chain that meets specific temperature requirements. the findings of young dogs of less than 6 months of age showing significantly lower seroconversion than adults agree with gunatilake and colleagues 76 veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 rabies awareness among dog owners and detection of antibody levels against rabies in dogs ishola et al. that the antibody levels against rabies among the entire dog population in abeokuta, ogun state could even be lower. there is therefore an urgent need for more effective and efficient strategy for rabies vaccination that would attain 70% to 80% of dog population coverage. a radical approach to rabies vaccination programmes is therefore necessary, as there should be sustained increase in mass vaccination. vaccinations should involve the use of potent rabies vaccines that are handled and stored under cold-chain. more importantly, the antibody responses of dogs to rabies vaccination should be routinely monitored through consistent sero-profiling at a regular post-vaccination interval to ensure successful vaccination programmes. conclusion dog owners in abeokuta had high awareness level of rabies. dogs vaccinated against rabies had low seroconversion while unvaccinated dogs had prevalence of rabies antibodies; these portend public health risk. there is therefore a critical need for more effective and efficient strategy for rabies vaccination that would attain 70% to 80% of dog population coverage with potent vaccine. there is a need for enforcement of compulsory pre-exposure vaccination compliance and annual booster vaccinations using properly stored immunogenic vaccines against rabies in dogs as part of responsible dog ownership. animal health authorities should conduct regular rabies sero-profiling of dogs, both those presented and vaccinated at veterinary hospitals and the general dog population in abeokuta, ogun state. these interventions should be given urgent attention in order to achieve the goal of zero rabies prevalence by the year 2030. acknowledgements we thank the management of the nigeria security and civil defence corps, the veterinary teaching hospital of the federal university of agriculture, abeokuta, and veterinary hospital, ministry of agriculture and natural resources, abeokuta ogun state for their support during the study. hazard in rabies epidemiology in this ecological region. while this study is limited to dogs presented to veterinary clinics, the overall consequence when the total dog population is considered could be worse if rural (less cared for) dogs are included. this strongly identifies this phenomenon as a challenge in the control of rabies in ogun state, nigeria. the higher presence of alsatian breed among the screened dogs is attributed to the great popularity of this breed and the common use of these dogs for security guard purposes as indicated by owners. the high percentage of the dog owners indicating knowledge of how rabies could be transmitted, suggests considerable high level of awareness of the disease. this could have contributed to the high percentage of the vaccinated dogs as observed in the study in abeokuta metropolis, ogun state, nigeria. the same may not be the case with the dog population (mainly local breeds) in rural areas whose management is mainly free-roaming and where veterinary services are less available (oluwayelu et al. 2015). analysis of the post vaccination period in this study showed no significant difference in the antibody levels although there was a slightly higher prevalence at 6-12 months period. it is noteworthy that 3.6% of screened dogs that were unvaccinated showed seroconversion. while this could be due to passive maternal antibody in some of these unvaccinated dogs that were young puppies of less than 6 months of age, there should be caution with regards to occurrence of antibody against rabies in unvaccinated adult dogs. since this may indicate field exposure and possible latent infection in such dogs, as rabies virus has been recovered from apparently healthy dogs (aghomo et al. 1986). taiwo and colleagues (taiwo et al. 1998) had reported 4 out of 11 puppies less than 3  months of age, that died of rabies as confirmed by histopathology. a similar finding was reported earlier (ezebuiro et  al. 1980). these findings are of public health significance. despite the sampling limited to veterinary hospital visiting dogs only, which represent dogs that are receiving better veterinary health care and attention from their owners, the low antibody levels against rabies observed in this study suggest 77 ishola et al. rabies awareness among dog owners and detection of antibody levels against rabies in dogs veterinaria italiana 2021, 57 (1), 71-77. doi: 10.12834/vetit.1787.9429.2 aghomo h.o., oduye o.o. & bobade p.a. 1986. occurrence of rabies virus antibodies in unvaccinated dogs in ibadan. afr j clin microbiol, 1, 119-122. barecha c.b., girzaw f., kandi v. & pal m. 2017. epidemiology and public health significance of rabies. persp med res, 5, 55-67. dairo d.m. & osizimete o.e. 2016. factors affecting vaccine handling and storage practices among immunization service providers in ibadan, oyo state, nigeria. afr health sci, 16, 576-583. ezebuiro e.o., fagbami a.h. & anosa v.o. 1980. a survey of rabies in man and animals in nigeria. federal livestock department, lagos, 142 pp. fooks a., horton d., muller t., freuling c. & rupprecht c. 2012. rabies. in manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds, bees). 7th ed. paris, office international des epizooties, 263-282. hurisa b., mengesha a., lemma h., nigussie d., godana a., adhanom a., kebeda g., kerga s., newayesilassie b., gebrewold g., bankovisky g., metlin a., kebede a. & urga k. 2015. antibody response of dogs to ethiorab rabies vaccine. int j vaccines vaccin, 1 (2), 00008. kennedy l.j., lunt m., barnes a., mcelhinney l., fooks a.r., baxter d.n. & ollier w.e. 2007. factors influencing 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dis poverty, 4, 3. 103 veterinaria italiana 2022, 58 (1), 103-109. doi: 10.12834/vetit.2124.12936.1 accepted: 10.09.2020 | available on line: 16.11.2022 1university of padova, via 8 febbraio 2, 35122 padova, italy. 2istituto zooprofilattico sperimentale delle venezie, viale dell’università 10, 35020 legnaro (pd), italy. *corresponding author at: university of padova, via 8 febbraio 2, 35122 padova, italy. e‑mail: michele.berlanda@unipd.it. michele berlanda1, carlotta valente1, carlo guglielmini1, patrizia danesi2, barbara contiero1 and helen poser1 keywords dermatology, dog, malassezia, yeast count, skin disease. summary the present study describes malassezia populations in clinically healthy dogs (hd) and dogs with malassezia overgrowth (mo), and evaluates the correlation with clinical signs and previous treatments. thirteen clinically hd and 84 dogs with mo were enrolled. clinical history and previous treatments were recorded. after a complete physical and dermatological examination, canine atopic dermatitis extent and severity index_03 scores were calculated. samples for cytology and mycological cultures were obtained from four body regions and from skin lesions. malassezia overgrowth was diagnosed by cytology. a global score (gs) for quantitative evaluation of the population of malassezia was calculated. in dogs with mo, the highest frequency of yeast detection was found in skin lesions (82%, p < 0.001). sum of gs (gss) obtained from dogs with mo (68, 0-621) was significantly higher compared to those of hd (3, 0-48, p < 0.001). gss in dogs previously treated with antibiotics (312.5, 30-975) was significantly higher compared to those of dogs that not have received antibiotics (80, 0-975, p = 0.015). no difference was found between dogs treated and those not treated with steroids. malassezia overgrowth in dogs in northern italy: frequency, body distribution, clinical signs and effects of pharmacologic treatments treatment (bond and loyd 1997). pruritus is a major clinical sign of malassezia dermatitis. other symptoms include erythema, alopecia, greasy exudation, and scaling. hyperpigmentation and lichenification are usually observed in chronic cases. frequently, affected body regions include lips, ear canals, ventral neck, axillae, groin, interdigital webs, perianal area, and intertriginous regions (miller et al. 2012). the exact pathogenesis of skin inflammation caused by m.  pachydermatis is still unclear. a key role seems to be played by phospholipase production (coutinho and paula 2000, teramoto et al. 2015) and by hypersensitivity against yeast antigens (bond et  al. 2002, bond et  al. 2006a, bond et  al. 2006b, khosravi et  al. 2007, layne and deboer 2016). many skin disorders, especially atopic dermatitis, keratinization defects, recurrent bacterial pyoderma, and endocrine diseases alter sebum production and cause a disruption of the epidermal barrier, favouring yeast proliferation (bond and ferguson 1996). it has also been proposed by some authors that long-term introduction malassezia sp. yeasts are normal inhabitants of the mammalian skin surface and are usually considered opportunistic pathogens (guillot and bond 1999). the taxonomic classification divided the genus malassezia into 18 species (gupta et  al. 2004, batra et  al. 2005, cabañes et  al. 2007, castellà et  al. 2014, theelen et  al. 2018, lorch et  al. 2018). all species within the genus are non-mycelial, unipolar, and budding. furthermore, all species except malassezia pachydermatis are lipid-dependent due to their inability to synthesize some fatty acids and their requirements for lipid supplementation for in vitro growth. m.  pachydermatis is the most common species of this genus in dogs and is frequently isolated from ear canals, skin, and mucosal surfaces (oral and anal), and less commonly from the anal sacs and vagina (guillot and bond 1999, bond et al. 1995). malassezia dermatitis is a term used to describe skin diseases associated with malassezia overgrowth (mo) in affected regions that show a good clinical and cytological response to appropriate antifungal 104 veterinaria italiana 2022, 58 (1), 103-109. doi: 10.12834/vetit.2124.12936.1 malassezia overgrowth in dogs berlanda et al. in sterile saline (0.9%) solution on a skin area of approximately 1 cm2 in all the above-mentioned cutaneous sites. cytology each tape-strip was stained with modified diff-quick® method skipping the first step of alcohol fixation, and examined microscopically at 40x magnification. ear samples were fixed and stained with the diff-quick® method and examined as skin specimens. yeasts were identified according to their typical morphology (cell shape, size, and budding pattern). the number of yeasts was assessed in 5  random fields at 40x magnification (cafarchia et  al. 2005), and number of yeasts per field was graded as follows: 0, no yeasts found; a, 1-5 yeasts; b, 6-10  yeasts; c, 11-50 yeasts; d, 51-100 yeasts; and e,  >  100 yeasts. malassezia overgrowth was diagnosed when more than 2 and 10 yeasts were counted in 5 random fields at 40x magnification in skin and ear cytology, respectively (mauldin et  al. 1997, bond et al. 1993). mycological culture swabs were preserved not more than 24 hours at 4  °c before mycological cultures were performed. swabs were plated directly onto petri dishes containing sabouraud dextrose agar (sda) and modified dixon’s agar (da) medium and incubated at 32 °c for 15 days. the positivity threshold was set at 70 ufc. identification colonies were identified as malassezia based on microscopic and macroscopic morphology and were suspected to belong to the non-lipid dependent m.  pachydermatis species when grown on the sda medium. identification was confirmed by pcr and sequencing of the large-subunit (26s) ribosomal dna gene as previously reported (kurtzman and robnett 1997). statistical analysis the statistical analyses were carried out using the statistical analysis system version 9.0 (sas inst. inc., cary, nc, usa). distribution of data was assessed by use of the shapiro-wilk’s normality test. normally and non-normally distributed data were reported as mean ± sd and median (range), respectively. frequencies of the detection from the considered body regions were reported as a percentage and compared using the chi-square test. due to the non-normal distribution of the yeast population sizes, a global score (gs) was calculated by summing administration of glucocorticoids or antibiotics may increase malassezia populations (bond and ferguson 1996, ihrke et  al. 1993, mauldin et  al. 1997). a significant effect from prednisone and cyclosporine on cutaneous malassezia populations in dogs with atopic dermatitis has recently been excluded (widmer et  al. 2018). lastly, studies providing evidence on the effect of antibiotic treatments on the malassezia population are lacking in dogs. the present study describes the frequency of detection and body distribution of malassezia yeasts in dogs in italy. we also examined the clinical signs, grade of lesions, and predisposing factors of malassezia dermatitis with particular attention on the effects of previous pharmacological treatments. materials and methods study design dogs with skin and ear diseases and cytological evidence of mo were prospectively enrolled in a veterinary teaching hospital (vth) in northern italy for a period of three years. a control group of healthy dogs (hd) was also recruited, including dogs in good general health that were presented for routine visits or vaccinations. these animals were free of clinical signs and had no history of skin and ear diseases. moreover, these dogs had not received any medication during the previous three months. clinical examination clinical history was collected from all dogs with a special focus on therapies in the previous three months. in addition, to complete physical and dermatological examination, the canine atopic dermatitis extent and severity index-03 score (cadesi-03) was calculated in dogs with mo (olivry et  al. 2007, machado et  al. 2011). client’s informed consent was obtained for each dog before examination. sampling procedures samples were obtained from all dogs from axilla, ventral interdigital webs of the forelimb, ear pinna, and ear canal of the right side. furthermore, in animals with dermatitis or otitis, additional samples were collected from specific lesion. samples for cytologic examination were collected from the skin with a tape-strip technique (bond et  al. 1994) and from the ear canal using cotton swabs rolled on a glass slide. samples for mycological cultures were collected by rubbing a sterile swab moistened 105veterinaria italiana 2022, 58 (1), 103-109. doi: 10.12834/vetit.2124.12936.1 berlanda et al. malassezia overgrowth in dogs and in five sampling sites in dogs with mo (n = 84). in the hd group, malassezia yeasts were detected by cytology in 8/13 dogs (62%) without any significant difference (chi-square  =  4.42, p  =  0.22) in the frequency of yeasts among the four anatomical areas. in dogs with mo, the greatest frequency of detection of malassezia spp. was recorded from the cutaneous lesions (82%, p < 0.001). the frequency of yeasts was significantly higher on the ear canal compared to the axilla (43% vs 23%, p < 0.001), while interdigital webs and ear pinna showed intermediate values (33% and 26%, respectively; p < 0.001). the comparison of the frequencies of cytological detection of malassezia species in different affected regions among mo and hd showed a tendency for significant differences regarding ear canal and ear pinna (43% vs 15%, chi-square = 3.6 p = 0.05; and 26% vs 54% chi-square = 4.1 p = 0.05, respectively) (table ii). global score (gs) although the gs was not available in 53.6% (45/84) of dogs with mo, the gss obtained from four sampled anatomical regions in hd (3, 0-48) were significantly lower compared to those of 39/84 dogs with mo (68, 0-612, p < 0.001) (table iii). in dogs with mo, the gs (312.5, 30-975) of the 12 dogs treated with systemic antibiotics in the previous three months was significantly higher compared to that of the 27 not treated dogs (80, 0-975, p = 0.015) (figure 1). the gs was not significantly affected by treatment with systemic or topical steroids (p = 0.35) (figure 2). the corresponding scores of various sampled regions. in particular, the score of the sampled regions was calculated using the following formula: score = 3*number of microscopic fields a + 8* number of microscopic fields b + 30*number of microscopic fields c + 75*n° microscopic fields d + 100*number of microscopic fields e. the coefficients were determined considering the median of the number of yeasts per field per each grade, except for the e grade, as all fields were considered having 100 yeasts per field. the association between the the sum of gs (gss) of malassezia and the severity of skin lesions (cadesi-03 scores) among the dogs with mo was also evaluated by use of the spearman’s correlation index. the effect of pharmacological therapies (systemic antibiotics and steroids in the previous three months of examination) on non-normally distributed data was tested with the mann-whitney test. normally distributed data were analysed using an analysis of variance (anova). the statistical models included the fixed effects of group of dogs (healthy dogs vs dogs with mo), sex, clinical problem (pruritus, multifocal alopecia, diffuse alopecia, and otitis externa), and treatments (topical and systemic corticosteroid, topical, and systemic antibiotic). a value of p ≤ 0.05 was considered to be statistically significant. results population and clinical data a total number of 240 dogs with skin problems was presented at the vth in the enrolment period. among these, 97 dogs fitted the inclusion criteria and were enrolled in the study. they were grouped as follows. 1. healthy dogs (hd) (n = 13). 2. dogs with mo (n = 84). the population included 52 females (2 hd and 50 with mo) and 45 males (11 hd and 34 with mo). all animals lived in northern italy. the median age at first examination of hd and dogs with mo was 58 months (3-132 months) and 71 months (4-192 months), respectively. in dogs with mo, 31 received systemic antibiotics (11 cefalexin, 8  amoxicillin + clavulanic acid, 3 benzylpenicillin + dihydrostreptomycin, 1 enrofloxacin, 1 clindamycin and 1 enrofloxacin + metronidazole, 6 not specified) in the previous three months. table i summarizes the list of presenting complaint in dogs with mo. frequency of cytological detection table ii summarizes the results of detection of malassezia spp. in four sampling sites in hd (n = 13) table i. main presenting complaints (%) in 84 dogs with malassezia overgrowth. problem % of cases* pruritus 73%a otitis externa 18%b diffuse alopecia 5%b multifocal alopecia 5%b *values with different letters along column are significantly different (chi square = 80.0; p < 0.001). table ii. cytological detection of malassezia spp. yeasts in four and five sampling sites in 13 clinically healthy dogs (hd) and 84 dogs with malassezia overgrowth (mo), respectively. sampling site hd° dogs with mo* p ear canal (%) 15% 43%b 0.05 interdigital webs (%) 38% 33%bc 0.72 ear pinna (%) 54% 26%bc 0.05 axilla (%) 31% 23%c 0.52 skin lesions (%) 82%a °chi square = 4.42, p = 0.22; *values with different letters along column are significantly different (chi square = 80.0; p < 0.001). 106 veterinaria italiana 2022, 58 (1), 103-109. doi: 10.12834/vetit.2124.12936.1 malassezia overgrowth in dogs berlanda et al. lesion scores in 39/84 dogs with mo, the cadesi-03 score was 18 (0-43). no significant difference was found in cadesi-03 score between male and female (p  =  0.163). neither antibiotics nor corticosteroids significantly influenced the cadesi-03 score. no correlation (r = 0.48; p = 0.0021) was found between cadesi-03 scores and modified gss. since the cadesi-03 score does not consider ear canals, the gs of this area was not considered in this single analysis. culture and identification of the malassezia yeasts from 142 swabs taken from 52 animals (39 with mo and 13 hd), 26 samples (18.3%) were considered positive. all isolates were able to grow on sda (at 32  °c) and were identified as non-lipid dependent malassezia yeasts. sequencing of the 26s rdna gene confirmed malassezia pachydermatis in all isolates. 1000 900 800 700 600 500 400 300 no antibiotics antibiotics 200 100 0 -100 g ss figure 1. box plot showing the sum of global score (gss) of 12 and 27 dogs that received (antibiotics) and did not receive (no antibiotics) systemic antibiotic therapy in the previous three months before examination. boxes represent the interquartile range (25th to 75th percentile). the horizontal line in each box represents the median. whiskers represent the 5th and 95th percentiles. outliers are plotted separately as circles. values are significantly different, p = 0.015. table iii. global scores (gs) of the four and five sampling sites in 13 clinically healthy dogs (hd) and 39 dogs with malassezia overgrowth (mo), respectively. data are reported as median (range). sampling site hd mo p ear canal 0 (0-30) 0 (0-475) 0.132 interdigital webs 0 (0-3) 0 (0-500) 0.196 ear pinna 0 (0-39) 0 (0-500) 0.748 axilla 0 (0-39) 0 (0-35) 0.525 skin lesions 12 (0-475) sum of gs* 3 (0-48) 68 (0-621) < 0.001 *without gs of lesions. genbank® (http://www.ncbi.nlm.nih.gov/genbank/) accession numbers were mn198166, mn198167, mn198171, mn198176, mn198172, mn198173, mn198174, mn198175, mn198177, mn198179, mn198170, mn198168, mn198178 and mn198169. discussion the main result of the present study is the observed increased number of malassezia yeast in a canine population treated with systemic antibiotic therapy. therefore, this is the first time that the effect of antibiotic treatments on the number of cutaneous malassezia yeasts is evidenced by a clinical study. the gs and then the number of malassezia yeasts in dogs that had received antibiotics were indeed significantly higher compared to those of non-treated dogs. in fact, the association between malassezia dermatitis and previous antibiotic therapy has been reported in the canine literature without clinical evidence (miller et  al. 2012). a recent study evidenced that the skin microbiota can be influenced by topical antimicrobial therapy, but the effect of systemic antibiotic treatment was not considered (chermaprai et  al. 2019). there are at least two possible explanations for our results. first, the changes in the skin microbiota following antibiotic treatment may predispose it to malassezia overgrowth giving rise to the thought that staphylococcus and malassezia species might have a symbiotic relationship. second, there may be some common predisposing factors that can favour both the secondary bacterial 1000 900 800 700 600 500 400 300 steroids no steroids 200 100 0 -100 g ss figure 2. box plot shows the sum of global score (gss) of 10 and 27 dogs that receive (steroids) and did not receive (no steroids) systemic steroid therapy during the previous three months before examination. the horizontal line in each box represents the median. boxes represent the interquartile range (25th to 75th percentile). whiskers represent the 5th and 95th percentiles. outliers are plotted separately as circles. values are not significantly different, p = 0.35. 107veterinaria italiana 2022, 58 (1), 103-109. doi: 10.12834/vetit.2124.12936.1 berlanda et al. malassezia overgrowth in dogs differences or correlations were found in the evaluation of cadesi-03 scores (olivry et  al. 2007, olivry et  al. 2008). however, it has to be said that cadesi-03 scores were developed and validated only for canine atopic dermatitis and may not be valid for malassezia dermatitis. gs also has not been validated. this could represent a weakness of the present study. it is generally accepted that mo is secondary to hypersensitivity disorders, endocrine diseases, and defects of keratinization (miller et al. 2012). therefore, in the present study only a part of the dogs could have had hypersensitivity disorder and, in particular, atopic dermatitis. this could also represent a study limitation, as already highlighted in a previously published study that uses the cadesi-03 scores for evaluating the difference between dogs with malassezia dermatitis and dogs with cutaneous lesions but without evidence of malassezia dermatitis (machado et  al. 2011). moreover, the present study is based on the count of yeasts, which is probably a relevant factor, but does not consider that some strains could express different virulence factors such as phospholipase productions (coutinho et al. 2000, teramoto et al. 2015, buommino et al. 2016, cafarchia and otranto 2004), or that the susceptibility to this organism could differ between hosts (e.g. malassezia hypersensitivity) (bond et al. 2006a, bond et al. 2006b). in both cases, clinical signs of malassezia dermatitis would likely develop also with low numbers of yeasts. finally, secondary bacterial infections that may have influenced the cadesi score should have been considered. this represents, as mentioned before, another study limitation. in conclusion, the present study provides helpful insights into the frequency of detection and body distribution of malassezia in healthy dogs and in dogs with skin disease. in particular, our results highlight the importance of the number of yeasts resulting from the skin cytology for the diagnosis of mo, even though the different pathogenicity of the involved yeast strains needs also to be considered. previous antibiotic treatments may represent a predisposing factor for the increased number of malassezia yeast supporting what has previously been assumed but without clinical evidence. however, further studies are necessary to confirm this hypothesis, also considering the potential effect of other factors not evaluated in the present study such as the concurrent predisposition to both mo and bacterial infections. infections, which justify the previous antibiotic treatments, and the overgrowth of malassezia. some changes in the skin barrier and immunological status have demonstrated a predisposition to bacterial and yeast overgrowth in dogs with atopic dermatitis (santoro et al. 2015). in the present study, 61 (73%) dogs with malassezia overgrowth were examined for pruritus with a presumptive diagnosis of allergic dermatitis, confirming the findings of other investigations concerning this association (machado et  al. 2011). therefore, these subjects could have been predisposed to skin bacterial secondary infections and to malassezia overgrowth. many patients of the present study were referred by other practices and so the lack of information about prior bacterial infections was consequently a limitation of the present study. further studies are necessary to understand whether there may be an altered competition with the resident microbial flora or if other mechanisms, not considered in the present study, can explain the presence of a greater number of malassezia in dogs treated with antibiotics (plant et  al. 1992, bond and ferguson 1996). in contrast to the effect of antibiotic therapy, glucocorticoids treatment showed no significant influence on gs. recently, widmer and colleagues (widmer et  al. 2018) have shown no significant impact of prednisone on canine cutaneous microbiota. thus, previous steroid therapy does not seem to affect the composition of the canine skin flora. the cytological examination revealed that the number of yeasts on five random fields can differ considerably in areas within the same slide. for this reason, we studied the global score (gs), which permits to overtake the effect of this variability and enhance the importance of fields with a high number of yeasts. as expected and reported in previous studies, the yeast population size (expressed by the gs) differed significantly between the healthy and diseased animals (crespo et al. 2002, nardoni et  al. 2004, yurayart et  al. 2011, cafarchia et  al. 2005). furthermore, in the present study, the frequency of yeast detection from dogs with skin disease was significantly higher when compared to that of clinically hd; this was also evident from areas not interested by lesions, although the frequency of yeast detection was higher in areas with skin lesions, as previously reported (bond and lloyd 1997, maachado et  al. 2011, nardoni et  al. 2004, yurayart et  al. 2011). despite the differences in gs and in the frequency of yeast detection, no significant 108 veterinaria italiana 2022, 58 (1), 103-109. doi: 10.12834/vetit.2124.12936.1 malassezia overgrowth in dogs berlanda et al. batra r., boekhout t., guého e., cabañes f.j., dawson t.l. jr & gupta a.k. 2005. malassezia baillon, emerging clinical yeasts. fems yeast res, 5, 1101-1113. bond r. & ferguson e. 1996. factors associated with 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associated with canine seborrheic dermatitis and healthy skin. vet microbiol, 148, 356-362. with and prevalence of high malassezia pachydermatis numbers on dog skin. j am vet med assoc, 201, 879-882. santoro d., marsella r., pucheu-haston c.m., eisenschenk m.n., nuttall t. & bizikova p. 2015. review: pathogenesis of canine atopic dermatitis: skin barrier and host-micro-organism interaction. vet dermatol, 26, 84-e25. teramoto h., kumeda y., yokoigawa k., hosomi k., kozaki s., mukamoto m. & kohda t. 2015. genotyping and characterisation of the secretory lipolytic enzymes of malassezia pachydermatis isolates collected from dogs. vet rec open, 21, 1-8. 363 1istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, teramo, italy. 2istituto zooprofilattico sperimentale della puglia e della basilicata, foggia, italy. 3istituto zooprofilattico sperimentale del piemonte, liguria e valle d'aosta, torino, italy. 4istituto zooprofilattico sperimentale del mezzogiorno, portici (na), italy. 5institute of animal health, university of las palmas de gran canaria, veterinary school, arucas, las palmas, spain. 6faculty of veterinary medicine, university of teramo, teramo, italy. *corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: + 39 0861 332420, e-mail: g.difrancesco@izs.it parole chiave brucella ceti, immunoistochimica, neurobrucellosi, stenella coeruleoalba. riassunto i batteri del genere brucella causano la brucellosi, una malattia infettiva comune all’uomo e ai mammiferi terrestri e acquatici. dal 1994 diversi casi d‘infezione da brucella sono stati segnalati nei mammiferi marini in tutto il mondo. i mammiferi marini infetti mostrano reperti lesivi analoghi a quelli osservati nei mammiferi terrestri con presenza di aborti, natimortalità, orchite e neurobrucellosi. se da un lato i dati siero-epidemiologici suggeriscono che l’infezione da brucella spp. è cosmopolita, la rilevazione mediante immunoistochimica di antigeni brucellari nei tessuti di animali infetti è spesso problematica. obiettivo del presente studio è stato quello di valutare, mediante l’impiego di un anticorpo monoclonale nei confronti dell’antigene lps di brucella spp., l’immunoreattività del sistema nervoso centrale (snc) in esemplari di stenella striata (stenella coeruleoalba) b. ceti-infetti affetti da neurobrucellosi. l'anticorpo in questione si è dimostrato capace di riconoscere immunoistochimicamente le brucelle lisce sia nel snc dei succitati animali b. ceti-infetti sia in più tessuti di ruminanti domestici brucella spp.-infetti, essendo stato parimenti caratterizzato mediante elisa e western blotting. in conclusione, i risultati di questo studio hanno rilevanza ai fini sia della diagnosi immunoistochimica sia della definizione patogenetica dell'infezione da b. ceti. indagine immunoistochimica in esemplari di stenella striata (stenella coeruleoalba) brucella ceti-infetti con sintomi di neurobrucellosi keywords brucella ceti, immunohistochemistry, neurobrucellosis, stenella coeruleoalba. summary bacteria of the genus brucella cause brucellosis, an infectious disease common to humans as well as to terrestrial and aquatic mammals. since 1994 several cases of brucella spp. infection have been reported in marine mammals worldwide. while sero-epidemiological data suggest that brucella spp. infection is widespread globally, detecting brucella spp.associated antigens by immunohistochemistry (ihc) in tissues from infected animals is often troublesome. the present study was aimed at investigating, by means of ihc based upon the utilization of an anti-brucella lps monoclonal antibody (mab), the central nervous system (cns) immunoreactivity shown by b. ceti-infected, neurobrucellosis-affected striped dolphins. the aforementioned mab, previously characterized by means of elisa and western blotting techniques, was able to immunohistochemically detect smooth brucellae both within the cns from b. ceti-infected striped dolphins and within a range of tissues from brucella spp.infected domestic ruminants. in conclusion, the results of the present study are of relevance both from the b. ceti infection's diagnostic and pathogenetic standpoints. gabriella di francesco1*, antonio petrini1, anna rita d'angelo1, ludovica di renzo1, mirella luciani1, tiziana di febo1, enzo ruggieri1, antonio petrella2, carla grattarola3, barbara iulini3, osvaldo matteucci1, giuseppe lucifora4, eva sierra5, antonio fernández5, roberto giacominelli stuffler6, clotilde angelucci6, marina baffoni6, giovanni di guardo6 and manuela tittarelli1 immunohistochemical investigations on brucella ceti-infected, neurobrucellosis-affected striped dolphins (stenella coeruleoalba) veterinaria italiana 2019, 55 (4), 363-367. doi: 10.12834/vetit.1920.10224.2 accepted: 30.10.2019 | available on line: 31.12.2019 short communication 364 nick title di francesco et al. veterinaria italiana 2019, 55 (4), 363-367. doi: 10.12834/vetit.1920.10224.2 subsequently enhanced, in recent years, through the inclusion of 9 cases of b. ceti infection in striped dolphins, 8 of which were found stranded between 2012 and 2019 along the italian coastline, while the remaining individual was found beached ashore in canary islands (spain) in 2004. the dolphin tissues were collected during post mortem examination, in tight agreement with the investigation protocols to be performed in the framework of the italian national stranding network (insn) for standard laboratory investigations on stranded cetacean specimens. positive and negative controls were included in each ihc run, with the positive ones being represented by the lung, liver and placental tissues from ovine and bovine fetuses either naturally or experimentally infected by brucella  spp. the brain from a dolphin morbillivirus-infected striped dolphin was additionally used as negative control tissue. further negative controls were represented by tissue sections obtained from the 7 immunohistochemically positive, b.  ceti-infected striped dolphins under study, from which the primary anti-brucella  ab was omitted. more in detail, brucella ihc was carried out using the mab 4b5a against lps brucella diluted 1:10 to 1:100. tissue sections were previously heat treated for antigen retrieval (at 121  °c for 8 minutes) in 0.01 m citrate buffer, ph 6.0. immunoreactions were then visualized by means of a peroxidase technique (envision plus kit, dako at izsam and vectastain elite abc kit standard vector at the faculty of veterinary medicine, university of teramo, italy). the brucella  spp. isolation and identification procedures were performed in accordance with the technique described in the oie manual of diagnostic tests and vaccines (world organisation for animal health 2017). with the only exception of the two individuals in wich b.  ceti infection was diagnosed only by means of biomolecular and ihc techniques (id 1.5, table i). all the tissue samples brucella ceti was first isolated from an aborted bottlenose dolphin (tursiops truncatus) fetus in 1994 (ewalt et  al. 1994) and, since then, several cases of infection have been reported among free-ranging cetaceans worldwide (guzman-verri et al. 2012). the first case of b. ceti infection in the mediterranean was recorded only in 2009 (isidoro-ayza et al. 2014), with the first case of b. ceti infection having been recorded along the italian coastline in 2012 (alba et  al. 2013). besides the “classical” species, some recently discovered brucella species have also demonstrated a zoonotic potential, as in the case of b. ceti (whatmore et  al. 2008, de massis et  al. 2019). brucella  spp. infection in marine mammals is characterized by a pathogenicity similar to that of terrestrial mammals. in addition, a documented involvement of the central nervous system (cns) in the striped dolphin (stenella coeruleoalba), similarly to what described in the human species, has been reported (guzman-verri et  al. 2012), with neurobrucellosis having not been recorded in bovine, caprine, ovine, swine, or canine hosts. nevertheless, this syndrome is a relatively common feature in non-treated human brucellosis-affected patients (obiako et  al. 2010). therefore, cetacean neurobrucellosis may serve as an interesting comparative neuropathology and neuropathogenesis model to understand how the bacterium is capable to cross the blood-brain barrier, thereby giving rise to host’s cns invasion. while sero-epidemiological data suggest that brucella  infection is widespread globally (nymo et  al. 2011), detecting brucella  spp.-associated antigens by immunohistochemistry (ihc) in tissues from naturally or experimentally infected animals is often troublesome the present study was aimed at investigating, by means of an anti-brucella  lps monoclonal antibody (mab), the cns immunoreactivity (ir) shown by b. ceti-infected, neurobrucellosis-affected striped dolphins, along with its comparative evaluation in a range of fetal tissues from b.  abortusand b.  melitensisinfected ruminants. a mab raised against brucella  lps was produced at istituto zooprofilattico sperimentale dell’abruzzo e molise ‘g. caporale’ (izsam) and characterized by western blotting (wb) and indirect elisa according to di febo and colleagues (di febo et  al. 2012) and portanti and colleagues (portanti et al. 2006), being subsequently characterized against b.  abortus rb51, b.  pinnipedialis and b.  ceti, which were not tested in the past experiments. samples of lung, liver and placental tissues from 16 ovine fetuses originating from 15 ewes experimentally infected with b.  melitensis biotype 3, along with samples of lung, liver and placental tissues from 6 additional aborted fetuses carried by sheep belonging to brucella-free flocks, were preliminarly investigated, 20 years ago, against brucella  spp. the study was table i. results of brucella ceti. ihc in the cns from infected striped dolphins (stenella coeruleoalba). id ihc(izsam) ihc (unite) bovine fetal lung tissue (positive control) 1_430 es 2004 positive positive positive 2_3479 it 2012 negative negative positive 3_4555 it 2012 positive positive positive 4_5566it 2014 negative negative positive 5_16769it 2017 positive positive positive 6_346it 2017 positive positive positive 7_202it 2018 positive positive positive 8_47465it 2018 positive positive positive 9_2785it2019 positive positive positive 365 di francesco et al. nick title veterinaria italiana 2019, 55 (4), 363-367. doi: 10.12834/vetit.1920.10224.2 the different neuro-topographical concentrations of b.  ceti organisms, not coincident with that of the microscopic field under study. an additional factor to be considered refers to the experimental conditions used in a portion of this work, that are counterparted by the ‘field conditions’ under which post mortem examinations are routinely carried out on stranded cetacean specimens, including also the adverse effects exerted by post mortem autolysis. based upon the herein presented results, brucella spp. ihc should be regarded as a laboratory procedure which is useful not only when analyzing were routinely processed for histopathology and brucella  immunohistochemistry (ihc), whose reliability and reproducibility were also evaluated by means of an ‘inter-laboratory comparison’, which involved two independent pathologists (based at izsam and at faculty of veterinary medicine, university of teramo, italy, respectively) (table i). the results of the characterization of 4b5a mab are shown in table ii (i-elisa) and figure 1 (wb). both i-elisa and wb confirmed that the aforementioned mab reacted with brucella smooth strains, with the typical lps-ladder pattern exhibited in wb analysis. conversely, the same mab did not react with rough brucella  strains (table i). the results of ihc investigations are reported in tables ii and iii. more in detail, brucella  spp.-associated antigens were detected in pulmonary necrotic cell debris as well as in the cytoplasm of both alveolar macrophages and neutrophils from the b. abortus-infected bovine fetuses (figure 2a) as well as in liver cells from the b.  melitensis-infected ovine fetuses under study (figure  2b). within the cns from b.  ceti-infected dolphins, macrophage-like cells were seen harbouring more or less consistent loads of microbial antigen (figure 2, c and d). neither background staining nor artifacts or positive ir were observed in negative control tissues. the results obtained in the present study clearly showed a strong ir against brucella  lps in tissues from all the brucella  spp.-infected, herein investigated bovine, ovine and striped dolphins (7 out 9 individuals) (figure 2, c and d). in this respect, the negative ihc results observed in the cns from 2 b. ceti-infected dolphins may be due either to the low sensitivity of brucella spp. ihc when low bacterial concentrations are present in infected tissues, or to table ii. indirect elisa: cross-reactivities of mab 4b5a anti-brucella lps. bacterial strain mab 4b5a* brucella melitensis biovar 2 positive brucella melitensis biovar 1 16m positive brucella melitensis biovar 1 rev.1 positive brucella abortus strain s19 positive brucella abortus strain s99 positive brucella abortus strain s99 (lps) positive brucella abortus biovar 2 positive brucella abortus biovar 3 positive brucella abortus biovar 6 positive brucella suis biovar 1 positive brucella ovis negative brucella abortus rb51 negative brucella ceti positive brucella pinnipedialis positive table iii. brucella melitensis detection by immunohistochemistry in infected animals. ovine tissues tested ihc pos bovine fetal lung tissue (positive control) fetal lung 26 16 ok controls 6 0 ok fetal liver 26 16 ok controls 6 0 ok total 64 32 figure 1. western blotting of mab 4b5a vs brucella abortus s99 (lane 1), brucella melitensis 16m (lane 2), brucella suis biotype 1 (lane 3), brucella ceti (lane 4), brucella pinnipedialis (lane 5). 1 2 3 4 5kda 260 160 110 80 60 50 40 30 20 15 10 366 nick title di francesco et al. veterinaria italiana 2019, 55 (4), 363-367. doi: 10.12834/vetit.1920.10224.2 health viewpoint, considering the documented zoonotic potential of brucella microorganisms. moreover, mab 4b5a anti brucella lps could represent a diagnostic and research laboratory reagent, whose use may be highly recommended also for the ihc diagnosis and pathogenetic characterization of b. ceti and b. pinnipedialis infections in cetaceans and in pinnipeds, respectively. acknowledgements this work was carried out within the framework of an ad hoc research project on brucella ceti infection, funded by the italian ministry of health and headed/ coordinated by istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ (grant code izs am 03/16 rc). ovine and bovine infected tissues, but also in the case of b.  ceti-infected, neurobrucellosis-affected striped dolphin cns tissue specimens (in which macrophage-like cells were seen harbouring more or less consistent loads of bacterial antigen), providing a method capable of achieving a direct and reliable ihc diagnosis of brucella infection. furthermore, and not less important, the consistent background and the non-specific reactions observed when using an anti-brucella  -polyclonal ab were not seen when mab 4b5a was used. the additional knowledge provided by this study on the detection of brucella infection in cetaceans may be helpful not only from a diagnostic standpoint but also for increasing our awareness on the (neuro)pathogenesis of brucella infection in aquatic mammals and, not less important, also from a public figure 2. brucella spp.-associated antigen positive immunohistochemical labeling in bovine fetal lung (a), in ovine fetal liver (b) as well as in cns (cervical spinal cord) tissues (c, d) from neurobrucellosis-affected, b. ceti-infected striped dolphins. brucella spp. ihc with mab 4b5a, mayer’s hematoxylin counterstain, different magnifications. a b c d 367 di francesco et al. nick title veterinaria italiana 2019, 55 (4), 363-367. doi: 10.12834/vetit.1920.10224.2 alba p., terracciano g., franco a., lorenzetti s., cocumelli c., fichi g., eleni c., zygmunt m.s., cloeckaert a. & battisti a. 2013. the presence of brucella ceti st26 in a striped dolphin (stenella coeruleoalba) with meningoencephalitis from the mediterranean sea. vet microbiol ,164, 158-163. baucheron s., grayon m., zygmunt m.s. & cloeckaert a. 2002. lipopolysaccharide heterogeneity in brucella strains isolated from marine mammals. res microbiol, 153, 277-280. de massis f., zilli k., di donato g., nuvoloni r., pelini s., sacchini l., d’alterio n. & di giannatale e. 2019. distribution of brucella field strains isolated from livestock, wildlife populations, and humans in italy from 2007 to 2015. plos one, https://doi.org/10.1371/ journal.pone.0213689. di febo t., luciani m., portanti o., bonfini b., lelli r. & tittarelli m. 2012. development and evaluation of diagnostic tests for the serological diagnosis of brucellosis in swine. vet it, 48, 145-156. ewalt d.r., payeur j.b., martin b.m., cummins d.r. & miller w.g. 1994. characteristics of a brucella species from a bottlenose dolphin (tursiops truncatus). j vet diagn invest, 6 (4), 448-452. groussaud p., shankster s.j., koylass m.s. & whatmore a.m. 2007. molecular typing divides marine mammal strains references of brucella into at least three groups with distinct host preferences. j med microbiol, 56, 1512-1518. guzmán-verri c., gonzález-barrientos r., hernández-mora g., morales j.a., baquero-calvo e., chaves-olarte e. & moreno e. 2012. brucella ceti and brucellosis in cetaceans. front cell infect microbiol, 2, 3. pmid:22919595. isidoro-ayza m., ruiz-villalobos n., pérez l., guzmán-verri c., muñoz p.m., alegre f., barberán m., chacón-díaz c., olarte e.c., barrientos r.g., moreno e., blasco j.m. & domingo m. 2014. brucella ceti infection in dolphins from the western mediterranean sea. bmc vet res, 10, 206. nymo i.h., tryland m. & godfroid j. 2011. a review of brucella infection in marine mammals, with special emphasis on brucella pinnipedialis in the hooded seal (cystophora cristata). vet res, 42, 93. portanti o., tittarelli m., di febo t., luciani m., mercante m.t., conte a. & lelli r. 2006. development and validation of a competitive elisa kit for the serological diagnosis of ovine, caprine and bovine brucellosis. j vet med b, 53, 494-498. world organisation for animal health (oie) 2019. brucella abortus, b. melitensis and b. suis (infection with b. abortus, b. melitensis and b. suis). in manual of diagnostic tests and vaccines for terrestrial animals, part 3, chapter 3.1.4. (version adopted in may 2016), oie paris, 355-398. 41 introduction mastitis is one of the most frequent and multifactorial disease causing major losses in the dairy industries (halasa et  al. 2007, vliegher et  al. 2012). economic losses mainly depend on early slaughter of cows (injured mammary parenchyma and decreased milk production) commercial devaluation of animals, microbiological diagnosis of pathogenic agent and use of in large amount of antibiotics without regard for resistance (halasa et  al. 2007, ema 2017). the economic losses caused by bovine mastitis in turkey reach to about 28 million dollars (tekeli 2005). streptococcal species are known to be significant causative pathogens of bovine mastitis (facklam 2002, freney et  al. 1992, fortin et  al. 2003, leigh 1999). streptococcal mastitis can be classified according to the source of infectious organisms, either contagious mastitis (streptococcus agalactiae) or environmental mastitis (streptococcus uberis, streptococcus dysgalactia, streptococcus bovis, streptococcus parauberis, streptococcus equi and streptococcus canis) (leigh 1999, oliver et  al. 1998). although s. agalactiae was one of the main pathogens causing mastitis (oliver et al. 1998, keefe 1997), environmental streptococci have consistently been reported as a leading cause of both subclinical and clinical mastitis throughout the world (phuektes et al. 2001, teng et al. 1998). antimicrobial therapy is commonly used for mastitis for more than fifty years but an efficient, safe, and economical treatment is still lacking (guérin-faublée et  al. 2002, hendriksen et  al. 2008). proper use of drug, dairy husbandry, sanitation procedures and the stage of the disease are among the most common reasons for this situation (keefe 1997). thus, the emergence of antibiotic resistance of infectious agent during the department of microbiology, burdur mehmet akif ersoy university, faculty of veterinary medicine, turkey *corresponding author at: department of microbiology, burdur mehmet akif ersoy university, faculty of veterinary medicine, turkey. e‑mail: ozlem‑sahan@hotmail.com. keywords antimicrobial resistance, cattle, lancefield, mastitis, streptococcus sp. summary streptococcal species are known to be responsible for bovine mastitis. the aim of the present study was to determine antimicrobial drug resistance patterns of hemolytic streptococci distributed according to lancefield serogrouping. streptococcus sp. strains were isolated from 124 bovine milk samples from 31 cows with subclinical or clinical mastitis submitted to mehmet akif ersoy university faculty of veterinary medicine, department of microbiology laboratory in burdur province, turkey from january 2015 to january 2017. a total of 63 streptococcus sp. were isolated and the most frequently obtained isolates were classified as lancefield’s serogroup b (84.13%), the remaining isolates as serogroup f (15.87%). out of 63 isolates, 53  (84.13%) showed beta-hemolytic activity whereas 10  (15.87%) alpha-hemolytic activity. antimicrobial resistance was assessed by disk diffusion test against the most common antibiotics used in the field. among the 63 streptococcus  sp. tested, the highest antimicrobial resistance patterns were observed for neomycin (95.24%), trimethoprim sulphamethoxazole (87.30%) and gentamicin (69.84%). none of the isolates showed resistance to amoxicillin-clavulanic acid, except for one serogroup f isolate. the resistance rates for the other antimicrobials ranged from 1.59% to 38.04%. a total of 50 isolates exibited multi-drug resistance to ≥ 3 antimicrobial agents tested. overall, our results suggested that there is an urgent need to enhance awareness among the dairy farmers in choosing the appropriate drug for treating mastitis. özlem şahan yapicier*, ezgi sababoglu, dilek ozturk, hulya turutoglu, faruk pehlivanoglu and mehmet kaya lancefield classification and antimicrobial resistance of hemolytic streptococci isolated from bovine mastitis veterinaria italiana 2021, 57 (1), 41-47. doi: 10.12834/vetit.1855.9879.3 accepted: 26.06.2020 | available on line: 27.07.2021 42 antimicrobial resistence of streptococcus sp. in turkey şahan yapicier et al. veterinaria italiana 2021, 57 (1), 41-47. doi: 10.12834/vetit.1855.9879.3 antimicrobial susceptibility test antimicrobial susceptibility test was carried out by the disk diffusion method on mueller-hinton agar (oxoid ltd, hampshire, uk) supplemented with 5% sheep blood according to the guidelines from clinical and laboratory standards institute (clsi 2017). the following antibiotics commonly used in veterinary medicine were selected: amoxicillin (10  μg; ax, oxoid, uk), amoxicillin clavulanic acid (30  μg; amc, oxoid, uk), cephaperazone (30  μg; cfp, oxoid, uk), cephalexin (30  μg; cl, oxoid, uk), ciprofloxacin (5 μg; cip, oxoid, uk), enrofloxacin (5 μg; enr, oxoid, uk), erythromycin (15  μ; e, oxoid, uk), gentamicin (10  μg; cn, oxoid, uk), lincomycin (10  μg; l, oxoid, uk), neomycin (30 μg; n, oxoid, uk), oxytetracycline (30  μg; ot, oxoid, uk), penicillin (10  units; p, oxoid, uk), trimethoprim sulphamethoxazole (25  μg; ts, oxoid, uk). the results were obtained by measuring the diameter of the growth inhibition zone around the antibiotic disc for each isolated bacteria and recorded as sensitive, intermediate and resistant according to the interpretive standards of clsi and antimicrobials manufacturers’ instructions. isolates displaying resistance to ≥  3 antimicrobial agents tested were defined as exhibiting multi-drug resistance (mdr) (schwarz et al. 2010, tenover 2006). results isolation and identification a total of 63 streptococci were characterised in this study. all isolates were aerobic, catalasi negative gram positivi cocci. they also demonstrated haemolytic activity. beta-hemolysis (84.13%) was observed in 53  of isolates. alpha hemolysis was instead evident in the remaining 10 isolates (15.87%). out of 57 (90.47%) camp-positive isolates, 4 (6.34%) showed alpha and 53  (84.12%) beta hemolysis. six  alfa-haemolitic streptococcus  sp. exhibited an esculin-positive reaction when subcultured on edward’s media. according to serological analysis, of the 63 isolates, 53 (84.13%) were grouped as lancefield’s serogroup b streptococci (gbs) and 10  (15.87%) as lancefield’s serogroup f streptococci  (gfs). camp and esculine hydrolysis results were summarized in table i. antimicrobial resistance among the 63 streptococcus sp. tested, the highest antimicrobial resistance patterns were observed for neomycin (95.24%), trimethoprim sulphamethoxazole (87.30%) and gentamicin (69.84%). except for one gfs strain, none of the isolates showed resistance to amoxicillin-clavulanic acid. the resistance rates for the other antimicrobials bacteriological examination of milk samples is an important basis for the selection of the appropriate chemotherapeutic agents and reduction of the therapeutic failures (schwarz et al. 2010). although streptococci are generally sensitive to beta-lactams and macrolides (hendriksen et  al. 2008, denamiel et  al. 2005, haenni et  al. 2010, kalmus et  al. 2011, tenhagen et  al. 2006), the increasing resistance of streptococci to some antimicrobials as tetracyclines and macrolids with the broad spectrum commonly used in veterinary was reported (aarestrup et  al. 1998, blowey et  al. 1995a, palmieri et  al. 2011, zhang et al. 2008). the main objective of the current study was to determine the phenotypic antimicrobial resistance profiles of the hemolytic streptococci distributed according to lancefield serogrouping. materials and methods bacterial isolates a total of 63 streptococcus isolates from subclinical or clinical bovine mastitis cases in burdur province of turkey were included in the present study. the area is the crossing point of the aegean, central anatolia and mediterranean parts of turkey. the isolates were obtained from 124 milk samples of 31 cows submitted to mehmet akif ersoy university, faculty of veterinary medicine, department of microbiology laboratory in burdur from january 2015 to january 2017. each of streptococci was isolated from an individual mammary gland of cow. isolation and identification the milk samples (10 μl) were streaked on 5% defibrinated sheep blood agar and edward’s media (oxoid, uk). the inoculated plates were incubated at 37 °c for 24-48 h aerobically. suspected colonies were morphologically characterized and examined by gram stain, catalase test, oxidase test, hemolytic activity, camp-reaction and esculin hydrolysis (akan 2006, barrow et al. 1993, quinn et al. 2004). lancefield serogrouping serological grouping of isolates was performed with a commercial latex agglutination kit (strep test kit, plasmatec, uk) for the identification of streptococcal groups a, b, c, d, f and g. streptococci were tested using the broth method described by the manufacturer. isolates indicated the lancefield group f were defined as s. anginosus group (also known as the s. milleri group) (facklam 1977, facklam 2002). 43 şahan yapicier et al. antimicrobial resistence of streptococcus sp. in turkey veterinaria italiana 2021, 57 (1), 41-47. doi: 10.12834/vetit.1855.9879.3 tel et  al. 2009, ekin et  al. 2011, gurturk et  al. 1998, ak 2000, ergun et al. 2004, acar et al. 2012, bal et al. 2010, macun et al. 2011, ikiz et al. 2013). as expected, in our study, gbs (84.12%) were the predominant serogroup and the rate of beta-hemolytic gbs (94.34%) was higher than alpha-hemolytic gbs (5.66%). however, the frequency of the gbs isolation was higher than that reported in the previous studies (keefe 1997, guérin-faublée et  al. 2002, ekin et  al. 2011, akay et al. 1993). as already reported (teixeira et  al. 2003), also in this study gbs beta-hemolytic strains are most commonly identified than the alpha haemolytic ones. to our knowledge the first case of bovine mastitis caused by gfs belonging the s. anginosus group (also known as the s. milleri group). isolates of this group can demonstrate alpha, beta and gamma hemolysis patterns and be both camp and esculine-hydrolysis positive (facklam 1977, facklam 2002, ruoff 1988, spellerberg et  al. 2015). we also found that some isolates have camp, esculine and beta or alpha hemolytic activity. streptococcus anginosus group members have been implicated as etiologic agents in a variety of purulent infections (tissue abscesses, such as brain, dental and hepatic, occasional endocarditis and wound infections), but clinical significance still remains unclear (gossling 1998, whitworth 1990). in this study, a wide diffusion of antibiotic resistance to most of antimicrobials tested was revealed even more concerning was the high prevalence and the mdr exibited by most of the isolated gbs and gfs. the generated data set allowed us to get better were determined in varying rates from 1.59% to 38.04% (table ii). a total of 50 isolates including 40  (80%) gbs and 10 (20%) gfs, were defined as exibiting mdr (table iii). discussion milk production from cattle is 250.000 tons / a year. only 20% of the product is processed in burdur while the remaining 80% in the other provinces of turkey (elmaz et  al. 2010). decrease in milk yield due to mastitis is then a very important problem involving the whole country. staphyloccoccus  sp. was referred to be the most frequent agent isolated in case of bovine mastitis world-wide (turutoglu et  al. 2002, turutoglu et  al. 2006, shitandi et  al. 2004, gianneechini et  al. 2002, savasan et  al. 2017, tel et  al. 2009, minst et  al. 2012). over the last 20 years’ period, the rate of streptococcal mastitis varied from 3.86% to 40% in different provinces of turkey (turutoglu et  al. 2002, table i. camp and esculine hydrolysis results of hemolytic streptococci isolated from bovine mastitis and serogrouped according to lancefield's group. camp test (+) esculine hydrolysis (+) lancefield b, α-hemolytic 3 (4.76 %) 0 lancefield b, β-hemolytic 50 (79.36 %) 0 lancefield f, α-hemolytic 1 (1.58 %) 6 (9.52%) lancefield f, β-hemolytic 3 (4.76 %) 0 total 57 (90.47 %) 6 (9.52 %) table ii. antimicrobial resistance of 63 hemolytic streptococci from bovine mastitis cases. antimicrobials gbs (n = 53) gfs (n = 10) total (n = 63) s n (%) i n (%) r n (%) s n (%) i n (%) r n (%) s n (%) i n (%) r n (%) ot 31 (58.50) 7 (13.20) 15 (28.30) 1 (10) 2 (20) 7 (70) 32 (50.79) 9 (14.29) 22 (34.92) l 34 (64.15) 3 (5.66) 16 (30.19) 7 (70) 0 3 (30) 41 (65.08) 3 (4.76) 19 (30.16) n 3 (5.67) 0 50 (94.33) 0 0 10 (100) 3 (4.76) 0 60 (95.24) cfp 47 (88.68) 4 (7.55) 2 (3.77) 8 (80) 1 (10) 1 (10) 55 (87.30) 5 (7.94) 3 (4.76) ts 3 (5.67) 2 (3.77) 48 (90.56) 2 (20) 1 (10) 7 (70) 5 (7.94) 3 (4.76) 55 (87.30) amc 52 (98.11) 1 (1.89) 0 9 (90) 0 1 (10) 61 (96.82) 1 (1.59) 1 (1.59) enr 22 (41.51) 16 (30.19) 15 (28.30) 4 (40) 1 (10) 5 (50) 26 (41.27) 17 (26.98) 20 (31.75) p 40 (75.48) 2 (3.77) 11 (20.75) 6 (60) 0 4 (40) 46 (73.02) 2 (3.17) 15 (23.81) cn 14 (26.42) 0 39 (73.58) 5 (50) 0 5 (50) 19 (30.16) 0 44 (69.84) ax 41 (77.36) 7 (13.21) 5 (9.43) 6 (60) 0 4 (40) 47 (74.60) 7 (11.11) 9 (14.29) cl 38 (71.69) 7 (13.21) 8 (15.10) 6 (60) 0 4 (40) 44 (69.84) 7 (11.11) 12 (19.05) cip 24 (45.28) 8 (15.10) 21 (39.62) 5 (50) 2 (20) 3 (30) 29 (46.03) 10 (15.88) 24 (38.09) e 35 (66.03) 10 (18.87) 8 (15.10) 3 (30) 3 (30) 4 (40) 38 (60.32) 13 (20.63) 12 (19.05) gbs = group b streptococci; gfs = group f streptococci; s = sensitive; i = intermediate; r = resistant; ot = oxytetracycline; l = lincomycin; n = neomycin; cfp = cephaperazone; ts = trimethoprim sulphamethoxazole; amc = amoxicillin clavulanic acid; enr = enrofloxacin; p = penicillin; cn = gentamicin; ax = amoxicillin; cl = cephalexin; cip = ciprofloxacin; e = erythromycin. 44 antimicrobial resistence of streptococcus sp. in turkey şahan yapicier et al. veterinaria italiana 2021, 57 (1), 41-47. doi: 10.12834/vetit.1855.9879.3 insights in to the antibiotic resistance of hemolytic streptococci. the highest resistance to neomycin (95.24%), trimethoprim sulphamethoxazole (87.30%) and gentamicin (69.84%) was not surprising for streptococcus  sp. as it has an intrinsic resistance to aminoglycosides and sulphonamides (swedberg et  al. 1998). on the other hand, except for one gfs isolate, all isolates showed high susceptibility to the amoxicillin-clavulanic acid. similar antibiogram patterns were observed by ikiz and colleagues (ikiz et  al. 2013). tel and colleagues (tel et  al. 2009) reported high gentamicin resistance (78.3%) as well. conversely, the high resistance rate to penicillin (23.81%) and amoxicillin (14.29%) was in contrast to what observed by other researchers who reported lower resistance rates (tenhagen et  al. 2006, ekin et  al. 2011). beta-lactam antibiotics were widely used in cows for the treatment and prevention of diseases; a high rate of resistance to these antibiotics was therefore not unexpected. on the other hand, it has stated that the cephalexin (first generation cephalosporins) and cephaperazone (third generation cephalosporins) have greater anti-streptococcal activities than other beta-lactams (ceniti et al. 2017). also in this study a lower resistance to cephalexin (19.05%) and cephaperazone (4.76%) was observed (rügsegger et al. 2014, zhang et al. 2018). increasing resistance of streptococci to commonly used broad-spectrum antimicrobials including tetracyclines (up to > 90%) and macrolides (up to > 70%) has been reported worldwide since the 1980s (aarestrup et  al. 1998, blowey et  al. 1995a, palmieri et al. 2011, zhang et al. 2008). in the present study, the isolated gfs and gbs showed similar resistance rates (30%) against lincomycin, whereas the isolated gfs were found to be highly resistant to oxytetracycline (70%) and erythromycin (40%). these results were similar to what has been previously described in other countries (kalmus et  al. 2011, petrovski et  al. 2006). although fluoroquinolones are considered among the most effective drugs, increasing the risk of quinolone-resistant bacteria should not to be ignored (lopez et  al. 2015). in this study, the resistance rates to enrofloxacin and ciprofloxacin were high in both gbs (28.30% and 39.62%) and gfs (50% and 30%). this was in agreement with other reports in turkey that streptococci have variable resistance to fluoroquinolones (acar et  al. 2012, macun et al. 2011). mdr was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories (schwarz et  al. 2010, tenover 2006). in this study, multiple resistances to three or more than three antimicrobial agents tested were observed. of the 63 streptococcus isolates, 50 (79.36%) were found as mdr. mdr rates of gbs and gfs isolates were 75.47% (40/53) and 100% (10/10), respectively. both, the isolated gbs and gfs were particularly resistant table iii. antimicrobial resistance profiles of 50 multi-drug resistant streptococcus sp. isolated from bovine mastitis. antimicrobials gbs (n = 40) gfs (n = 10) n % n % resistance to 3 antimicrobials cl, cn, cip 0 0 1 10 n, ts, cn 7 17.5 0 0 ot, n, ts 1 2.5 0 0 cl, cip, ax 1 2.5 0 0 resistance to 4 antimicrobials n, ts, enr, cn 4 7.5 0 0 ts, enr, p, cn 1 2.5 0 0 n, ts, p, cn 1 2.5 0 0 n, ts, enr, p 0 0 1 10 ot, n, ts, cn 0 0 2 20 ot, l, cn, cip 1 2.5 0 0 ot, l, n, e 1 2.5 0 0 n, ts, cn, cip 1 2.5 0 0 n, ts, cl, cip 1 2.5 0 0 l, n, ts, cn 0 0 1 10 resistance to 5 antimicrobials n, ts, enr, cn, cip 1 2.5 0 0 ot, ts, enr, cn, cip 1 2.5 0 0 ot, n, ts, cn, e 4 10 0 0 ot, n, ts, cl, cip 1 2.5 0 0 ot, n, ts, enr, cip 1 2.5 0 0 ot, l, n, ts, e 1 2.5 1 10 resistance to 6 antimicrobials l, n, ts, enr, cn, cip 1 2.5 0 0 ot, n, ts, cl, cn, cip 1 2.5 0 0 resistance to 7 antimicrobials l, n, ts, enr, cn, cip, e 1 2.5 0 0 ot, n, ts, enr, cn, ax, e 0 0 1 10 ot, l, n, ts, p, cn, cip 2 5 0 0 l, n, ts, cl, p, cn, cip 1 2.5 0 0 resistance to 8 antimicrobials l, n, ts, enr, p, cn, cip, ax 1 2.5 0 0 l, n, ts, enr, p, cn, cip, e 2 5 0 0 ot, n, ts, cl, enr, p, cn, cip 1 2.5 0 0 ot, cfp, n, ts, cl, cn, cl, e 1 2.5 0 0 resistance to 9 antimicrobials l, n, ts, cl, enr, p, cn, cip, ax 0 0 1 10 ot, l, n, ts, cl, p, cn, cip, ax 1 2.5 0 0 l, n, amc, cl, enr, p, cip, ax, p 0 0 1 10 resistance to 10 antimicrobials ot, l, cfp, n, cl, enr, p, cn, ax, e 0 0 1 10 gbs = group b streptococci; gfs = group f streptococci; ot = oxytetracycline; l = lincomycin; n = neomycin; cfp = cephaperazone; ts = trimethoprim sulphamethoxazole; amc = amoxicillin clavulanic acid; enr = enrofloxacin; p = penicillin; cn = gentamicin; ax = amoxicillin; cl = cephalexin; cip = ciprofloxacin; e = erythromycin. 45 şahan yapicier et al. antimicrobial resistence of streptococcus sp. in turkey veterinaria italiana 2021, 57 (1), 41-47. doi: 10.12834/vetit.1855.9879.3 conclusions streptococcal mastitis is a common cause of economic loss in dairy herds in turkey as well as throughout the world. the economic losses could be irreversible because of late and improper diagnosis of the main etiological agent. there is an urgent need to enhance awareness among the dairy farmers in choosing the appropriate drug for treating mastitis. this should be done keeping in mind the emergence of mdr strains. in addition to treatment, control measures should be improved and put in place in accordance with contagious transmission and environmental exposure. to gentamicin, oxytetracycline, trimethoprim sulphamethoxazole and neomycin. meanwhile, the isolated gbs and gfs were resistant to ≥  3 antimicrobials in varying rates from 2.5 to 17.5% and from 10 to 20%, respectively. nevertheless, one alpha-hemolytic gfs strain showed resistance against 10 antimicrobials. as observed in other countries, our results indicated the presence of streptococcus sp. isolates with high level of mdr (nam et al. 2009, ding et al. 2016). aarestrup f.m., rasmussen s.r., artursson k. & jensen n.e. 1998. trends in the resistance to antimicrobial agents of streptococcus suis isolates from 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(in turkish). tel o.y., keskin o., zonturlu k.a. & kaya n.b.a. 2009. subclinical mastitis prevalence and determination of the antibiotics susceptibility in şanlıurfa region. fırat univ sag bil vet derg, 23, 101-106. teng l.j., hsueh p.r., cheng y.c., ho s.w. & luh k.t. 1998. antimicrobial susceptibility of viridans group streptococci in taiwan with an emphasis on the high rates of resistance to penicillin and macrolides in streptococcus oralis. j antimicrob chemother, 41, 621-627. tenhagen b.a., koster g., wallmann j. & heuwieser w. 2006. prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in brandenburg, germany. j dairy sci, 89, 2542-2551. tenover f.c. 2006. mechanisms of antimicrobial resistance in bacteria. am j med sci, 119, 3-10. turutoglu h., ercelik s. & ozturk d. 2006. antibiotic resistance 213 #these authors contributed equally to this work. 1department of life sciences and biotechnology, university of ferrara, via l. borsari 46, 44121 ferrara, italy. 2veterinary clinic, via reale 124p, 48011 alfonsine, ravenna, italy. 3department of the sciences of agriculture, food and environment, university of foggia, via napoli 25, 71122 foggia, italy. 4department of biology, ecology and earth sciences, university of calabria, via p. bucci, 87036 arcavacata di rende, cosenza, italy. *corresponding author at: department of life sciences and biotechnology, university of ferrara, via l. borsari 46, 44121 ferrara, italy. e‑mail: marco.pezzi@unife.it. keywords chronic enteritis, domestic rabbit, italy, lucilia sericata, urogenital myiasis. summary the report describes a case of urogenital myiasis in a domestic rabbit oryctolagus cuniculus l. (lagomorpha: leporidae) caused by lucilia sericata (meigen; diptera: calliphoridae) in region emilia-romagna (northern italy). the case, occurring in june 2018, is the first one involving l. sericata as an agent of myiasis in a domestic rabbit in italy. species identification was based on morphological investigations of males through identification keys. the rabbit developed the urogenital myiasis as a consequence of chronic enteritis causing an accumulation of faeces in the perianal and perineal region. marco pezzi1*, marilena leis1, maria gabriella marchetti1, elisabetta mamolini1, carlo nicola francesco del zingaro2, annagiulia zanardi1, chiara scapoli1, annunziata giangaspero3# and teresa bonacci4# urogenital myiasis caused by lucilia sericata (diptera: calliphoridae) in a domestic rabbit in italy veterinaria italiana 2020, 56 (3), 213-215. doi: 10.12834/vetit.2061.10978.2 accepted: 18.05.2020 | available on line: 31.12.2020 owned in alfonsine (ravenna, emilia-romagna, northern italy) and affected by chronic enteritis, was brought on 19 june 2018 to the local veterinary clinic. during examination, the veterinarian detected an infestation by dipteran larvae in the vaginal vestibulus (figure 1). no lesions were apparently visible on the mucosa. after taking photographs of the infestation site, the veterinarian mechanically removed the larvae, collecting a total of 18 live larvae and prescribed a therapy with antibiotics (enrofloxacin) to treat the enteritis. the larvae were stored in a plastic test tube sealed with a clean cotton cloth. the larvae, 9.36 ± 0.62 mm long, were brought to the laboratories of the department of life sciences and biotechnology, university of ferrara (ferrara, italy) for morphological investigations. three larvae were killed by quick immersion in hot water (about 90 °c), fixed and stored in 80% ethanol. the other larvae were reared to adults in plastic boxes containing about 90g of ground beef, at 25 ± 2 °c 50% relative humidity and a 16/8 (l/d) photoperiod. once reached the adult stage, torpor was induced into introduction myiasis is commonly defined as a form of parasitism of vertebrates (including humans) by larvae of diptera actively feeding on live or dead host tissue (zumpt 1965). according to the anatomical localization, it is classified as auricular, cutaneous, gastrointestinal, nasopharyngeal, ophthalmic and urogenital (hall and smith 1993, scholl et  al. 2009, francesconi and lupi 2012) or on the basis of parasite-host relationship as accidental, facultative or obligatory (hall and smith 1993, scholl et al. 2009). in domestic rabbits, several cases of traumatic myiasis have been reported worldwide – mostly caused by species of the family calliphoridae (bisdorff and wall 2006, cousquer 2006, druce 2015). this report describes a rare case of urogenital myiasis in a domestic rabbit diagnosed in northern italy. case report a 6-year-old privately-owned female rabbit, oryctolagus cuniculus l. (lagomorpha: leporidae), case report 214 urogenital myiasis in a rabbit pezzi et al. veterinaria italiana 2020, 56 (3), 213-215. doi: 10.12834/vetit.2061.10978.2 most frequently causing myiasis in rabbits, with devastating and sometimes fatal consequences (turner et  al. 2018). in italy, l. sericata has been described as an agent of myiasis in cats (principato and cioffi 1996, pezzi et  al. 2015, pezzi et  al. 2017), dogs (principato and cioffi 1996, bonacci and brandmayr 2016) and humans (majocchi 1920, dutto et  al. 2010, berlot and calderan 2017, dutto and vanin 2018). only another case of myiasis in a rabbit has been reported to date in italy, involving another species of the genus lucilia, lucilia caesar l. (principato and cioffi 1996). the myiasis was localized in the auricular region. a third species of the genus, lucilia eximia (wiedemann) was reported as agent of myiasis in rabbits in brazil, causing myiasis in the perineal region (moretti and thyssen 2006). for egg laying, females of l. sericata are attracted by soiled hair and skin (cousquer 2006). in domestic rabbits, accumulation of faeces and urine in the perineal region may attract dipteran females and contribute to develop myiasis (druce 2015). a case of debilitation by pasteurella multocida (proteobacteria: pasteurellaceae) with accumulation of faeces in the perineal region has been described as causing urogenital myiasis in the vulva (‘vulvomyiasis’) of a rabbit in austria (hinaidy and niebauer 1979). post-partum problems may also underlie urogenital myiasis (hall 1979). urogenital myiasis can be classified as external or internal (francesconi and lupi 2012); in the case reported here, the condition favouring the development of external urogenital myiasis was chronic enteritis leading to faeces accumulation around the anus. although all larvae were mechanically removed and a systemic antibiotic therapy was applied, the debilitated rabbit died after two days. myiasis are serious parasitic diseases whose prevention involves proper management of hygiene and animal health (choe et  al. 2016). the case of myiasis by l. sericata described here in a domestic rabbit in italy emphasizes the need for synergies between veterinarians and entomologists, not only for a correct identification of the species and for a proper therapy, but also to develop suitable and effective educational programs aimed to pet owners. acknowledgements the authors wish to thank paola ancarani for valuable technical assistance. professor annunziata giangaspero and professor teresa bonacci contributed equally to this work flies by exposure to co 2 . each fly was placed in an individual test tube, killed by exposure to -  20  °c and stored in a freezer. species identification was based on morphology of males examined under a nikon smz 800 stereomicroscope (nikon instruments europe, amsterdam, the netherlands), using the identification key of szpila (szpila 2012). based on morphological investigations according to identification keys, the species causing the myiasis was identified as lucilia sericata (meigen; diptera: calliphoridae). discussion this is the first case of myiasis by l. sericata ever reported in a rabbit in italy. myiasis by l. sericata have been reported in rabbits in other european countries, such as netherlands (leegwater-van der linden 1976), austria (hinaidy and niebauer 1979, hinaidy and frey 1990) and united kingdom (bisdorff and wall 2006, turner et al. 2018), and also in turkey (i̇pek and i̇pek 2012) and usa (hall 1979). in united kingdom l. sericata is recognized by veterinarians as the species figure 1. urogenital myiasis in a domestic rabbit by lucilia sericata. genital region showing the vaginal vestibulus with dipteran larvae. the inlay shows a detail of the genital opening with dipteran larvae. 215 pezzi et al. urogenital myiasis in a rabbit veterinaria italiana 2020, 56 (3), 213-215. doi: 10.12834/vetit.2061.10978.2 berlot g. & calderan c. 2017. ocular, nasal and aural myiasis in an intoxicated patient: a case report. clin med rev case rep, 4, 156. bisdorff b. & wall r. 2006. blowfly strike prevalence in domestic rabbits in southwest england and wales. vet parasitol, 141, 150-155. bonacci t. & brandmayr p. 2016. primi dati sui ditteri che causano miasi canine in calabria. in proc. of the 25th national italian congress of entomology, padua, 20-24 june 2016, 315. choe s., lee d., park h., jeon h.k., kim h., kang j.h., jee c.h. & eom k.s. 2016. canine wound myiasis caused by lucilia sericata (diptera: calliphoridae) in korea. korean j parasitol, 54, 667-671. cousquer g. 2006. veterinary care of rabbits with myiasis. in pract, 28, 342-349. druce k. 2015. myiasis in domestic rabbits. vet nurs j, 30, 199-202. dutto m., pomero f., migliore e. & fenoglio l. 2010. cutaneous myiasis in a geriatric patient. parassitologia, 52, 435-438. dutto m. & vanin s. 2018. cutaneous myiasis by lucilia sericata (meigen) 1826 (diptera: calliphoridae) and failure to application of the isomegalen for minimum colonization time. minerva medicoleg, 138, 30-33. francesconi f. & lupi o. 2012. myiasis. clin microbiol rev, 25, 79-105. hall m.j.r. & smith k.g.c. 1993. diptera causing myiasis in man. in medical insects and arachnids. (r.p. lane & r.w. crosskey, eds). chapman and hall, london, 429-469. hall r.d. 1979. the blow flies of missouri: an annotated checklist (diptera: calliphoridae). trans mo acad sci, 13, 33-36. hinaidy h.k. & frey h. 1990. neue myiasis-fälle bei tieren in österreich. mitt österr ges tropenmed parasitol, 12, 111-120. hinaidy h.k. & niebauer g.w. 1979. fakultativmyiasis bei einem kaninchen und einem meerschweinchen. wien tierärztl mschr, 66, 384-386. i̇pek d.n. & i̇pek p. 2012. a case of traumatic myiasis in a domestic rabbit (oryctolagus cuniculus) caused by lucilia sericata. turkiye parazitol derg, 36, 54-56. references leegwater-van der linden m.e. 1976. myiasis caused by lucilia sericata (meigen) in a rabbit. tijdschr diergeneesk, 101, 431. majocchi d. 1920. sopra due nuovi casi di dermato-myiasis muscosa da lucilia sericata. memorie r accad sci ist cl sci fis bologna, 7, 237-252. moretti t.c. & thyssen p.j. 2006. miíase primária em coelho doméstico causada por lucilia eximia (diptera: calliphoridae) no brasil: relato de caso. arq bras med vet zootec, 58, 28-30. pezzi m., whitmore d., chicca m., lanfredi m. & leis m. 2015. traumatic myiasis caused by an association of sarcophaga tibialis (diptera: sarcophagidae) and lucilia sericata (diptera: calliphoridae) in a domestic cat in italy. korean j parasitol, 53, 471-475. pezzi m., whitmore d., bonacci t., del zingaro c.n.f., chicca m., lanfredi m. & leis m. 2017. facultative myiasis of domestic cats by sarcophaga argyrostoma (diptera: sarcophagidae), calliphora vicina and lucilia sericata (diptera: calliphoridae) in northern italy. parasitol res, 116, 2869-2872. principato m. & cioffi a. 1996. notes on the incidence of the lucilia genus (diptera: calliphoridae) in umbria, central italy. a case of myiasis by lucilia ampullacea (villen 1922) in testudo graeca. in proc. 20th international congress of entomology, florence, 25-31 august 1996, 769. scholl p.j., catts e.p. & mullen g.r. 2009. myiasis (muscoidea, oestroidea). in medical and veterinary entomology, 2nd  ed. (g. mullen & l. durden, eds). academic press, san diego, 309-338. szpila k. 2012. key for identification of european and mediterranean blowflies (diptera, calliphoridae) of medical and veterinary importance adult flies. in forensic entomology, an introduction, 2nd  ed. (d. gennard, ed). wiley-blackwell, west sussex, 77-81. turner r., arsevska e., brant b., singleton d.a., newman j., noble p.m., jones p.h. & radford, a.d. 2018. risk factors for cutaneous myiasis (blowfly strike) in pet rabbits in great britain based on text-mining veterinary electronic health records. prev vet med, 153, 77-83. zumpt f. 1965. myiasis in man and animals in the old world. butterworth & co., london, 267 pp. 289 1department of veterinary medicine, university of bari “aldo moro”, bari, italy. 2ministry of health, directorate-general for hygiene, food safety and nutrition (dgisan), rome, italy. 3department of agricultural and environmental science, university of bari “aldo moro”, bari, italy. *corresponding author at: department of veterinary medicine, university of bari “aldo moro”, food safety section, strada provinciale per casamassima km 3, 70010 valenzano (bari), italy. e-mail: roberta.barrasso@uniba.it. parole chiave benessere animale, consumatore, disponibilità a pagare, questionario. riassunto l'interesse dei cittadini europei verso il benessere degli animali risente di diverse variabili: la tipologia di consumatore e il suo paese di appartenenza, ad esempio. al fine di valutare il benessere degli animali nell’azienda agricola, è essenziale sviluppare parametri oggettivi, ovvero animal-based measures, riguardanti il comportamento, la salute e la fisiologia degli animali. la determinazione di indicatori validi e affidabili di benessere animale rappresenta l’obiettivo chiave di diversi programmi di ricerca. agli strumenti utilizzati vanno aggiunte, inoltre, indagini rivolte agli agricoltori, a esperti del settore e ai consumatori di carne. l’obiettivo dello studio è stato indagare le abitudini alimentari degli italiani rispetto al consumo di carne e di valutare la loro conoscenza del benessere degli animali correlandola con la disponibilità a pagare un prodotto etichettato animal-friendly. durante lo studio è stato sottoposto un questionario composto di ventitré domande a risposta chiusa a cinquecento intervistati. i risultati dell’indagine sono stati, quindi, elaborati attraverso analisi statistiche multivariate. la variabile che è risultata in grado di influenzare maggiormente il prezzo di acquisto della carne è il luogo di acquisto, con un’ampia oscillazione tra la grande distribuzione organizzata (g.d.o.) e i piccoli esercizi commerciali che propongono prodotti biologici. inoltre, si evince che l'interesse verso il tema del benessere degli animali è direttamente correlato alla disponibilità economica e al grado d’istruzione del consumatore. interesse dei consumatori nei confronti del benessere animale e loro disponibilità a pagare keywords animal welfare, consumer, questionnaire, willingness to pay. summary the interest of european consumers towards animal welfare can be influenced by several variables, both related to the consumers themselves and to the different countries of the eu. in order to assess animal welfare at farm level, it is essential to develop animal-based measures in accordance with the animals’ actual welfare state in terms of their behaviour, health and physiology. the search for valid and reliable indicators is a key objective of several research programs especially for assessing welfare at farm level and the tools may include surveys addressed to farmers. however, there is a need to guarantee financial support for farmers who breed animals in accordance with such welfare conditions, to cover their additional costs. the aim of the study was to investigate the eating habits of italian consumers regarding meat consumption linked to their knowledge of animal welfare and to their willingness to pay. we investigated consumers’ understanding of animal welfare using a questionnaire (based on a list of twenty-three closed-ended questions) designed for collecting data from large numbers of respondents and multivariate statistical analysis. the data in our study showed that the variable with the greatest influence on purchase price was the place of meat purchase. as regards level of education, it appears that people with a high level of education are more concerned about animal welfare and, consequently, are willing to spend a higher price when buying meat. consumer attention to the animal-welfare issue is on the rise and, in parallel with this growth, there is also a greater willingness to pay, i.e. a surcharge for the products obtained in the respect of animal welfare. this growth is influenced by the awareness and knowledge of the characteristics of animal welfare. giancarlo bozzo1, roberta barrasso1*, claudia annarita grimaldi2, giuseppina tantillo1 and rocco roma3 consumer attitudes towards animal welfare and their willingness to pay veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 accepted: 19.07.2019 | available on line: 22.10.2019 290 veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 animal welfare and willingness to pay bozzo et al. european commission, consumers were asked to rate the importance of farm animal protection on a scale from 0 to 10, with their answers averaging 7.8. this shows that the food quality opinion is established, along with the complete nature and safety of the final product, also by the welfare of the animals involved (blokhuis et  al. 2008, napolitano et al. 2007). in order to assess animal welfare at farm level, it is essential to develop animal-based measures founded on the animals’ actual welfare state in terms of their behaviour, health and physiology (blokhuis et  al. 2003). the search for valid and reliable indicators is a key objective of several research programs, especially for assessing welfare at farm level – the tools may include surveys addressed to farmers; in this case, the methodology used to conduct interviews is decisive. studies conducted by heise and theuvsen (heise and theuvsen 2015) and heise and colleagues (heise et  al. 2015) clearly indicated that different methodological approaches (open-ended and closed-ended questions) can lead to substantial differences in the perceptions of farm animal welfare (faw) of farmers and veterinarians. a similar pattern might also exist regarding consumers’ definitions and appreciation of animal welfare. differences in approaches complicate the development of a common assessment framework for animal welfare, that would be unanimously accepted by the various stakeholders. most scientific concepts defining faw are actually criticized for not adequately addressing public conceptions of faw (fraser 2008, vanhonacker and verbeke 2014). the animal welfare concept is characterized by scientific, ethical, economic, cultural and religious dimensions that continue to evolve (fraser 2009, green and mellor 2011). knowledge about the public’s understanding of faw should be augmented by encouraging the scientific dialogue between citizens and stakeholders along the food supply chain and by developing animal-welfare programs. indeed, one of the key goals of new eu-funded projects is to develop a concept that adequately considers society’s definition of animal welfare (heise and theuvsen 2017). previous studies (de greef et  al. 2006, lassen et  al. 2006, marie 2006) showed that consumers strongly associate faw with outdoor access, adequate space requirements and the ability of animals to engage introduction welfare quality® is an eu-funded project aimed at achieving ‘integration of animal welfare in the food quality chain: from public concern to improved welfare and transparent quality’. recent european research (european parliament committees 2017) has shown considerable and growing interest among european consumers in the intangible characteristics of products, such as environmental protection, social equity and animal welfare (sassatelli 2006). indeed, large parts of western societies, where some events (bse, avian influenza, etc…) have raised awareness of the effects that animal husbandry has on meat safety, believe that animal welfare standards in livestock production need to be improved (de jonge and van trijp 2013, ec 2007). however, considerable variability has been observed between different parts of the world (kjørstad 2005) and even across the eu (nocella et  al. 2010), where consumers in northern eu member countries seem to be more concerned with animal welfare problems (nocella et  al. 2010). german consumers, for example, rate animal-welfare aspects very highly, with 61% feeling that it is important to protect farm animal-welfare. in contrast, only 34% of polish citizens agree with this statement [european commission (ec) 2016]. little is known about the views of consumers from some developing countries, such as brazil, regarding animal production systems (clark et al. 2016). state of the art in recent years, the eating habits of the population have changed. indeed, some people respond to their growing concerns over animal welfare by eating less meat or by becoming vegetarians or even vegans (vanhonacker et al. 2010). additionally, the number of consumers who source meat from more animal-friendly production systems has increased constantly (lusk and norwood 2012, schulze et al. 2008). as a result, a number of animal welfare programs have been developed, introducing so-called “animal welfare products” onto the market (heise and theuvsen 2017). furthermore, consumers are requesting not only for safe and quality foods, but also for a certification that animals had been bred and slaughtered ethically (salamano et  al. 2013). in a 2007 study by the infine, dallo studio emerge la necessità di garantire un sostegno finanziario agli agricoltori che allevano gli animali in conformità al loro benessere, per coprire i costi aggiuntivi necessari ad assicurare tali condizioni di allevamento. 291veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 bozzo et al. animal welfare and willingness to pay and cross-cultural differences (nocella et  al. 2010, lagerkvist and hess 2011). for instance, a study showed that eu countries such as germany, france and great britain have a higher wtp for animal-welfare attributes than spanish, danish and italian consumers. accordingly, a higher wtp has been observed for salmon and pigs reared in improved welfare conditions, although the presence of unsuitable products (over-pale salmon from organic farming and boar-tainted pork from uncastrated male pigs) decreased wtp (lagerkvist et al. 2006, liljenstolpe 2008, olesen et al. 2010). studies based on choice experiments and cost estimates showed that animal-friendly practices may be economically sustained by consumers’ increased wtp; further studies are necessary to verify whether the rise in wtp at least covers the increased costs to farmers. thus, consumer wtp provided a useful tool to obtain information about the real value that consumers give to animal welfare and they could sustain the implementation of the corresponding animal-friendly practices as assessed by benefit-cost estimates (carlsson et  al. 2007). however, wtp is often over-estimated because of hypothetical bias and social desirability effects in the answers (dransfield et al. 2005, napolitano et al. 2010). in contrast to the large number of studies regarding wtp for animal welfare or sustainability attributes (verain et  al. 2012), relatively few studies have been conducted segmenting consumers based on their preferences for a broader range of production-related attributes (animal welfare, environmental impact, health and safety) and for more traditional product characteristics (i.e. colour, fat content, country of origin, price). some studies (dransfield et  al. 2005, swanson and mench 2000) indicated that consumer intent to pay, measured through questionnaires, was higher for products obtained using animal-friendly farming techniques. in particular, people appeared to be prepared to pay an average 5% extra for pork from outdoor-raised pigs, with one-fifth of consumers claiming to be willing to pay 20% extra (dransfield et  al. 2005). in another study conducted on citizens from the 25 eu member states, the majority of respondents (57%) stated that they were prepared to pay more for eggs from animal welfare-friendly production systems: 25% could accept a 5% increase, 21% declared that an increment of 10% would be acceptable and 11% were prepared to pay 25% extra or more (ec 2005). similar results were obtained in the usa, where 44% of respondents expressed the intent to pay 5% more for food from animals raised humanely and 20% said they were prepared to pay up to 10% more (swanson and mench 2000). the aim of the study was to investigate the eating habits of italian consumers regarding meat in natural innate behaviour. other frequently named criteria related to feed and water supply and naturalness of feed. meuwissen and colleagues (meuwissen et  al. 2004) found that citizens rated space, medicines and living surface as the most important indicators of the level of animal welfare. miele and colleagues (miele et  al. 2011) found that citizens define faw based on 12 established criteria known as the “welfare quality” approach. in response to this public endorsement, an increasing number of regulations have been issued on the welfare of farm animals during growth (ec 2016), transportation (ec 2005)1 and slaughter (ec 2009)2. legislation on animal protection (italian legislative decree no. 146)3, although necessary in order to provide a minimum level of welfare to animals, does not guarantee to farmers sufficient revenues to sustain the increased costs, in spite of the subsidies introduced by the regional rural development programme (rdp). despite this public drive towards increased farm animal welfare standards, many farmers, practitioners and research groups are concerned about the extra costs arising from increased levels of animal welfare. they claim that this may lead to a reduced market competitiveness: for instance, without this increased cost, it has been estimated that farmers’ added value for meat is only 19% (economic research service 2004). the results of the surveys of eu and non-eu operators suggest that the application of aw legislation/standards implies higher production costs for operators, regardless of their geographical position, to achieve and maintain compliance with aw legislation/ standards (european commission 2017). conversely, such added value may be offset by raising the price of certified meat. a recent study (napolitano et al. 2008) on willingness to pay (wtp) for yogurt revealed that consumers were influenced by information about low standards of animal welfare and moved their wtp towards their expectations. however, the difference between expectancy and wtp was not totally correlated with faw, because wtp was also associated with other aspects such as the sensory properties of the products (napolitano et  al. 2008) and different meat types (i.e. species of origin) (carlsson et al. 2007). studies show that wtp tends to be influenced by national policy, the awareness of food scandals 1 european commission (ec) 2005. council regulation (ec) no. 1/2005 of 22 december 2004 on the protection of animals during transport and related operations and amending directives 64/432/eec and 93/119/ec and regulation (ec) no 1255/97. off j, l 3, 05/01/2005, 1-44. 2 european commission (ec) 2009. council regulation (ec) no. 1099/2009 of 24 september 2009 on protection of animals at the time of killing. off j, l 303, 18/11/2009, 1-30. 3 decreto legislativo 26 marzo 2001, no. 146 riguardante l’attuazione della direttiva 98/58/ce relativa alla protezione degli animali negli allevamenti. off j, 95, 24/04/2001. 292 veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 animal welfare and willingness to pay bozzo et al. table i. an outline of the questions put forward to the respondents. questions 1) age <25 14) are you concerned with animal welfare? yes 25-40 i am quite interested 40-60 no >60 i never thought about it 2) gender male 15) when you buy meat, do you think about how the animals were raised and slaughtered? yes, most of the time female yes, sometime 3) highest academic qualification primary school/middle school no, never high school i don’t know graduate 16) which of these animals, according to you, has the worst quality of life? chickens and laying hens postgraduate qualification cattle 4) place of residence small town (up to 5.000) pigs town (5.000-30.000) sheep city (more than 30.000). 17) what characteristics should cattle breeding have to ensure animal welfare? animals are bred in a healthy and natural way 5) do you eat meat? yes animals are free to move around an open area no animals are free to behave naturally 6) if you answered no to question 5, please state why? ethical and religious reasons animals are bred to ensure a good yield meat is harmful to the human health 18) how do you keep yourself updated on animal welfare? newspaper and magazines meat production is unsustainable for the environment internet 7) how do you consider your meat consumption in the last years? increased tv shows about animal unchanged i don’t normally keep updated, but if i find news about it, i pay attention decreased i am not interested 8) how often do you eat beef? often 19) what do you think about products made following animal welfare? they are healthier foods sometimes they are higher quality foods never they are more profitable for the farmer 9) how often do you eat pork? often they are more sustainable for the environment sometimes 20) are you willing to buy more expensive meat that has been produced following animal welfare measures? yes, up to 10% more never yes, up to 20% more 10) how often do you eat sheep? often no, i’m not willing to pay more sometimes i don’t know never 21) how should the product obtained according to animal welfare be tagged? informative labels on the pack 11) how often do you eat poultry? often rating and scoring system sometimes a logo on the package never realistic picture about the livestock 12) where do you usually buy meat? butcher’s 22) in your opinion, who should guarantee animal welfare? producers and farmers supermarket veterinarians discount store european commission other (producer or organic shops) italian government 13) when buying, does the meat price influence your choice? yes, a lot 23) do you consider the information about livestock and animal slaughter exhaustive on current labels? yes, information is clear and sufficient yes, enough no, information is not sufficient no no, information is not fully clear i don’t know i don’t know. 293veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 bozzo et al. animal welfare and willingness to pay variables collected and the wtp. in the multiple regression model, we assume that there is a linear relationship between the dependent variable y and xn independent variables. the independent variables are sometimes called both explanatory variables, because they are able to explain the y statistical variation, and predictor variables, due to the predictive capability of the y  value (daniel and cross 2013). in our study, the dependent variable was the consumer’s willingness to pay (wtp), while the independent variables included all information collected from the interviewees. after a frequency analysis and a statistical correlation index of all the data, eight variables were chosen: (i) type of consumer; (ii) concern; (iii) consumer’s qualification; (iv) place of meat purchase4; (v) age; (vi) gender; (vii) residence and (viii) trend of meat consumption in the last years. as regression method, we used the forward type in which the independent variables were entered one at a time according to the probability that they affect the significance of the obtained model. according to the highest level of determination coefficient (r2), the best fitting model was obtained. results the percentages of respondents who often eat chicken meat and beef were respectively 33.8% and 27.2%, whilst the consumption frequency of pork meat was 21.9% and of sheep meat was only 2.32%. as results of the former regression model, based on the eight variables listed above, r2 was very low due to the high outlier presence that influenced the total variability of the sample. several statistical methods reduced this effect. we used the percentage difference (pd) between the observed value of wtp and the calculated value by the regression model. in the final regression, we used 147 observations in which the pd was higher than -  25% and lower than + 25% so that a high r2 was obtained (table ii). the fitting model chose four of the original variables as significant: (i) type of consumer; (ii) concern; (iii) consumer’s qualification and (iv) place of meat purchase. conversely, the remaining variables: (i) age; (ii) gender; (iii) residence and (iv) trend of meat consumption in the last years, did not affect the model’s significance. by looking at the fourth box in table ii, it is clear that consumption linked to their knowledge of animal welfare, using willingness to pay (wtp) as a proxy of their behaviour. material and methods sampling and data collection we investigated consumers’ understanding of animal welfare using a questionnaire designed for collecting data from large numbers of respondents and multivariate statistical analysis. the questionnaire was based on a list of twenty-three closed-ended questions (table i). five hundred respondents compiled the questionnaire and data were collected in the months of september and october 2018. the opportunity to fill out the questionnaire both in paper format and in digital version was provided in order to interview more people, and thereby achieve better statistical representativeness. given the ubiquitous presence of smartphones, a link was created whereby people could also answer the questionnaire from their computers or phones. a total of three hundred respondents used social media and internet links to respond to the questionnaire; conversely, others were interviewed in areas adjacent to supermarket butchers’ counters. the questionnaire was divided into three different parts, according to the nature of the questions: • socio-economic data: information on social, cultural and economic issues (gender, age, place of residence); this information allowed us to segment the sample and to study different meat consumption behaviours. • meat consumption habits: characteristics of spending habits (frequency and place of purchase, quality, price ratio), regarding meat consumption. • animal welfare interest: knowledge and awareness of respondents about animal welfare, ranging from a description of animal welfare, its importance and how clearly they were able to interpret labelling information to recognize that a given food had been produced respecting animal welfare. questions about the wtp surcharge for meat products compliant with animal welfare criteria were asked at the end of the questionnaire. statistical analysis a multiple linear regression analysis was carried out to predict the wtp for the sample and to assess specific forms of relationship between 4 the significance of the four variables used in the analysis is as follows: the variable ‘type of consumer’ was related to the frequency of meat consumption; ‘concern’ indicated consumer sensitivity to various aspects of animal welfare; ‘consumer’s qualification’ regarded the educational level (primary school, middle school, high school, degree or postdegree); ‘place of meat purchase’ was butcher’s, supermarket, discount store, producer or organic shops. 294 veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 animal welfare and willingness to pay bozzo et al. purchase. indeed, from our results, it emerged that if the meat is bought in a supermarket or in a discount store, the consumer has a lower wtp; on the other hand, if the meat is bought in a butcher’s, from the producer or in an organic shop, consumers raise their wtp. this difference could be explained by the several kinds of customer in retail settings whose purchases are strictly linked to quality-price ratio. as regards the second variable, it is clear that the greater the consumers' interest in animal welfare, the higher their wtp. conversely, frequency of meat purchases negatively affected wtp. finally, as regards level of education, it appears that people with a high level of education are more concerned about animal welfare and, consequently, are willing to spend a higher price when buying meat. the data in our study showed that educational background influences experts’ views on certain animal-welfare aspects. these results are in agreement with those from other studies (nøhr et al. 2016, bracke et al. 2008) investigating the influence of expert education and current profession in regards to their opinion on the validity of welfare measures. moreover, results involving 196 european experts showed that current profession was more pivotal than educational background in their approach to welfare measures and criteria. however, rodenburg and colleagues (rodenburg et  al. 2008) showed that welfare scientists including ethologists and veterinarians presumably are better qualified than lay people to make judgements on the overall animal-welfare state whenever the welfare judgement is to be based on a complex dataset on various welfare indicators. according to a previous study conducted by the the place of meat purchase was the most significant variable. the second variable that may influence the consumer’s wtp was concern, the third variable was the type of consumer while the fourth was the level of education. the linear equation obtained (table iii) was as follows: wtp = ‑2.9 (constant) + 3.6 (place of purchase) + 1.3 (concern) ‑ 1.4 (type of consumer) + 1.9 (qualification) discussion the limit of our study is represented by the part of the population which answered the questionnaire (table iv). as in previous studies (carlucci et  al. 2009, grunert and valli 2001), we considered mainly younger subjects with a higher level of education. indeed, since the questionnaire was also disseminated via internet, the people who responded were mainly young people and students, as they were the most frequent social media users at the time. table iii showed that the variable with the greatest influence on purchase price was place of meat table iii. coefficients. model unstandardized coefficients standardized coefficients t p b standard deviation (sd) beta 1) (constant) 5.652 0.697 8.107 0.000 place of meat purchase 5.014 0.379 0.738 13.218 0.000 2) (constant) -0.521 0.828 -0.629 0.531 place of meat purchase 3.733 0.322 0.550 11.595 0.000 concern 1.399 0.142 0.467 9.854 0.000 3) (constant) 2.831 0.791 3.577 0.000 place of meat purchase 3.606 0.266 0.531 13.573 0.000 concern 1.309 0.117 0.437 11.147 0.000 type of consumer -1.346 0.161 -0.301 -8.348 0.000 4) (constant) -2.944 0.971 -3.033 0.003 place of meat purchase 3.590 0.221 0.529 16.261 0.000 concern 1.275 0.098 0.420 12.858 0.000 type of consumer -1.426 0.134 -0.319 -10.614 0.000 consumer’s qualifications 1.858 0.230 0.241 8.093 0.000 table ii. the sample coefficient of determination. model r r-squared adjusted r-squared standard deviation (sd) 1 0.738 0.545 0.542 3.344 2 0.853 0.727 0.724 2.597 3 0.903 0.816 0.812 2.139 4 0.935 0.874 0.870 1.778 295veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 bozzo et al. animal welfare and willingness to pay animal-welfare understanding with regard to many socio-demographic variables. gender, age, place of residence and children significantly influenced the perceived importance of the 12 animal-welfare criteria, while education level had no significant effect on consumers’ animal welfare definitions (tuyttens et  al. 2010). conversely, in our study, the consumer’s place of residence was excluded among the variables able to influence consumer wtp. the results of our study showed that only 7.9% of the respondents were unwilling to pay extra costs for products obtained in respect of animal welfare; 33.7% said they were willing to pay up to 10% more and as many as 58.4% of the persons interviewed claimed to be prepared to meet an increase of up to 20% more than normal (figure 1). conclusions using social media for public investigation appears to be a rapid and effective method to reach people and explain the nature and aims of the survey; moreover, this method saves time for collecting and inputting data. the statistical procedure we used is very common in marketing and consumer studies and its results have also been used for commercial purposes. as regards our results, it is clear that consumer attention to the animal-welfare issue is on the rise and, in parallel with this growth, there is also a greater wtp, i.e. a surcharge for the products obtained in the respect of animal welfare. this growth is influenced by the awareness and knowledge of the characteristics of animal welfare. for this purpose, more efforts should be made to clarify to consumers what are exactly the animal-welfare criteria, chiefly by public bodies, to raise awareness among citizens. interestingly, in our study the level of income clearly affected consumer wtp. this was particularly evident between customers of supermarkets and of discount stores. it is also clear that the farmers who breed animals in institute of grocery distribution (igd 1999), a strong influencing factor on the knowledge of, and interest in, food production is where the consumer lives, in particular whether they are of rural or urban origin (table i). tuyttens and colleagues (tuyttens et  al. 2010) also showed significant differences in 0 10 20 30 40 50 60 no extra costs 7.9% 33.7% 58.4% up to 10% more up to 20% more % figure 1. consumer willingness to pay. table iv. characteristics of the consumers interviewed. questions answers (%) 1) age <25 13.5 25-40 47.0 40-60 31.4 >60 8.0 2) gender male 36.5 female 63.5 3) highest academic qualification primary school/middle school 1.1 high school 9.7 graduate 41.8 postgraduate qualification 47.5 4) place of residence small town (up to 5.000) 18.1 town (5.000-30.000) 27.6 city (more than 30.000). 54.2 5) do you eat meat? yes 96.0 no 4.0 6) if you answered no to question 5, please state why? ethical and religious reasons 60.9 meat is harmful to the human health 21.7 meat production is unsustainable for the environment 17.4 7) how do you consider your meat consumption in the last years? increased 7.8 unchanged 48.9 decreased 42.6 8) where do you usually buy meat? butcher’s 39.2 supermarket 47.9 discount store 1.5 other (producer or organic shops) 8.9 9) how do you keep yourself updated on animal welfare? newspaper and magazines 16.0 internet 17.3 tv shows about animal 12.0 i don’t normally keep updated, but if i find news about it, i pay attention 45.6 i am not interested 8.6 10) are you concerned with animal welfare? yes 69.8 i am quite interested 19 no 1.3 i never thought about it 9.3 296 veterinaria italiana 2019, 55 (4), 289-297. doi: 10.12834/vetit.1823.9669.2 animal welfare and willingness to pay bozzo et al. products of animal origin subject to stringent welfare criteria with the label “animal welfare” on their packaging; ii) an institutional subsidies system for farmers who choose animal welfare would help achieving a good quality/price ratio, increasing the supply of animal-welfare certified products and, conversely, lowering their market price. institutional subsidies are essential to cover the farmer expenses required to obtain the necessary certifications. accordance with the appropriate welfare conditions, deserve financial support to cover the additional costs. to achieve this, two actions in particular are 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demonstrated by the detection of specific leishmania cellular immunity (cabral et  al. 1998, cardoso et al. 1998) and by the detection of parasite's dna in seronegative dogs (solano‑gallego et  al. 2001, fernández‑bellon et  al. 2008). in fact, the majority of dog population is exposed and becomes infected without showing clinical evidence of disease or even antibodies (baneth et al. 2008). sicily is a region highly endemic for canl (brianti et  al. 2014, brianti et  al. 2016) and approximately 47% of the sicilian population lives in areas at risk for visceral leishmaniasis (cascio et al. 2002), thus making an early diagnosis, coupled with vector control strategies in cat, human and dog populations, necessary. several studies have also been conducted on sicilian islands, such as in lampedusa island (foglia manzillo et  al. 2018). this study showed that more than 50% of dogs tested was sieropositive while in lipari and vulcano islands, prevalence was 41.7% and 23.6%, respectively (otranto et al. 2017). materials and methods study area the present study was carried out in pantelleria, a small island (80 km2) (36°47’27”n 11°59’38”e) during february 2017. this island is located the southwest 1istituto zooprofilattico sperimentale della sicilia, centro di referenza nazionale per le leishmaniosi, via g. marinuzzi 3, 90129 palermo, italy. 2section of dermatology, department of health promotion, maternal-infant, internal medicine and specialization of excellence “g. d’alessandro” (promise) university of palermo, palermo, italy. *corresponding author at: istituto zooprofilattico sperimentale della sicilia, centro di referenza nazionale per le leishmaniosi, via g. marinuzzi 3, 90129 palermo, italy. tel.: +39 091 6565348, e-mail: fabrizio.vitale@izssicilia.it. keywords leishmania infantum, pantelleria island, canine leishmaniasis. summary dogs are the major reservoir of leishmania infantum, the causative agent of canine visceral and cutaneous human leishmaniasis in the mediterranean basin. canine and human leishmaniosis are endemic in italy, particularly in central and southern regions, including islands. here we show a preliminary, clinical, serological and molecular study carried out in pantelleria island during 2017. in this study, we clinically examined 136 dogs for the presence of symptoms compatible with leishmaniasis, determined the titer of anti‑leishmania antibodies, and investigated leishmania dna by real time pcr in blood and/or lymph node of each dog. the prevalence of disease was equal to 27% with 95% ci [21%; 32%], lower than prevalence obtained in the other sicily islands (lampedusa, lipari). we observed that enlarged lymph nodes was more positivitely associated with canine leishmaniasis (canl) than other clinical signs. the results obtained showed that in an endemic area, such as sicily, diagnosis of canl needs to be carried out by including an immunological, molecular clinical approach. fabrizio vitale1*, federica bruno1, antonella migliazzo1, antonella galante1, angela vullo1, raffaele graziano1, salvatore d’avola1, valentina caputo2 and germano castelli1 cross-sectional survey of canine leishmaniasis in pantelleria island in sicily veterinaria italiana 2020, 56 (2), 103‑107. doi: 10.12834/vetit.2059.10976.3 accepted: 02.01.2020 | available on line: 31.12.2020 104 veterinaria italiana 2020, 56 (2), 103‑107. doi: 10.12834/vetit.2059.10976.3 cross‑sectional survey of canine leishmaniasis in sicily vitale et al. was carried out in a lightcycler® 96 (roche life science) using 1  ×  taqman universal master mix (applied biosystems, monza, italy) and performed as previously described (vitale et al. 2004). statistical analysis a descriptive analysis of the data was performed according to 11 variables that have been collected to evaluate possible associations with the presence of l.  infantum in dogs. data is showed in table i. variables were analyzed in the logistic regression model using the stata 9.2 software (statacorp lp, college station, texas). results characteristics of the dog study population in pantelleria island were summarized in table ii: 63.24% were owners dog (36.76% owned by the kennel); 58.96% of the dogs examined were males, of mixed breed (70.58%); 58.65% of the dogs in the sample were aged less than or equal to 5 years, while 71.97% of the dogs lived with one or more dogs and 89.71% underwent regular repellent tools (collars, spot‑on and spray formulations). the results of the evaluation of typical clinical signs of canl were expressed in percentages (%) and are shown in table iii. clinically, 30/136 (25.73%) of the animals showed at least one of sicily and 60 km (37 miles) east away from the tunisian coast. dogs largely represent the most abundant domestic animals present on the island; less than 1,000 dogs have been estimated. sampling the population study comprised 136 stray and domestic dogs. the dogs were selected based on the owners’ willingness to have their pet included in the survey. dogs were of different sex, breed and age. clinical assessment was performed and all data was recorded including signaling, anamnestic history, repellents used and clinical examination. finally, a dermatologic examination for ectoparasites and changes compatible with canine leishmaniosis (e.g., alopecic, nodular, ulcerative, crusty, or scaly dermatitis) was conducted. for each dog a blood sample was performed, while from 102/136 dogs needle aspirates of lymph nodes were collected. all samples (serum, whole blood, lymph node aspirate) were sent to the national reference centre for leishmaniosis (c.re.na.l.). anti‑leishmania antibody detection by ifat the collected serum samples were tested by ifat for the l.  infantum antibodies (bio merieux spa, florence, italy). dna extraction and real time pcr assays total dna was extracted from edta blood and lymph node aspirate using an e.z.n.a tissue dna kit (omega biotech vwr, norcross, ga, usa) following the manufacturer’s instructions. the real‑time polymerase chain reaction (rt‑pcr) table i. variables considered in the analysis. variable description and coding ifat serological test result (0 = negative; 1 = positive) dog owner 0 = domestic dog; 1 = kennel sex 0 = male; 1 = female age ≤ 5 years; > 5 years dog breed 0 = purebreed dog; 1 = mixed-breed dog repellent tools 0 = never/ occasionally; 1 = regularly cohabitation with other dogs 0 = no; 1 = 1 or more slimming 0 = no; 1 = yes skin signs 0 = no; 1 = yes enlarged lymph nodes 0 = no; 1 = yes ocular signs 0 = no; 1 = yes table ii.characteristics of the canine population in pantelleria and variables considered in the study, absolute frequencies and percentage values. absolute frequency % dog dog owners 86 63.24 kennel 50 36.76 sex male 79 58.96 female 55 41.04 breed purebred dog 40 29.42 mixed breed 96 70.58 age ≤ 5 years 78 58.65 > 5 years 55 41.35 repellent tools never/ occasionally 14 10.29 regularly 122 89.71 cohabitation with other dogs yes 95 71.97 no 37 28.03 105veterinaria italiana 2020, 56 (2), 103‑107. doi: 10.12834/vetit.2059.10976.3 vitale et al. cross‑sectional survey of canine leishmaniasis in sicily ml) than lymph node. out of 29 dogs qpcr positive, 13 showed no symptoms of leishmaniasis (weight loss, skin and ocular signs and enlarged lymph nodes); the remaining 16 dogs showed one or more symptoms mentioned above. the prevalence of the infection was 27% (95% ci, 21%‑32%). discussion canl constitutes a considerable veterinary challenge, as well as an important public health problem. pantelleria island (sicily) is featured by optimal conditions to study a well‑defined population of animals and pathogens. the prevalence of leishmania infection, estimated from the model, was 27% (95% ci, 21%‑32%). this prevalence was lower than lipari island (41.7%) but similar to the vulcano island (23.6%) (otranto et al. 2017), while seroprevalence (30.88%) was lower than lampedusa island seroprevalence (54%) (foglia manzillo et al. 2018). this variability in canine seroprevalence in sicilian islands could be due to differences in disease surveillance and, importantly, for the presence of a kennel in pantelleria which surely promotes stray dog population control. this finding, however, could be also related to the differences of the population under examination. repellent tools (collars, spot‑on and spray formulations) performed on 90% of the dog study population had surely a protective effect. repellent typical clinical sign of canl: 6.62% of dogs showed weight loss, 11.85% cutaneous signs, 3.73% ocular signs while 16.18% of examined dogs had enlarged lymph nodes 30.88% of the dogs examined (42/136) were positive at ifat with titers ranging from 1:160 to 1:5,120 and 51 of 136 exhibiting an antibody titer from 1:40 to 1:80. table iv shows the results obtained in the logistic regression after applying the stepwise backward variable selection method. the results obtained from the analysis showed that the age and the enlarged lymph nodes were positively associated with serum‑positivity to ifat, ie dogs over 5 years of age had a probability of being positive with l.  infantum 3 times higher compared to older dogs less than or equal to 5 years; while dogs with enlarged lymph nodes were 4 times more likely to be seropositive to l.  infantum than dogs with no enlarged lymph nodes. real time pcr assay (qpcr) was carried out from 136 blood and 102 lymph node samples, providing useful information to support the tissue of choice for diagnosis of canl. among qpcr 73/102 negative dogs 27 samples showed negative ifat titre (< 1:40), 24 displayed a threshold titre (from 1:40 to 1:80) and 22 dogs were positive (≥ 1:160) (table  v). twenty‑nine dogs (28.43%) tested positive for l. infantum dna by qpcr from lymph node matrices with different parasite load. only one dog tested qpcr positive (0.73%) in blood showing higher parasite load (> 1000 leishmania/ table iii. clinical signs in absolute frequencies and percentage values observed in dog. absolute frequency % clinical signs yes 30 25.73 no 106 74.27 weight loss yes 9 6.62 no 127 93.38 cutaneous signs yes 16 11.85 no 119 88.05 enlarged lymph nodes yes 22 16.18 no 114 83.82 ocular signs yes 5 3.73 no 126 96.27 cohabitation with other dogs yes 95 71.97 no 37 28.03 table iv. coefficient and or for logistic regression on ifat and age, enlarged lymph nodes. variables coefficient p-value or 95% ci for or lower upper age 0.981 0.017 2.67 1.192 5.961 enlarged lymph nodes 1.357 0.017 3.89 1.277 11.825 ci = confidence interval; or = odds ratio. table v. detection and accurate parasite quantification of the qpcr in lymph nodes aspirates. lymph node aspirates qpcr negative ifat titre (< 1:40) threshold ifat titre (1:40 and 1:80) positive ifat titre (≥ 1:160) total negative 27 24 22 73 1-10 leishmania/ml 0 2 4 6 10-100 leishmania/ml 0 3 2 5 100-1,000 leishmania/ml 1 4 2 7 > 1,000 leishmania/ml 0 2 9 11 102 106 veterinaria italiana 2020, 56 (2), 103‑107. doi: 10.12834/vetit.2059.10976.3 cross‑sectional survey of canine leishmaniasis in sicily vitale et al. one dog showed parasite's dna in blood, this study also suggests that blood matrices is inadequate for qpcr, since hematogenous dissemination occurs only rarely. on the other hand, these results indicated that lymph nodes were the matrix of choice for molecular detection of canl in dogs (martínez et al. 2011, moreira et al. 2007). overall, the combination of serology, direct detecton of dna and clinical examination should be performed in order to reach a correct diagnosis. the absence of reports of clinical case of human cutaneous/visceral leishmaniasis infection in pantelleria island during the period of our study could be related to asymptomatic leishmania infections in immunocompetent hosts without clinically evident disease. acknowledgements this research was granted by ministry of health rc izs si 01/2017. tools remains the most effective antivectorial method to control l.  infantum. furthermore, the presence of seropositivity of dogs older than 5 years (30.88%), was positively associated with leishmania infection. during investigation, cutaneous and ocular signs and weight loss observed were related to leishmania disease in dogs, since percentages of association obtained are very low. only enlarged lymph nodes were positively associated with leishmania infection, even if only 16,18 % of dogs presented this clinical sign (table iv). early canl diagnosis was crucial to identify infectious dogs through several diagnostic tests, but the correct interpretations of these were of great importance to make an accurate diagnosis of the disease. ifat test ‘gold standard’ technique for mass screening dogs, could however generate false positive results due to both serological cross‑reactivity with other pathogens and low sensitivity in asymptomatic dogs. the use of qpcr for the assessment of parasite load could be more informative. however, as 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plos negl trop dis, 10 (9), e0004987. busani l., gras l.m., romi r., boccolini d., severini f. & bongiorno g. 2012. zanzare, flebotomi e zecche: atlante bibliografico delle specie d’interesse sanitario in italia (1985‑2009). rapporti istisan 12/22. roma, istituto superiore di sanità. cabral m., o’grady j.e., gomes s., sousa j.c., thompson h. & alexander j. 1998. the immunology of canine leishmaniosis: strong evidence for a developing disease spectrum from asymptomatic dogs. vet parasit, 76, 173‑180. cardoso l., neto f., sousa j.c., rodrigues m. & cabral m. 1998. use of a leishmanin skin test in the detection of canine leishmania‑specific cellular immunity. vet parasitol, 79, 213‑220. cascio a., colomba c., antinori s., orobello m., paterson d. & titone l. 2002. pediatric visceral leishmaniasis in western sicily, italy: a retrospective analysis of 111 cases. eur j clin microbiol infect dis, 21 (4), 277‑282. fernández‑bellon h., solano‑gallego l., rodríguez‑cortés a., ferrer l., gallego m., alberola j. & ramis a. 2008. little evidence of seasonal variation of natural infection by leishmania infantum in dogs in spain. vet parasitol, 155, 32‑36. foglia manzillo v., gizzarelli m., vitale f., montagnaro s., torina a., sotera s. & oliva g. 2018. serological and entomological survey of canine leishmaniasis in lampedusa island, italy. bmc vet res, 14 (1), 286. references gramiccia m., scalone a., di muccio t., orsini s., fiorentino e. & gradoni l. 2013. the burden of visceral leishmaniasis in italy from 1982 to 2012: a retrospective analysis of the multi‑annual epidemic that occurred from 1989 to 2009. euro surveillance, 18, 20535. martínez v., quilez j., sanchez a., roura x., francino o. & altet l. 2011. canine leishmaniasis: the key points for qpcr result interpretation. parasit vectors, 4, 57. molina r., amela c. & nieto j. 1994. infectivity of dogs naturally infected with leishmania infantum to colonized phlebotomus perniciosus. trans r soc trop med hyg, 88, 491‑493. moreira m.a.b., luvizotto m.c.r., garcia j.f., corbett c.e.p. & laurenti m.d. 2007. comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs. vet parasitol, 145, 245‑252. otranto d., napoli e., latrofa m.s., annoscia g., tarallo v.d., greco g., lorusso e., gulotta l., falsone l., basano f.s., pennisi m.g., deuster k., capelli g., dantas‑torres f. & brianti e. 2017. feline and canine leishmaniosis and other vector‑borne diseases in the aeolian islands: pathogen and vector circulation in a confined environment. vet parasitol, 15 (236), 144‑151. solano‑gallego l., miró g, koutinas a., cardoso l., pennisi m.g., ferrer l., bourdeau p., oliva g. & baneth g. 2011. leishvet guidelines for the practical management of canine leishmaniosis. parasit vectors, 4, 86. solano‑gallego l., morell p., arboix m., alberola j. & ferrer l. 2001. prevalence of leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using pcr on several tissues and serology. j clin microbiol, 39, 560‑556. vitale f., reale s., vitale m., petrotta e., torina a. & caracappa s. 2004. taqman‑based detection of leishmania infantum dna using canine samples. ann n y acad sci, 1026, 139‑143. world organization for animal health (oie). 2018. chapter. 3.1.11 – leishmaniosis (most recent updates adopted in 2014). in manual of diagnostic tests and vaccines for terrestrial animals, oie, paris. https:// www.oie.int/fileadmin/home/eng/health_standards/ tahm/3.01.11_leishmaniosis.pdf. 265 introduction q fever is a zoonosis with a world‑wide distribution (except new zealand) caused by coxiella burnetii, an obligatory intracellular bacterium. q or ‘query’ fever was first observed in slaughterhouse workers in brisbane, queensland, australia in 1933 and was initially described by derrick as a self‑limiting febrile illness of unknown etiology (derrick 1937). at that time, the etiological agent was considered to be a virus and all trials to isolate the pathogen by inoculating guinea pigs with blood or urine of infected patients were unsuccessful (derrick 1937). burnet and freeman (burnet and freeman 1937) isolated an intracellular bacterium from guinea pigs that had been previously injected with blood or urine from the infected slaughterhouse workers and named it rickettsia burnetii. in the same period, a laboratory‑acquired fever infection occurred in the rocky mountain laboratory in hamilton, montana, laboratory of hygiene of foods of animal origin, faculty of veterinary medicine, university of thessaly, greece * corresponding author at: laboratory of hygiene of foods of animal origin, faculty of veterinary medicine, university of thessaly, greece. e‑mail: apexara@vet.uth.gr. parole chiave coxiella burnetii, ruminanti domestici, febbre q, sieroprevalenza. riassunto la febbre q è una zoonosi causata dal batterio gram‑negativo coxiella burnetii, un patogeno intracellulare obbligato. i principali serbatoi e fonte di infezione umana sono i ruminanti ma l'infezione da c. burnetii è stata dimostrata in molte specie animali. nei ruminanti è spesso asintomatica ma è stata anche associata a infertilità e aborti; nell'uomo, è stata considerata prevalentemente un rischio professionale, determinata dal contatto con prodotti infetti come placenta, urina, feci o latte. la febbre q è tornata ad essere un'emergenza per la salute pubblica dopo la vasta epidemia avvenuta nei paesi bassi tra il 2007 e il 2009. nonostante la sieroprevalenza di c. burnetii nei ruminanti sia comunemente rilevata dai vari test diagnostici utilizzati nei laboratori, non è ancora disponibile un metodo ufficiale per questo patogeno. le numerose indagini condotte in vari paesi mostrano percentuali elevate di infezione nei ruminanti domestici specie nelle pecore e nella capre. l'unico paese con una prevalenza apparente zero è la nuova zelanda. febbre q e sieroprevalenza di coxiella burnetii nei ruminanti domestici keywords coxiella burnetii, domestic ruminants, q fever, seroprevalence. summary q fever is a zoonosis caused by coxiella burnetii, an obligate intracellular gram‑negative bacterium. infection by c. burnetii has been demonstrated in many animal species, but ruminants are the major reservoirs and the main sources of human infection. in ruminants, c. burnetii infection is often asymptomatic, but it has been also associated with infertility and abortions. in humans, q fever was considered predominately an occupational hazard due to close contact with infected ruminants by means of their contaminated birth products, urine, feces or milk. q fever has recently gained renewed attention after the large outbreak in the netherlands in 2007‑2009, indicating its importance as an emerging public health threat. the seroprevalence of c. burnetii in ruminants is commonly detected by various tests but no official standard technique is still available. according to surveys conducted in many countries of the five continents, a relatively high proportion of farm ruminants are found seropositive to c. burnetii. the only country with an apparent zero prevalence is new zealand. the seroprevalence in goats and sheep is usually higher than cattle. andreana pexara*, nikolaos solomakos and alexander govaris q fever and seroprevalence of coxiella burnetii in domestic ruminants veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 accepted: 01.12.2016 | available on line: 31.12.2018 266 veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 primary infection more than half of the patients remain asymptomatic. q fever may manifest as acute or chronic q  fever with long‑term sequelae. acute q  fever usually develops as a non‑specific febrile illness, pneumonia or hepatitis (karakousis and trucksis 2006). atypical pneumonia and hepatitis are usually the most classic forms of q  fever. hepatitis may be expressed as infectious‑like hepatitis or fever of unknown origin (fuo) with characteristic hepatic granulomas on liver biopsy. less common manifestations of acute q fever include myocarditis, pericarditis, meningoencephalitis and skin rash (angelakis and raoult 2010). the chronic infection can manifest itself as endocarditis, chronic fatigue syndrome and problems related to pregnancy (arricau‑bouvery and rodolakis 2005). q  fever was considered predominately an occupational hazard and close contact with ruminants appeared to be strongly associated with the disease in humans (psaroulaki et al. 2006). q fever has recently gained renewed attention after the largest‑ever recorded outbreak which involved over 4,000 human cases in the netherlands in 2007‑2009 (vanderburg et  al. 2014). this outbreak highlighted the importance of q  fever as an emerging public health threat. moreover, the widespread distribution of c.  burnetii in food producing animals and its occurrence in foods of animal origin, particularly in milk, necessitates the investigation of food as a significant vehicle for the transmission of this zoonotic bacterium to humans. unpasteurized milk is the most significant source of c.  burnetii. there are epidemiological indications that consumption of milk and/or milk products containing c.  burnetii has been associated with sero‑conversion in humans. moreover, unpasteurized milk and derived dairy products have been proposed by several authors as sources of human infection (fishbein and raoult 1992, hatchette et  al. 2001, maltezou et  al. 2004). however, the contribution of milk ingestion, mainly drinking unpasteurized milk, to q fever infection in humans is difficult to establish. moreover, c. burnetii was detected in animal products such as raw‑milk cheese and butter prepared from raw milk as well as in the meat of infected animals (capuano et  al. 2012, hirai et al. 2012, eldin et al. 2013, hilbert et al. 2015). the pathogen was detected even in chicken eggs from japan and iran (tatsumi et  al. 2006, rahimi and doosti 2012). in addition to the adverse effects to human health, c.  burnetii infection in animals can result in the decrease of the livestock production with important socioeconomic effects (perry et  al. 2011). this review summarizes the current knowledge on c.  burnetii infection in domestic ruminants with special focus on serological prevalence. usa (davis and cox 1938). as dermacentor andersoni was collected near the infected guinea pigs with a febrile illness and enlarged spleens in nine mile creek, montana, it was concluded that the fever was acquired by means of possible vectors (cox 1938). the causative agent with filterable properties was characterized as the ‘nine mile agent’. the organism was observed intravacuolarly in infected tissue cultures and could be also transmitted to humans (cox 1938). the american and australian research groups then demonstrated that the australian q fever and the nine mile agents were in fact isolates of the same microorganism which was classified as rickettia burnetii (maurin and raoult 1999). in 1948, philip re‑classified r. burnetii according to cultural and biochemical characteristics. to honor both cox and burnet, the q fever pioneers, they re‑named it as coxiella burnetii (philip 1948). in europe, q  fever was first reported in humans in greece during the second world war, when the microorganism was detected in sera of german soldiers who had febrile illness, known as the ‘balkan flu’ (caminopetros 1946). in 1945, american soldiers who returned to usa from italy, developed an acute febrile illness accompanied by pneumonia. the cause of the epidemic was identified by serological test as ricketsia of q  fever (commission on acute respiratory diseases 1946). q  fever is listed within the category of multiple species diseases in the world organisation for animal health list (oie 2016). several domestic and wild animals as well as birds, reptiles and arthropods (particularly ticks) can harbour the pathogen, but cattle, goats and sheep are the main reservoirs. in most animals the infection is asymptomatic, but abortions or stillbirths may occur. the bacteria are spread to the environment by secretions of infected animals (urine, feces and milk) but predominantly via the birth products (more than 109 bacteria/g placenta) (arricau‑bouvery and rodolakis 2005). in humans, the airborne pathway is the main mode of transmission. the infection is usually caused by inhalation of infectious aerosols directly from birth fluids or via inhalation of dust contaminated by dried placental material, birth fluids and excreta of infected animals (tissot‑dupont and raoult 2008). the bacterium can become airborne, traveling on wind currents for miles, resulting in outbreaks (tissot‑dupont et  al. 2004). humans can also be infected by direct contact with infected animals particularly during abortion and parturition. the infection in humans by ingestion of unpasteurized milk or dairy products, has been also recorded (tissot‑dupont and raoult 2008). in addition to ruminants, cats and dogs are also able to shed the organism. in humans, the main characteristic of q  fever is its clinical polymorphism. following coxiella burnetii in domestic ruminants pexara et al. 267veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 remain viable for several months in dairy products, meat and meat products, water, aborted foetuses, manure, wool, hay, equipment, and clothing during conditions of high humidity, low temperatures, and no sunlight (efsa 2010). for example, c. burnetii can survive 12  to  16 months in wool, 120  days in dust, 49  days in dried urine and 30  days in dried sputum (nabc 2010). c.  burnetii has two distinct antigenic phases, phase i and phase ii. phase i and ii variants are morphologically identical, but differ in some biochemical characteristics including their lipopolysaccharide (lps) composition. lps  ii is of rough type in contrast to lps  i, which is phenotypically smooth and contains a noticeable amount of two sugars virenose and dihydrohydroxystreptose (narasaki and toman 2012). organisms isolated from infected animals or humans express phase i antigens and are highly infectious. organisms expressing phase ii antigens are less infectious and are obtained by repeated in ovo or in vivo passages. in experimentally infected animals, antibodies to phase ii antigens are initially produced, while antibodies to phase i antigens are produced in later stages. c. burnetii is able to survive permanently inside the macrophages, causing an infection after an acute episode (gwida et al. 2012). q fever in domestic ruminants domestic ruminants are the primary animal reservoirs of c. burnetii. q  fever is an airborne disease and inhalation of infected aerosols and dust is the main route of infection of domestic ruminants (tissot‑dupont et  al. 2004). also, ruminants may become infected by ingestion of contaminated pastures, hay and straw (maurin and raoult 1999). it is likely that c. burnetii contaminated manure plays a role on the maintenance of infection in animal populations (efsa 2010). the pathogen has been isolated in several tick species (maurin and raoult 1999). ticks appear to play an important role in enzootic transmission cycles in domestic ruminants (beaman and hung 1989). a strong correlation has been reported between seropositivity and ticks’ infestation in animals (psaroulaki et al. 2006). the presence of q fever in animals is also related to the characteristics of certain c.  burnetii strains, and in particular infectivity, virulence and resistance to environmental conditions (barberio 2015). maintenance of c.  burnetii infection in animal populations may be also affected by other factors such as manure management (capture, storage, treatment and utilization), farm characteristics (herd/flock size, animal and herd/flock density) and farm environmental conditions (temperature and relative humidity) (efsa 2010). characteristics of the bacterium coxiella burnetii c.  burnetii is a small pleomorphic rod (0.2‑0.4  mm wide, 0.4‑1.0  mm long) with a membrane similar to that of a gram‑negative bacterium (maurin and raoult 1999). it replicates to high numbers within a parasitophorous vacuole of eukaryotic host cells, with an estimated doubling time of 20‑45  h (mertens and samuel 2007). c.  burnetii was originally classified in the rickettsiales order, the rickettsiaceae family, and the rickettsiae tribe together with the genera rickettsia and rochalimaea. to date, following philogenetic investigations based on genome comparison and 16s rrna sequence analysis, the bacterium was reclassified from the order rickettsiales to legionellales, and falls in the gamma group of proteobacteria (raoult et al. 2005). the microrganism produces resistant spore‑like forms (coleman et  al. 2004). this ability has been attributed to the existence of c.  burnetii cycle variants described in in vitro studies: large‑cell variants (lcv), small‑cell variants (scv), and small dense cells (sdc) (coleman et  al. 2004). the lcv is the larger and the metabolically active intracellular form of c.  burnetii. it undergoes sporogenic differentiation and produces the resistant, spore‑like forms. the sdc and scv represent the small morphological variants of the bacteria likely to survive extracellularly as infectious particles, a trait that is important for persistence in the environment and transmission (ecdc 2010, kersh et al. 2010). the environmentally stable scv (or endospore) is the form phagocytosed by macrophages during early infection and the form associated to food‑borne risk (efsa 2010). the endospores display a tropism for reproductive organs including the mammary gland, are secreted in the milk of infected animals, both from clinical cases and asymptomatic carriers and are also excreted in the detritus of normal births and abortions as well as in the urine and faeces of infected animals. endospores are released after mother cell lysis and since they are metabolically inactive, they remain stable in soil and dust over many years (angelakis and raoult 2010) and can be spread in dust or windborne aerosols for up to 11 miles (18 km) (hawker et al. 1998). c.  burnetii is resistant to acids (up to ph 4.5), temperature (62°c for 30 min), uv light and pressure (up to 300,000 kpa) (frangoulidis 2010). furthermore, the organism can survive for more than 6 months in 10% salt solutions. c.  burnetii is killed by exposure for 30 min to 5% h 2 o 2 , 0.5% hypochlorite, 70% ethanol, for less than 30  min to 5% chloroform or formaldehyde gas (in a 80% humidified environment). pasteurization of milk (71.66°c for 15 s) is effective in killing c. burnetii (frangoulidis 2010). endospores can pexara et al. coxiella burnetii in domestic ruminants 268 veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 coxiella burnetii in domestic ruminants pexara et al. in faeces (rodolakis et  al. 2007). in asymptomatic herds, apparently healthy goats and cows may shed the bacterium in milk for several months or years (rodolakis et  al. 2007). c.  burnetii has been also isolated in bull semen but the role of males in the persistence of infection has not extensively examined (kruszewska et al. 1997). q fever diagnosis in domestic ruminants since c.  burnetii infection is frequently subclinical, q fever disease in domestic ruminants remains unclear and not properly investigated. reproductive disorders should trigger an investigation, considering q  fever among the differential diagnoses. in many countries, diagnosis of q fever in domestic ruminants still relies mainly on modified ziehl‑neelsen (mzn)‑stained smears of placental material from aborted fetuses, supplemented by immunohistochemistry (ihc) where appropriate, although polymerase chain reaction (pcr) is increasingly being used for disease confirmation in developed countries (jones et  al. 2010). pcr can be used to detect c.  burnetii dna in a wide range of samples, including placenta tissues, faeces, vaginal mucus and milk (horigan et  al. 2011). high level of specifity and sensitivity were acquired by pcr method applied with the primers consisting of repetitive transposon‑like element (kırkan et al. 2008). real‑time pcr is now also commonly used to support a diagnosis of c. burnetii abortion/stillbirth in animals faeces, vaginal mucus and milk (horigan et al. 2011). a variety of indirect methods (serologic assays) have been used to detect c.  burnetii antibodies in animal serum samples, including complement fixation test (cft), enzyme linked immunosorbent assay (elisa), microagglutination test (ma), indirect immunofluorescence assay (ifa) and indirect fluorescent antibody test (ifat) (roest et  al. 2013). the cft is weakly sensitive and the antigen used in this test frequently fails to detect antibodies in sheep or goats (horigan et  al. 2011). the elisa is more sensitive than the cft and is able to test a higher number of animals and flocks (rodolakis 2006). a combination of both direct and indirect methods is recommended in current protocols to detect q  fever on herd level; however, no official standard technique is still available (roest et al. 2013). efsa outlined the need for harmonized schemes for the passive and active monitoring/reporting of q  fever in animals so that its prevalence/incidence could be compared over time and between countries (efsa 2010). recently, some proposals have been elaborated for the development of harmonized monitoring and reporting schemes for the seroprevalence of c. burnetii in various countries. clinical signs of q fever in domestic ruminants although c. burnetii infection in domestic ruminants is common, clinical disease is rather rare. the infection is generally asymptomatic; but sometimes it can induce reproductive disorders, which differs among ruminant species. infected sheep may deliver live or dead lambs as well as large abortion waves, mainly at the end of gestation, without specific signs until abortion is imminent (roest et al. 2013). in contrast to ewes, goats remain chronically infected and can abort twice following an infection (berri et  al. 2007). martinov (martinov 2007) has described also experimentally induced q fever with respiratory manifestations in sheep. in cattle, yet the symptoms described have so far been inconsistent. factors linked to the disease in cattle have been infertility, abortion and metritis and mastitis in many studies (arricau‑bouvery and rodolakis 2005, barlow et  al. 2008). also, correlation between c.  burnetii seropositivity and fertility and a low abortion risk has been reported (lopez‑gatius et  al. 2012, garcia‑ispierto et  al. 2013). however, the presence of c. burnetii in dairy herds has been not yet clearly demonstrated to negatively affect reproductive performance. in fact, a recent study showed that seropositive shedding cows had better reproduction than non‑infected cows (garcia‑ispierto et al. 2013). both symptomatic and asymptomatic infected ruminants shed c.  burnetii in large amount in the environment. shedding of c.  burnetii into the environment mainly occurs during abortion and parturition; placentas of infected small ruminants can contain over 109 hamster infective doses or bacteria per gram of tissue (fournier et  al. 1998). goats may shed the bacterium in placenta and vaginal mucus in two successive parturitions after a q fever infection (berri et al. 2007). at their first kidding, young goats shed more c. burnetii cells than adults (de cremoux et  al. 2012). a similar pattern is observed in cattle herds (guatteo et  al. 2008). in contrast, ewes are usually show one abortion and did not shed the pathogen in vaginal mucus at subsequent lambing (berri et  al. 2002). the pathogen excretion can also occur in faeces, milk and urine. c.  burnetii shedding can persist for a long time. in goats, shedding of c.  burnetii in vaginal mucus, faeces, and milk lasted 1 to 5 weeks, 2 to 5 weeks, and 1 day to 6 weeks, respectively. in sheep, the shedding lasted 71 days, 8 days after lambing and 8  days in vaginal mucus, faeces, and milk, respectively. in cows, longest observed duration of excretion of c. burnetii in faeces and milk was 14 days and 13  months, respectively (arricau‑bouvery and rodolakis 2005). goats and cows mostly shed c.  burnetii in milk (guatteo et  al. 2006, rodolakis et al. 2007) whereas ewes shed the pathogen mostly 269veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 pexara et al. coxiella burnetii in domestic ruminants summarized in table i. in the netherlands, according to an early (1987) survey of seroprevalence of q fever by using elisa showed values of 3.5% and 1% in sheep and goats, respectively (houwers and richardus 1987). more recently, according to results of a serological study in dutch cattle, c.  burnetii antibodies were present in 16% of lactating cows, but only in 1.0% of young animals (muskens et  al. 2011). in switzerland, magouras and colleagues (magouras et  al. 2017) reported a herd‑level seroprevalence of 5.0% in sheep flocks and 11.1% in goat farms. animal‑level seroprevalence was 1.8% in sheep and 3.4% in goats. in germany (southern bavaria), seroprevalence of c.  burnetii in cattle tested by elisa was estimated 11.8% at animal‑level and 81.0% at herd‑level (rehácek et al. 1993). in another study conducted in germany, 7.8% of cattle, 1.3% of sheep and 2.5% of goats were found to be infected with c. burnetii (hartung 1999). in a study conducted in france, seroprevalence was found in 13/14 and 24/28 of dairy goat herds in 2006 and 2008, respectively (dubuc‑forfait et  al. 2009). in demark, serum samples taken from 164  high‑risk cows with abortion problems showed a prevalence of 10% by cft and 18% by elisa, in 2003. in denmark, given the lack of a standard technique, efforts are encouraged both for the validation of the methods and for development of reference reagents for quality control, proficiency and harmonization purposes. the seroprevalence of c. burnetii in domestic ruminants q fever in animals has been detected worldwide, whilst the only country with an apparent zero prevalence is new zealand (guatteo et  al. 2011). although a great number of c.  burnetii seroprevalence studies have been conducted throughout the world, the extent of disease in productive ruminants is difficult to quantify. data reported are mostly based on herds with high increase in abortion (georgiou 2013). published data revealed differences in seroprevalence in the animal population in various countries. europe accomplished data on the serological prevalence of c.  burnetii in domestic ruminants from published studies conducted in european countries are table i. the serological prevalence of c. burnetii in domestic ruminants from published studies conducted in european countries. — cont’d country year of study animal species number tested % positive method reference animals (n) herds (n) animals herds albania 1995‑1997 cattle 311 ‑ 10.9 ‑ elisa cekani et al. 2008 1995‑1997 sheep 350 ‑ 8.8 ‑ elisa cekani et al. 2008 1995‑1997 goat 443 ‑ 8.9 ‑ elisa cekani et al. 2008 1999 cattle 552 ‑ 8.5 ‑ elisa cekani et al. 2008 1999 sheep 292 ‑ 12.3 ‑ elisa cekani et al. 2008 1999 goat 260 ‑ 4.2 ‑ elisa cekani et al. 2008 austria ‑ sheep ‑ 70 ‑ 2.86 cft wagner et al. 2005 ‑ goat ‑ 30 ‑ 16.7 cft wagner et al. 2005 bulgaria 2002‑2006 cattle 15,866 ‑ 8.53 ‑ cft martinov 2007 2002‑2006 sheep 8,727 ‑ 11,59 ‑ cft martinov 2007 2002‑2006 goat 3,928 ‑ 13,69 ‑ cft martinov 2007 cyprus ‑ cattle 75 ‑ 24 ‑ ifa psaroulaki et al. 2006 ‑ sheep 481 ‑ 18.9 ‑ ifa psaroulaki et al. 2006 ‑ goat 417 ‑ 48,2 ‑ ifa psaroulaki et al. 2006 denmark 2003 cattle 164 10 cft christoffersen 2007 18 elisa 2004 cattle 80 ‑ 35 ‑ elisa christoffersen 2007 2006 cattle 266 ‑ 25 ‑ elisa christoffersen 2007 france 2006 goat 359 14 36 92.9 elisa dubuc‑forfait et al. 2009 2008 goat 1,057 28 32 85.7 elisa dubuc‑forfait et al. 2009 germany 1991 cattle 1,095 ‑ 12 ‑ elisa rehácek et al. 1993 1998 cattle 21,191 ‑ 7.8 ‑ elisa hartung 1999 1998 sheep 1,346 ‑ 1.3 ‑ elisa hartung 1999 1998 goat 278 ‑ 2.5 ‑ elisa hartung 1999 ifa = indirect immunofluorescence assay; ifat = indirect fluorescent antibody test; elisa = enzyme linked immunosorbent assay; cft = complement fixation test; mat = microagglutination test. continued 270 veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 coxiella burnetii in domestic ruminants pexara et al. 38% and 47% of sheep and goat herds, respectively (masala et  al. 2004). in northern italy, 44.9% out of 650  cattle with experienced abortion were seropositive for c.  burnetii (cabassi et  al. 2006). in northwest italy, the animal level seroprevalence was 15.5% in sheep and 16.2% in goats. the sheep‑farm and goat‑farm seroprevalence was 38.7% and 19.5%, respectively (rizzo et al. 2016). in northern ireland, 6.2% of cattle and 48.4% of tested herds were seropositive in 2009 (mccaughey et  al. 2010). in great britain, estimates of animal and flock/herd seroprevalences were 0.9% and 10.2%, respectively, for sheep and 0.8% and 3%, respectively, for goats (lambton et al. 2016). a seroprevalence of 35% was found in blood samples from 80 cattle in 2004, and 25% in 266 cattle in 2006 (christoffersen 2007). in austria, wagner and colleagues (wagner et  al. 2005) examined blood samples from 70 styrian sheep by cft and found that 1.5 % of the samples contained antibodies to c. burnetii. in italy (emilia‑romagna region), the q  fever seroprevalence in cattle was 13.1% at herd level and 4.4% at animal level (martini et al. 1994). in italy (campania), capuano and colleagues (capuano et  al. 2004) reported a seroprevalence of 11.8% in sheep, 6.3% in goats and 14% in cattle. in sardinia, italy, antibodies to c. burnetii have been detected in table i. the serological prevalence of c. burnetii in domestic ruminants from published studies conducted in european countries.— cont’d country year of study animal species number tested % positive method reference animals (n) herds (n) animals herds greece ‑ sheep 554 ‑ 10.5 ‑ ifa pape et al. 2009 ‑ goat 61 ‑ 6.6 ‑ ifa pape et al. 2009 italy ‑ cattle 711 99 4.4 13.1 cft martini et al. 1994 ‑ cattle ‑ ‑ 14 ‑ ifat capuano et al. 2004 ‑ sheep ‑ ‑ 11.8 ‑ ifat capuano et al. 2004 ‑ goat ‑ ‑ 6.3 ‑ ifat capuano et al. 2004 1999‑2002 sheep ‑ 675 ‑ 38 elisa masala et al. 2004 1999‑2002 goat ‑ 82 ‑ 47 masala et al. 2004 ‑ cattle 650 ‑ 44.9 ‑ elisa cabassi et al. 2006 2012 sheep 2,553 111 15.5 38.7 elisa rizzo et al. 2016 2012 goat 3,185 206 16.2 19.5 elisa rizzo et al. 2016 montenegro ‑ sheep 954 ‑ 5.03 ‑ mat & ifa lausevic 2001 netherlands 1987 sheep 3,603 191 3.5 27.2 elisa houwers and richardus 1987 1987 goats 594 ‑ 1 ‑ elisa houwers and richardus 1987 2008 cattle 2,936 ‑ 16 ‑ elisa muskens et al. 2011 2008 cattle (young animal) 1,831 ‑ 1 ‑ elisa muskens et al. 2011 spain 2007‑2008 cattle 626 46 6.7 43 elisa ruiz‑fons et al. 2010 2007‑2008 sheep 1,379 42 11.8 74 elisa ruiz‑fons et al. 2010 2007‑2008 goat 115 11 8.7 45 elisa ruiz‑fons et al. 2010 2005 sheep 1,011 34 8.9 67.6 elisa garcia‑perez et al. 2009 2009‑2010 cattle 1,306 ‑ 12.3 ‑ elisa astobiza et al. 2012 slovakia 2000 sheep 269 ‑ 37.22 ‑ elisa dorko et al. 2010 2009 sheep 269 ‑ 58.42 ‑ elisa dorko et al. 2010 switzerland ‑ sheep ‑ 100 1.8 5 elisa magouras et al. 2015 ‑ goat ‑ 72 3.4 11.1 elisa magouras et al. 2015 poland ‑ goat 98 ‑ 79.6 ‑ mat platt‑samoraj et al. 2005 2011‑2012 cattle 169 ‑ 11.83 ‑ elisa bielawska‑drózda et al. 2014 2011‑2012 cattle 169 ‑ 10.65 ‑ cft bielawska‑drózda et al. 2014 uk (ireland) 2009 cattle 5,182 273 6.2 48.4 elisa mccaughey et al. 2010 ‑ sheep 1,022 58 12.3 62.1 ifa mccaughey et al. 2010 ‑ goat 54 7 9.3 42.9 ifa mccaughey et al. 2010 uk (great britain) ‑ goat 5791 384 0.9 10.2 elisa lambton et al. 2016 sheep 522 145 0.8 3 elisa lambton et al. 2016 ifa = indirect immunofluorescence assay; ifat = indirect fluorescent antibody test; elisa = enzyme linked immunosorbent assay; cft = complement fixation test; mat = microagglutination test. 271veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 pexara et al. coxiella burnetii in domestic ruminants in 2000 and 58.42% in 2009 (dorko et  al. 2010). in poland, platt‑samoraj and colleagues (platt‑samoraj et  al. 2005) examined serum samples from a goat farm with animals affected with reproductive disorders and found that 79.6% of the samples had c. burnetii antibodies in a more recent survey 11.83% and 10.65% of cattle had c.  burnetii antibodies when serum samples were tested by elisa and cft method, respectively (bielawska‑drózda et al. 2014). in greece, pape and colleagues (pape et  al. 2009) found c. burnetii antibodies in 10% of the animals examined. the seroprevalence was higher in sheep flocks (10.4%) compared to goats’ herds (6.5%). in cyprus, the prevalence of antibodies against c.  burnetii was 48.2% in goats, 18.9% in sheep, and 24% in cattle (psaroulaki et al. 2006). asia, africa, america and oceania data on seroprevalence of c.  burnetii in domestic ruminants collected from studies conducted in countries in asia are presented in table ii. in turkey, cetinkaya and colleagues (cetinkaya et al. 2000) in 1998, found a seroprevalence in spain (basque region), a serosurvey conducted during the years 2007‑2008 showed a prevalence of 11.8%, 8.7% and 6.7% in sheep, goats and beef cattle, respectively (ruiz‑fons et  al. 2010). in the same study, a c.  burnetii prevalence of 74%, 45% and 43% for ovine, caprine and bovine herds, respectively, was also recorded (ruiz‑fons et  al. 2010). in northern spain, 8.9% of the sheep were seropositive, 67.6% of the flocks had at least one seropositive animal, but only 14.7% of them showed a seroprevalence higher than 25% (garcia‑perez et al. 2009). in northern spain, in a study conducted by astobiza et al. (2012) cows showed a statistically significantly higher seroprevalence (12.3%) than heifers (1.1 %) and calves (0%) (astobiza et al. 2012). in albania, cekani and colleagues (cekani et  al. 2008) conducted a survey on c.  burnetii and reported that the seroprevalence detected in sheep and goats (9.8%) was higher than in cattle (7.9%). in bulgaria, martinov (martinov 2007) found that the seropositivity for c.  burnetii was 8.53% in cattle, 11.59% in sheep and 13.69% in goats. in montenegro, lausevic (lausevic 2001) reported a 5.03% seroprevalence of c.  burnetii in sheep. in slovakia, the seropositivity in sheep was 37.22% table ii. the serological prevalence of c. burnetii in domestic ruminants from published studies conducted in asian countries. — cont’d country year of study animal species number tested % positive method reference animals (n) herds (n) animals herds bangladesh 2009‑2010 cattle 620 ‑ 0.65 ‑ elisa haider et al. 2015 2009‑2010 goats 529 ‑ 0.76 ‑ elisa haider et al. 2015 china ‑ cattle 1140 19 33 84 elisa el‑mahallawy et al. 2016 2011‑2013 sheep 2112 ‑ 14.39 ‑ elisa yin et al. 2015 iran 2008 cattle 93 12 10.75 16.6 elisa khalili and sakhaee 2009 2008 goats 76 9 65.78 100 elisa khalili and sakhaee 2009 2009 sheep 85 29.42 elisa sakhaee and khalili 2010 2010 cattle 246 19 22.3 78.9 elisa azizzadeh et al. 2011 2010‑2011 sheep 253 ‑ 23.7 ‑ elisa mostafavi et al. 2012 2011‑2012 sheep 1100 ‑ 19.5 ‑ elisa asadi et al. 2013 2011‑2012 goat 180 ‑ 27.2 ‑ elisa asadi et al. 2013 ‑ sheep 255 29 36.5 89.6 elisa keyvani et al. 2014 ‑ goat 205 28 29.8 78.5 elisa keyvani et al. 2014 2014 cattle 120 10 0.83 10 elisa edalati‑shokat et al. 2015 2014 sheep 200 10 27.5 100 elisa edalati‑shokat et al. 2015 2014 goat 50 10 54 100 elisa edalati‑shokat et al. 2015 ‑ sheep 253 33.6 87.50 elisa esmaeili et al. 2014 japan 1982‑1991 cattle 562 ‑ 46.6 ‑ ifat htwe et al. 1992 1974‑1989 sheep 256 ‑ 28.1 ‑ ifat htwe et al. 1992 1974‑1989 goat 85 ‑ 23.5 ‑ ifat htwe et al. 1992 korea 2010 cattle 1000 ‑ 1.3 ‑ elisa jang et al. 2011 2010‑2013 cattle 1,095 ‑ 6.2 ‑ elisa kim et al. 2014 ‑ goat 575 ‑ 19.1 ‑ elisa jung et al. 2014 ifa = indirect immunofluorescence assay; ifat = indirect fluorescent antibody test; elisa = enzyme linked immunosorbent assay; cft = complement fixation test; mat = microagglutination test. continued 272 veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 coxiella burnetii in domestic ruminants pexara et al. of c. burnetii of 5.8% and 35.4% in cattle at herd and animal level, respectively, and a seroprevalence of 10.5% and 44.7% at flock and animal level, respectively. in the same country, kalender (kalender 2001) reported a c. burnetii seropositivity of 38.59% in aborted ewes and of 11.01% in non aborted ewes. in western turkey, kilic and colleagues (kilic et  al. 2005) found a relative low seropositivity in sheep (3%). in the same country, according to a study of seyitoğlu and colleagues (seyitoğlu et al. 2006), the seropositivity was 5.6% in healthy cattle and 22.6% in cattle with an abortion history. in eastern turkey, the seropositivity was found to be 16.3% in cattle and 5.4% in sheep (ceylan et  al. 2009). two other separate studies in sheep showed a seroprevalence of c.  burnetii of 21.07% (karaca et  al. 2009) and 20% (kennerman et  al. 2010). more recently, a seroprevalence of 12.4% was found in dairy cattle (gazyagci et al. 2011) and of 20.0%, 29.0% and 21.0% in cattle, sheep and goats, respectively, when tested by elisa. in the same animals the prevalence was of 22.0%, 29.0% and 23.0%, respectively when tested by ifa (parin and kaya 2015). in southeast iran, in 2008, a significantly higher average seroprevalence (65.78%) was observed in goats than in cattle (10.75%) (khalili and sakhaee 2009). the seroprevalence of c.  burnetii was 29.42% and 23.7% in sheep in two studies conducted in southeast iran (sakhaee and khalili 2010) and northen iran, respectively (mostafavi et  al. 2012). in eastern iran, 22.3% of dairy cattle were found seropositive to c.  burnetii according to a study conducted by azizzadeh and colleagues (azizzadeh et  al. 2011), in 2010. in northeastearn iran, seroprevalence of c.  burnetii at animal level was 36.5% for sheep and 29.8% for goat populations (keyvani et  al. 2014). in northwestern iran, 33.6% of sheep and 87.50% of sheep herds were found positive for c. burnetii (esmaeili et al. 2014). recently, in western iran, antibodies to c.  burnetii were detected in 27.5% of sheep, in 54% of goats and in 0.83% of dairy cattle (edalati‑shokat et  al. 2015). in iran, according to a study conducted by asadi and colleagues (asadi et  al. 2013), the seroprevalence of c.  burnetii in sheep and goats with a history of abortion was 19.5% and 27.2%, respectively. in pakistan, seroprevalence was 26.9% and 34.9% for sheep and goat, respectively (zahid et  al. 2016). in bangladesh, a low seropositivity in cattle and goats (0.65% and 0.76%, respectively) was recorded by haider and colleagues (haider et al. 2016). in japan, a survey on prevalence of c. burnetii antibodies in healthy ruminant farms showed values of 25.4% in cattle, 28.1% in sheep and 23.5% in goats. in this study, the seroprevalence reached values of 84.3% in bovine herds with reproductive disorders (htwe et  al. 1992). in china, c.  burnetii seropositive cattle (33% of studied animals) were detected in 13 of the 15 surveyed provinces and in 16 of the 19 herds (84%) (el‑mahallawy et  al. 2016). in the same table ii. the serological prevalence of c. burnetii in domestic ruminants from published studies conducted in asian countries. — cont’d country year of study animal species number tested % positive method reference animals (n) herds (n) animals herds pakistan ‑ sheep 271 ‑ 26.9 ‑ elisa zahid et al. 2016 ‑ goat 271 ‑ 34.9 ‑ elisa zahid et al. 2016 turkey 1998 cattle 416 48 5.8 35.4 ifat cetinkaya et al. 2000 1998 sheep 411 47 10.5 44.7 ifat cetinkaya et al. 2000 ‑ sheep 184 ‑ 38.59 ‑ ifa kalender 2001 ‑ sheep 224 ‑ 11.01 ‑ ifa kalender 2001 ‑ cattle 177 ‑ 5.6 ‑ elisa seyitoğlu et al. 2006 ‑ cattle 53 22.6 elisa seyitoğlu et al. 2006 2006‑2008 cattle 92 ‑ 26.3 ‑ elisa ceylan et al. 2009 2006‑2008 sheep 92 ‑ 5.4 ‑ elisa ceylan et al. 2009 ‑ sheep 465 ‑ 21.07 elisa karaca et al. 2009 2001‑2004 sheep 743 42 20 81 elisa kennerman et al. 2010 2002 sheep 100 3 cft kilic et al. 2005 ‑ cattle 200 ‑ 20 elisa parin and kaya 2015 ‑ sheep 200 ‑ 29 elisa parin and kaya 2015 ‑ goats 200 ‑ 21 elisa parin and kaya 2015 ‑ cattle 200 ‑ 22 ifa parin and kaya 2015 ‑ sheep 200 ‑ 29 ifa parin and kaya 2015 ‑ goats 200 ‑ 23 ifa parin and kaya 2015 ifa = indirect immunofluorescence assay; ifat = indirect fluorescent antibody test; elisa = enzyme linked immunosorbent assay; cft = complement fixation test; mat = microagglutination test. 273veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 pexara et al. coxiella burnetii in domestic ruminants 2003). in gambia, goats had a significantly higher seroprevalence (24.2%) than sheep (18.5%) (klaasen et al. 2014). in a serological study conducted in egypt, 22.5% of sheep and 16.5% of goats were found seropositive to c. burnetii (mazyad and hafez 2007). in egypt, nahed and khaled (nahed and khaled 2012) reported a seroprevalence of 13%, 32.7%, and 23.3% in cattle, sheep and goats, respectively. more recently, in egypt, anti‑coxiella antibodies were detected in 13.2% of cattle (gwida et al. 2014). data on the serological prevalence of c.  burnetii in animals from published studies conducted in countries of america and oceania are summarized in table iv. in the usa, goats were found to have a significantly higher average seroprevalence (41.6%) than sheep (16.5%) or cattle (3.4%) (mcquiston and childs 2002). in california (usa), deforce and cone (deforce and cone 2006) estimated the seroprevalence of c.  burnetii in bighorn sheep at a level of 10%. in missouri (usa), blood samples from boer goats were tested by elisa and animal and herd‑level seroprevalence estimates for c.  burnetii were 1.2% and 4.2%, respectively (baker and pithua country, 14.39% of the examined tibetan sheep were c. burnetii seropositive (yin et al. 2015). in korea, two separate studies in cattle showed a seroprevalence of c. burnetii of 1.3% (jang et al. 2011) and 6.2% (kim et  al. 2014). in korea, the estimated seroprevalence in native goats was 19.1% (jung et al. 2014). in africa, summarized data on seroprevalence of c. burnetii in animals are shown in table iii. in nigeria, an antibody prevalence of 59.8% was detected among 306 dairy cows (adesiyun et  al. 1984). in zimbabwe, serological evidence of q fever infection was found in 39% of cattle and in 10% of goats (kelly et  al. 1993). in south africa’s transvaal province, 8,900 cattle were examined for antibodies to c. burnetii and 7.78% were found positive (gummow et  al. 1987). in cameroon, q  fever in cattle had a seroprevalence of 31.3% (scolamacchia et al. 2010). in sudan, hussein and colleagues (hussein et  al. 2012) reported a c.  burnetii prevalence of 24.22% in caprine serum samples from 8 states. in togo, dean and colleagues (dean et  al. 2013) reported a c.  burnetii seroprevalence of 14.8%, 14.4% and 8.3% in cattle, sheep and goats, respectively. in chad, the seroprevalence was 4%, 11% and 13% in cattle, sheep and goats, respectively (schelling et al. table iii. the serological prevalence of c. burnetii in domestic ruminants from published studies conducted in african countries. country year of study animal species number tested % positive method reference animals (n) herds (n) animals herds cameroon 2000 cattle 13,377 146 31.3 68 elisa scolamacch et al. 2010 chad 1999‑2000 cattle 195 ‑ 4 ‑ elisa schelling et al. 2003 1999‑2000 sheep 142 11 ‑ elisa schelling et al. 2003 1999‑2000 goat 134 ‑ 13 elisa schelling et al. 2003 ‑ goat 72 ‑ 16.8 ‑ ifa mazyad and hafez 2007 ‑ cattle 54 ‑ 13 ‑ elisa nahed and khaled 2012 ‑ sheep 55 ‑ 32.7 ‑ elisa nahed and khaled 2012 ‑ goat 30 ‑ 23.3 ‑ elisa nahed and khaled 2012 egypt ‑ sheep 89 ‑ 22.5 ‑ ifa mazyad and hafez 2007 2012‑2013 cattle 1,194 ‑ 13.2 ‑ elisa gwida et al. 2014 2012‑2013 cattle 1,194 9 13.2 100 elisa gwida et al. 2014 gambia 2012 sheep 398 ‑ 18.5 ‑ elisa klaasen et al. 2014 2012 goat 490 ‑ 24.2 ‑ elisa klaasen et al. 2014 japan 1982‑1991 cattle 562 ‑ 46.6 ‑ ifat htwe et al. 1992 1974‑1989 sheep 256 ‑ 28.1 ‑ ifat htwe et al. 1992 1974‑1989 goat 85 ‑ 23.5 ‑ ifat htwe et al. 1992 nigeria ‑ cattle 306 ‑ 59.8 ‑ cat adesiyun et al. 1984 transvaal 1985‑1986 cattle 8,900 ‑ 7.78 ‑ cft gummow et al. 1987 sudan 2010‑2011 goat 460 ‑ 24.22 ‑ elisa hussien et al. 2012 togo 2011 cattle 242 ‑ 14.8 ‑ elisa dean et al. 2013 2011 sheep 207 ‑ 14.4 ‑ elisa dean et al. 2013 2011 goat 198 ‑ 8.3 ‑ elisa dean et al. 2013 zimbabwe ‑ cattle 180 ‑ 39 ‑ ifa kelly et al. 1993 ‑ goat 180 ‑ 10 ‑ ifa kelly et al. 1993 ifa = indirect immunofluorescence assay; ifat = indirect fluorescent antibody test; elisa = enzyme linked immunosorbent assay; cft = complement fixation test; mat = microagglutination test. 274 veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 coxiella burnetii in domestic ruminants pexara et al. the seroprevalence was higher in dairy sheep (24.3%) than in meat sheep (10.2%). in the same study, 48.6% of farms had at least one seropositive sheep. in australia (queensland), in a study conducted by cooper and colleagues (cooper et al. 2011), 16.8% beef cattle were positive to c.  burnetii antibodies. in mexico, the 28% of the dairy cattle, 10% of beef cattle, 40% of sheep and 35% of goats were seropositive (salinas‑malendez et  al. 2002). in colombia, lorbacher de ruiz (lorbacher de ruiz 1977) found that 25% and 17% of tested dairy cows and beef cattle heifers, respectively, were seropositive. in venezuela, a high seropositivity (60.23%) was recorded in goats belonging to herds with a history of high percentage of abortions (oropeza et  al. 2010). in ecuador, the prevalence of c. burnetii reached 12.6% with a herd prevalence of 46.9% (carbonero et al. 2015). in australia, extremely low prevalence values (0.67% and 0.5%) were recorded in cattle by banazis and colleagues (banazis et  al. 2010) and hore and kovesdy (hore and kovesdy 1972), respectively; 2014). in a study conducted in washington state (usa), the results identified c.  burnetii antibodies in 8.0% of goat serum samples, 8.6% of goat herds, and 25.8% of counties (sondgeroth et  al. 2013). in an epidemiological investigation of a q  fever outbreak conducted in washington, montana, and oregon (usa), anderson and colleagues (anderson et al. 2015) tested 567 goats from 17 herds finding a c. burnetii seroprevalence of 12%. in canada (newfoundland), 55.8% of the examined goats were seropositive to c.  burnetii (hatchette et  al. 2001). in the same area of canada, hatchette and colleagues (hatchette et  al. 2002) found that seropositivity increased in sheep from 3.1% in 1997 to 23.5% in 1999‑2000. they also observed a seroprevalence of c.  burnetii of 24% and 15.6% in examined cows and goats, respectively. in canada (ontario), the seroprevalence of c.  burnetii ranged between 33 and 82% in cattle herds and between 0 and 35% in sheep flocks (martin and innes 2002). recently, also in ontario, in a study conducted by meadows and colleagues (meadows et  al. 2015), 14.7% of sheep were found infected with c. burnetii. table iv. the serological prevalence of c. burnetii in domestic ruminants from published studies conducted in countries of america and oceania. country year of study animal species number tested % positive method reference animals (n) herds (n) animals herds america canada 1997 sheep 234 ‑ 3.1 ‑ mif hatchette et al. 2002 1999 goat 147 ‑ 55.8 ‑ ifa hatchette et al. 2001 2000‑2001 cattle 75 ‑ 24 ‑ mif hatchette et al. 2002 2000 sheep 34 ‑ 23.5 ‑ mif hatchette et al. 2002 2000 goat 64 ‑ 15.6 ‑ mif hatchette et al. 2002 2010‑2012 sheep 2,363 72 14.7 48.6 elisa meadows et al. 2015 colombia ‑ cattle 357 ‑ 25 ‑ cft lorbacher de ruiz 1977 ‑ cattle 125 ‑ 17 ‑ cft lorbacher de ruiz 1977 ecuador 2008‑2010 cattle 2,668 386 12.6 46.9 elisa carbonero et al. 2015 mexico ‑ cattle na ‑ 28 ‑ elisa salinas‑malendez et al. 2002 ‑ cattle na ‑ 10 ‑ elisa salinas‑malendez et al. 2002 ‑ sheep na ‑ 40 ‑ elisa salinas‑malendez et al. 2002 ‑ goat na ‑ 35 ‑ elisa salinas‑malendez et al. 2002 usa (california) 1992 ‑1999 sheep 268 ‑ 10 ‑ cft deforge and cone 2006 (washington) 2010‑2011 goat 1794 105 8 8.6 elisa sondgeroth et al. 2013 (missouri) 2012 goat 249 24 1.2 4.2 elisa baker and pithua 2014 (washington, montana, and oregon) goat 567 ‑ 12 ‑ elisa anderson et al. 2015 venezuela ‑ goat 315 ‑ 60.63 ‑ elisa oropeza et al. 2010 oceania australia (victoria) 1970 cattle 1576 49 0.5 12.2 cft hore and kovesdy 1972 australia (western) na cattle 329 ‑ 0.61 ‑ elisa banazis et al. 2010 na sheep 50 ‑ 0 ‑ elisa banazis et al. 2010 new zealand 1990‑1992 cattle 2181 ‑ 0 ‑ cft hilbink et al. 1993 ifa = indirect immunofluorescence assay; ifat = indirect fluorescent antibody test; elisa = enzyme linked immunosorbent assay; cft = complement fixation test; mat = microagglutination test. 275veterinaria italiana 2018, 54 (4), 265‑279. doi: 10.12834/vetit.1113.6046.3 pexara et al. coxiella burnetii in domestic ruminants of domestic ruminants and, in particular, sheep and goats. vaccination of domestic ruminants is reported to be effective in preventing abortion and reducing bacterial shedding, especially after several years of administration (roest et  al. 2013). the implementation of good hygiene and other management practices including manure management and risk materials handling, may also reduce the environmental load and, in turn, may result in a decrease of c. burnetii human and animal infection (roest et al. 2013). conversely, no seropositive sheep were found in 2009 (banazis et  al. 2010). in 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author at: department of veterinary medicine, university of bari, 70010 valenzano, italy. e‑mail: giuseppe.passantino@uniba.it. keywords blood, broiler, ileal microflora, immunity, sumac. summary sumac (rhus coriaria l.) is a plant species belong to anacardiaceous family that is worldwide diffused. the sumac seed power (ssp), produced by grinding dried fruits, is recognized to have defensive and beneficial effects on numerous health‑related problems. in this study, ssp was included in broilers basal‑diet to investigate the comparative effects of different levels of ssp on performance, carcass characteristics, blood parameters, immune system and ileal microorganisms. a total of 225, one day‑old male broilers (ross 308) were randomly assigned to the five dietary treatments with three replicates per treatment. the experimental diets were: basal‑diet (bd); and bd including 0.05, 0.10, 0.15 and 0.20% ssp, respectively. during the whole feeding period (42 days), birds fed corn‑based grower (1‑21  days) and finisher (22‑42 days) diets, respectively. results indicated that supplementing ssp had no effect on broiler body weight gain, feed intake and feed conversion as well as carcass characteristics (p > 0.05). similarly, blood total protein, albumin, glucose and triglyceride were not influenced by dietary ssp. conversely, serum total cholesterol and ldl‑cholesterol levels were decreased, while hdl‑cholesterol increased in all ssp fed groups compared to control (p < 0.05). in this study the addition of ssp in broilers diets did not show any effect on blood heterophils and lymphocyte. moreover, the lactobacillus count remained unaffected by dietary treatments, while e.  coli count in broiler ileal content was lower when fed 0.10% ssp than the other groups (p < 0.05). thus, the present findings indicated a positive effect of feeding ssp (especially at 0.10% diet) on blood cholesterol levels and e. coli count in broiler chickens. maryam azizi1, giuseppe passantino2*, yeasmin akter3, faramin javandel1, alireza seidavi1, bojlul bahar4, cormac j. o'shea5, vito laudadio6 and vincenzo tufarelli6 effect of sumac (rhus coriaria l.) seed powder on growth, carcass traits, blood parameters, immune system and selected ileal microorganisms of broilers veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 accepted: 03.09.2019 | available on line: 31.12.2020 plants and their products, including plant extracts, fruits or seeds, in animal nutrition and health may include the stimulation of appetite and intake, the improvement of endogenous digestive enzyme secretion, activation of immune response, as well the antibacterial, antiviral and antioxidant action (nouzarian et al. 2011). over the past few decades, a number of studies have focused on the biological activity of sumac extract. sumac (rhus coriaria l.) is a plant belonging introduction in recent years, phytogenic and herbal products have been accepted by consumers as natural additives and have received increased attention (landy et  al. 2011, dhama et  al. 2015). a variety of herbal supplements have been widely used to sustain and improve health of humans (freeman et  al. 1995) and animals (gardzielewska et  al. 2003). beneficial effects of bioactive medicinal or herbal 186 effect of sumac on broilers azizi et al. veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 crude protein (cp). energy levels were adjusted with vegetable oil and digestible amino acid levels with soybean meal, fish meal and synthetic amino acids. the composition and nutrient specifications of diets are reported in table i. chicks had ad libitum access to water and feed. day old chicks were brooded using heaters, while humidity was kept at 55 to 65% by adding water to floor. the temperature and humidity program were set according to the instructions for ross 308 broilers (aviagen). the lighting program was based as suggested by the ross management manual (aviagen) and comprised 23:1 h light:darkness until slaughter at day 42. birds were vaccinated against bronchitis disease (at 1 day of age), newcastle disease (at 11 and 21 days of age), influenza disease (9 days of age) and gumboro disease (14 and 23 days of age). growth performance and carcass traits average individual bw and average daily gain (adg), and cage average daily feed intake (adfi) as well feed conversion ratio (fcr) were recorded on a weekly basis. the fcr values were corrected for to anacardiaceous family, growing worldwide, especially in temperate and sub‑tropical regions. traditionally, sumac has been used as medicine (zargari 1997) by native north americans for the treatment of bacterial diseases, such as syphilis, gonorrhea, dysentery, and gangrene (erichsen‑brown 1989). research indicated that sumac extracts have various health‑promoting benefits, due to a multitude of compounds with antifibrogenic, antifungal, antinflammatory, antimicrobial, antimutagenic, antioxidant, antiviral, cytotoxic, hypoglycaemic and leukopenic properties (rayne and mazza 2007, marech et  al. 2018). sumac fruits contain flavonols, phenolic acids, hydrolysable tannins, anthocyanins, and organic acids such as malic, citric and tartaric acids (jung 1998, greathead 2003, ozcan and haciseferogullari 2004). more recently, there has been a resurgence in interest in sumac seed due to the aforementioned potential bioactive properties (rayne and mazza 2007). some researchers have proved an increase in body weight and decrease in feed efficiency, when supplementing sumac in broilers diet (gulmez et al. 2006, ghasemi et al. 2014). therefore, keeping in view the positive effects of sumac or its potential bioactive components, the present research was designed to evaluate the impact of different levels of sumac seed powder (ssp) on growth performance, carcass traits, hematology, immunity, and ileal microflora of broiler chickens. materials and methods animals, diets and management all experimental procedures conducted were approved by the islamic azad university (rasht branch, rasht, iran) in accordance with the institutional animal welfare procedure for experimental rearing and handling of poultry. a total of 225 one day‑old male chicks of ross 308 strain (aviagen) were allotted to five dietary treatment groups with three replicates per treatment. groups were formed by animals with similar mean body weight (bw). the sumac was locally purchased and it was ground to a fine powder and then mixed with the basal diet. the dietary treatments were as follows: a basal diet (bd) without ssp as control, and the bd including 0.05, 0.10, 0.15 and 0.20% ssp, respectively. during the whole experimental period (42 days), birds were fed corn‑based grower (1‑21  days) and finisher (22‑42  days) diets, and covering the nutrient requirements as suggested by the ross breeder manual (aviagen). diets provided similar metabolizable energy (me kcal/kg) and table i. ingredients and nutrient analysis of diets fed to broiler chickens. ingredients (%) starter finisher corn 56.9 58.7 soybean meal (43% cp) 33.1 30 fish meal 3.4 3.5 vegetable oil 2.0 3.5 dicalcium phosphate 1.55 1.55 oyster shell 1.03 1.18 dl-methionine 0.01 0.01 vitamin premix* 0.5 0.5 mineral premix** 0.5 0.5 salt 0.26 0.26 sand 0.75 0.75 nutritional content me (kcal/kg) 2,910 3,030 crude protein (%) 20.1 19 crude fat (%) 4.60 6.14 ca (%) 0.95 0.9 total p (%) 1.23 1.06 available p 0.45 0.36 meth 0.50 0.38 lys 1.01 1.01 met + cys 0.83 0.71 * vitamin a, 7.2 mg; d3, 1.6 mg; vitamin e, 14.4 mg; vitamin k3, 1.6 mg; vitamin b1, 0.72 mg; vitamin b2, 3.13 mg; vitamin b3, 4 mg; vitamin b6, 1.2 mg; vitamin b9, 0.5 mg; vitamin b12, 6 mg; vitamin b5, 12 mg; h2, 2 mg; choline chloride, 3 mg and antioxidant; 10 mg. ** mn, 13227 mg; fe, 100 mg; zn, 4235 mg; cu, 16 mg; i, 0.64 mg and se, 0.2 mg. 187 azizi et al. effect of sumac on broilers veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 reaction among gluconic acid, hydrogen peroxide, a phenolic compound, and 4‑aminoantipyrine forms a red‑violet colored quinoneimine, and the absorbance of quinoneimine chromagen, measured by spectrophotometry, is directly associated with the amount of glucose in the sample. immunity response humoral immune response of broiler chickens to the newcastle vaccine at days 28 and 42 was assessed by hemagglutination inhibition (hi) test. sheep red blood cells (srbc) were used as a test antigen to quantify specific antibody responses. on days 21 and 35, broiler chickens were injected with srbc and sampled at days 28 and 42 to assess the humoral immune response. for the srbc injection, initially 1 ml of pbs along with 10 ml of srbc was mixed, and 0.5 ml of the obtained solution was drawn into the syringe and injected under the skin of the broiler chicken's breast. three birds per treatment group were randomly selected for blood sampling from the brachial vein for measuring antibody titres against newcastle disease virus (seidavi et al. 2014). ileal microflora at the end of the trial, three birds from each repetition were euthanized and ileums removed for further cultures. the ileal contents were placed on agar plates for determination of bacterial growth and colony counts. the culture media were prepared 24 h before collection as follows: man rogosa sharpe (mrs, 1.10660.500) agar was used to culture lactobacilli, eosin methylene blue (emb, 1.01347.0500) agar to culture e. coli, respectively. the cultures of lactobacillus and e. coli bacteria were made an aerobically form. the plates were incubated at 37.5 °c for 48 h. a colony counter was used to count bacterial colonies on plates. after counting the number of colonies in each plate, the number so obtained was multiplied by inverse of the dilution and the result was stated as the number of colony forming unit (cfu) in 1 g of sample (downes and ito 2001). statistical analysis data were analyzed using a completely randomized experimental design involving five treatments, and subjected to statistical analysis using the glm and linear and quadratic response procedures of the statistical analysis system v8 (spss). differences among main effect means were assessed via duncan’s multiple range test. statements of significance were set at p ≤ 0.05. the bw of any bird that died during the experiment. at the age of 42 days after 4 h of fasting for complete evacuation of the gut, one bird from each replicate was selected. care was taken to choose the most representative male birds with respect to bw compared to the group mean bw. these animals were used for measuring carcass yield and gastrointestinal tract characteristics. birds were fully plucked using a dry plucking method. feet were separated from the carcass at the tibio‑tarsal joint. neck, wing tips, gut and liver were removed, and the empty or edible carcass was weighed, and intestinal segments were recorded. the carcass parts were dissected and separately weighted. blood biochemical parameters before blood collection, feed was removed from all birds for a period of 4 h in an attempt to allow stabilization of plasma constituents, and blood sampling was done in the morning to further reduce the variability of plasma traits. at 42 days of age, a 5 ml venous blood sample was collected from the basilic vein in the wing of three birds taken from each replicate. care was taken to choose the most representative male birds with respect to bw compared to the group mean bw. the whole blood sample was transferred into a tube coated with 10  mg of the anticoagulant ethylene diamin‑etetra acetic acid (edta). blood samples were centrifuged at 3,000 rpm × 20 min and plasma was collected and stored at ‑  20  °c until analyses. plasma cholesterol and triglyceride levels were determined using enzymatic methods (teifazmoon pars, co., tehran, iran), and hdl‑ and ldl‑cholesterol were measured using diagnostic kits (teifazmoon pars co, tehran, iran). the colorimetric determination of cholesterol in plasma samples involved the use of cholesterol oxidase procedure of barham and trinder (barham and trinder 1972), which is based on the formation of a colored red‑purple quinoneimine dye, produced by oxidative condensation of a phenolic compound with 4‑aminoantipyrine in the presence of hydrogen peroxide. the absorbance of the quinoneimine dye, measured spectrophotometrically, has a direct relationship with the amount of cholesterol in the sample. plasma triglycerides were measured using a series of coupled reactions in which triglycerides are hydrolyzed to produce glycerol. the glycerol is converted to pyruvate and then to lactate. decreased absorbance, measured spectrophotometrically, is proportional to the triglyceride concentration in the sample (schmid and forstner 1986). a glucose oxidase kit (teifazmoon pars, co., tehran, iran), based on oxidase‑peroxidase procedure was used to measure plasma glucose. in this assay, glucose is oxidized in the presence of the glucose oxidase catalyst into h 2 o 2 and gluconic acid. the 188 effect of sumac on broilers azizi et al. veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 weight such as liver, gizzard and abdominal fat pad of broilers were not affected by dietary ssp (p  >  0.05). in agreement with our results, sharbati and colleagues (sharbati et  al. 2013) reported no significant differences in carcass characteristics in broilers fed diets including sumac extract. as shown in figure i, at the end of the experimental period, blood total protein, albumin, triglycerides and glucose were not influenced (p > 0.05) by treatments, while lower blood cholesterol (p < 0.05) was found in ssp supplemented groups. the lower blood cholesterol level in broilers fed ssp supplemented diets might be due to its polyphenolic components. some studies have demonstrated that polyphenols could have beneficial effects on cardiovascular disease (hertog et al. 1995, laudadio et al. 2015, tufarelli et al. 2017), and could be regarded as bioactive compounds with a high potential health‑promoting capacity. in previous studies, tebib and colleagues (tebib et al. 1994), bravo (bravo 1998) and mansoob (mansoob 2012) also reported that polyphenols have been shown to depress the reverse cholesterol transport, reduce the intestinal cholesterol absorption and even increase bile acid excretion. similarly, valiollahi and colleagues results and discussion the effects of feeding ssp on growth performance of broilers are presented in table ii. sumac seed powder supplemented diets had no effect on body weight, fcr, and feed intake of chickens from 1‑21 (grower phase) and 22‑42 (finisher phase) days of age (p > 0.05). similarly, the growth performance of birds considering the overall rearing period (1‑42  days of age) was not significantly influenced by ssp‑supplemented diet. our findings are confirmed by a recent study of cakmak and colleagues (cakmak et al. 2017), reporting no effect (p > 0.05) of sumac powder on body weight gain, feed intake and efficiency; conversely ghasemi and colleagues (ghasemi et al. 2014) reported significant (p < 0.05) influence of sumac extract on growth traits of broiler chickens. these results might be explained by differences in form and levels of sumac supplemented in poultry diet. the effects of dietary ssp on broiler chicks carcass characteristics are shown in table iii. carcass traits, such as hot carcass, cooked carcass and meat cut (breast, thigh and wing) weight, were not influenced by dietary ssp (p > 0.05). similarly, internal organ table ii. effects of different levels of sumac seed powder (ssp) on growth performance of broiler chickens. item control 0.05% ssp 0.10% ssp 0.15% ssp 0.20% ssp sem p-value day 1-21 body weight (g/bird) at day 21 715.8 697.8 682.1 712.5 683.1 15.84 0.450 average feed intake (g) 1,002.3 974.0 958.6 996.0 962.3 20.46 0.481 feed conversion ratio (fcr) 1.40 1.39 1.40 1.39 1.40 0.008 0.890 day 21-42 body weight (g/bird) at day 42 1,241.3 1,061.0 1,208.3 1,072.0 1,178.6 97.30 0.602 average feed intake (g) 2,512.6 2,162.3 2,418.0 2,254.3 2,379.6 170.00 0.634 feed conversion ratio (fcr) 2.02 2.04 2.01 2.10 2.02 0.037 0.461 day 1-42 body weight (g/bird) 1,957.1 1,758.8 1,890.5 1,784.50 1,861.8 90.6 0.561 average feed intake (g) 3,515.0 3,136.3 3,376.6 3,250.3 3,342.6 160.6 0.563 feed conversion ratio (fcr) 1.79 1.78 1.78 1.82 1.79 0.016 0.522 table iii. effects of different levels of sumac seed powder (ssp) on carcass characteristics (g) of broiler chickens. item control 0.05% ssp 0.10% ssp 0.15% ssp 0.20% ssp sem p-value linear quadratic hot carcass 1,713 1,440 1,516 1,534 1,509 104 0.470 0.216 0.276 cooked carcass 1,129 1,078 1,165 1,139 1,148 66 0.902 0.943 0.980 breast 391 345 408 378 374 39 0.837 0.957 0.957 thighs 173 185 184 188 191 9.5 0.740 0.555 0.736 wings 50 49 56 49 57 4.7 0.615 0.975 0.823 liver 54 54 54 55 57 5.4 0.988 0.830 0.752 gizzard 40 44 45 47 45 3.74 0.738 0.291 0.385 abdominal fat 67 33 34 33 66 5.74 0.255 0.783 0.939 189 azizi et al. effect of sumac on broilers veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 demonstrated that dietary ssp had important influences on plasma ldl and hdl of broilers (p < 0.05). after consumption of ssp, the level of ldl decreased, whereas hdl increased in ssp supplemented group compared to control (p < 0.05). these findings are in accordance with kheiri and colleagues (kheiri et  al. 2015) who demonstrated that blood hdl concentrations were significantly increased and ldl decreased in ssp‑treated groups. (valiollahi et  al. 2014) and kheiri and colleagues (kheiri et  al. 2015) found a lower blood cholesterol level in broiler chickens fed sumac powder. furthermore, research indicated that d‑limonene, a monocyclic monoterpene component of sumac, has hypocholesterolemic effects in the body (kurucu et al. 1993, marshall 1995, santiago et al. 2011). results of plasma ldl and hdl cholesterol in the current study are presented figure i. data a. cholesterol cholesterol: p-value 0.003; linear 0.005; quadratic 0.011 dietary levels of ssp (%) c h o le st er o l m g /d l 150 140 130 120 110 100 0 0.05 0.1 0.15 0.2 a b b b b b. glucose cholesterol: p-value 0.389; linear 0.172; quadratic 0.121 dietary levels of ssp (%) g lu co se m g /d l 350 300 250 200 100 150 50 0 0 0.05 0.1 0.15 0.2 c. triglycerides triglycerides: p-value 0.173; linear 0.396; quadratic 0.366 d. hdl hdl: p-value 0.003; linear 0.001; quadratic 0.001 dietary levels of ssp (%) h d l m g /d l b 90 80 85 75 70 60 65 55 50 0 0.05 0.1 0.15 0.2 a a a a e. ldl ldl: p-value 0.001; linear 0.001; quadratic 0.001 dietary levels of ssp (%) ld l m g /d l 90 70 50 30 10 0 0.05 0.1 0.15 0.2 a bc c bc b f. albumin albumin: p-value 0.948; linear 0.965; quadratic 0.973 g. total protein total protein: p-value 0.243; linear 0.031; quadratic 0.025 dietary levels of ssp (%) to ta l p ro te in g /d l 4 3.5 3 2.5 2 1.5 0 0.05 0.1 0.15 0.2 dietary levels of ssp (%) tr ig ly ce ri d es m g /d l 80 40 50 60 70 30 20 10 0 0 0.05 0.1 0.15 0.2 dietary levels of ssp (%) a lb u m in m g /d l 2 1.8 1.6 1.4 1.2 1 0.4 0.6 0.8 0.2 0 0 0.05 0.1 0.15 0.2 figure 1. effect of dietary sumac seed powder (ssp) on blood biochemistry in broiler chickens. 190 effect of sumac on broilers azizi et al. veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 present in many plant species (tufarelli et al. 2017), including sumac, whose tannins have remarkable antimicrobial activity. a direct correlation between total phenols and antimicrobial activity is well documented, and research indicated that sumac is effective against both gram positive and negative bacteria (ahmadian‑attari et  al. 2007). the effect of dietary ssp on intestinal microbial population is presented in table v. findings of the present study indicated a lowest e. coli counts in 0.10% ssp supplemented group (p < 0.05), while lactobacillus population was unchanged in all dietary groups (p > 0.05). in agreement with mansoob (mansoob 2012), sumac has significant antimicrobial activity that can reduce the pathogenic bacteria in gut tract. therefore, the differences in e. coli populations under ssp treatments may possibly be attributed to the bioactive components of plant product; however, it is necessary to conduct further research regarding the effect of ssp on poultry, specially on the effect on broiler gut morphology. in conclusion, this study demonstrated that dietary supplementation of sumac seed powder influenced positively blood cholesterol in broiler chickens, supporting their growth and carcass traits. moreover, a significant reduction of e. coli in small intestine by supplementing the plant extract was found, and this could be interesting as it may have the potential to be an alternative natural feed additive for broiler chickens. acknowledgments financial support by rasht branch, islamic azad university, and grant number 17.16.4.8774 is gratefully acknowledged. moreover, it was assessed a negative correlation between the dietary sumac powder consumption and plasma total cholesterol and ldl concentrations in broiler chickens (golzadeh et al. 2012). it was also demonstrated that high level of sumac consumption might have a protective effect on atherosclerosis and oxidative stress (setorki et  al. 2012). studies indicated a strong relationship between total fat intake and cellular cholesterol concentration and several diseases, including atherosclerosis, cancer, diabetes, depression in human (leaf and kang 1998, katan 2000, ayerza et  al. 2002). low cholesterol containing diet has become an important concern for people with atherosclerosis, and poultry meat is one of the main products consumed, given its low in fat and cholesterol content compared to red meat. the effects of dietary ssp on immune related parameters are presented in table iv. addition of sumac had no effect on some selected haematological parameters (lymphocyte and monocyte) of broilers (p > 0.05). we have not found other evidences in literature related to the effect of sumac on haematological traits in chickens. in the present study, the heterophil to lymphocyte (h/l) ratio was unaffected by dietary ssp (p > 0.05). as well demonstrated, heterophils increased and lymphocytes decrease when birds are stressed, so that the h/l ratio is a valuable index of response to a stressor (maxwell and robertson 1998). results of our study indicated that dietary supplementation of ssp had no harmful effects on birds health status. natural inhibitors for pathogenic microorganisms have been explored in many plants (al‑zoreky 2009). among plant constituents, polyphenols have received a great deal of attention in recent years, due to their biological functions. tannins are high molecular weight phenolic compounds, which are table vi. effects of different levels of ssp on blood parameters (mg/dl) of broiler chickens. item control 0.05% ssp 0.10% ssp 0.15% ssp 0.20% ssp sem p-value linear quadratic heterophil 28.3 32.7 29.3 34.0 33.7 3.14 0.620 0.728 0.909 monocyte 2.33 3.33 3.67 3.67 2.00 0.745 0.408 0.055 0.048 lymphocyte 69.3 64.0 67.0 62.3 64.3 3.33 0.616 0.443 0.572 h/l 42.2 51.5 44.3 55.2 53.1 7.41 0.674 0.704 0.866 h/l = heterophil to lymphocyte ratio. table v. effects of different levels of ssp on ileum microflora (log cfu/g) of broiler chickens. item control 0.05% ssp 0.10% ssp 0.15% ssp 0.20% ssp sem p-value linear quadratic e. coli 6.63 6.43 5.16 6.67 5.54 0.294 0.013 0.432 0.590 lactobacilli spp. 7.02 7.63 7.30 7.42 7.13 0.269 0.553 0.200 0.192 l:e 1.05 1.19 1.41 1.11 1.29 0.168 0.082 0.365 0.289 l:e = lactobacilli to e. coli ratio. 191 azizi et al. effect of sumac on broilers veterinaria italiana 2020, 56 (3), 185‑192. doi: 10.12834/vetit.1892.10049.3 ahmadian‑attari m., amin g.h., fazeli m.r. & jamalifar h. 2007. a review on the antibacterial and effects of sumac fruit. med plants, 7, 1‑9. al‑zoreky n.s. 2009. antimicrobial activity of pomegranate (punica granatum l.) fruit peels. int. j food microbiol, 134, 244‑248. ayerza r., coates w. & lauria m. 2002. chia seed (salvia hispanica l.) as an ω‑3 fatty acid source for broilers: influence on fatty acid composition, cholesterol and fat content of white and dark meats, growth performance and sensory characteristics. poult sci, 81, 826‑837. barham d. & trinder p. 1972. an improved colour reagent for the determination of blood glucose by oxidase system. analyst, 97, 142‑145. bravo l. 1998. polyphenols: chemistry, dietary sources, 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oxidative stress‑induced liver injury in high‑fat diet and l‑name‑treated rats. eur j nutr, 51, 57‑68. schmid m. & forstner v. 1986. laboratory testing in veterinary medicine diagnosis and clinical monitoring. boehringer mannheim gmbh, mannheim. 253 pp. seidavi a., asadpour l., dadashbeiki m. & payan‑carreira r. 2014. effects of dietary fish oil and green tea powder supplementation on broiler chickens immunity. acta sci vet, 42, 1205. setorki m., rafieian m., heidarian e., ghatreh k., shahinfard n., ansari r. & forouzandeh z. 2012. effect of rhus coriaria consumption with high cholesterol food on some atherosclerosis risk factors in rabbit. j babol univ med sci, 14, 38‑45. 404 not found 119 veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 accepted: 15.07.2020 | available on line: 31.12.2021 department of veterinary science, university of parma, parma, italy *corresponding author at: department of veterinary science, university of parma, parma, italy. e-mail: mara.bertocchi@unipr.it. clotilde silvia cabassi, mara bertocchi*, costanza spadini, laura denti, sara flisi, emiliana schiano, sandro cavirani, enrico parmigiani and simone taddei keywords medical honey, minimal bactericidal concentration, non-traditional pets, wound. summary in recent years, due to the growing phenomenon of antimicrobial resistance, the search for alternative strategies to antibiotic treatments is increasing and a considerable interest for the use of medical honey in clinical practice has emerged. honey has been used for the treatment of skin lesions, in both humans and animals. however, knowledge concerning the use of medical honey in non-traditional companion animals is scarce. the aim of this study was to assess the antibacterial activity of a standardized medical honey (revamil, bfactory) against bacterial strains isolated from skin lesions of non-traditional companion animals. the minimum bactericidal concentration (mbc) of revamil honey against seventeen clinical isolates and three reference strains was established. the medical honey showed antimicrobial activity against both gram-positive and gram-negative bacteria. growth was inhibited for all the strains at concentrations of medical honey ranging from 10 to 40%. pseudomonas oryzihabitans and alcaligenes faecalis showed the lowest mbc (10%). the reference strain staphylococcus aureus atcc25923 showed a higher sensitivity to 20% honey compare to the corresponding clinical isolate (p = 0.001). the observed results suggest that revamil could represent an effective therapeutic aid, useful for the reduction of antibiotic use, in case of pathological skin infections in non-traditional companion animals. antimicrobial activity of a standardized medical honey on bacterial isolates from infected skin lesions of non-traditional companion animals (bowler et  al. 2001). however, antibiotic resistance is considered one of the most serious public health problems of our century and the growing antibiotic resistance in veterinary medicine is a current threat to human health (prestinaci et  al. 2015, tang et  al. 2017). according to the international guidelines on the prudent use of antimicrobials in veterinary medicine1, honey and other alternative therapies were used for the treatment of skin lesions, in both humans and animals (bowler et  al. 2001, carnwath et al. 2014, di ianni et al. 2015b, olofsson et al. 2016, pelizzone et al. 2014, subrahmanyam 1991). however, knowledge concerning the use of medical honey in non-traditional companion animals is lacking. honey is produced by honey bees using the nectar of flowers or honeydew and is mostly composed introduction the loss of integrity of the skin barrier, caused by mechanical, thermal or chemical injuries, facilitates bacterial contamination of the underlying tissues, which can lead to wound colonization or, at worst, to invasive infection. complications related to bacterial contamination of the wound and bacterial interactions with the damaged tissues can cause impaired wound healing. non-healing wounds frequently show pathologic inflammation and even suppurative discharge (guo and dipietro 2010, rosique et  al. 2015). this what normally occurs in all vertebrate classes including reptiles. the methods used to house captive reptiles generally predispose these animals to a variety of opportunistic microbial pathogens and reptile wounds are frequently contaminated with both gram-positive and gram-negative bacteria (mitchell et  al. 2004). therefore, broad-spectrum antibiotic therapy may be required to control microbial populations contaminating the wounds 1 commission notice, guidelines for the prudent use of antimicrobials in veterinary medicine (2015/c299/04), official journal of the european union, 11/09/2015 c299, s. 7. 120 veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 antimicrobial activity of honey on bacteria from non-traditional pets cabassi et al. systemic and multi-organ involvement may result in death (harkewicz 2001). the aim of this study was to assess the antibacterial activity of a standardized medical honey (revamil, bfactory) against bacterial strains isolated from skin lesions of pet reptiles and other non-traditional companion animals. materials and methods tested product, bacterial strains and reagents the tested product was a standardized medical honey in gel formulation (revamil, bfactory), consisting of glucose oxidase (gox) positive 100% pure honey. samples for bacterial isolation were collected by swabs from infected skin lesions of 17 captive animals, brought to the veterinary teaching hospital of the department of veterinary science of the university of parma to be treated for different injuries. the animals were kept as pets and sample collection was part of the normal diagnostic process. the swabs were immediately plated onto tryptose agar (oxoid) containing 5% of bovine erythrocytes and macconkey agar (difco) and incubated aerobically for 24 hours at 37 °c. identification of bacterial isolates was based on their growth and colony characteristics, gram staining, cellular morphology, catalase and oxidase reactions. species identification was carried out using api biochemical test systems (biomérieux), as well as conventional biochemical tests (quinn et al. 1994). clinical bacterial strains are reported in table i. three bacterial reference strains, staphylococcus aureus atcc25923, escherichia coli atcc25922 and pseudomonas aeruginosa atcc27853, were also evaluated. the following reagents were used for the antimicrobial activity evaluation: bacto agar (becton dickinson, sparks, usa), bacto brain heart infusion (bhi) broth (becton dickinson, sparks, usa), mueller hinton (mh) broth (becton dickinson, sparks, usa), phosphate buffer (pb). agarized medium for colony-forming unit (cfu) counts was prepared by the addition of 1.5% of bacto agar (w/v) to mh broth. sterility control was performed for all the prepared media by incubation for 24 hours at 37 °c in air. evaluation of the minimum bactericidal concentration (mbc) the standardized medical honey was dissolved in pb at a 50% (v/v) concentration by stirring with a magnetic stir bar at room temperature. eighty microliters of the 50% emulsion were serially of glucose and fructose. it also contains vitamins, minerals, amino acids, enzymes, organic acids and other compounds. the beneficial properties of honey were known since ancient times (molan and rhodes 2015) and its therapeutic use remained popular until the advent of antibiotics (langemo et  al. 2009). the antibacterial activity of honey was reported in numerous studies (basualdo et  al. 2007, mandal and mandal 2011, subrahmanyam 1991, vandamme et  al. 2013). honey exerts bacteriostatic and bactericidal activities (vandamme et  al. 2013). many enzymes are present in an internal pouch of the bee called “crop” and are added to honey. the glucose oxidase catalyzed the glucose oxidation to form gluconic acid and hydrogen peroxide. gluconic acid lowers the ph and the hydrogen peroxide boosts the bactericidal action (minden-birkenmaier and bowlin 2018, molan and rhodes 2015). the lowering of ph at 3.5-4 causes a series of events essential to the process of tissue repairing: reduction in protease activity in the wound site, increasing of oxygen release from hemoglobin and stimulation of fibroblast and macrophage activity. furthermore, the hydrogen peroxide stimulates the production of the vascular endothelial growth factor (vegf) and sterilize the wound site (minden-birkenmaier and bowlin 2018, molan and rhodes 2015). in addition to glucose oxidase, the invertase produced by the bee increases the strength of the osmotic potential of the honey dividing sucrose into fructose and glucose (minden-birkenmaier and bowlin 2018, molan and rhodes 2015). fluids into the wound are drawn out of damaged tissues leading to drying of cellular tissues and bacterial death (molan and rhodes 2015). in addition, phenolic compounds, organic acids, vitamins and flavonoids exert antioxidant activities and boost the antimicrobial effect of the honey. flavonoids neutralize free radicals produced by the hydrogen peroxide (minden-birkenmaier and bowlin 2018, molan and rhodes 2015). however, despite the increase of studies on the use of honey for the wound healing of either traumatic or surgical origin, only a few studies on its use on infected wounds were done. some authors analyzed the effect of the honey on the growth of selected intestinal bacteria (shin and ustunol 2005) and against pathologic bacteria frequently isolated from skin wounds of mammals, including humans (basualdo et al. 2007). the number of pet reptiles or other non-traditional companion animals is steadily increasing, leading to greater scientific interest in the medical and reproductive aspects of these animals (bertocchi et  al. 2018, di ianni et  al. 2014, di ianni et  al. 2015a, taddei et  al. 2010). in reptiles, bacteria can cause skin diseases, secondary to traumatic wounds or management errors (mitchell et  al. 2004) and infected wounds that are not promptly treated may rapidly evolve causing sepsis or septic shock. the 121veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 cabassi et al. antimicrobial activity of honey on bacteria from non-traditional pets performed. growth controls were performed by testing the bacterial strains with the same procedure as above, but in absence of standardized medical honey. agarized mueller hinton plates were read by counting the number of cfu after 24 hours of incubation in air at 37 °c. the test was considered valid when no contaminant growth was present in sterility controls and growth (cfu count of at least 400 cfu/ml) was visible onto growth control mueller hinton plates. the mbc was defined as the lowest concentration of standardized medical honey at which there is no growth of the organism. statistical analysis differences between treatments were analyzed by heteroscedastic one-way anova. homogeneity of variances was assessed by levene’s test. multiple comparisons were performed by games-howell test. comparisons between the different strains of the same bacterial species at each concentration of honey were performed by t test. differences at p < 0.05 were considered statistically significant. statistical analysis was performed by spss version 26 software (ibm). results all replicates of each bacterial strain showed reproducibility of results. mbc values were expressed as percent concentration of standardized medical honey and are reported in table ii. diluted in a microtiter plate to obtain the following concentrations of standardized medical honey in pb: 50%, 25%, 12.5%, 6.25% and 3.125%. for each bacterial strain, three to five colonies from fresh agar plates were inoculated into tubes containing bhi broth. tubes were briefly vortexed using a vortex mixer and incubated at 37 °c in a shaker at 225 revolution per minute (r.p.m.) for 3-4 hours to reach the log-growing phase. bacterial suspension was centrifuged at 1,000 g for 20 min and gently resuspended in pb. bacterial suspension was adjusted spectrophotometrically at 600 nm with 1 cm path length to an optical density value in the range 0.08-0.13, containing approximately 108 cfu/ ml in pb. the bacterial suspension was further diluted to reach a bacterial concentration of 2.5x106 cfu/ ml. twenty microliters of this bacterial suspension were inoculated into wells containing 80 µl of pb at increasing concentrations of standardized medical honey and into control wells. final concentrations of standardized medical honey were therefore as follow: 40%, 20%, 10%, 5% and 2.5%. final bacterial concentration was 5 x 105 cfu/ml. only for gram-positive strains, 2% of mh broth was present in the final suspension to allow bacterial growth. conversely, for gram-negative strains no addition was required. inoculated wells were incubated in air at 37 °c for 24 hours. after incubation, 20 microliters from each tube were serially diluted and plated onto agarized mueller hinton, to perform the cfu count. for each strain and for each standardized medical honey concentration, three independent experiments, each with three replicates, were table i. clinical bacterial isolates. sample origin species lesion bacterial isolate api® identification s1 turtle trachemis scripta skin wound morganella morganii api 20 e 0174000 s2 turtle testudo hermanni skin wound klebsiella oxytoca api 20 e 5245773 s3 turtle testudo hermanni skin wound pseudomonas oryzihabitans api 20 e 0200000 s6 rat rattus norvegicus skin abscess staphylococcus aureus api staph 6736353 s7 turtle trachemis scripta skin wound staphylococcus xylosus api staph 6736452 s8 snake python regius infected skin burns micrococcus spp. api staph 0006000 s9a snake python regius stomatitis pseudomonas aeruginosa api 20 ne 1154575 s9b snake python regius stomatitis klebsiella oxytoca api 20 e 5255773 s10 snake python regius necrotic stomatitis stenotrophomonas maltophilia api 20 e 5202000 s11 turtle testudo hermanni skin wound staphylococcus auricularis api staph 6300000 s12/1 duck anas platyrhynchos skin wound pseudomonas aeruginosa api 20 ne 0154575 s12/2 duck anas platyrhynchos skin wound escherichia coli api 20 e 5144572 s13/2 snake etherodon nasicus skin abscess alcaligenes faecalis api 20 ne 0000057 s14/1 snake epicrates cenchria skin wound citrobacter braakii api 20 e 3644553 s14/2 snake epicrates cenchria skin wound pseudomonas aeruginosa api 20 ne 0154575 s15 snake epicrates cenchria skin wound pseudomonas aeruginosa api 20 ne 0554575 s16 snake python regius skin wound pseudomonas aeruginosa api 20 ne 0554575 122 veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 antimicrobial activity of honey on bacteria from non-traditional pets cabassi et al. atcc25922, all the p. aeruginosa tested strains, morganella morganii and citrobacter braakii. finally, p. oryzihabitans and a. faecalis were even more sensitive to honey, since their growth was completely inhibited by the concentration of 10%. for each bacterial strain, logarithmic (log) reduction of cfu/ml as a function of medical honey concentration, compared to growth in the absence of honey, was evaluated and reported in figures  1 and 2. at honey concentrations ranging from 10 to 40%, depending on the bacterial strain, growth was inhibited for all the strains (figures 1 and 2). among the tested bacterial strains, those which showed the lowest mbc (10%) were the clinical isolates pseudomonas oryzihabitans and alcaligenes faecalis (figure 2b and 2h, table ii). reference strains showed a pattern of sensitivity to the presence of medical honey similar to those of clinical isolates of the same bacterial species, especially at low concentrations (figures 1a and 2a, c, d). however, s.  aureus atcc25923 and e.  coli atcc25922 showed a lower mbc compared to the corresponding clinical isolates (figures 1a and 2c, table ii), the difference was statistically highly significant for s. aureus at 20% revamil (p=0.001). moreover, several statistically significant differences between the different p.  aeruginosa strains for all the concentrations of honey in the range 0-10% were found (with p values of significant differences ranging from < 0.001 to 0.049). therefore, the considered standardized medical honey was able to completely inhibit bacterial growth of all the tested strains at the concentration of 40%. some strains were completely inhibited also in presence of a lower concentration of medical honey (20%), notably s. aureus atcc25923, micrococcus spp., staphylococcus auricularis, e. coli a. staphylococcus aureus c. staphylococcus auricularis b. staphylococcus xylosus d. micrococcus sp. strain 0 2 4 6 8 10 12 0 2.5 5 10 20 40 lo g c fu /m l revamil (%) p=0.045 p=0.003 p=0.004 p=0.026 p=0.021 0 2 4 6 8 10 12 0 2.5 5 10 20 40 lo g c fu /m l revamil (%) p=0.003 p=0.009 p=0.015 p=0.015 p=0.003 p=0.009 p=0.005 p=0.005 p=0.003 p=0.005 p=0.002 p=0.002 p=0.003 p=0.005 p=0.006 p=0.006 0 2 4 6 8 10 0 2.5 5 10 20 40 lo g c fu /m l revamil (%) 0 2 4 6 8 10 12 14 16 0 2.5 5 10 20 40 lo g c fu /m l revamil (%) atcc25923 s6 p=0.023 p=0.015 p=0.004 p=0.001 p=0.001 p=0.007 p<0.001 figure 1. concentration-dependent inhibition of gram-positive bacteria by medical honey. the experiments were performed at least in triplicate and the error bars indicate ± 1 standard deviation. statistically significant differences between treatments and p values are showed on graph. table ii. minimum bactericidal concentration results. mbc value reference strains staphylococcus aureus atcc25923 20% escherichia coli atcc25922 20% pseudomonas aeruginosa atcc27853 20% gram-positive isolates s6 staphylococcus aureus 40% s7 staphylococcus xylosus 40% s8 micrococcus spp. 20% s11 staphylococcus auricularis 20% gram-negative isolates s1 morganella morganii 20% s2 klebsiella oxytoca 40% s3 pseudomonas oryzihabitans 10% s9a pseudomonas aeruginosa 20% s9b klebsiella oxytoca 40% s10 stenotrophomonas maltophilia 40% s12/1 pseudomonas aeruginosa 20% s12/2 escherichia coli 40% s13/2 alcaligenes faecalis 10% s14/1 citrobacter braakii 20% s14/2 pseudomonas aeruginosa 20% s15 pseudomonas aeruginosa 20% s16 pseudomonas aeruginosa 20% 123veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 cabassi et al. antimicrobial activity of honey on bacteria from non-traditional pets discussion the development of antibiotic resistance in bacteria is a global emergency and infections caused by resistant bacteria are increasingly common in many different animal species (sørum and sunde 2001, szmolka and nagy 2013). together with the search for more effective antimicrobials, increasing efforts to develop alternative therapies could help in reducing the use of antibiotics and limiting the spread of antibiotic resistance. alternative therapies may find useful application especially in mild infections. the antibacterial properties of honey have long been known (vandamme et  al. 2013). honey is widely used in human medicine for the management of acute, chronic, traumatic and post-surgical wounds (ahmed et  al. 2003), but a. pseudomonas aeruginosa c. escherichia coli b. pseudomonas oryzihabitans d. klebsiella oxytoca e. morganella morganii g. citrobacter braakii f. stenotrophomonas maltophilia h. alcaligenes faecalis strain strain p=0.001 p=0.001 p=0.003p=0.003 0 2 4 6 8 10 12 14 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) p=0.001 p=0.001 p=0.004 p=0.004p=0.004p<0.001p=0.002 p=0.001p=0.001 p=0.001p=0.001 p=0.003 p=0.034 p=0.003 p<<0.001p=0.002 p<<0.001p=0.002 p=0.007 0 2 4 6 8 10 12 14 16 18 20 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) p=0.010 p=0.002p=0.003p=0.003 0 2 4 6 8 10 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) p=0.002 p=0.001 p<<0.001 p<<0.001 p=0.002 p=0.001 p=0.001 p=0.001p=0.005p<<0.001 p=0.004 0 2 4 6 8 10 12 14 16 18 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) p=0.002 p=0.009 p=0.005 p=0.005p=0.005p=0.009 p=0.002p=0.002 p=0.009p=0.009 0 2 4 6 8 10 12 14 16 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) p<<0.001 p<<0.001 p<<0.001 p<<0.001 p<<0.001 p<<0.001 p<<0.001 p<<0.001p<<0.001p<<0.001 p<<0.001 strain 0 2 4 6 8 10 12 14 16 18 20 22 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) atcc27853 s9a s12/1 s14/2 s15 s16 0 2 4 6 8 10 12 14 16 18 20 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) atcc25922 s12/2 p=0.001 p<0.001 p=0.001 p<<0.001 p<<0.001 p<<0.001 p=0.019 p<<0.001p=0.004p=0.002 p=0.001 0 2 4 6 8 10 12 14 16 18 20 0 2,5 5 10 20 40 lo g c fu /m l revamil (%) s2 s9b p<0.001 p<0.001 p<0.001 p<<0.001 p<<0.001 p<<0.001 p=0.016 p<<0.001p<<0.001p<<0.001 p<<0.001 p=0.033 figure 2. concentration-dependent inhibition of gram-negative bacteria by medical honey. the experiments were performed at least in triplicate and the error bars indicate ± 1 standard deviation. statistically significant differences between treatments and p values are showed on graph. 124 veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 antimicrobial activity of honey on bacteria from non-traditional pets cabassi et al. observed between p. aeruginosa strains in absence of honey or at low honey concentrations. in particular, statistically significant differences between p.  aeruginosa atcc27853 and some of the other strains were found at concentration 0% and 2.5% of revamil only. this could be due to differences in the bacterial concentration of the inoculum. anyway, the bactericidal activity of revamil was similar for all the strains of p. aeruginosa (mbc  =  20%). the mbcs showed by the reference strains of s. aureus and e. coli were lower than those of the corresponding clinical isolates, although only for s. aureus the difference was statistically significant. this is in agreement with what reported by voidarou and colleagues (voidarou et al. 2011), who found a higher resistance of clinical isolates of s. aureus, e. coli, salmonella typhimurium, streptococcus pyogenes, bacillus cereus and bacillus subtilis compared to their corresponding reference strains. in general, medical honey acts primarily as a hyperosmolar medium, but it also represents an important physical barrier because of its considerable viscosity. its immunomodulatory effects together with the anti-inflammatory and antioxidant properties of its components improve wound healing (majtan 2014). moreover, the high content of nutrients promotes epithelialization and angiogenesis (molan 2001). in particular, an important source of nutrients for the tissues is represented by the presence of carbohydrates, mostly glucose and fructose, with maltose, sucrose and isomaltose in smaller quantities. carbohydrates represent about 80% of the honey components (carnwath et al. 2014, cavanagh et al. 1970, cooper et  al. 2002, minden-birkenmaier and bowlin 2018). the rapid bactericidal activity of revamil honey is primarily linked to the presence of bee defensin-1 and the gox enzima. this enzyme turns the honey sugar into gluconic acid and 3‰ hydrogen peroxide, effective against bacteria but not harmful to tissues (kwakman et  al. 2010). in conclusion, our result regarding antimicrobial activity of revamil honey suggest that it could represent an effective therapeutic aid, useful for the reduction of antibiotic use, in case of pathological skin infections in non-traditional companion animals. acknowledgements thanks to angela andreoli and raffaele boselli of bfactory italia for providing the standardized medical honey. also for ulcers, burns, eye diseases, skin diseases, oral mucosa problems, necrotic areas (al-waili 2004, bardy et  al. 2008, biswal et  al. 2003, molan and rhodes 2015, subrahmanyam 1991). moreover, cases of positive therapeutic response to honey in patients unresponsive to traditional treatments were reported (bardy et  al. 2008, dunford and hanano 2004, efem 1988, schumacher 2004). regarding veterinary medicine, the effectiveness of different types of honey in the treatment of equine infected wounds was reported (carnwath et al. 2014). however, to our knowledge no data are available on regarding non-traditional pets. with this study, we assessed the antimicrobial activity of a standardized medical honey against bacteria isolated from non-traditional companion animals, mostly reptiles. moreover, three reference bacterial strains, belonging to the most representative species among the isolates, were tested. all bacterial strains were completely inhibited at honey concentrations between 10% and 40%, depending on the strain (figure 1 and 2). considering the s.  aureus strains, our results agree with the literature (almasaudi et al. 2017, cooper et al. 1999, cooper et al. 2002, lu et al. 2014). lu and colleagues (lu et al. 2014) have showed an important inhibition by honey on the formation of s. aureus biofilm. other authors have found a growth inhibition of both methicillin-sensitive s.  aureus (mssa) and methicillin-resistant s.  aureus (mrsa) (almasaudi et  al. 2017, cooper et  al. 1999, cooper et  al. 2002). moreover, the bactericidal activity of medical honey against some important resistant bacteria, such as mrsa, can be increased by the addition of a synthetic bactericidal peptide (kwakman et  al. 2011). furthermore, some authors showed an inhibitory activity sustained by honey against streptococcus pyogenes, streptococcus mutans, proteus mirabilis, p. aeruginosa, enterococcus faecium and enterobacter cloacae (kwakman et  al. 2008, kwakman et  al. 2010, majtan et  al. 2014). in general, as reported by almasaudi and colleagues (almasaudi et al. 2017), the antibacterial activity of medical honey was found both against gram-positive (s. aureus, bacillus subtilis, bacillus cereus, enterococcus faecalis, micrococcus luteus) and gram-negative (e. coli, p. aeruginosa, and salmonella typhi) bacteria (gupta et  al. 1993, jeddar et  al. 1985, mohapatra et  al. 2011). this study confirmed what found by other authors. a similar pattern of sensitivity to low honey concentrations was found between reference strains and clinical isolates of the same species. significant differences , however, were 125veterinaria italiana 2021, 57 (2), 119-126. doi: 10.12834/vetit.1964.12937.1 cabassi et al. antimicrobial activity of honey on bacteria from non-traditional pets ahmed a., hoekstra m.j., hage j.j. & karim r.b. 2003. honey-medicated dressing: transformation of an ancient remedy into modern therapy. ann plas surg, 50 (2), 143-148. https://doi.org/10.1097/01. sap.0000032306.44107.c1. almasaudi s.b., al-nahari a.a.m, abd el-ghany e.s.m., barbour e., al muhayawi s.m., al-jaouni s., azhar e., qari m., qari y.a. & harakeh s. 2017. antimicrobial effect of different types of honey on staphylococcus aureus. saudi j biol sci, 24 (6), 1255-1261. https://doi. org/10.1016/j.sjbs.2016.08.007. al-waili n.s. 2004. natural honey lowers 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karapanou a., mantzourani i., stavropoulou e., fotou k., tzora a., skoufos i. & bezirtzoglou e. 2011. antibacterial activity of different honeys against pathogenic bacteria. anaerobe, 17 (6), 375-379. https://doi.org/10.1016/j. anaerobe.2011.03.012. 211 parole chiave evoluzione biologica, canine distemper virus, cani, europa, genomica, emoagglutinina. riassunto il canine distemper virus (cdv) è l’agente eziologico del cimurro nei cani. mostra un elevato potenziale di superamento delle barriere di specie, infettando un ampio range di carnivori selvatici e domestici. dei suoi geni codificanti, l’emoagglutinina (h) mostra alta eterogeneità ed è stata usata per determinare la relazione tra i ceppi di cdv, per via della sua variabilità e il ruolo chiave nel determinare il tropismo cellulare, il passaggio di specie e la capacità di elicitare una risposta immunitaria protettiva. questo studio ha analizzato l’intera sequenza del gene h dei ceppi di cdv artici identificati in italia da cani durante un periodo in cui è stata osservata un’aumentata diffusione di cdv. sono stati descritti i cambiamenti comuni degli aminoacidi e le caratteristiche dei ceppi cdv artici collezionati dal 2011 al 2016 in europa, fornendo un’analisi aggiornata delle caratteristiche genomiche. per valutare l’aumento della divergenza genomica rispetto ai ceppi di cdv artico di campo, è stata effettuata una comparazione con i ceppi vaccinali di cdv. questo studio restituisce un’analisi completa ed aggiornata della corrente circolazione dei ceppi del lineage artico e le principali variazioni degli aminoacidi nella sequenza del gene dell’emoagglutinina circolanti in italia. presenta, inoltre, nuove informazioni relative all’evoluzione dei più recenti ceppi di cdv del lineage artico collezionati in europa. aggiornamento sui ceppi di canine distemper virus (cdv) del lineage artico rilevati nei cani in italia veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.1455.7862.2 accepted: 17.07.2018 | available on line: 30.09.2018 keywords biological evolution, canine distemper virus, dogs, europe, genomics, hemagglutinin. summary canine distemper virus (cdv) is the etiologic agent of distemper in dogs. it exhibits an elevated potential of crossing species barriers, infecting a wide range of wild and domestic carnivores. of its encoding genes, hemagglutinin (h) shows high heterogeneity, and it was used to determine the relationship between cdv strains due to its variability and key role in determining cell tropism, host shift, and in eliciting a protective immune response. this study analysed the full-length h gene sequence of arctic-like cdv strains collected from dogs in italy during a period in which an increased activity of cdv diffusion was observed. the common amino acid changes and features of arctic-like cdv strains collected from 2011 to 2016 in europe were described, providing an updated analysis of the genomic features. a comparison with cdv vaccine strains was carried out to evaluate the increased genomic difference with cdv arctic-like field strains. this study provides a complete and updated analysis of the current spreading strains of arctic-like lineage and the main amino acid variations in the hemagglutinin gene sequence circulating in italy. moreover, it provides novel information regarding the evolution of the most recent cdv arctic-like lineage strains collected in europe. 1 istituto zooprofilattico sperimentale della sicilia “a. mirri”, via gino marinuzzi 3, 90129 palermo, italy. 2 dipartimento di scienze mediche veterinarie, università di bologna, via tolara di sopra 50, 40064 ozzano emilia (bo), italy. * corresponding author at: istituto zooprofilattico sperimentale della sicilia “a.mirri”, via gino marinuzzi 3, 90129 palermo, italy. tel.: +39 0916565447, e-mail: dottoremira@gmail.com. francesco mira1*, giuseppa purpari1*, santina di bella1, domenico vicari1, giorgia schirò1, patrizia di marco1, giuli macaluso1, mara battilani2 and annalisa guercio1 *francesco mira and giuseppa purpari should be considered joint first author update on canine distemper virus (cdv) strains of arctic-like lineage detected in dogs in italy 212 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx distinguishable on the basis of additional critera: the nucleotide (nt) identity of at least 98% for the strains belonging to the same clade, and a clear separation in the phylogenetic tree, with high bootstrap values (budaszewski et al. 2014). the circulation of 3 distinct lineages have been reported in italy: europe (also called europe-1/ south america-1), europe wildlife, and arctic-like lineages (balboni et  al. 2014, di francesco et  al. 2012, di sabatino et  al. 2014, marcacci et  al. 2014, martella et al. 2006, martella et al. 2002, monne et al. 2011). arctic-like lineage cdv strains have also been detected in animal hosts other than dogs, such as wolves (di sabatino et al. 2014), badgers (di sabatino et al. 2016), and tigers (seimon et al. 2013). an adequate host immune response against h protein may prevent cdv infections (martella et al. 2008). however, despite the fact that the incidence of cd in dogs has been reduced by the use of modified live virus (mlv) vaccines (blixenkrone-møller 1993), outbreaks of cd have also been reported in vaccinated dogs in italy as well as in other countries (balboni et al. 2014, decaro et al. 2004, di francesco et  al. 2012, martella et  al. 2007, martella et  al. 2006, scagliarini et  al. 2003). genetic and antigenic heterogeneity between the vaccine and field strains was hypothesised to be responsible for cd in vaccinated dogs (bae et  al. 2013, di francesco et  al. 2012, zhao et  al. 2010). however, as has also been hypothesised, some cases of cd in dogs vaccinated shortly before the onset of disease were more likely because of the presence of residues of maternally derived antibodies, and/or to the incorrect handling or management of the immune prophylaxis (blixenkrone-møller et al. 1993, zhao et al. 2010). in this study we analysed and characterised the h gene sequences of the cdv strains collected from dogs during a period in which increased activity of cdv was observed in italy. we also reconstructed the geographic and temporal evolutions of the cdv arctic-like lineage. for this purpose, the recent field strains were sequenced and their sequences were compared with those available in the genbank in order to elucidate the genetic divergences of circulating cdv strains. the findings in this study reveal features in common with the most recently collected strains in europe and provide novel information regarding the evolution and diffusion of the cdv arctic-like lineage. materials and methods samples the cdv strains analysed in this study were identified from samples of 91 dogs in southern italy that exhibited introduction canine distemper (cd) is a highly contagious and often fatal viral disease, characterised by respiratory, gastrointestinal and nervous signs (elia et  al. 2006, von messling et  al. 2003). the severity of clinical signs is influenced by strain virulence, environmental conditions, host age and immune status (espinal et  al. 2014). canine distemper virus (cdv), the causative agent of cd, belongs to the morbillivirus genus within the paramyxoviridae family (de vries et  al. 2015). canine distemper virus was first described by h. carré in 1905 (carré 1905) and, over time, it has been reported in a broad range of terrestrial and aquatic carnivores (carvalho et  al. 2012) as well as in non-carnivorous species (qiu et  al. 2011, yoshikawa et  al. 1989). the genome consists in negative-sense, single-stranded rna encoding for 6 structural proteins: nucleocapsid (n), matrix  (m), fusion (f), hemagglutinin (h), polymerase (l), and phosphoprotein (p) (martella et  al. 2008). the h  glycoprotein is the most variable integral membrane protein of the viral envelope, and mediates the binding of the virus to receptors on the host cell in the first step of infection (martella et  al. 2008, von messling et  al. 2001). moreover, it plays an essential role in cell tropism; therefore, sequence variations may affect the virulence, host range, and neutralisation-epitopes of cdv (ke et  al. 2015). the h protein is a transmembrane glycoprotein composed of a short n-terminal cytoplasmatic tail followed by a transmembrane domain and a large c-terminal ectodomain. the ectodomain is structured as a stalk and a 6-blade (b1-b6) beta-propeller fold, and each blade module contains 4-stranded anti-parallel beta-sheets (s1-s4) (ke et al. 2015). the h gene represented a suitable target for the molecular typing of cdv strains since it showed the greatest variability in the viral genome. in fact, on the basis of the genetic divergence in the h gene (bonami et  al. 2007), distinct genetic lineages were recognised. the cdv strains belonged to the same lineage if they clustered together in the same clade and showed an amino acid (aa) divergence of less than 4% (martella et al. 2006, mochizuki et al. 1999). therefore, on the bases of these criteria, 6 lineages (america-1 and -2, asia-1 and -2, europe, and arctic) were recognised for many years (martella et al. 2008). in recent years, intense and extensive molecular studies have led to the evidence of new, additional lineages. to this end, twelve genetic lineages have now been described (america-1 and -2, arctic-like, asia -1, -2, and -3, europe wildlife, europe-1/south america-1, south america-2, south america-3, south africa, rockborn-like) (balboni et  al. 2014, espinal et  al. 2014). more recently, in addition to these molecular typing criteria, it has been proposed that, within each genotype, subgenotypes were cdv strains in dogs in italy mira et al. veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 213 screened using a reverse transcription pcr (rt-pcr) assay, which amplified a 429-bp fragment of the p  gene (barrett et  al. 1993). the rt-pcr was carried out using the qiagen® onestep rt-pcr kit (qiagen s.r.l., milan italy) in a 50 μl reaction mix consisting of 10 μl of 5x buffer, 2 μl of deoxynucleotide (dntp) mix, 0.3 μl of rnase inhibitor (30 u/μl), 1 μl of each primer dmv1-dmv2 (20 μm) (table i), 2 μl of enzyme mix, 31.2 μl of nuclease free water, and 2.5 μl of rna extract. cdv rna (strain bussell) and nuclease-free water were used as positive and negative controls, respectively. reverse transcription and amplification were carried out in a single-step protocol under the following thermal conditions: 50  °c for 30 minutes to synthesise the first cdna, 95  °c for 15 minutes to inactivate the reverse transcriptase, followed by 35  cycles each of 94  °c for 45 seconds, 56  °c for 45 seconds, 72 °c for 1 minute, and a final extension of 72 °c for 10 minutes. sequencing and phylogenetic analysis positive samples from 10 screened dogs (table  ii), selected on the basis of different geographical areas of collection and origin, underwent a second previously described rt-pcr using primer pair c (demeter et  al. 2007). this process amplified the entire cdv h gene sequence (table i). the cdv rna was amplified in a single-step pcr protocol using the qiagen® onestep rt-pcr kit (qiagen s.r.l.) in a 100 μl reaction mix consisting of 20 μl of 5x buffer, 4  μl of dntp mix, 0.6 μl of rnase inhibitor (30 u/μl), 1.6 μl of each primer c-forward/c-reverse (20 μm) (table i), 4 μl of enzyme mix, 63.2 μl of nuclease-free water, and 5 μl of rna extract. reverse transcription and amplification were carried out under the following thermal conditions: 50  °c for 30 minutes, 95  °c for 15 minutes, followed by 40 cycles each of 94  °c for 45 seconds, 52 °c for 45 seconds, 72 °c for 2 minutes, and a final extension of 72 °c for 10 minutes. suspected clinical signs of distemper (neurological, respiratory, or enteric signs). the samples were collected from february 2015 to october 2016 and were sent to the istituto zooprofilattico sperimentale della sicilia ‘a. mirri’ (palermo, italy) for diagnostic purposes. the specimens included conjunctival, nasal, and rectal swabs or organs (brain, lungs, spleen, kidneys, liver, intestine). the swabs and autoptic specimens were both processed using homogenisation (10% w/v) in eagle’s minimum essential medium (emem, lonza, biowhittaker, slough, uk) supplemented with antibiotics and antimycotic (1,000  u/ml penicillin g sodium salt, 1 mg/ml streptomycin sulfate, 2.5 μg/ml amphotericin b) (paa laboratories gmbh, pasching, austria). the homogenates were centrifuged at low speed (1,500  x  g for 15 minutes at 4  °c); the supernatants were then collected, incubated at 37 °c for 1 hour, and were finally stored at -  80  °c until processed. molecular detection of cdv viral rna was extracted from 140 μl of homogenate using a qiaamp® viral rna mini kit (qiagen s.r.l., milan, italy) according to the manufacturer’s instructions. the presence of the cdv genome was mira et al. cdv strains in dogs in italy table i. oligonucleotide primers used in the pcr assays. pr im er se qu en ce (5 '-3 ') fr ag m en t ta rg et re fe re nc e dmv-1 5'-atgtttatgatcacagcggt-3' 429 bp gene p barrett et al. 1993 dmv-2 5'-attgggttgcaccacttgtc-3' b-forward 5'-aggccgtacatcaccaagtc-3' 1110 bp gene h demeter et al. 2007 b-reverse 5'-tggtaagccatccggagttc-3' c-forward 5'-aacttagggctcaggtagtc-3' 2023 bp c-reverse 5'-agatggacctcagggtatag-3' table ii. list of samples and accession numbers. strain sample age origin vaccinated signs death accession n. cdv_izssi_37465ct_2016 nasal swab 4 years shelter unknown g-r no mf663673 cdv_ izssi_36770pa_2016 rectal swab 10 months private yes g-r no mf663674 cdv_ izssi_3853ct_2016 lung 5 years shelter no g – r – n yes kx943321 cdv_ izssi_3315ct_2016 spleen 3 months stray dog unknown g – r yes kx943322 cdv_ izssi_980pa_2016 ocular swab 2 months private no g – r no kx943320 cdv_ izssi_285pa_2016 ocular swab 12 years private yes g – r – n yes kx943319 cdv_ izssi_47586c8_2015 ocular swab 1,5 years shelter no g – r – n yes kx943326 cdv_ izssi_45182_2015 lung 8 years shelter no g – r – n yes kx943325 cdv_ izssi_25130_2015 nasal swab 8 years private no r – n yes kx943324 cdv_ izssi_8387cucciolo_2015 lung 2 months stray dog unknown r yes kx943323 g = gastrointestinal signs; r = respiratory signs; n = nervous signs. nick title first author et al. 214 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx (gq214373) from 1988 to 2015. the h  gene sequences of america i (onderstepoort-af305419; convac-z35493; lederle-ef418782; snyder hillaf259552) and rockborn-like (rockborn candurgu266280; rockborn 46th-gu810819; lesser panda-af178039; vanguard-ef095750) lineage vaccine strains were also included in the dataset. nucleotide and deduced amino acid sequences were aligned and analysed using bioedit ver. 7.0.0 software (hall 1999). the prediction of potential n-linked glycosylation sites was carried out using netnglyc 1.0 web-based programme (www.cbs.dtu. dk/services/netnglyc/). the phylogenetic analysis was carried out with mega5 software (tamura et  al. 2011) using the neighbor-joining method according to the tamura 3-parameter model, modelled using a gamma distribution with 5 rate categories (boostrap 1,000 replicates). the best-fit model of nt substitution was selected using the find best dna/protein model function included with the mega5 software. the phocine distemper virus strain ulster/88 (d10371) was used as the outgroup. the h gene sequences of the cdv arctic-like lineage available from the sequence databases were different lengths. for this reason the phylogenetic analysis was carried out a representative pcr product of each of the 10 dogs was purified using an illustra™ gfx™ pcr dna and gel band purification kit (ge healthcare life sciences, amersham, buckinghamshire, uk) and was sent to bmr genomics srl (padova, italy) for direct sanger sequencing with a set of 4 primers (primer pairs b-forward/reverse and c-forward/reverse) (table i). according to an overlapping strategy, the sequences were assembled using the abi chroma align web-based programme. assembled nt sequences were entered into the blast (www. blast.ncbi.nlm.nih.gov/blast.cgi) and the ena (www.ebi.ac.uk/ena) web-based programmes to search for related sequences in the public domain sequence databases. a dataset of 122  cdv h  gene sequences from worldwide cdv strains was retrieved from the genbank/embl databases. the dataset included 59 h gene sequences of arctic-like cdvs, collected from domestic and wild animals in italy (dq226087-88, kf914669, kc966928-29, km115532-36, kx024708-09, kf184985-87, kf184989-91, kc992186-87, hm443706, hm443710-19, hm443720-22, hm443724), hungary (dq889178-86), switzerland (kr002657-61), the u.s.a. (ay964108-12), china (af172411, ef445052), greenland (z47760), russia (x84998), and austria table iii. nucleotide and deduced amino acid (in brackets) variations in h gene sequence in analyzed cdv strains. nt position aa position 114 145 (49) 173 (58) 197 (66) 240 249 360 423 516 (172) 528 583 (195) 818 (273) 843 897 929 (310) 1026 1059 reference strains(1) g a (ile) t (val) g (ser) a c a c g (leu) c g (val) g (val) g g g (gly) c c strains italy 2015/16(2) g a (ile) t (val) g (ser) a/g c a t t (phe) c g (val) t (ile) a g a (asp) a c strains swiss(3) . . c(ala) . g t . . g (leu) . . . . . . . strains 2011/13(4) . . . . g . g . g (leu) . a (ile) . . . . t wolves(5) . . . . g . g . g (leu) . a(ile) . . . . t badgers(6) a g(val) . a (asn) g . g . g (leu) t a (ile) c (thr) . a . . t nt position aa position 1071 1117 (373) 1250 (417) 1278 1303 (435) 1333 (445) 1524 1527 1626 1645 (549) 1663 (555) 1675 (559) 1754 (585) 1807 (603) reference strains(1) a g (glu) t (ile) a g (asp) t (leu) c t c t (tyr) a (thr) c (pro) t (ile) a (asn) strains italy 2015/16(2) g g (glu) c (thr) g/a a (asn) a/t (leu/ met) t t c t(tyr) a (thr) t (ser) t/c (ile/thr) a/c (asn/ his) strains swiss(3) . a(lys) . g . t (leu) . . . . . . t (ile) a (asn) strains 2011/13(4) . . . g . a (met) . . t t/c (tyr/his) a/g (thr/ ala) . t(ile) a (asn) wolves(5) . . . g . a(met) . . t . . . t (ile) a (asn) badgers(6) . . . g . a(met) . c t c (his) . . t (ile) a (asn) (1) reference strains (dq226087-dq226088), (2) strains analyzed in this study, (3) strains swiss (kr002657-58), (4) strain 2011/13 (kf184989-91, km115532-36, kj567090-93, kf914669), (5) wolves (kc966928-29), (6) badgers (kx024708-09). first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 215 complete identity between 3 cdv sequences (cdv_ izssi_8387cucciolo_2015, cdv_izssi_45182_2015, and cdv_izssi_36770pa_2016), while the other sequences showed high identity rates (99.94-99.67%). comparison with related reference sequences showed high reciprocal identity rates with cdv arctic-like lineage reference sequences (>  96.3%) and, of these, the highest identity rates were obtained with other european strains: strains from dogs (99.78-99.45%) collected in switzerland in 2013 and from dogs (99.67-99.23%), wolves (99.72-99.45%), and badgers (99.28-99.01%) collected in italy in 2013 and 2015. the nt and aa identity rates of the cdv strain (cdv_izssi_3853ct_2016) with the closely related onderstepoort (af305419) and rockborn (gu266280) vaccine strains were 91-89% and 95-94%, respectively. comparison of the sequences in this study with the first complete h gene sequences of arctic-like lineage reported in italy (dq226087-88) showed 17 single-nucleotide polymorphisms (snps), including 9 synonymous and 8 non-synonymous substitutions. the point mutations observed were 13 transitions and 4 transversions. of these snps, g516t was common to the strains collected in italy in 2000 (hm443714-16-17) while t1754c and a1807c were characteristic only of the samples in this study, including only complete cdv h gene sequences (1824 nts), of which 27 sequences were from cdv artic-like lineage strains collected in europe from 2003 to 2015, and 68 sequences of other lineages. in order to identify the relationships with previously available cdv arctic-like lineage sequences and to compare the higher number of h gene sequences available at the time of this study, an additional phylogenetic analysis including 53 sequences of 861 nts (from nt residue 469 to 1329) was carried out using the same previous statistical model without gamma distribution and outgroup. the h gene sequences of the italian cdv strains analysed in this study were submitted to the genbank/embl/ddbj databases under the accession numbers reported in table ii. results a 429-bp fragment of the p gene was detected in samples from 33 of the dogs that were tested (36.3%). amplicons obtained from the samples of 10  dogs that tested positive (table ii) were sequenced, and the full-length h gene sequence (1824 nts) was determined. the deduced aa sequences (607 aas) were also inferred. the reciprocal comparison of the sequences showed table iv. amino acid variations of analysed viral strains shared with arctic-like lineage and america i/rockborn-like lineage vaccines sequences. strains (accession n.) h gene amino acid residues 172 195 310 417 435 445 559 585 603 2004 italy(1) (dq226087) leu val gly ile asp leu pro ile asn 2005 italy(1) (dq226088) cdv_izssi_37465ct_2016 phe val asp thr asn ser thr cdv_izssi_36770pa_2016 phe val asp thr asn ser cdv_izssi_3853ct_2016 phe val asp thr asn ser cdv_izssi_3315ct_2016 phe val asp thr asn ser thr cdv_izssi_980pa_2016 phe val asp thr asn ser his cdv_izssi_285pa_2016 phe val asp thr asn met ser his cdv_izssi_47586c8_2015 phe val asp thr asn ser cdv_izssi_45182_2015 phe val asp thr asn ser cdv_izssi_25130_2015 phe val asp thr asn met ser cdv_izssi_8387cucciolo_2015 phe val asp thr asn ser 2013 swiss(2) asp thr asn ser 2012-15 italy(2) ile asp thr asn met ser 2008 italy(1) (hm443706) asp thr asn 2000 italy(2) phe 1988-2006(2) arctic-like lineage -(*) 1994-2007(2) america i lineage vaccines val 1999-2009(2) rockborn-like lineage vaccines strains were named (1) including year of collection/submission, country and accession number or (2) were grouped (2013 swiss: kr002657-61; 2012-15 italy: kx024708-09, kc966928-29, kf914669, km115532-36; 2000 italy: hm443714, hm443716, hm443717; 1988-2006 arctic-like lineage: dq889178-86, hm443710-13, hm443715, hm443719-22, hm443724, ef445052, ay964112, ay964108, gq214373, af172411, z47760, x84998; america i lineage vaccines: af259552, ef418782, z35493, af305419; rockborn-like lineage vaccines: ef095750, af178039, gu810819, gu266280) and titled including year of collection/submission and country or lineage. (*) except isolate 3148-03 (2003, austria, gq214373): ans603ser. nick title first author et al. 216 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx a non-synonymous substitution that changed the first aa (asn603his). a non-synonymous substitution affects the second aa position (310asp) of the potential glycosilation site 309-311. sequences of and they were all non-synonymous substitutions (leu172phe, ile585thr, and asn603his, respectively). twelve snps (a240g, c423t, g843a, g929a, c1026a, a1071g, t1250c, a1278g, g1303a, t1333a, c1524t, c1675t) were common to the sequences of the arctic-like lineage collected beginning in 2011 (7  synonymous and 5 non-synonymous). of these, 8 snps (at nt residues 240, 423, 843, 929, 1026, 1196, 1250, 1303) were also common in strains collected in italy in 2008 (hm443706). compared to the sequences of arctic-like lineage collected beginning in 2011, 4 snps characteristics of sequences collected only in italy from 2011 to 2015 (3 synonymous – a360g, c1059t, c1626t – and 2 non-synonymous – g583a, a1663g) and 2 characteristics of strains collected only in switzerland in 2013 (non synonymous – t173c, g1117a) were observed. nucleotide changes and the predicted amino acid sequence substitutions are shown in table iii. a total of 5 aa changes was shared by our strains and the arctic-like cdv reference strains collected after 2011: gly310asp, ile417thr, asp435asn, leu445met, pro559ser. another 3 aa changes were observed: change leu172phe, common to the 3 strains collected in italy in 2000 (hm443714-16-17), and the aa substitution ile585thr and asn603his, never previously reported. other critical aa residues were 195val and 445leu, in common with the strains in this study and arctic cdv strains collected in switzerland in 2013 (kr002657-61). amino acids at these residues were different from the most recent italian arctic cdv strains collected from 2011 to 2013, except for strains cdv_izssi_285pa_2016 and cdv_ izssi_25130_2015, which showed the aa  met  (m) at residue 445. none of the sequences in this study showed change thr555ala, observed in some italian strains collected in 2012/13 (km115532-36). predicted amino acid sequence changes shared with arctic-like cdv reference sequences are shown in table iv. alignment with related sequences of cdv vaccine strains belonging to america i and rockborn-like lineages showed at least 42 and 32 main aa differences, respectively. these aa changes increased the genomic divergence between field arctic-like strains and vaccine strains (table iv). in all the strains in this study, aa asn (n) and aa tyr (y) were observed at residues 530 and 549, respectively. amino acid residues crucial for receptor interactions (slam and nectin-4) were conserved (aa residues from 454 to 555). a total of 8 potential glycosilation sites were predicted at aa residues: 19-21, 149-151, 309-311, 391-393, 422-424, 456-458, 587-589, and 603-605, with the exception of strains cdv_izssi_285pa_2016 and cdv_izssi_980pa_2016, which lacked the glycosilation site in the aa positions 603-605, due to figure 1. neighbour-joining tree based on the full-length hemagglutinin (h) gene sequences (1,824 nucleotides) of canine distemper virus (cdv) displaying the genetic relationships between cdv arctic-like lineage strains and 68 cdv strains of other lineages (bootstrap 1000 replicates; bootstrap values shown greater than 80%). black dot markings (•) indicate cdv strains analysed in this study. cdv arctic-like lineage strains were indicated with: year and country of collection, host, and accession number. cdv strains of other lineages were grouped using the following accession numbers in parentheses: europe/south america i (af478543, af478547, z47761, dq494317, z77673, dq494319, dq494318, jf810111, dq889177, ay386315, hm563059, gq214376, gq214384, fj392652, jn215474, jn215476), south america iii (kf835420, kf835414, kf835425), south america ii (fj392651), europe wildlife (dq889188, dq889187, z47759, gq214374, gq214369, dq228166, jn153022, jn153021, jn153023), america ii (ay498692, af164967, ay526496, z47763, z47764, z47762, z47765), asia i (ab212965, d85754, ab329581, fj409464, hq540293, af178038), rockborn-like (ay964114, fj461702, gu266280, af178039, gu810819, fj705238), south africa (fj461714, fj461698, fj461720, fj461724), asia ii (ab040767, ab040768, eu252149, ab025270, ab252718) and america i (af305419, af014953, dq903854, af378705, z35493, am903376, ay548109, ay466011, af259552, ef418782, dq778941). first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 217 collected in italy in 2000 (hm443714-16-17), which also showed 32 residues, but in different positions. the main difference among the proline residues in the strains collected before 2011 was the lack of the aa due to the change at residue 559 (pro559ser). cysteine residues (12) were maintained within the arctic lineage. the asn603his substitution described was located immediately adjacent to the cysteine residue at position 602. in this study the phylogenetic tree showed the relationship between the sequences and the complete h gene sequences retrieved from genbank (figure 1). all strains segregated into distinct branches according to 11 of the previously reported lineages (budaszewski et  al. 2014, espinal et  al. 2014): europe/south america-i, south america-ii, south america-iii, america-i, america-ii, asia-i, asia-ii, rockborn-like, arctic-like, south africa, europe wildlife. all of our italian strains clustered within the arctic lineage (intra-lineage variation was < 3.7%) in a clade that included european strains collected after 2011, were clearly distinct from those of the vaccine strains (america i and rockborn-like lineages) that were used for comparison. the aa variation with respect to the other lineages was constantly > 4%. in the additional phylogenetic tree of cdv strains only of arctic-like lineage (figure 2), the strains in this study clustered together with strains collected in italy and switzerland from 2012 to 2016, and strains collected in italy in 2000 and 2008. strains collected in austria, italy, hungary, and the usa from 2000-2006 segregated in a separate cluster. other strains, including the first arctic-like strains from russia and greenland, segregate separately. the selected nt range for this phylogenetic tree involved 5 (residues 172, 195, 310, 417, 435) of the 9 aa residues described. the strains in this study clustered within a main clade including strains collected in china, europe, and the usa after 1999, and this clade showed an nt identity rate greater than 98  % when compared to the first strains of this lineage collected in russia (96.7 %) and greenland (97.3 %). discussion despite the introduction and extensive use of modified live virus vaccines, canine distemper virus still remains one of the most dangerous viruses for dogs. similar to other members of the genus morbillivirus, cdv is highly contagious, can cause severe disease, and can result in up to 100 % mortality (sawatsky & von messling 2010). of the cdv lineages spreading in italy, the arctic-like cdv strains represent an interesting case of transmission among different host species and of spreading through different countries. the first reports of vaccine strains belonging to the america i lineage lacked glycosilation site 309-311 due to an aa substitution at residue 309 (aa ser in ondersterpoort and convac strains; aa arg in the lederle and snyder hill strains) while the onderstepoort strains also lacked the potential glycosilation site 456-458. in this study the sequence alignment of all the arctic cdv strains showed a content of 32 proline residues, as were observed in all the arctic cdv strains collected after 2011. previously collected strains showed a higher number (33-35 residues) of proline residues, with the exception of 3 strains figure 2. neighbour-joining tree based on a 861 nucleotides (nt 469-1329) fragment of h gene sequences of cdv arctic-like lineage strains (bootstrap 1000 replicates; bootstrap values shown greater than 70%). black dots markings (•) indicates cdv strains analysed in this study. each sequence was indicated with: year and country of collection, host and accession number. nick title first author et al. 218 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx if these strains had permanently been established in italy (balboni et  al. 2014, martella et  al. 2006). almost 10 years later, the first cases of an arctic-like cdv strain affecting wolves, dogs, and badgers in a central region of italy were reported (di sabatino et  al. 2014, lorusso & savini 2014, di sabatino et  al. 2016). a close connection between strains from dogs and wolves in this area was pointed out, suggesting that the domestic dog could act as a reservoir for a virus that was then passed on to wolves (di sabatino et  al. 2014, marcacci et  al. 2014). the circulation of this particular cdv lineage in this region of italy (abruzzo) had not been reported prior to 2009 (di francesco et al. 2012), which suggests a more recent introduction than had previously been hypothesised. in 2014, 6  artic-like cdv strains were reported in other central italian regions (emilia-romagna and lazio) (balboni et  al. 2014) and, as described in this study, a close genomic relationship was observed with the other cdv strains collected from dogs and wolves in central italian regions (lazio and abruzzo) beginning in 2011. the geographic and temporal evidence of cdv arctic-like lineage in europe is represented in figure 3. our study described the molecular features of some cdv strains of arctic-like lineage collected in arctic-like lineage strains date back to the late 1980s and include the phocine strain pdv-2 identified in 1988 from a phoca siberica of lake baikal (visser et  al. 1990), and a canine strain gr88 detected in the same year from a sledge dog population in northern greenland (blixenkrone-möller et al. 1992). the arctic-like lineage was then reported in the mid-1990s in china (af172411), in 2003 in austria (benetka et  al. 2011), in 2004 in the usa (pardo et  al. 2005), and from a fox in 2005 in china (zhao et  al. 2010) – far from the artic ecosystem. from 2004-2006, the arctic-like lineage appeared once again in europe; closely related cdv strains were reported by martella and colleagues (martella et  al. 2006) and demeter and colleagues (demeter et  al. 2007) in italy and hungary, respectively. a possible explanation for the origin of these strains in separate geographic areas is the introduction of dogs imported from eastern europe or northern asia to italy, in addition to an intensified phenomenon of uncontrolled trading of low cost and high value breed pets (martella et al. 2006, demeter et al. 2007). a retrospective study report identified the spread of arctic-like cdv strains into italy as early as 2000 (monne et al. 2011) but it remained unclear whether this evidence represented occasional findings or figure 2. geographic distribution of the arctic-like cdv strains from dogs and wild animals in europe. each country (red: switzerland; yellow: austria; green: hungary) or italian region (blue: trentino-alto adige, friuli-venezia giulia, veneto; pink: emilia-romagna; grey: abruzzo; brown: lazio; orange: sicily) and year of collection are indicated using colour coding, according to the following references: (a) monne et al. 2011; (b) balboni et al. 2014; (c) benetka et al. 2011; (d) martella et al. 2006; (e) demeter et al. 2007; (f ) di sabatino et al. 2014; lorusso & savini 2014; marcacci et al. 2014; (g) willi et al. 2015; (h) di sabatino et al. 2016; (i) present study. first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 219 temporal patterns were also observed in strains collected in switzerland (willi et  al. 2015) in 2013 (val58ala, glu373lys) and in italy (km115532-35) in 2012/13 (thr555ala). these specific changes could contribute to trace cdv spread among different regions or countries. the temporal evidence of the aa changes of cdv arctic-like lineage is represented in figure 4. according to a previous description (ke et al. 2015), except for changes at residues 172 and 603, all these changes were located in blades b2, b3, b4, and b6 of the β-propeller fold, and some (417, 445) underwent epistatic interactions. some changes (310, 559, 603) affected the proline residues and n-linked glycosilation sites. these changes could have played an evolutionary role, due to their potential impact on the protein structures and the unique characteristics of the n-linked site among the wild-type strains at residues 309-311 (ke et al. 2015). in fact, n-linked glycosylation sites showed that they were important for the correct folding, transport, and functioning of other paramyxovirus fusion and attachment glycoproteins (sawatsky & von messling 2010); therefore, additional studies are necessary to better address any potential biological role. the intense sampling in italy in recent years has confirmed the drift acting on the h gene/ glycoprotein, and that the spread was mainly driven by a geographic pattern. this supports the theory that the introduction of these most recent cdv strains were carried by dogs imported from eastern european countries (balboni et  al. 2014, demeter et al. 2007, martella et al. 2006). given the hungarian origin of the dogs affected in switzerland and the high identity rates with the sequences of this study, a potential common origin could be possible. the transportation of dogs still plays an important role in the introduction of novel or re-emerging viral strains, as has already been observed for cdv (martella et  al. 2006) as well as for other canine viruses (decaro et  al. 2007, mira et  al. 2018). this evidence also suggests the potential spread to other nearby european countries and, therefore an additional epidemiological survey is necessary. our strains clustered within a clade of strains collected after 2008 in italy and switzerland (figure  2) that showed an nt similarity greater than 98  % when compared to the first strains of this lineage collected in china, russia, and greenland before 2000. these strains are clearly separated in the phylogenetic tree and, based on these criteria (budaszewski et  al. 2014), they could be argued to belong to a different subgenotype within the artic-like lineage. the differences were however consistent with the geographical origins of the strains, which are themselves a result of separate disseminations across the artic ecosystem and 2015-2016 from dogs in southern italy. due to the different lengths of some sequences in genbank, it was possible to integrally compare the entire cdv h gene with most, but not all, arctic-like cdv sequences available. nonetheless, it was possible to point out common and interesting features. compared to strains circulating from 2000 to 2006, the strains in this study showed different h gene aa profiles at specific residues: 172phe, 195val, 310asp, 417thr, 435asn, 445leu/met, 559ser, 585ile/thr, 603asn/his. the majority of these aa residues (195, 310, 417, 435, 445, 559) described a pattern shared by all the sequences of arctic-like lineage found in italy since 2011 (balboni et al. 2014, di sabatino et al. 2016), and some of these changes (gly310asp, ile417thr, asp435asn, pro559ser) have been constantly observed in all recent european sequences of this lineage, suggesting common ancestors. differences were observed at residues 195 and 445, and these 2 amino acid changes represented critical aa differences between both the strains in this study and the swiss cdv strains with respect to recent strains observed in 2013 in italy (di sabatino et  al. 2016). in specific combination with a few other amino acids that were analysed, residue 195 has relevant and compensational effects in the attachment protein of cdv (sattler et  al. 2014); therefore additional studies regarding the potential role of these changes are necessary. the leu172phe change was only observed in the strains considered in this study, as well as in 3 strains collected in 2000 (monne et  al. 2011). changes to ile585thr and asn603his were unique for only 4 of our strains. interestingly, changes at residues 585 and 603 were observed in strains from different geographical areas, while the change at residue 172 was a constant change in all the sequences in this study, irrespective of the collection area, and could therefore be considered a potential synapomorphy. similarly, specific aa changes with geographic and figure 4. first evidence of h gene sequence amino acid changes of cdv arctic-like lineage strains, indicated with year, host, and amino acid residue. positions were based on reference sequences with the accession numbers dq226087 and dq226088, according to the following references: (a) monne et al. 2011; (b) martella 2013 (unpubl.), balboni et al. 2014, di sabatino et al. 2014, lorusso & savini 2014, marcacci et al. 2014; (c) di sabatino et al. 2016; (d) present study. nick title first author et al. 220 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx a large body of literature has reported the propensity of cdv to jump species barriers into canine and non-canine hosts, seals, and non-carnivores (ludlow et  al. 2014). in fact, recent cases of cdv infection in wolves (di sabatino et  al. 2014) and badgers (di sabatino et al. 2016) in italy and tigers in russia (seimon et al. 2013) have been reported. wolves and tigers are subspecies listed as endangered by the international union for the conservation of nature (www.iunc.org). as has previously been suggested (cleaveland et  al. 2000), domestic dogs have been considered to be a likely source of cdv infection; therefore, additional studies are necessary to evaluate any potential cdv spread into wild animals in the same italian areas. in conclusion, our findings contribute to knowledge regarding the arctic-like cdv lineage in italy and describe cdv strains with specific aa profiles that are spreading in europe; thus, additional immunisation programmes to improve vaccine coverage among susceptible dogs are necessary to control cdv (rikula et  al. 2007). even if complete h gene amplification from field samples is difficult due to the scale of the task size and its transcription level (panzera et  al. 2014), complete h gene sequence and related metadata still remain useful in obtaining the complete comparison of circulating strains. this, in turn, facilitates a better understand of the epidemiological features of cdv. acknowledgments we acknowledge dott. calogero di bella and mr. michele di gesaro from izs della sicilia for kindly supplying the map in figure 3. central-eastern europe. thus is similar to reports of other subgenotypes (budaszewski et al. 2014). samples from live dogs were collected by non-invasive sampling (oculo-conjuntival or rectal swabs, stools, blood) and the effectiveness of this type of sampling for diagnostic molecular assays has previously been demonstrated (di francesco et  al. 2012, elia et  al. 2015). this is also a valid diagnostic approach to evaluating the viral shedding of suspect or stray dogs, especially before contact with other susceptible animals. rectal swabs positive for cdv can be collected from asymptomatic dogs (budaszewski et  al. 2014) and, together with sub-clinical infected dogs, they may transmit the virus, acting as cdv reservoirs for a long time (greene & appel 2006). the cdv is quickly inactivated in the environment, and transmission mainly occurs by aerosol infection from the infected animal (martella et al. 2008). a non-invasive sampling approach could contribute to check viral shedding from clinically and subclinically infected dogs in order to avoid the spread of cdv. it was not possible to obtain information from the positive dogs regarding their immune status before infection; therefore, it was not possible to confirm the real occurrence of cd in protected dogs. as suggested in the guidelines of the world small animal veterinary association (wsava) (day et  al. 2016), inclusion of the antibody specific for cdv should be considered in the vaccination schedule in order to evaluate the effectiveness of vaccination and to better programme cdv prophylaxis. moreover, the increased divergence observed between analysed and vaccine strains in the last 10 years has suggested the need for antigenic mapping in order to better evaluate the effectiveness of current vaccines. first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 221 bae c.w., lee j.b., park s.y., song c.s., lee n.h., seo k.h., kang y.s., park c.k. & choi i.s. 2013. deduced sequences of the membrane fusion and attachment proteins of canine distemper viruses isolated from dogs and 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distemper outbreak in rhesus monkeys, china. emerg infect dis, 17, 1541-1543. rikula u., nuotio l. & sihvonen l. 2007. vaccine coverage, herd immunity and occurrence of canine distemper from 1990-1996 in finland. vaccine, 25, 7994-7998. centro di referenza nazionale per il benessere animale rev 03 04/04/2014 pag. 1 data ora inizio n° id azienda (cod.asl) valutatore tel/mail dati azienda: personale: caratteristiche allevamento: tipo di allevamento intensivo biologico semiestensivo (presenza di aree esterne) ciclo aperto ciclo chiuso ingrasso svezzamento sito 1 sito 2 sito 3 goland danbred altro (specificare) pic hypor n° scrofe tot n° in gestazione n° in lattazione n° magroni e tempo permanenza management/personale addetto agli animali n° totale addetti si no corsi esterni (es: corsi obbligatori 146/2001) e' presente e conforme il registro di carico e scarico? si no e' presente e conforme il registro dei trattamenti? si no si no si no si no gli animali vengono abbattuti in allevamento? si no stordimento/iugulazione farmacologicamente stordimento/phiting esistono procedure scritte di eutanasia? si no capostalla scheda di valutazione benessere suini nome indirizzo telefono/e-mail proprietario gli animali vengono ispezionati almeno una volta al giorno? veterinario aziendale sistema produttivo linea genetica/incroci n° scrofette n° sottoscrofa (lattonzoli) n° suinetti in svezzamento e tempo di permanenza n °ingrasso n° verri tot n° verri per f.a. n° totale n° in gabbia n° in box anelli al naso (solo allev. aperto) il personale e' adeguatamente formato? si no corsi interni i registri sono conservati per il periodo stabilito dalla normativa? incisione auricolare per identificazione centro di referenza nazionale per il benessere animale pag. 2 dlgs 122/2011 conformita' n° gabbie n° box n° tipologie gabbie n° tipologie box si no si no si no si no si no si no si no si no si no si no si no si no si no si no si no si no si no si no % fibra da cartellino se disponibile % lipidi e kcal da cartellino se disponibile da cartellino se disponibile tutte le scrofe sono alimentate almeno una volta al giorno? l'alimentazione e' ad libitum? indicare metodo di alimentazione gestazione scrofe in box gestazione scrofe e scrofette vengono sottoposte a trattamento antiparassitario? gli animali gravidi ricevono un mangime ad alto tenore energetico in quantita' sufficiente? % lipidi e kcal da cartellino se disponibile scrofe in gabbia gestazione tutte le scrofe sono alimentate almeno una volta al giorno? l'alimentazione e' ad libitum? gli animali gravidi ricevono un mangime riempitivo o ricco in fibra in quantita' sufficiente? gli animali gravidi ricevono un mangime ad alto tenore energetico in quantita' sufficiente? indicare metodo di alimentazione % fibra da cartellino se disponibile da cartellino se disponibile gli animali gravidi ricevono un mangime riempitivo o ricco in fibra in quantita' sufficiente? gli impianti meccanici (alimentazione, areazione,...) vengono ispezionati almeno una volta al giorno? e' presente un recinto infermeria? il recinto infermeria e' dotato di lettiera confortevole? il recinto infermeria e' sufficientemente ampio affinche' gli animali possano girarsi? la ventilazione e' forzata? le scrofe e le scrofette sono allevate in gruppo tra 4 settimane post fecondazione e una settimana prima della data prevista del parto? indicare quanti gg post fecondazione sono messe in box se la ventilazione e' forzata e' presente un impianto di riserva? se la ventilazione e' forzata e' presente un sistema di allarme? vengono prese misure atte ad evitare lotte che vadano oltre il comportamento normale durante la formazione dei gruppi? indicare quali misure centro di referenza nazionale per il benessere animale pag. 3 dlgs 122/2011 conformita' n° box n° tipologie box si no si no si no la ventilazione e' forzata? si no verri si no indicare metodologia di alimentazione e' presente un recinto infermeria? il recinto infermeria e' dotato di lettiera confortevole? il recinto infermeria e' sufficientemente ampio affinche' gli animali possano girarsi? se presenti piu' verri nello stesso box, vengono prese misure atte ad evitare lotte che vadano oltre il comportamento normale durante la formazione dei gruppi? si no indicare quali misure tutti i suini sono alimentati almeno una volta al giorno? si no l'alimentazione e' ad libitum? gli impianti meccanici (alimentazione, areazione,...) vengono ispezionati almeno una volta al giorno? si no se la ventilazione e' forzata e' presente un impianto di riserva? si no se la ventilazione e' forzata e' presente un sistema di allarme? si no centro di referenza nazionale per il benessere animale pag. 4 dlgs 122/2011 conformita' n° sale parto n° gabbie per sala sala parto la ventilazione e' forzata? si no animali gravidi ricevono un mangime riempitivo o ricco in fibra in quantita' sufficiente? si no % fibra da cartellino se disponibile animali gravidi ricevono un mangime ad alto tenore energetico in quantita' sufficiente? si no % lipidi e kcal da cartellino se disponibile prima di essere sistemate nelle sale parto le scrofe vengono pulite? si no tutti le scrofe sono alimentate almeno una volta al giorno? si no gli impianti meccanici (alimentazione, areazione,...) vengono ispezionati almeno una volta al giorno? si no se la ventilazione e' forzata e' presente un impianto di riserva? si no se la ventilazione e' forzata e' presente un sistema di allarme? si no centro di referenza nazionale per il benessere animale pag. 5 dlgs 122/2011 conformita' n. box n. tipologie box si no si no si no si no l'alimentazione e' ad libitum? se l'alimentazione non e' ad libitum gli animali vengono alimentati almeno una volta al giorno? si no vengono prese misure atte ad evitare lotte che vadano oltre il comportamento normale durante la formazione dei gruppi? si no svezzamento indicare a che eta' viene effettuata la castrazione, se sono usati analgesici, anestetici e da chi viene effettuata (veterinario-operatore) eta': analgesia: si no anestesia: si no vet op indicare a che eta' viene effettuato il taglio della coda, a che lunghezza e da chi viene effettuata (veterinario-operatore eta': < 1,2 cm >1,2 cm vet op indicare a che eta' viene effettuato il taglio dei denti, con che metodo e da chi viene effettuata (veterinario-operatore eta': taglio limatura vet op lo svezzamento viene effettuato dopo i 28 gg di eta'? si no se no, indicare l'eta' e se sono presenti impianti specializzati e' presente un recinto infermeria? il recinto infermeria e' sufficientemente ampio affinche' gli animali possano girarsi? il recinto infermeria e' dotato di lettiera confortevole? indicare quali misure gli impianti meccanici (alimentazione, areazione,...) vengono ispezionati almeno una volta al giorno? si no se la ventilazione e' forzata e' presente un impianto di riserva? si no se la ventilazione e' forzata e' presente un sistema di allarme? si no la ventilazione e' forzata? si no centro di referenza nazionale per il benessere animale pag. 6 dlgs 122/2011 conformita' n. box n. tipologie box si no si no si no e' presente un recinto infermeria? il recinto infermeria e' dotato di lettiera confortevole? il recinto infermeria e' sufficientemente ampio affinche' gli animali possano girarsi? vengono prese misure atte ad evitare lotte che vadano oltre il comportamento normale durante la formazione dei gruppi? si no indicare quali misure tutti i suini sono alimentati almeno una volta al giorno? si la ventilazione e' forzata? si gli impianti meccanici (alimentazione, areazione,...) vengono ispezionati almeno una volta al giorno? si no se la ventilazione e' forzata e' presente un impianto di riserva? si no se la ventilazione e' forzata e' presente un sistema di allarme? si no no l'alimentazione e' ad libitum? si no magronaggio no indicare metodologia di alimentazione centro di referenza nazionale per il benessere animale pag. 7 dlgs 122/2011 conformita' n. box n. tipologie box si no si no si no la ventilazione e' forzata? si no si no indicare metodologia di alimentazione ingrasso e' presente un recinto infermeria? il recinto infermeria e' dotato di lettiera confortevole? il recinto infermeria e' sufficientemente ampio affinche' gli animali possano girarsi? vengono prese misure atte ad evitare lotte che vadano oltre il comportamento normale durante la formazione dei gruppi? si no indicare quali misure tutti i suini sono alimentati almeno una volta al giorno? si no l'alimentazione e' ad libitum? gli impianti meccanici (alimentazione, areazione,...) vengono ispezionati almeno una volta al giorno? si no se la ventilazione e' forzata e' presente un impianto di riserva? si no se la ventilazione e' forzata e' presente un sistema di allarme? si no centro di referenza nazionale per il benessere animale pag. 8 si no si no e' sporco? e' accessibile? e' utilizzato? si no si no si no gabbia tipo 3 lo spazio a disposizione e' sufficiente affinche' la scrofa riesca a muoversi nelle direzioni consentite, sdraiarsi ed alzarsi senza difficolta'? si no rumori continui < 85 db pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? agli animali? le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? superficie disponibile/pavimentazioni/fessurati gabbia gabbia gestazione dlgs 122/2011 conformita' lo spazio a disposizione e' sufficiente affinche' la scrofa riesca a muoversi nelle direzioni consentite, sdraiarsi ed alzarsi senza difficolta'? si no numero succhiotti(se più tipi di box indicare per ogni tipo) alimento e acqua ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? altro locali di stabulazione luce > 40 lux per 8 h la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 10-20 °c) altro (misurazioni) quantita' per box altro (catene, palle,... e' presente materiale per esplorazione e manipolazione? indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) gabbia tipo 2 gabbia tipo 3 gabbia tipo 1 gabbia tipo 2 lo spazio a disposizione e' sufficiente affinche' la scrofa riesca a muoversi nelle direzioni consentite, sdraiarsi ed alzarsi senza difficolta'? si no altro metodo materiali manipolabili gabbia tipo 1 si no legno torbapaglia fieno centro di referenza nazionale per il benessere animale pag. 9 gabbia gestazione parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 stato sanitario generale (buono medio scarso) igiene corpo (% corpo imbrattato di feci) <10% 10-30% >30% ferite (trauma contro le strutture) ferite corpo unghielli altro (specificare) condizione clinica bcs (1-3-5) rogna a:auricolare c:corpo ulcere spalla ascessi patologie respiratorie n° bursiti zoppia patologie enteriche scoli vulvari prolasso utero prolasso retto altro (specificare) stereotipie masticazione a vuoto tongue rolling morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) 8 10 11 * se ci sono diverse tipologie di gabbie valutare almeno una per tipo e indicare in quale gabbia è stabulato l'animale id animali n° scrofe in gestazione n. animali da campionare ≤ 10 11-20 12 13 14 21-30 31-60 61-200 > 200 centro di referenza nazionale per il benessere animale pag. 10 tipo tipo misure misure condizioni metereologiche nei giorni/settimane precedenti la valutazione superficie disponibile/pavimentazioni/fessurati scrofe 2,25 mq/scrofette 1,64 mq (<6+10%; >40-10%) lati recinto > 2,8 mt (< 6 animali: > 2,4 mt) tipo di pavimentazione (vedi allegato 2) mm fessura mm travetto mm fessura mm travetto pavimento pieno continuo: scrofe 1,3 mq /scrofette 0,95 mq (vedi allegato 2) ampiezza fessurati 20 mm ; larghezza travetti 80 mm tipo di pavimentazione (vedi allegato 2) tipo di pavimentazione (vedi allegato 2) mm fessura mm travetto misure esterne lati box superficie disponibile (no truogolo) larghxlungh misure esterne lati box superficie disponibile (no truogolo) larghxlungh box gestazione dlgs 122/2011 conformita' box tipo 6 n° animali tipo di pavimentazione (vedi allegato 2) mm fessura mm travetto se presenti parchetti esterni indicare tipologia (vedi allegato 2) tipo misure box tipo 4 n° animali misure esterne lati box superficie disponibile (no truogolo) larghxlungh misure esterne lati box superficie disponibile (no truogolo) larghxlungh misure esterne lati box superficie disponibile (no truogolo) larghxlungh misure esterne lati box superficie disponibile (no truogolo) larghxlungh tipo di pavimentazione (vedi allegato 2) mm fessura mm travetto tipo di pavimentazione (vedi allegato 2) box tipo 1 n° animali box tipo 2 n° animali box tipo 3 n° animali mm fessura mm travetto n° animali box tipo 5 centro di referenza nazionale per il benessere animale pag. 11 si no si no e' sporco? e' accessibile? e' utilizzato? si no si no si no e' presente materiale per esplorazione e manipolazione? box tipo 1 paglia fieno quantita' per box altro (catene, palle,... box tipo 3 box tipo 4 box tipo 6 si no legno torba box tipo 2 box tipo 5 materiali manipolabili lunghezza truogolo (se più tipi di box indicare per ogni tipo) pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? ogni scrofa ha accesso agli alimenti contemporaneamente agli altri componenti del gruppo? indicare metodo (se più tipi di box indicare per ogni tipo) il sistema utilizzato per l'alimentazione garantisce che si evitino aggressioni? alimento e acqua altro zona per coricarsi asciutta e pulita che consenta a tutti gli animali di stare sdraiati contemporaneamente locali di stabulazione altro (misurazioni) luce > 40 lux per 8 h rumori continui < 85 db la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 10-20 °c) gli animali tenuti al di fuori dei fabbricati sono dotati di un riparo? (solo per allevamenti all'aperto) (solo per allevamenti le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? numero succhiotti(se più tipi di box indicare per ogni tipo) altro metodo indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) centro di referenza nazionale per il benessere animale pag. 12 box gestazione parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 stato sanitario generale (buono medio scarso) igiene corpo (% corpo imbrattato di feci) <10% 10-30% >30% lesioni da aggressione vulva coda resto del corpo ferite (trauma contro le strutture) ferite corpo unghielli altro condizione clinica bcs (1-3-5) rogna a:auricolare c:corpo ulcere spalla ascessi patologie respiratorie n° bursiti zoppia patologie enteriche scoli vulvari prolasso utero prolasso retto altro (specificare) stereotipie masticazione a vuoto tongue rolling morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) comportamenti aggressivi aggressivita' verso altre scrofe aggressione vulva grooming altro (specificare) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) id animali interazioni positive tra gli animali 31-60 61-200 > 200 *valutare un minimo di due box. se ci sono diverse tipologie di box valutarne almeno una per tipo. valutare un minimo di 3 animali per box e indicare in che tipo di box sono stabulati gli animali valutati n. animali da campionare n° scrofe in gestazione 8 10 11 12 13 14 ≤ 10 11-20 21-30 centro di referenza nazionale per il benessere animale pag. 13 si no si no si no e' sporco? e' accessibile? e' utilizzato? si no si no si no numero succhiotti(se più tipi di box indicare per ogni tipo) indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) la superficie libera a disposizione e' almeno di 6 mq? se il recinto e' usato per l' accoppiamento ha una superficie libera al suolo di almeno 10 mq? box tipo 1 n° animali il recinto e'costruito in modo tale che il verro possa girarsi, ed avere contatto olfattivo, uditivo, visivo con altri animali? locali di stabulazione se presenti parchetti esterni indicare tipologia (vedi allegato 2) tipo misure il recinto usato per l'accoppiamento e' libero da ostacoli? condizioni metereologiche nei giorni/settimane precedenti la valutazione altro (misurazioni) le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? box verri dlgs 122/2011 conformita' superficie disponibile/pavimentazioni/fessurati n° animali misure tipo di pavimentazione mm fessura mm travetto box tipo 2 n° animali misure tipo di pavimentazione mm fessura mm travetto box tipo 3 box tipo 3 altro metodo materiali manipolabili zona per coricarsi asciutta e pulita che consenta a tutti gli animali di stare sdraiati contemporaneamente la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 10-20 °c) e' presente materiale per esplorazione e manipolazione? si no legno torba paglia fieno quantita' per box altro (catene, palle,... box tipo 1 misure tipo di pavimentazione mm fessura mm travetto box tipo 2 alimento e acqua ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? luce > 40 lux per 8 h tipo tipo misure misure misure tipo di pavimentazione rumori continui < 85 db gli animali tenuti al di fuori dei fabbricati sono dotati di un riparo? (solo per allevamenti all'aperto) centro di referenza nazionale per il benessere animale pag. 14 box verri parametri animal based (valutare l'intero animale) (per verro si intende qualsiasi maschio intero) 1 2 3 4 5 6 7 8 9 10 <10% 10-30% >30% testicoli resto del corpo ferite corpo unghielli altro (specificare) bcs (1-3-5) rogna a:auricolare c:corpo ulcere spalla ascessi patologie respiratorie atrofia del grugno forme neurologiche n° bursiti zoppia patologie enteriche prolasso retto altro (specificare) masticazione a vuoto tongue rolling morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) aggressivita' verso altri verri grooming altro (specificare) id animali comportamenti aggressivi interazioni positive tra gli animali (se presenti piu' verri nello stesso box) stereotipie igiene corpo (% corpo imbrattato di feci) ferite (trauma contro le strutture) lesioni da aggressione (se più animali per box) condizione clinica stato sanitario generale (buono medio scarso) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) n° verri n. animali da campionare ≤ 10 11-20 21-30 31-60 8 10 11 12 centro di referenza nazionale per il benessere animale pag. 15 si no si no si no e' sporco? e' accessibile? e' utilizzato? si no si no si no gabbia tipo 3 materiali manipolabili e per la nidificazione e' presente materiale per esplorazione e manipolazione? altro (misurazioni) alimento e acqua ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? numero succhiotti(se più tipi di box indicare per ogni tipo) la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 24 °c) pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? si no legno torba paglia fieno quantita' per box altro (catene, palle,... gabbia tipo 1 altro metodo rumori continui < 85 db lo spazio a disposizione e' sufficiente affinche' la scrofa riesca a muoversi nelle direzioni consentite, sdraiarsi ed alzarsi senza difficolta'? si no le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? dietro alla scrofa c'e' uno spazio libero per il parto naturale o assistito? si no se le scrofe sono stabulate in stalli parto dove possono muoversi liberamente ci sono apposite strutture atte a proteggere i suinetti? locali di stabulazione luce > 40 lux per 8 h superficie disponibile/pavimentazioni gabbia gabbia tipo 3 gabbia tipo 2 gabbia parto: scrofa dlgs 122/2011 conformita' indicare materiale si no nella settimana prima della data prevista del parto viene fornito materiale per la nidificazione fruibile dalla scrofa in quantita' sufficiente? gabbia tipo 2 indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) gabbia tipo 1 lo spazio a disposizione e' sufficiente affinche' la scrofa riesca a muoversi nelle direzioni consentite, sdraiarsi ed alzarsi senza difficolta'? si no lo spazio a disposizione e' sufficiente affinche' la scrofa riesca a muoversi nelle direzioni consentite, sdraiarsi ed alzarsi senza difficolta'? si no centro di referenza nazionale per il benessere animale pag. 16 parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 indicare gg dal parto stato sanitario generale (buono medio scarso) <10% 10-30% >30% vulva mammelle/capezzoli ferite corpo ferite testa unghielli altro (specificare) bcs (1-3-5) rogna a:auricolare c:corpo ulcere spalla ascessi patologie respiratorie n° bursiti zoppia patologie enteriche mastite prolasso utero prolasso retto altro (specificare) masticazione a vuoto tongue rolling morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) aggressivita' verso i suinetti cannibalismo valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) gabbia parto: scrofa id animali comportamenti aggressivi igiene corpo (% corpo imbrattato di feci) ferite (trauma contro le strutture) lesioni da aggressione da parte dei suinetti condizione clinica stereotipie 8 10 21-30 31-60 61-200 > 200 * se ci sono diverse tipologie di gabbie valutare almeno una per tipo e indicare in quale gabbia è stabulato l'animale n° scrofe in lattazione n. animali da campionare≤ 10 11-20 11 12 13 14 centro di referenza nazionale per il benessere animale pag. 17 si no si no si no e' sporco? e' accessibile? e' utilizzato? si no si no si no indicare materiale pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? alimento e acqua ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? numero succhiotti o tazze (se più tipi di box indicare per ogni tipo) la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 24 °-34° c) le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? mm travetto valutare solo se in calcestruzzo: ampiezza fessurati 11 mm/larghezza travetti 50 mm locali di stabulazione altro (misurazioni) luce > 40 lux per 8 h rumori continui < 85 db gabbia 1 mm fessura mm travetto gabbia 3 superficie disponibile/pavimentazioni gabbia gabbia tipo 3 gabbia tipo 2 gabbia tipo 1 si no legno torba paglia fieno quantita' per box altro (catene, palle,... materiali manipolabili e' presente materiale per esplorazione e manipolazione? altro gabbia parto: suinetti dlgs 122/2011 conformita' gabbia 2 mm fessura mm fessura mm travetto e' presente una parte di pavimento sufficientemente ampia per consentire agli animali di riposare insieme contemporaneamente, piena o ricoperta da un tappetino, paglia o altro materiale adeguato? centro di referenza nazionale per il benessere animale pag. 18 parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 stato sanitario generale (buono medio scarso) orecchie tronco arti coda ferite corpo unghielli carpo altro (specificare) sottopeso splay leg patologie respiratorie ascessi deambulazione difficoltosa patologie enteriche forme neurologiche ernie atresia ano altro (specificare) suzione dell'ombelico suzione del prepuzio suzione/morsicatura dell'orecchio bell nosing accalcamento morsicatura vulva scrofa altro (specificare) grooming gioco altro (specificare) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) numerosita' nidiata condizione clinica lesioni da aggressione ferite (trauma contro le strutture) rilievi comportamentali interazioni positive tra gli animali n° nidiate ≤ 10 11-20 gabbia parto: suinetti 21-30 31-60 61-200 > 200 valutare l'intera nidiata. indicare la numerosità della nidiata e per ogni voce indicare il numero dei suinetti 12 13 14 n. animali da campionare 8 10 11 centro di referenza nazionale per il benessere animale pag. 19 si no si no e' sporco? e' accessibile? e' utilizzato? si no si no si no mm travetto rumori continui < 85 db misure tipo di pavimentazione mm fessura box svezzamento dlgs 122/2011 conformita' superficie disponibile/pavimentazioni/fessurati altro (misurazioni) n° animali misure tipo di pavimentazione mm fessura mm travetto box tipo 3 n° animali locali di stabulazione luce > 40 lux per 8 h misure tipo di pavimentazione mm fessura mm travetto box tipo 2 n° animali 10-20 kg 0,20 mq/ 20-30 kg 0,30 mq valutare solo se in calcestruzzo: ampiezza fessurati 14 mm/larghezza travetti 50 mm box tipo 1 le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? gli animali tenuti al di fuori dei fabbricati sono dotati di un riparo? (solo per allevamenti all'aperto) la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 18-32 °c) e' presente materiale per esplorazione e manipolazione? ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? si no legno torba paglia fieno quantita' per box altro (catene, palle,... box tipo 1 numero succhiotti(se più tipi di box indicare per ogni tipo) indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) se l'alimentazione e' razionata, il sistema utilizzato garantisce che si evitino aggressioni? zona per coricarsi asciutta e pulita che consenta a tutti gli animali di stare sdraiati contemporaneamente alimento e acqua altro metodo materiali manipolabili altro lunghezza truogolo (se più tipi di box indicare per ogni tipo) indicare metodo (se più tipi di box indicare per ogni tipo) se l'alimentazione e' razionata, ogni animale ha accesso agli alimenti contemporaneamente agli altri componenti del gruppo? box tipo 2 box tipo 3 centro di referenza nazionale per il benessere animale pag. 20 box svezzamento parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 stato sanitario generale (buono medio scarso) < 10% 10-30% orecchie tronco arti coda ferite corpo unghielli altro (specificare) > 30% sottopeso ascessi patologie respiratorie n° bursiti zoppia patologie enteriche forme neurologiche ernie prolasso retto altro (specificare) morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) suzione dell'ombelico suzione del prepuzio suzione/morsicatura dell'orecchio bell nosing morsicatura coda attacchi (laterali, frontali) accalcamento altro (specificare) grooming gioco-corse altro (specificare) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) *valutare un minimo di due box. se ci sono diverse tipologie di box valutarne almeno una per tipo. valutare un minimo di 3 animali per box e indicare in che tipo di box sono stabulati gli animali valutati interazioni positive tra gli animali id animali condizione clinica lesioni da aggressione igiene corpo (% corpo imbrattato di feci) ferite (trauma contro le strutture) stereotipie comportamenti aggressivi e altri rilievi comportamentali 31-60 61-200 > 200 8 10 11 12 13 14 n° animali in svezzament o n°animali da campionare ≤ 10 11-20 21-30 centro di referenza nazionale per il benessere animale pag. 21 si no locali di stabulazione altro (misurazioni) condizioni metereologiche nei giorni/settimane precedenti la valutazione gli animali tenuti al di fuori dei fabbricati sono dotati di un riparo? (solo per allevamenti all'aperto) luce > 40 lux per 8 h rumori continui < 85 db zona per coricarsi asciutta e pulita che consenta a tutti gli animali di stare sdraiati contemporaneamente le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 15°-25° °c) tipo misure misure (vedi allegato 2) tipo di pavimentazione mm fessura mm travetto tipo misure tipo misure misure (vedi allegato 2) tipo di pavimentazione mm fessura mm travetto box tipo 3 n° animali se presenti parchetti esterni indicare tipologia (vedi allegato 2) valutare solo se in calcestruzzo: ampiezza fessurati 18 mm/larghezza travetti 80 mm box tipo 1 n° animali misure (vedi allegato 2) tipo di pavimentazione mm fessura mm travetto box tipo 2 n° animali superficie disponibile/pavimentazioni/fessurati 20-30 kg 0,30 mq/ 30-50 kg 0,40 mq/ 50-85 kg 0,55 mq box magronaggio dlgs 122/2011 conformita' centro di referenza nazionale per il benessere animale pag. 22 si no e' sporco? e' accessibile? e' utilizzato? si no si no si no box tipo 3 box tipo 1 box tipo 2 materiali manipolabili e' presente materiale per esplorazione e manipolazione? si no legno torba paglia fieno quantita' per box altro (catene, palle,... altro metodo indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? numero succhiotti(se più tipi di box indicare per ogni tipo) lunghezza truogolo (se più tipi di box indicare per ogni tipo) se l'alimentazione e' razionata, il sistema utilizzato garantisce che si evitino aggressioni? indicare metodo (se più tipi di box indicare per ogni tipo) alimento e acqua altro se l'alimentazione e' razionata, ogni animale ha accesso agli alimenti contemporaneamente agli altri componenti del gruppo? centro di referenza nazionale per il benessere animale pag. 23 box magronaggio parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 stato sanitario generale (buono medio scarso) <10% 10-30% orecchie tronco flanks biting arti coda ferite corpo unghielli altro (specificare) >30% sottopeso rogna a:auricolare c:corpo ascessi patologie respiratorie n° bursiti zoppia patologie enteriche forme neurologiche ernie prolasso retto altro (specificare) masticazione a vuoto morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) bell nosing suzione dell'ombelico suzione del prepuzio morsicatura orecchie morsicatura fianco morsicatura arti morsicatura tronco morsicatura coda altro (specificare) grooming altro (specificare) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) id animali condizione clinica interazioni positive tra gli animali lesioni da aggressione ferite (trauma contro le strutture) igiene corpo (% corpo imbrattato di feci) stereotipie comportamenti aggressivi > 200 14 n° animali in magronaggi n°animali da campionar ≤ 10 8 11-20 10 21-30 11 *valutare un minimo di due box. se ci sono diverse tipologie di box valutarne almeno una per tipo. valutare un minimo di 3 animali per box e indicare in che tipo di box sono stabulati gli animali valutati 31-60 12 61-200 13 centro di referenza nazionale per il benessere animale pag. 24 si no zona per coricarsi asciutta e pulita che consenta a tutti gli animali di stare sdraiati contemporaneamente le concentrazioni di gas (es.ammoniaca)sono mantenute entro limiti non dannosi per gli animali? pavimenti e recinzioni sono tenuti in uno stato che non provochino lesioni agli animali? gli animali tenuti al di fuori dei fabbricati sono dotati di un riparo? (solo per allevamenti all'aperto) la temperatura e' mantenuta entro limiti non dannosi per gli animali? (zona di comfort termico 15°-25° °c) locali di stabulazione altro (misurazioni) luce > 40 lux per 8 h tipo misure condizioni metereologiche nei giorni/settimane precedenti la valutazione se presenti parchetti esterni indicare tipologia (vedi allegato 2) rumori continui < 85 db tipo misure tipo misure misure (vedi allegato 3 per truogolo) tipo di pavimentazione mm fessura mm travetto box tipo 2 n° animali misure (vedi allegato 3 per truogolo) tipo di pavimentazione mm fessura mm travetto box tipo 3 n° animali box tipo 1 n° animali misure (vedi allegato 3 per truogolo) tipo di pavimentazione mm fessura mm travetto superficie disponibile/pavimentazioni/fessurati 85-110 kg 0,65 mq/> 110 kg 1 mq box ingrasso dlgs 122/2011 conformita' ampiezza fessurati 18 mm/larghezza travetti 80 mm centro di referenza nazionale per il benessere animale pag. 25 si no e' sporco? e' accessibile? e' utilizzato? si no si no si no box tipo 3 box tipo 1 box tipo 2 materiali manipolabili e' presente materiale per esplorazione e manipolazione? si no legno torba paglia fieno quantita' per box altro (catene, palle,... se sono utilizzati i succhiotti questi sono funzionanti? altro metodo indicare metodo e intervalli di tempi di somministrazione (se più tipi di box indicare per ogni tipo) ogni animale dispone in permanenza di acqua fresca (pulita) sufficiente? numero succhiotti(se più tipi di box indicare per ogni tipo) lunghezza truogolo (se più tipi di box indicare per ogni tipo) se l'alimentazione e' razionata, il sistema utilizzato garantisce che si evitino aggressioni? indicare metodo (se più tipi di box indicare per ogni tipo) alimento e acqua altro se l'alimentazione e' razionata, ogni animale ha accesso agli alimenti contemporaneamente agli altri componenti del gruppo? centro di referenza nazionale per il benessere animale pag. 26 box ingrasso parametri animal based (valutare l'intero animale) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 stato sanitario generale (buono medio scarso) <10% 10-30% >30% orecchie tronco flanks biting arti coda ferite corpo unghielli altro (specificare) sottopeso rogna a:auricolare c:corpo ascessi patologie respiratorie n° bursiti zoppia patologie enteriche forme neurologiche ernie prolasso retto altro (specificare) masticazione a vuoto morsicatura barre morsicatura mangiatoia gioco con l'acqua grattamento contro strutture cane seduto altro (specificare) bell nosing suzione dell'ombelico suzione del prepuzio morsicatura orecchie morsicatura fianco morsicatura arti morsicatura tronco morsicatura coda altro (specificare) grooming altro (specificare) valutazione complessiva benessere dell'animale (inserire valore da 0 a 10) id animali condizione clinica interazioni positive tra gli animali lesioni da aggressione ferite (trauma contro le strutture) igiene corpo (% corpo imbrattato di feci) stereotipie comportamenti aggressivi > 200 14 n° animali in ingrasso ≤ 10 11-20 10 21-30 11 8 n°animali da campionare *valutare un minimo di due box. se ci sono diverse tipologie di box valutarne almeno una per tipo. valutare un minimo di 3 animali per box e indicare in che tipo di box sono stabulati gli animali valutati31-60 12 61-200 13 centro di referenza nazionale per il benessere animale pag. 27 note /osservazioni relative alla visita data ora fine osservazione cognome e nome valutatore/i recapito telefonico/mail 149 department of veterinary medical sciences, alma mater studiorum university of bologna, bologna, italy *corresponding author at: department of veterinary medical sciences, alma mater studiorum university of bologna, bologna, italy. tel.: +39 333 448 5003, e-mail: mariana.roccaro2@unibo.it. keywords age determination, dog, forensic medicine, puppy trade, teeth examination, veterinary legislation. summary age determination of puppies represents a significant issue of animal welfare and forensic medicine, particularly for what concerns trade and imports of dogs. despite the movement of puppy dogs before the age of 15 weeks is forbidden by regulation (eu) no 576/2013, the occurrence of illegal transport of younger puppies is not uncommon. the illegal trade of puppies increases instances of falsified documentation, the counterfeit of vaccine certificates and discrepancies between the declared age and the real age of the puppies. consequently, determining the exact age of animals and evaluating their welfare become legally crucial. dental examination currently represents the most common approach to estimate the age of a puppy in clinical practice and in forensic investigations. in this work we addressed the legal, health and welfare issues associated with dogs’ trade and import and we reviewed the existing literature referring to the assessment of age in dogs by dental examination. the imprecision and inaccuracy of this method make it poorly convincing in legal proceedings. the reasons for such vagueness are to be ascribed both to the lack of standardization and to many variability factors (size, breed, sex, diet, etc.) which influence dental eruption and development. mariana roccaro* and angelo peli age determination in dog puppies by teeth examination: legal, health and welfare implications, review of the literature and practical considerations veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 accepted: 26.06.2019 | available on line: 31.12.2020 they are transported to the distribution countries, often when they are too young to be moved. data collected within a project on the movement of pet animals funded by the italian ministry of health and carried out by the istituto zooprofilattico sperimentale dell’abruzzo e del molise revealed that, in 2014, 86% of imported puppies were under three months old (arena et al. 2015). consequently, determining the exact age of animals becomes legally crucial. it follows that the method used to assess age has to be reliable, precise and accurate. the methods available for assessing the age in dogs are different. examination of teeth has long been used to age in man and domestic animals, it is non-invasive and requires no special equipment. instrumental analyses such as the radiographic evaluation of limbs’ ossification centres (hare 1959, hare 1960, seoudi 1947, sumner‐smith 1966, thrall and robertson 2015) and dental radiography to assess pulp cavity/tooth width ratio (kershaw et  al. 2005, mbizah et  al. 2016) have been developed. introduction age determination in dogs is of great importance both in clinical veterinary practice and in veterinary forensic medicine; it is called in question in the purchase of animals as well as for their movement for non-commercial or trading purposes and this is especially the case of young animals. in this regard, the movement of pet animals, including dogs, within the european union (eu) has to comply with clearly defined rules that prevent them from being transported before they have reached a certain age. despite the legislation currently in force, the growing demand of purebred puppies at low prices has been fostering the illegal puppy trade from eastern european countries to western europe. according to the four paws international’s report on puppy trade in europe, dog traffic in italy and france is estimated at 43 million euros (four paws international 2013). puppies are bred in extremely poor conditions in so-called ‘puppy mills’ in eastern europe, where low enforcement of transport, health and welfare legislation allows the cheap prices; later 150 veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 legal and welfare implications of age determination by dental examination roccaro & peli for what concerns the non-commercial movement of dogs within the eu, the relevant legislation is regulation (eu) no 576/20132, together with commission implementing regulation (eu) no 577/20133. in order to be moved into an eu country from another eu country, the dog must be marked by the implantation of a transponder or by a clearly readable tattoo (applied before july 2011) and subsequently it must be vaccinated against rabies by an authorised veterinarian, no earlier than 12 weeks of age. the period of validity of the vaccination starts not less than 21 days from the completion of the vaccination protocol for the primary vaccination. finally, the pet animal must be accompanied by a passport completed and issued by an authorised veterinarian documenting the alpha-numeric code displayed by the transponder, and the details of the vaccination against rabies. the intra-union pet trade must comply with the requirements of the directive 92/65/eec4 (implemented in italy by legislative decree no 633/19965), according to which the animals must come from registered establishments. dogs must be marked, vaccinated against rabies, be accompanied by a passport and a health certificate issued by an official veterinarian, who notifies the movement to the competent authorities of destination through the community trade control and expert system (traces). finally, dogs must undergo a clinical examination in order to verify that they show no signs of diseases and are fit to be transported for the intended journey, in accordance with regulation (ec) no 1/20056 on the protection of animals during transport. this regulation also states that puppies younger than 8 weeks of age are unfit for transport unless accompanied by their mother. according to the over mentioned laws, the movement of dogs, either for non-commercial or trading purposes, is strictly dependent on the completion of the rabies vaccination protocol; therefore, puppies cannot be moved before 15 weeks of age. however, eu countries have discretion whether or not they allow the introduction onto their territory of ‘young dogs’, i.e. dogs which are less than 12 weeks old however, radiographic investigations have not found wide application outside the research field due to the cost of the required equipment, the exposure of the practitioner and the animal to x-rays, the eventual need for sedation and the lack of a standardized protocol. ocular lens examination, dental abrasion and tartar can be used in adult dogs, but are subject to a considerable individual variability (gesierich et  al. 2015, tobias et  al. 2000). lastly, the histologic investigation of dental cementum deposition, widely used in wild animals, proved to be unreliable for age determination in dogs (van lancker et al. 2005). this work aims to address the issues associated with age determination in dog puppies by dental examination, which currently represents the most common approach to estimate the age of a puppy in clinical practice and in forensic investigations. the first part focuses on the regulatory framework that requires knowledge of the dog’s age and on the consequences of trade and movement of dogs that are too young; the second part starts with a brief summary of dental development, there follows an overview of the existing literature referring to the assessment of eruption of deciduous and permanent teeth; finally, the last part identifies limits and areas of improvement for the application of such methodology in practice. legal, health and welfare implications the laws that imply the knowledge of the dogs’ age essentially concern the identification, the trade and the movement of animals for non-commercial and trading purposes. in italy, law no 281/19911 established an obligation to identify and register dogs in the national database; the definition of manner and timing was left to the individual regions. the identification requirements were subsequently harmonized by the state-regional agreement of 24 january 2013, according to which dogs must be identified and registered within two months of age and the sale or transfer of unidentified dogs is forbidden. 1 l. 14 august 1991, no. 281. “legge quadro in materia di animali di affezione e prevenzione del randagismo”. off j, 203, 30/08/1991, 3-5. 2 regulation (eu) no. 576/2013 of the european parliament and of the council of 12 june 2013 on the non-commercial movement of pet animals and repealing regulation (ec) no. 998/2003. off j, l 178, 28/06/2013, 1-26. 3 commission implementing regulation (eu) no. 577/2013 of 28 june 2013 on the model identification documents for the non-commercial movement of dogs, cats and ferrets, the establishment of lists of territories and third countries and the format, layout and language requirements of the declarations attesting compliance with certain conditions provided for in regulation (eu) no. 576/2013 of the european parliament and of the council. off j, l 178, 28/06/2013, 109-148. 4 council directive 92/65/eec of 13 july 1992 laying down animal health requirements governing trade in and imports into the community of animals, semen, ova and embryos not subject to animal health requirements laid down in specific community rules referred to in annex a (i) to directive 90/425/ eec. off j, l 268, 14/09/1992, 54-72. 5 d.lgs. 12 november 1996, no. 633. “attuazione della direttiva 92/65/cee che stabilisce norme sanitarie per gli scambi e le importazioni nella comunità di animali, sperma, ovuli ed embrioni non soggetti, per quanto riguarda le condizioni di polizia sanitaria, alle normative comunitarie specifiche di cui all’allegato a, sezione i, della direttiva 90/425, cee”. off j, 296, 18/12/1996, ordinary supplement no. 222. 6 council regulation (ec) no. 1/2005 of 22 december 2004 on the protection of animals during transport and related operations and amending directives 64/432/eec and 93/119/ec and regulation (ec) no. 1255/97. off j, l 3, 05/01/2005, 1-44. 151veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 roccaro & peli legal and welfare implications of age determination by dental examination 12 weeks of age on and becomes effective 21 days after the completion of the vaccination protocol. the trade of puppies that are too young to be vaccinated effectively represents a great danger not only for the animals but also for humans, especially if they are born from a non-vaccinated mother and they come from a not rabies-free country. other welfare issues include the inadequate socialization of puppies in their countries of origin before being sold and the early maternal separation, which may play a role in the development of behavioural disorders. compared to puppies separated from their mother and littermates at two months of age, puppies separated at 30 to 40 days of age are more likely to develop a variety of behavioural problems as an adult, including excessive barking, destructiveness, attention-seeking, fearfulness on walks, noise reactivity and aversion to strangers (pierantoni et  al. 2011). all of these factors lead to societal costs in terms of new owners needing to treat their sick animals and to manage potential behavioural problems, such as aggression, which also has a significant impact on public health (sacks et al. 1996, méndez gallart et al. 2002, langley 2009, rosado et al. 2009). besides, aggression is one of the main causes of abandon and euthanasia of dogs (reisner et al. 1994, overall and love 2001). dental development in dogs: general features and literature overview the assessment of age through the observation of deciduous and permanent eruption and succession of teeth has been used for a long time in veterinary practice, starting with production animals. this non-invasive method requires no special equipment and can be performed both in living and dead animals. clinical examination of teeth includes the assessment of number, integrity, shape and colour of the teeth, as well as the potential presence of tartar, halitosis and bleeding. therefore, it requires a thorough knowledge of the anatomy of the teeth and the physiology of their development. dogs, like other mammals, are characterized by the possession of a diphyodont dentition. at birth, a puppy is toothless; within a few weeks, deciduous teeth, also known as ‘milk teeth’, develop, which are later replaced by permanent teeth. teeth in the upper dental arcade normally erupt a few days earlier than the ones in the lower arcade (girard 1845, balasini 1995). the permanent set is deemed to be complete by the seventh month. the most accredited canine dental formulas are, for the deciduous dentition id3/3, cd1/1, pd3/3 and for the permanent dentition i3/3, c1/1, p4/4, m2/3; where ‘i’ stands for incisor, ‘c’ for canine, ‘p’ for premolar, ‘m’ for molar and ‘d’ for deciduous (nickel et  al. 1979, and have not received an anti-rabies vaccination, or between 12 or 16 weeks old and have received an anti-rabies vaccination but are not yet fully protected. this is possible for non-commercial movements from another eu country or a third country and for trade purposes within eu countries. it must be noted that countries like italy, germany, france, united kingdom, spain, the netherlands do not admit this exception. on the contrary, imports from non-eu countries of young dogs not vaccinated against rabies are not allowed under any circumstances. as stated above, eu legislation and traces guidelines are often violated. puppies are transported when they are still too young, with no identification, without or with incorrect vaccinations, dog-passports and transport papers, in unsanitary conditions and with little regard for their wellbeing (benini 2008). in italy, law no 201/20107 establishes the punishment for the illegal trade and import of pet animals, with a harsher punishment if the animals have less than 12 weeks of age. the measure of punishment for illegal puppy trafficking is severe enough on paper, but rarely results in a conviction in court. therefore, the deterring function of the punishment is close to nil. the difficulty in determining the exact age of the animals, in case the transport papers lack or in order to verify their regularity, is undoubtedly the most critical element that arises in this kind of judicial proceedings. the trade and movement of dogs that are too young have not only legal implications, but also impacts on animal health and welfare. it has been observed that removal from the litter prior to eight weeks of age may cause severe distress (serpell and jagoe 1995), and the separation of dog puppies from their mother at six weeks of age impairs their physical condition and weight gain, with an increase of disease susceptibility and mortality (slabbert and rasa 1993). the health risk is increased by transport and promiscuity, therefore puppies often become ill and die during the transport or soon after. an inquiry of the italian veterinary councils federation (benini 2008) on control of pet import reveals that 52% of dog puppies were found to be sick: 34% were infested with endoparasites, 23% were infected with parvovirus, 17% had fungal infections, 16% had scab and 10% were carriers of distemper. the risk of infectious disease spread is therefore very high and becomes critical when zoonoses, such as rabies, are involved. rabies vaccination of puppies can be regularly performed only from 7 l. 4 november 2001, no. 201. “ratifica ed esecuzione della convenzione europea per la protezione degli animali da compagnia, fatta a strasburgo il 13 novembre 1987, nonché norme di adeguamento dell’ordinamento interno”. off j, 283, 03/12/2010, 1-27. 152 veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 legal and welfare implications of age determination by dental examination roccaro & peli as few are the observed animals. mellanby (mellanby 1929) obtained the eruption times for deciduous and permanent teeth through the observation of 17 and 4 puppies, respectively, of different types of dog. deciduous teeth erupted 3 days earlier in larger dogs compared to the smaller ones, while permanent teeth erupted one week in advance. in a later study, carried out by arnall (arnall 1960), the sample included 5 bull terrier puppies. eruption times for deciduous teeth fell between 20-35 days of age, while permanent dental eruption began at 105-125 days. however, as the author himself admitted, such a limited study can only allow to draw general principles of eruption and any detailed conclusions can only apply to the breed of dog observed. regarding to the sample size, an exception is represented by the works of shabestari and colleagues (shabestari et  al. 1967) and kremenak (kremenak 1969). in order to study dental eruption chronology, shabestari and colleagues used 106  closely related purebred beagle puppies. based on previously published data, the authors conclude that in dog dental eruption times vary more between breeds than among individuals of the same breed. kremenak’s study on deciduous dental eruption was based on the daily observation of 32 purebred (16 beagles, 10 labradors, 6 pointers) and 48 mixed-breed puppies of known age, for a total of 40 male and 40 female puppies. on the average, all teeth erupted from 22 to 34 days of age, and this is quite in accordance with arnall’s results (arnall 1960). males preceded females in eruption of 21 of the 28 teeth, but this sex difference was found not statistically significant. when comparing puppies of different breeds, results supported the view that deciduous teeth eruption in beagles occurs later than in strains of larger dog breeds. interestingly, additional data from the observation of eight female mixed-breed puppies from the same litter allowed the author to notice a rather wide variability among individuals sharing the same sex and bloodline, with ranges of up to nine days. it is widely recognised that dental eruption is affected by several factors, such as general health state, diet, sex, breed and body size. several authors agree that teeth erupt earlier in the larger dog strains (girard 1845, mellanby 1929, piérard 1967, kremenak 1969, barone 1981, sisson and daniels grossman 1982, evans and de lahunta 2013, dyce et al. 2018). breed as well influences the timing of dental eruption, development and wear. there are in fact significant differences among dog breeds in terms of head size and shape: brachycephalic and dolichocephalic breeds represent the two extremes of such variability. for this reason, nickel and colleagues (nickel et  al. 1979) explicitly refers to the german shepherd’s dentition as a prototype, as it is the closest to the evans and de lahunta 2013, dyce et al. 2018). there are no deciduous precursors for the first premolar or the molar teeth in dogs. on the basis of the changes that take place in the evolution and alteration of the teeth, the life of an animal can be divided in four periods: eruption of the deciduous teeth, wearing of the deciduous teeth, eruption of the permanent teeth and wearing of the permanent teeth. this work consists of a historical overview of the existing literature pertaining to the timing of dental eruption in dogs. the information was searched through the catalogue of national library service and the data-base available which included the collections of: directory of open access journals (doaj), medline/pubmed (nlm), proquest collection (web of science), science direct journals (elsevier), wiley (crossref ), wiley online library. the search key words were as follow: age determination and dog; age assessment and dog; age and dog; teeth and dog; dentistry and dog; dental eruption and dog. the literature search included 24 anatomy, dentistry, paediatrics and zoognostic textbooks and 8 papers. the literature search on the data-base gave unsatisfying results, therefore a scientometric approach was not possible and we opted to proceed with a retrospective analysis of the literature. most of the bibliographic material on dental eruption and development is dated and sometimes hard to trace. the first documents on age assessment by teeth date back to the late 1800s (girard 1845, liautard 1885, huidekoper 1891). however, we decided to exclude these sources from the present work because both of them state that, in puppies, the incisors are already present at birth. we have not been able to find any reasonable explanation for such a statement, other than a sloppiness in the description, as it seems unlikely that selection could have led to the creation of substantially different dogs in just two hundred years. the timing of dental eruption has been reported in numerous anatomy, dentistry, paediatrics and zoognostic textbooks (cornevin and lesbre 1894, miller 1952, bourdelle and bressou 1953, silver 1963, ferrara 1965, nickel et  al. 1979, barone 1981, harvey and emily 1993, balasini 1995, bonetti 1995, hoskins 2001, vaissaire 2001, squarzoni 2003, reece 2009, veggetti and falaschini 2009, van de wetering 2011, evans and de lahunta 2013, gorrel 2013, veronesi et al. 2013, dyce et al. 2018) on the basis of previous published data or presumably from direct observations on the animals, but often there is no remind to original data or references. some journal articles merely cite data published in books (barton 1939, fulton et  al. 2014, hale 2005, piérard 1967), while research papers that explicitly declare the number of dogs used in the study are very few, as well 153veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 roccaro & peli legal and welfare implications of age determination by dental examination all incisors, premolars, etc.). very few authors give separate timing for upper and lower teeth (mellanby 1929, arnall 1960, shabestari et  al. 1967, kremenak 1969, bonetti 1995). more importantly, it became evident that there is a wide disagreement in the chronology of dental development among the authors. in order to highlight this variability, we provide a diagram illustrating the timing of deciduous and permanent dental eruption according to the different authors (supplementary table i and ii). data were derived from 8 manuscripts and 21 of the 24 consulted textbooks, which were selected for providing separate information for at least each tooth class. diagrams summarizing the earliest and latest ages of deciduous and permanent teeth eruption (figure 1) are given below. in this case, only sources indicating a time range for each single tooth were selected, otherwise the resulting ranges would have been wide to the point of losing usefulness. the first deciduous teeth to erupt are the canines. some authors placed their appearance at 15-20 days (miller 1952, ferrara 1965, balasini 1995), while for the majority of the sources they do not erupt before 3 or 4 weeks of age. incisors follow immediately after, usually starting from corner incisors (i3), then intermediate (i2) and lastly central incisors (i1). their eruption window is quite variable, ranging from 3-5  days for some authors (ferrara 1965, barone 1981, balasini 1995) up to 15-20 days (miller 1952, arnall 1960, kremenak 1969, veronesi et  al. 2013). the deciduous premolars begin to erupt at the turn of the second and the third week of age, normally in this order: p3, p4, p2. for most authors the eruption of the deciduous dentition is complete by 6 weeks of age, but for others it extends up to 8 (miller 1952, original wild ancestor. moreover, some breeds are known to be predisposed to dental anomalies, which can make age assessment by dental examination even more difficult. hypodontia (missing teeth) is most common in small-breed dogs (van de wetering 2011, lobprise 2012), but it also occurs in brachycephalic breeds (akers and denbow 2008, dyce et  al. 2018) and in large breeds such as dobermann, rottweiler and german shepherd (van de wetering 2011). the premolar teeth are the most commonly missing. additional teeth are common in brachycephalic dogs (boxer, bulldog) and mastiff (van de wetering 2011, lobprise 2012). delayed eruption has been observed in tibetan terrier, irish soft coat wheaten terrier, portuguese water dog and chinese crested dog (hoskins 2001, lobprise 2012). for what concerns sex, contrarily to kremenak's (kremenak 1969) findings, according to harvey and emily (harvey and emily 1993) teeth of female dogs erupt earlier. moreover, they state that season also affects the time of dental eruption, and teeth of dogs born in the summer erupt earlier. in addition to these factors, the dog’s diet and eating habits determine significant variations in tooth wear, therefore affecting the estimated age (girard 1845, piérard 1967, barone 1981, balasini 1995, veggetti and falaschini 2009, liebich et al. 2014). the analysis of the available sources on dental eruption and development revealed terminological discrepancies in the described phenomena; for example, some authors include the monophyodont first premolar among the deciduous teeth even if it does not shed, others number the deciduous premolars 1, 2 and 3, which can be confusing. differences also exist in the degree of detail of the provided data, both in relation to the timing (days vs. weeks) and the tooth classes (time ranges available for each single tooth or, more generally, for 2 3 4 5 6 7 8 i1 i2 i3 canine c p1 p2 p3 p4 premolars months weeks 1 incisors 2 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 72 3 4 5 6months weeks incisors premolars molars a. deciduous teeth b. permanent teeth figure 1. a. the interval between the earliest and the latest age of deciduous dental eruption (mellanby 1929, bourdelle and bressou 1953, ferrara 1965, shabestari et al. 1967, kremenak 1969, balasini 1995, vaissaire 2001, veronesi et al. 2013.) b. the interval between the earliest and the latest age of permanent dental eruption (mellanby 1929, miller 1952, bourdelle and bressou 1953, arnall 1960, silver 1963, nickel et al. 1979, shabestari et al. 1967, barone 1981, balasini 1995, vaissaire 2001, reece 2009, evans and de lahunta 2013, dyce et al. 2018). 154 veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 legal and welfare implications of age determination by dental examination roccaro & peli moreover, variability due to body size, breed and sex cannot be overlooked and shall be taken into consideration when assessing the dog’s age. research studies on dental eruption have been performed on medium and large dog breeds (mellanby 1929, arnall 1960, shabestari et al. 1967, kremenak 1969) and in most cases they only provide pooled-sex data. no specific data are available for small-sized dog breeds, which are known to have a delayed dental eruption (hoskins 2001, lobprise 2012). this dearth could be addressed by carrying out studies on a consistent sample including different dog breeds, with the aim of defining a ‘standard eruption chronology’ for each one of them. dental anomalies such as hypodontia or supernumerary teeth are relatively common, especially in purebred dogs, as a consequence of the genetic defect being perpetuated (hoskins 2001, akers and denbow 2008, van de wetering 2011, lobprise 2012). furthermore, individual variations in subjects of the same sex and bloodline have been observed (kremenak 1969). finally, but no less importantly, we cannot omit the inevitable observer variability, related to the subjectivity of judgement which is inbuilt in any visual assessment. the assessor should therefore be adequately trained, have full knowledge of the anatomy and the physiology of dental development, as well as be fully aware of the wide range of physiological and pathological variability. on the basis of the available information, the assessment of a puppy’s age by dental examination is subject to either an overestimation or an underestimation of no less than 2 weeks during the first 2-3 months of age, which is mainly due to the wide genetic variability among breeds. this uncertainty increases hand in hand with the dog’s growth as a result to the intervention of other factors, such as the environment and the individual habits. against this background, it is not surprising that dental examination cannot be considered a reliable method to determine the exact age of a dog, but at most to estimate it, as agreed by all the consulted authors. the correspondence between the real age and its assessment by dental examination is at most 41% (nickel et al. 1979). conclusions the so-called pet travel scheme (pets), introduced by regulation (ec) no 998/20038 (subsequently repealed by regulation (eu) no 576/2013), is a silver 1963, evans and de lahunta 2013), 10 (van de wetering 2011) or even 12 weeks (harvey and emily 1993, squarzoni 2003, hale 2005, fulton et al. 2014). time ranges for permanent dental eruption are even wider. the substitution process starts with incisors, this time in the opposite order (i1, i2, i3). data on incisor eruption times are quite divergent. according to miller (miller 1952) and evans and de lahunta (evans and de lahunta 2013) incisors begin to erupt at 2 months of age, for other authors the eruption window starts at 3 months (silver 1963, nickel et al. 1979, harvey and emily 1993, hale 2005, reece 2009, fulton et al. 2014), and the remaining sources place their appearance at 4 months. for most authors, canines erupt at 5-6 months, while for some of them they can appear one or two months earlier (arnall 1960, harvey and emily 1993, squarzoni 2003, gorrel 2013, fulton et  al. 2014). premolars erupt between 4-6 months of age, starting from p1, according to almost all the authors. only balasini (balasini 1995) and vaissaire (vaissaire 2001) placed the eruption of the first premolar at 3 months. according to half of the consulted sources, the first molar erupts at 4 months, while for the remaining half one month later. the other molars normally follow with a gap of one month between each one. at 7 months, the permanent set is deemed to be complete. practical considerations the analysis of the literature revealed that teeth eruption and development in dogs is far from being a uniform process. eruption of deciduous teeth should be completed by the sixth week of age, but for some authors it can last up to 10 or even 12 weeks (harvey and emily 1993, hoskins 2001, squarzoni 2003, hale 2005, van de wetering 2011). at 15-16 weeks, age at which a puppy can be legally moved within eu countries, according to some authors the eruption of i1 (miller 1952, silver 1963, nickel et al. 1979, harvey and emily 1993, hale 2005, reece 2009, evans and de lahunta 2013, dyce et al. 2018), c (squarzoni 2003, gorrel 2013, fulton et  al. 2014) and p1 (balasini 1995, vaissaire 2001) should be already in place, while for others it is just about to start (mellanby 1929, barton 1939, bourdelle and bressou 1953, arnall 1960, ferrara 1965, piérard 1967, shabestari et  al. 1967, barone 1981, veggetti and falaschini 2009, veronesi et  al. 2013). the eruption window extends up to 5-6 months for incisors, premolars and the first molar, up to 7 months for canines and the remaining molars. information provided by the consulted sources are often quite general, with wide time ranges frequently referred to the entire teeth class rather than the single tooth. therefore, they are unlikely to be useful in forensic investigations. 8 regulation (ec) no. 998/2003 of the european parliament and of the council of 26 may 2003 on the animal health requirements applicable to the non-commercial movement of pet animals and amending council directive 92/65/eec. off j, l 146, 13/06/2003, 1-9. 155veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 roccaro & peli legal and welfare implications of age determination by dental examination disagreement between assessments performed by different experts, makes it arduous to stand up in court. a method for age estimation in dog puppies which is simple, fast, non-invasive, reproducible and, over all, accurate, that can be systematically used in different academic and forensic scenarios, is currently lacking. our comprehensive analysis of the available information on the timing of tooth eruption in dogs has highlighted a wide disagreement in the chronology of dental development among the authors. age assessment by tooth examination in dogs is affected by a degree of uncertainty of no less than 4 weeks, attributable to the biological variability due to general health state, nutrition, sex and, above all, breed and body size. of all animal species, in fact, the canine species displays the widest range of phenotypic diversity. as a consequence, individuals of the same chronological age may show a range of different biological ages. this inevitable uncertainty clashes with the certainties demanded by legal sciences. system which allows animals to travel easily between member countries without undergoing quarantine. according to this regulation, puppy dogs cannot be moved within the member countries before 15  weeks of age, they must be identified and vaccinated against rabies. structural controls and law enforcement on the inner borders of the eu appear to be poorly implemented and the occurrence of illegal transport of younger puppies is not uncommon. the illegal trade of puppies increases instances of falsified documentation, the counterfeit of vaccine certificates and discrepancies between the declared age and the real age of the puppies. invariably, it also supports puppy farms in countries where welfare standards for animal breeding and husbandry may be of dubious quality. the difficulty in determining the exact age of the animals in case the transport papers lack or in order to verify their regularity, which often results in a wide akers r.m. & denbow d.m. 2008. teeth. in anatomy and physiology of domestic animals. blackwell publishing,  443-445. arena l., messori s., ferri n. & ruggieri e. 2015. introduzione di cuccioli dall’estero. 30 giorni, 5, 25-28. arnall l. 1960. some aspects of dental development in the dog ii. eruption and extrusion. j small anim pract, 1, 259-267. balasini d. 1995. l’età nel cane. in 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an esp pediatr, 56, 425-429. miller m.e. 1952. guide to the dissection of the dog, 3rd ed. ithaca, new york. nickel r., schummer a. & seiferle e. 1979. denti e dentatura dei carnivori. in trattato di anatomia degli animali domestici. ambrosiana, milano, 82-87. overall k.l. & love m. 2001. dog bites to humans: demography, epidemiology, injury, and risk. j am vet med assoc, 218, 1923-1934. pierantoni l., albertini m. & pirrone f. 2011. prevalence of owner-reported behaviours in dogs separated from the litter at two different ages. vet rec, 169, 468-468. piérard j. 1967. note on applied anatomy: determination of the age of dogs. can vet j, 8, 197-200. reece w.o. 2009. teeth. in functional anatomy and 157veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 roccaro & peli legal and welfare implications of age determination by dental examination annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 3; year: 2020; doi: 10.12834/vetit.1876.9968.2 supplementary table i. timing of deciduous dental eruption according to the different authors. —cont’d continued 1 2 3 2 3 4 5 6 7 8 9 10 11 12 incisors i canine c p2 p3 p4 i1 19-31d i2 20-27d i3 19-28d canine c 20-28d p2 28-39d p3 21-35d p4 24-37d incisors i canine c i1 i2 i3 canine c premolars p i1 30d i2 28d i3 25d canine c 21d p2 p3 p4 incisors i 20-35d canine c 20-35d premolars p 20-35d incisors i canine c premolars p i1 30-33d i2 28-29d i3 24-28d canine c 15-20d premolars incisors premolars incisors incisors premolars incisors age months weeks mellanby 1929 cornevin and lesbre 1894 author barton 1939 miller 1952 bourdelle and bressou 1953 vaissaire 2001 arnall 1960 silver 1963 ferrara 1965 158 veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 legal and welfare implications of age determination by dental examination roccaro & peli annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 3; year: 2020; doi: 10.12834/vetit.1876.9968.2 supplementary table i. timing of deciduous dental eruption according to the different authors. —cont’d continued i1 21-41d i2 19-35d i3 20-37d canine c 18-28d p2 27-40d p3 20-34d p4 25-41d incisors i canine c premolars p i1 30d i2 28d i3 25d canine c 21d premolars p i1 i2 i3 canine c p2 p3 p4 incisors i canine c premolars p incisors premolars incisors incisors premolars sisson and grossman 1982 harvey and emily 1993 squarzoni 2003 hale 2005 fulton 2014 nickel et al. 1979 gorrel 2013 dyce et al. 2018 barone 1981 kremenak 1969 incisors i canine c i1 21-31d i2 21-30d i3 20-28d canine c 19-25d p2 27-39d p3 22-33d p4 24-38 d incisors premolars shabestari et al. 1967 piérard 1967 1 2 3 2 3 4 5 6 7 8 9 10 11 12 age months weeks author 159veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 roccaro & peli legal and welfare implications of age determination by dental examination annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 3; year: 2020; doi: 10.12834/vetit.1876.9968.2 supplementary table i. timing of deciduous dental eruption according to the different authors. —cont’d i1 30-33d i2 25-30d i3 21-25d canine c 15-20d p2 28-34d p3 20-28d p4 20-28d incisors i 20-30d canine c 20-30d veggetti et al. 2009 incisors i incisors i canine c premolars p i1 i2 i3 canine c premolars p i1 i2 i3 canine c p2 p3 p4 incisors i canine c premolars p incisors premolars incisors incisors premolars liebich et al. 2014 evans and de lahunta 2013 veronesi et al . 2013 balasini 1995 bonetti 1995 peterson and kutzler 2011 1 2 3 2 3 4 5 6 7 8 9 10 11 12 age months weeks author 160 veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 legal and welfare implications of age determination by dental examination roccaro & peli annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 3; year: 2020; doi: 10.12834/vetit.1876.9968.2 supplementary table ii. timing of permanent dental eruption according to the different authors. —cont’d continued i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 incisors i canine c p1 p2 p3 p4 m1 m2 m3 incisors i canine c premolars p molars m i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 incisors i canine c incisors i canine c premolars p molars m incisors premolars molars premolars molars incisors premolars molars squarzoni 2003 barone 1981 harvey and emily 1993 balasini 1995 bonetti 1995 sisson and grossman 1982 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 age months weeks author 161veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 roccaro & peli legal and welfare implications of age determination by dental examination annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 3; year: 2020; doi: 10.12834/vetit.1876.9968.2 supplementary table ii. timing of permanent dental eruption according to the different authors. —cont’d continued i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 incisors i canine c premolars p molars m i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 incisors i molars m i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 incisors i canine c premolars p molars m hale 2005 vaissaire 2001 incisors premolars molars incisors premolars molars incisors premolars molars reece 2009 veggetti et al. 2009 evans and de lahunta 2013 gorrel 2013 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 age months weeks author 162 veterinaria italiana 2020, 56 (3), 149-162. doi: 10.12834/vetit.1876.9968.2 legal and welfare implications of age determination by dental examination roccaro & peli annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 56; issue: 3; year: 2020; doi: 10.12834/vetit.1876.9968.2 supplementary table ii. timing of permanent dental eruption according to the different authors. —cont’d incisors i canine c premolars p molars m incisors i canine c premolars p molars m i1 i2 i3 canine c p1 p2 p3 p4 m1 m2 m3 incisors premolars molars fulton 2014 dyce et al. 2018 liebich et al. 2014 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 age months weeks author 239 veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 accepted: 27.05.2020 | available on line: 31.12.2021 department of veterinary medical sciences (dimevet ), university of bologna, via tolara di sopra 50, ozzano dell’emilia, 40064 bologna, italy *corresponding author at: department of veterinary medical sciences (dimevet ), university of bologna, via tolara di sopra 50, ozzano dell’emilia, 40064 bologna, italy. e-mail: nicola.ellero3@unibo.it. nicola ellero*, aliai lanci, giancarlo avallone, jole mariella, carolina castagnetti, luisa vera muscatello, chiara di maio and francesca freccero keywords clostridium piliforme, foals, infectious necrotic hepatitis, tyzzer’s disease. summary seizures, coma and death rapidly appeared after admission in a one-month-old foal with a history of lethargy, fever and anorexia. severe icterus and necrotizing hepatitis were observed at necropsy. clinical signs, laboratory and postmortem findings were compatible with a suspect of clostridial hepatitis. tyzzer’s disease was confirmed by the presence of organisms morphologically consistent with clostridium piliforme in the hepatocytes at the margins of multiple areas of hepatic necrosis. to the authors’ knowledge, this is the first reported case of clostridial hepatitis caused by clostridium piliforme in a horse in italy. the first case of tyzzer’s disease in a young foal in italy: a case report tyzzer’s disease reported in a horse in italy. possible environmental and management risk factors are also considered. case description a thirty one-day-old quarter horse male foal, 96 kg in bodyweight, was referred to the perinatology and reproduction unit (equine clinical service, department of veterinary medical sciences) of the university of bologna, following the acute onset of depression and anorexia. the foal was born from a 10-year-old quarter horse mare, with attended foaling, and had assumed colostrum from the udder. the placenta was macroscopically normal. the breeding farm was located in a mountain area in the emilia-romagna apennines. the property, extended for 3.9 hectares, is bordered by a medium-sized torrent and thick forest. moreover, a little calf stable was present in the farm. the breeding farm included forty-five horses: 2 stallions, 14 show horses, 7 mares with their respective suckling foals and 15 yearlings. a group of six foals, with their dams, was housed in a paddock with free access to a three-sided outdoor stall. mares were fed with ad libitum hay and concentrate (13.0% protein, twice daily) diet during lactation and foals were also supplemented with 100 grams of concentrate (19.0% protein) for suckling introduction clostridium piliforme is the causative agent of tyzzer’s disease, an acute and fatal necrotizing hepatitis (duncan et  al. 1993). the number of species in which the disease has been reported, has rapidly increased in the last few years (sellon and long 2013a). it includes horse, cow, dog, cat, rat, mouse, hamster, gerbil, guinea pig, rabbit, muskrat, wombat, red panda, coyote, snow leopard, grey fox, raccoon and serval. this form of clostridial hepatitis is rarely observed in equids although some cases have been reported in foals (pulley and shively 1974, harrington 1975, harrington 1976, whitwell 1976, thomson et al. 1977, dickinson 1980, turk et al. 1981, brown et al. 1983, carrigan et al. 1984, copland et  al. 1984, nold et  al. 1984, scarrat et  al. 1985, van der lugt 1985, humber et  al. 1988, shirakawa et  al. 1989, peek et  al. 1994, appel and burdinski 1995, st denis et  al. 2000, borchers et  al. 2006). united states, united kingdom, canada, australia and new zealand are the most affected countries (pulley and shively 1974, harrington 1975, harrington 1976, turk et al. 1981, whitwell 1976, thomson et al. 1977, carrigan et  al. 1984, dickinson 1980). in this report, we describe the clinicopathological and histological features of a case of equine clostridial hepatitis caused by clostridium piliforme that, to the best of the authors’ knowledge, is the first case of case report 240 veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 tyzzer's disease in a foal ellero et al. μg/dl and fibrinogen concentration was normal (3.27 g/l, reference range 2.0 to 7.0 g/l; corley and stephen 2008). the biochemistry profile showed increased total bilirubin (7.52 mg/dl, reference range 0.5 to 1.7  mg/dl; corley and stephen 2008), triglycerides (594 mg/dl, reference range 45 to 155 mg/dl; corley and stephen 2008), aspartate aminotransferase (2,053  u/l, reference range 252 to 440 u/l; corley and stephen 2008), bile acids (42.6 µmol/l, reference range 7.4 to 19.4 µmol/l; barton and leroy 2007), creatine kinase (12,023 u/l, reference range 81 to 585 u/l; corley and stephen 2008), creatinine (2.25  mg/dl, reference range 1.1 to 1.8 mg/dl; corley and stephen 2008) and urea (46.71 mg/dl, reference range 6 to 21 mg/ dl; corley and stephen 2008). blood gas analysis revealed metabolic acidosis (ph  7.051, reference range 7.33 to 7.41; corley and stephen 2008) with hypocapnia (paco 2 34.7 mmhg, reference range 46 to 64  mmhg; corley and stephen 2008) and decrease in bicarbonates (hco 3 9.6  mmol/l, reference range 31.6 to 37.7  mmol/l; corley and stephen 2008). electrolyte imbalances included hyponatremia (133  mmol/l, reference range 136 to 154 mmol/l; corley and stephen 2008) and hypocalcemia (total calcium 0.9 mmol/l, reference range 2.7 to 3.3 mmol/l; corley and stephen 2008). blood culture was negative. to provide respiratory support, intranasal oxygen therapy was started at 8 litres per minute (lpm). intravenous (iv) therapies were administered through a jugular vein catheter. broad-spectrum antimicrobial therapy was initiated with sodium ampicillin (50 mg/kg iv qid) and amikacin sulfate (30 mg/kg iv sid). initial fluid therapy included 5% dextrose at 5.0 ml/kg/h and lactated ringer’s solution at 5.2 ml/kg/h. within thirty minutes of hospitalization, seizures started and diazepam (0.4 mg/kg iv) was administered. despite treatments, the clinical condition rapidly progressed to high fever (40.7 °c), severe dyspnea and further seizures. flunixin meglumine (1.1 mg/kg iv) and butorphanol (0.04 mg/kg iv) were administered and the foal was then intubated through the mouth with a sterile endotracheal silicone tube (14.3 mm in diameter, 55 cm in length). the tube was attached to a self-inflating resuscitation bag (ambu bag), connected to an oxygen supply (8 lpm), and supported ventilation was started at 10 breaths per minute. the condition deteriorated to refractory pyrexia (42.5 °c), cyanotic mucous membranes, shock and death within two hours of hospitalization. due to the onset of central nervous system (cns) signs, immediately after death a cerebrospinal fluid (csf) sample was collected through cisternal puncture method. the biochemistry profile showed increase in lactate dehydrogenase (90 u/l, reference range 0 to 8 u/l; corley and stephen 2008) and foals twice daily. the paddock was characterized by an earthy soil, without grass. the evening before hospitalization, the foal had shown less interest in following the mare and, in the next morning, it was found recumbent and too weak to stand up. the veterinarian referred that the foal was depressed and showed a low rectal temperature (34  °c). the foal was treated with dexamethasone (0.1 mg/kg iv), sodium hemisuccinate hydrocortisone (0.5  mg/ kg iv), equine plasma (one liter iv), dimethyl sulfoxide (1 g/kg, 5% solution) and lactated ringer’s solution (two liters iv). due to the worsening of the clinical condition, the foal was then admitted to the clinic. at admission, the foal was recumbent, severely depressed and presented bilateral horizontal nystagmus. the rectal temperature was 39.9  °c. the respiratory rate was 56  breaths/minute, with increased abdominal effort. the heart rate was 120 beats per minute (bpm), with weak peripheral pulse, cool limbs and prolonged capillary refill time (3 seconds). the oral mucous membranes were dark red and sticky. petechiae were present on ears and the upper lip. the right eye showed a severe hyphema and the sclerae were hyperaemic. venous blood was collected from jugular vein for bacteriology, haematology, biochemistry, serum immunoglobulin g (igg) determination and blood gas analysis. blood culture was performed using 10 ml of jugular blood withdrawn after clipping and aseptic preparation of the skin. the sampling needle was then discarded, and a new needle was used to inoculate the blood into the commercially available culture bottle (oxoid signal blood culture system, oxoid limited, basingstoke, hampshire, en, uk). hypoglycaemia (37 mg/dl, reference range 130 to 216 mg/dl; corley and stephen 2008) and hyperlactatemia (19.9 mmol/l, reference range 0.2 to 0.7 mmol/l; corley and sthepen 2008) were detected through rapid methods (medisense optium, abbott laboratories medisense products, bedford, ma, us and lactate scout+, gesellschaft zur entwicklung und herstellung, bioelektrochemischer sensoren mbh, leipzig, ge, eu, respectively). the adequate serum igg level was confirmed by immunoturbidimetric method (dvm rapid test ii, mai animal health, elmwood, wi, us): 1,618 mg/dl (reference range 930 to 1,930 mg/dl; perkins and wagner 2015). total leucocyte count appeared normal (7,770 cells/mm3, reference range 5,300 to 12,200 cells/mm3; corley and stephen 2008), but the differential cell count showed lymphocytosis (5,370 cells/mm3, reference range 1,730 to 4,850 cells/mm3; corley and stephen 2008), monocytosis (1,620 cells/mm3, reference range 50 to 630 cells/mm3; corley and stephen 2008) and neutropenia (480 cells/mm3, reference range 2,760 to 9,270 cells/mm3; corley and stephen 2008). serum amyloid a (saa) concentration was 282 241veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 ellero et al. tyzzer's disease in a foal millimeters in diameter, yellowish foci. additional gross lesions included a mild follicular pharyngitis (figure 2) and moderate meningeal hyperemia. histologically, the liver was characterized by multifocal to coalescing, randomly distributed, foci of coagulative necrosis infiltrated by small number of degenerated neutrophils (figure 3). within the cytoplasm of hepatocytes adjacent to necrotic foci, a moderate number of intracytoplasmic filamentous bacteria, evidenced with giemsa and warthin starry stains, were clear and were morphologically consistent with clostridium piliforme (figure 4). brain sections revealed multiple bilateral and asymmetrical perivascular microhemorrhages consistent with diffuse endothelial damage secondary to septicemia. discussion tyzzer’s disease is characterized by an acute necrotizing hepatitis caused by clostridium piliforme, a spore-forming, gram-negative, motile, obligate intracellular bacillus (duncan et  al. 1993). decrease in glucose (26 mg/dl, reference range 30 to 70 mg/dl; corley and stephen 2008), while total proteins were normal (112.41 mg/dl, reference range 10 to 120 mg/dl; corley and stephen 2008). at necropsy, a severe yellow discoloration was evident on mucosal surfaces, and multiple, disseminates petechiae were present on all serosal surfaces, being more evident on the epicardium (figure 1), and in the cns. hepatic parenchyma was characterized, on cut surface, by multifocal, few figure 1. foal, heart. epicardial surface is affected by multifocal petechial hemorrhages. figure 2. foal, pharynx. pharyngeal mucosa is diffusely and moderately thickened by follicular inflammatory infiltrate. figure 3. foal, liver. histological section of liver characterized by multifocal areas of necrosis bordered by degenerated neutrophils. hematoxylin and eosins stain. figure 4. foal, liver. numerous intracellular filiform bacteria are evidenced within the cytoplasm of hepatocytes adjected to necrotic areas. warthin starry stain. 242 veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 tyzzer's disease in a foal ellero et al. five other suckling foals have shown fever, which responded to ceftiofur (5 mg/kg im twice a day) for five days, with no further deterioration in their clinical condition. in some of the previously reported cases, foals were found dead on pasture without previous clinical signs but often infected foals showed weakness, pyrexia, tachypnea and icterus (carrigan et al. 1984, scarratt et al. 1985). some foals showed a yellowish liquid diarrhea. after ingestion, spores replicated in the gastrointestinal tract and reached the liver through hepatic portal system, where they caused necrotizing hepatitis. the subsequent septicemia was responsible for the myocarditis, colitis and pulmonary hemorrhage. hepatic encephalopathy finally ensued and death occurred after appearance of cns signs: seizures and coma rapidly appeared within 2 to 48 hours (swerczek et al. 1973, whitwell 1976, turk et  al. 1981). in the present case, the foal had shown depression and loss of suckle reflex the evening before hospitalization and has developed signs of sepsis (fever, tachycardia, tachypnea, hypoperfusion, petechiae) in the next twelve hours. cns signs, such as horizontal nystagmus, were present at admission, seizures appeared after thirty minutes and death occurred within two hours. in this case, histological findings consistent with hepatic encephalopathy were not evident, and neurological signs were more likely secondary to the microhemorrhages caused by the septicemic status. previously reported hematological and biochemical findings included leukopenia, elevated serum fibrinogen, hypoglycemia, metabolic acidosis, elevated bilirubin and increase in hepatic enzymes such as aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh) and alkaline phosphatase (alp) (brown et  al. 1983, humber et  al. 1988, swerczek 2013). in this case, severe neutropenia was present, reflecting the hyperacute course of the disease. hypoglycemia, hyperlactatemia likely due in part to hypoperfusion, metabolic acidosis and electrolyte imbalances were also present, which can all be attributed either to compromised liver function or septicemia. in the biochemistry profile, increased ast activity was associated with liver disease, such as acute liver failure or cholangiohepatitis, in association with increased bile acids, sign of decreased liver function (barton and leroy 2007), and increased total bilirubin, sign of intraor extrahepatic obstruction. no alterations were detected in alt, alp and γ-glutamyl transferase activities. csf  analysis showed increase in ldh and decrease in glucose, while total protein was normal. in human medicine, elevated ldh level was observed in the csf of patients with bacterial meningitis, because this enzyme may be secreted by granulocytes (lampl et al. 1990). csf glucose concentration was probably although the organism has often been reported to be gram-negative in tissue sections, it can also appear as gram-variable or gram-positive if fixation and staining are done under anaerobic conditions (duncan et  al. 1993). the organism can be found in form of spores in the environment and its spreading may be due to the movement of the soil. in equine species, adults are rarely affected but they may be sources and carriers of infection to susceptible foals. the most likely route of infection in foals is through ingestion of spores shed in the feces of adult carrier horses (swerczek et  al. 1973, humber et  al. 1988). foals normally consume freshly feces from their dams during the second to fifth week of age (francis-smith and wood-gush 1977). interestingly, foals are affected at 1 to 5 weeks of age (swerczek et al. 1973, chanter 1995, fosgate et al. 2002). others species susceptible to infection, such as rodents and rabbits, are possible source of environmental contamination, since the infection is confined to the gastrointestinal tract (ganaway et al. 1971). the breeding farm, in the presented case, is bordered by a torrent, on one side, and thick forest on three sides. in association with the presence of a little calf stable, it is not possible to exclude contacts between horses and wild animals, such as rodents. the predisposing factors for tyzzer’s disease include age of foals (9 to 30 days), time of the year of their birth (april to may), rainfall in the spring and high protein and nitrogenous diets fed to nursing mares (swerczek 2013). passive transfer of clostridium piliforme-specific antibodies through colostrum may play a role in foals’ protection (hook et  al. 1995). foals born to non-resident mares or less than 6 year-old mares, having a lower quality of colostrum (leblanc et  al. 1992), were more likely to develop tyzzer’s disease (fosgate et  al. 2002), suggesting that variability in colostral quality may be a potential risk factor. in the presented case, the colt was born in april and had been admitted to the clinic in may, at one-month of age. the authors do not have information about the level of serum igg after the ingestion of colostrum but at admission it was appropriate, considering the admin-istration of equine plasma by the referring veterinarian shortly before hospitalization. there is also no information about antibody status towards clostridium piliforme of the mare and the other members of the group. both the mare and the foal were supplemented with high protein concentrate but no information is available regarding the quality of the forage and the pasture composition. during the month before admission, the paddock was characterized by an earthy soil, without grass, and the weather was characterized by a mean temperature of 5.5 °c and a high level of humidity (91.1%), precipitation (25 rainy days out of 31) and wind (21.7 km/h). following the case described here, the owner reported that the 243veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 ellero et al. tyzzer's disease in a foal due to hypoglycaemia, as it was more than 50% of the serum glucose concentration (hussein and shafran 2000). normal level of csf total protein was probably due to the preserved permeability of the blood-brain barrier. neutrophilic pleocytosis and increased total protein are the most commonly reported findings in the csf of horses with infectious meningitis (corley and stephen 2008, furr 2008), although cases with normal csf cell counts have been described (pellegrini-masini and livesey 2006). in this case, total nucleated cell count and intracellular bacteria in the csf sample were not evaluated but it is not possible to exclude certainly a diagnosis of bacterial meningoencephalitis due to the histopathological evidence of multiple perivascular microhemorrhages in the cns. in the majority of cases, foals responded temporarily to fluid therapy, antimicrobial and anti-inflammatory drugs and parenteral nutrition, but then rapidly deteriorated and succumbed to the disease (scarratt et  al. 1985, humber et  al. 1988, st denis et  al. 2000). in the reported case, oxygen, antimicrobial and fluid therapies did not improve the clinical condition of the foal. pyrexia and seizures were refractory to nonsteroidal anti-inflammatory drugs and benzodiazepines, respectively. successful treatment has been reported only in one presumptive case (peek et al. 1994) and in one confirmed case (borchers et  al. 2006) of tyzzer’s disease, reflecting the poor prognosis. interestingly, the foal was treated with corticosteroids by the referring veterinarian before hospitalization. the effects of corticosteroids are numerous and include alteration of cytokine production, decreased adhesion molecule and immunoglobulin receptor expression, and decreased phagocytosis and cell migration (sellon and long 2013b). in horses, corticosteroids induce an increase in peripheral blood neutrophil and a decrease in lymphocyte concentrations (burguez et  al. 1983). from studies conducted in laboratory animals, the severity of tyzzer’s disease varies with the host immune status. the disease is more severe in young mice with immature immune systems than in adults, and iatrogenic immunosuppression increases susceptibility of all mice to clostridium piliforme infection (riley et  al. 1990). for these reasons, immune suppressive therapies seem to increase the severity of the disease even in infected foals. classical gross findings of tyzzer’s disease have been detected in the presented case, including hepatomegaly with multifocal areas of necrosis in the hepatic parenchyma (harrington 1975, harrington 1976, whitwell 1976, thomson et  al. 1977, carrigan et  al. 1984, nold et  al. 1984, scarratt et  al. 1985). the hepatic and mesenteric lymph nodes often appeared hyperplastic and edematous (pulley and shively 1974, harrington 1975, whitwell 1976, carrigan et al. 1984, nold et al. 1984, copland et  al. 1984, scarratt et  al. 1985, humber et  al. 1988). petechiae and ecchymosis featured the serosal surface of the diaphragm, heart, small and large intestine (whitwell 1976, turk et  al. 1981, scarratt et  al. 1985, van der lugt 1985, humber et  al. 1988, st  denis et  al. 2000). the gastrointestinal tract showed a semi-solid or liquid contents, often yellowish, and subcutaneous or visceral icterus was present (harrington 1975, harrington 1976, copland et al. 1984, humber et al. 1988, st denis et al. 2000). clinical signs, laboratory and postmortem findings suggested a presumptive diagnosis, which was confirmed by pathognomonic histopathologic findings of multifocal areas of liver necrosis and hepatitis (carrigan et  al. 1984, copland et  al. 1984, nold et  al. 1984, scarratt et  al. 1985, van der lugt 1985, humber et  al. 1988, paar et  al. 1993, st denis et  al. 2000). in the center of the necrotic areas, hepatocytes were destroyed and replaced by red blood cells, neutrophils and mononuclear cells. at the periphery of the necrotic areas, hepatocytes contained intracellular filamentous bacilli, highlighted with warthin-starry silver stains (ganaway et  al. 1971). histologic hepatic lesions in the presented case were consistent with this description. bacilli and microscopic inflammatory changes might be present in the hepatic lymph nodes, in the intestinal mucosal cells and in myocardial cells (whitwell 1976, carrigan et al. 1984, humber et  al. 1988), but were not detected in the present case. clostridium piliforme is very difficult to culture from clinical or postmortem samples (ganaway et  al. 1971, franklin et  al. 1994). in fact, in the presented case, blood culture was negative, in face of the evidence of hematogenous spread. the bacterium can be isolated in the yolk sac of developing chicken embryos (ganaway et al. 1971). a recently developed real-time polymerase chain reaction (qpcr) assay represents a valid diagnostic test (borchers et  al. 2006). the pcr, targeting 16s ribosomal ribonucleic acid (rrna) genomic sequences, can detect the organism in both antemortem and postmortem liver samples of foals with clinical signs and provides early diagnosis and opportune treatment. in this case, differential diagnosis were based on clinical signs, laboratory and postmortem findings and included acute hepatitis, bacterial septicemia and toxic hepatitis. tyzzer’s disease was confirmed by the presence of organisms morphologically compatible with clostridium piliforme in the hepatocytes, in association with anamnesis, clinical signs and age of the foal. this is the first reported case of tyzzer’s disease in a horse in italy but in the authors’ opinionmany other cases might have been misdiagnosed. foals found dead on pastures should undergo postmortem 244 veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 tyzzer's disease in a foal ellero et al. to isolate each foal from adult horses excepting their dams, to remove horse manure daily and to clean farm areas with one of the appropriate chemical disinfectants. environmental hygiene and high-quality colostrum remain the only ways of prevention tyzzer’s disease in foals. although the mortality rate of the disease is high, successful outcome seems possible if intensive care and antimicrobial therapy are initiated promptly (borchers et al. 2006). acknowledgments this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors wish to thank the referring veterinarian doctor andrea banchio. examination with the aim of detecting the causative agent specific qpcr or histopathological techniques. among the preventive strategies, paddock management is essential in horse farms with a history of tyzzer’s disease: young foals should graze in healthy environment characterized by well-grassed paddocks, free from potentially contaminated soil. excessive number of horses entering and leaving the farm and temporary holding fences used by a large number of horses should be avoided, because they can increase the incidence of infection. although there are no information about the survival of the endospores on open pastures, they are sensitive to exposure to 0.4% peracetic acid, 0.015% sodium hypochlorite, 1% idophol and 5% phenol (ganaway 1980, itoh et  al. 1987). in the present case, the foal shared the paddock with other foals and mares. for these reasons, the authors have advised the owner 245veterinaria italiana 2021, 57 (3), 239-246. doi: 10.12834/vetit.1983.12227.1 ellero et al. tyzzer's disease in a foal appel g. & burdinski k. 1995. tyzzer's disease in a pony foal from schleswig-holstein. dtsch tierarztl wochenschr, 102 (5), 204-205. barton m.h. & leroy b.e. 2007. serum bile acids concentrations in healthy and clinically iii neonatal foals. j vet int med, 21 (3), 508-513. borchers a., magdesian k.g., halland s., pusterla n. & wilson w.d. 2006. successful treatment and polymerase chain reaction (pcr) confirmation of tyzzer's disease in a foal and clinical and pathologic characteristics of 6 additional foals (1986-2005). j vet int med, 20 (5), 1212-1218. brown c.m., ainsworth d.m., 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tyzzer's disease in foals in england. equine vet j, 8 (3), 118-122. 1977. tyzzer's disease in the foal: case reports and review. canadian vet j, 18 (2), 41-43. turk m.a., gallina a.m. & perryman l.e. 1981. bacillus piliformis infection (tyzzer's disease) in foals in northwestern united states: a retrospective study of 21 cases. javma, 178 (3), 279-281. 233 veterinaria italiana 2021, 57 (3), 233-237. doi: 10.12834/vetit.1821.9620.3 accepted: 25.01.2021 | available on line: 31.12.2021 1department of veterinary preventive, faculty of veterinary, federal university of pelotas, pelotas/rs, brasil. 2center for technological development, faculty of biotechnology, federal university of pelotas/rs, brasil. *corresponding author at: department of veterinary preventive, faculty of veterinary, federal university of pelotas, pelotas/rs, brasil. e-mail: waller.stefanie@yahoo.com.br stefanie bressan waller1*, marcos roberto alves ferreira2, angelita dos reis gomes1, márcia kutscher ripoll1, anna luiza silva1, otávia de almeida martins1, luiza da gama osorio1, fabrício rochedo conceição2, renata osório de faria1 and mário carlos araújo meireles1 keywords aureobasidium pullulans, black yeasts, dematiaceous fungus, differential diagnosis, dog, opportunistic infection. summary a case of subcutaneous phaeohyphomycosis in a dog on the right limb during a post-operative period of castration was described for the first time. the macroscopic and microscopic characteristics of the fungal colonies growth on the sabouraud-dextrose agar were detailed. the fungus was identified as aureobasidium pullulans on the basis of phenotypic analysis and confirmed by sequencing of the internal transcribed spacers (its) region of rdna. the spontaneous remission of the lesion was observed in five weeks without antifungal treatment. this work highlights the importance of considering the pathogenic potential of this environmental fungus and the need of including it in the differential diagnosis of cutaneous lesions in dogs. subcutaneous phaeohyphomycosis due to aureobasidium pullulans infection in a dog subcutaneous phaeohyphomycosis in a dog due to aureobasidium pullulans infection. case report a swab from a cutaneous lesion of a 5-year-old labrador female dog, living in rio grande/rs (southern brazil), was sent to our laboratory with the clinical suspicion of sporotrichosis. the lesion was located on the femorotibial-patellar joint of the right posterior limb. it was a single, circumscribed exudative and ulcerative lesion with alopecic borders and dark-crust in the center, that, when removed, exposed a pinkish lesion with the blackened center (figure 1a). anamnestic data revealed that the animal had access to the courtyard of the house and residual medication of a recent ovariohysterectomy. the lesion in the limb appeared during the postoperative period, and no other clinical alterations were noted. mycological analysis the laboratory procedures were performed according to the solicited suspicious of introduction phaeohyphomycosis is an opportunistic infection caused by dematiaceous fungi, which include the aureobasidium genus. this fungal genus recognised as uncommon pathogen, is now known as hospital contaminant and emergent pathogen (chan et  al. 2011). most of the infections in humans and animals occur by the traumatic implantation from environment soil and plants (lloret et al. 2013, wang et al. 2019). aureobasidium pullulans is the main fungal species responsible for cutaneous (chen et  al. 2016, pikazis et al. 2009) and subcutaneous (de oliveira et al. 2013, joshi et  al. 2010) lesions in immunocompromised and immunosuppressed humans. this species also is reported causing systemic involvement, in particular at pulmonary level, difficult to differentiate from other important infections (hofman et al. 2008). in veterinary, the reports of a.  pullulans infections are rare and include canine cases of otitis (campbell et al. 2010) and disseminated infections (perkins et al. 2004). cases of cutaneous lesions in a porcupine (salkin et  al. 1976) and in a cat have also been observed (bernhardt et al. 2015). in this report we describe the first case of case report 234 veterinaria italiana 2021, 57 (3), 233-237. doi: 10.12834/vetit.1821.9620.3 aureobasidium pullulans infection in a dog waller et al. molecular analysis this isolate was named as ‘mv 2578’ and was genetically characterized by sequencing the internal transcribed spacer (its) using the primers its1 (5'-tcc gta ggt gaa cct tgc gg) and its4 (5'-tcc tcc gct tat tga tat gc) (irinyi et  al. 2015). it was identified by data bank analysis with ncbi blastn (identity 99%) and the sequence was submitted online at genbank number mg595273.1. for phylogenetic analysis, the sequences of the isolated strain were compared with the homologous sequences of a. pullulans reference strains and clinic relevant isolates obtained from genbank (figure 3). the multiple sequences alignment obtained from the isham its and genbank databases showed 95% identity between a.  pullulans mv2578 sequence its-4 and 15 human and veterinary clinical isolates. a.  pullulans mv2578 sequence showed also 82.5% identity with ay4 and 2712h strains, and 75% with hn4.4 strain. follow‑up of the procedures, and the risk factors in case of suspicious of aureobasidium  sp., the direct examination should be performed with 20% potassium hydroxide solution in skin samples for the search of multiple thick-walled and dark hypha with oval cells containing dark septa (chen et  al. 2016, larone 2011). although the swab was processed sporotrichosis. the direct examination of the sample did not show cigar-like cells in gram staining but oval structures arranged in strings. the sample was sown in duplicate on sabouraud-dextrose agar (sda) with chloramphenicol and in mycosel® agar. both agars were incubated at 25  °c and 37  °c to confirm the dimorphic characteristic of sporothrix sp. after six days, smooth and white colonies with creamy appearance were observed on the sda plates at both temperatures (figure 2a). the microscopic analysis showed unicellular and budding yeast-like cells, and consequently the sporotrichosis suspicious was discarded. the colonies were observed daily and, on the 10th day of incubation, they changed the smooth borders to an irregular appearance similar to a fringe and became dark, leading us to suspect infection by aureobasidium pullulans. the direct microscopic examination of lactophenol cotton blue (lcb) mount showed numerous hyaline yeasts cells with some dematiaceous oval cells (figure 2b). on the 15th day, these cells changed to hyaline or dematiaceous delicate hyphae with thin-walled, producing small blastoconidia and directly from the walls at certain fertile points (figure 2c). on the 19th  day, the colonies were almost totally dark (figure  2d) and the examination of lcb showed hyphae with thick-walled, dark, and septated segments with blastoconidia (figure 2e). figure 1. subcutaneous lesion located on femoro-tibial-patellar joint of the right posterior limb of an immunocompetent labrador female dog with aureobasidium pullulans infection. note alopecia on the borders of the exudative and ulcerative lesion and a dark-crust in the center (a), and the clinical spontaneous remission after five weeks (b). 235veterinaria italiana 2021, 57 (3), 233-237. doi: 10.12834/vetit.1821.9620.3 waller et al. aureobasidium pullulans infection in a dog lesion on the right posterior limb appeared after castration, although the surgical scar did not show cutaneous alterations. however, postoperative stress might have played an important role. it is known that the ovariohysterectomy deregulates b and t cells (nenadović et  al. 2017), affecting the immune system. the blood loss and the tissues trauma due to the surgery also stimulate proinflammatory (il-6 and tnf-α) and anti-inflammatory (il-10 and tgf-β) cytokines which block the cell-mediated immune responses (angele and faist 2002) and increase the susceptibility of the host to infection. it is believed that a traumatic inoculation related to the environmental contact (chen et  al. 2016, lloret et al. 2013, pikazis et al. 2009, wang et al. 2019) was the source of infection, since the animal had free access to the courtyard of the house. considering that aureobasidium pullulans is a black-yeast-like surface colonizer and is commonly associated as contaminant in hospital (wang et  al. 2019), it is important to be aware of the possible risk of false positives in the diagnosis. a histopathological analysis is recommended to demonstrate tissue invasion (perkins et al. 2004). in this case, however, it was not possible to perform histopathology because the owner did not allow the animal to undergo a new surgical procedure. various agents of subcutaneous or systemic phaeohyphomycosis are similar in appearance and, according to the clinical suspicious solicited by the veterinarian, in the gram staining we noted oval structures arranged in strings, which were compatible to the arrangement of fungal elements described by chen and colleagues (chen et al. 2016). aureobasidium pullulans was suspected when a dark fringe appeared on the border of colonies and the macromorphological and micromorphological characteristics of the fungal structures were similar to those described by larone (larone 2011). the diagnosis of phaeohyphomycosis is generally based on fungal detection by cytology and/or histology. cultures however provide definitive diagnosis and species identification (lloret et  al. 2013). the diagnosis of a.  pullulans relies on direct microscopic examination of clinical samples which may be misleading and should be confirmed by the molecular assay (chan et  al. 2011). in our case, the fungal species was confirmed through the its sequence polymorphism. according to the anamnaestic data, the subcutaneous figure 2. aureobasidium pullulans mv 2578 (mycology collection of centro de diagnóstico e pesquisa em micologia veterinária, ufpel, brazil). a. macroscopic appearance on sabouraud-dextrose agar after the 6 day of incubation at 37 ºc and microscopic characteristic using lactophenol cotton blue, note hyaline and dematiaceous oval cells (b, 1000×); on the 15th day, dematiaceous hyphae with fertile points were noted (c, 1000×); the colony appearance changed to dark (d) after 19 days with hyphae showing thick-walled and septated segments with blastoconidia (e, 1000×). 0.250 0.200 0.150 0.100 0.050 0.000 gq376094.1 aureobasidium pullulans strain uoa/hcpf 10936 hm130688.1 aureobasidium pullulans strain uoa/hcpf 688a kc253966.1 aureobasidium pullulans strain uoa/hcpf 12688a kc253967.1 aureobasidium pullulans strain uoa/hcpf 12768b kc253969.1 aureobasidium pullulans strain uoa/hcpf 14061 kc253964.1 aureobasidium pullulans strain uoa/hcpf 11686 kc253964.1 aureobasidium pullulans strain uoa/hcpf 11686 eu934759.1 aureobasidium pullulans strain uoa/hcpf 8352 hq215536.1 aureobasidium pullulans strain ay4 eu707927.1 aureobasidium pullulans strain 2712h dq680686.1 aureobasidium pullulans strain hn4-4 mg595273.1 aureobasidium pullulans strain mv2578 eu934757.1 aureobasidium pullulans strain uoa/hcpf 8227 kp131644.1 aureobasidium pullulans strain cnrma6.840 kp131642.1 aureobasidium pullulans strain cnrma11.985 kp131643.1 aureobasidium pullulans strain cnrma6.805 ay213639.1 aureobasidium pullulans strain uwfp 769 kp131645.1 aureobasidium pullulans strain wm 10.248 ef567985.1 aureobasidium pullulans strain wm 05.7 figure 3. neighbor-joining phylogenic analysis of its of aureobasidium pullulans mv 2578 and most significant culture collection strains and clinically relevant isolates. the optimal tree with the sum of branch length = 0.77464334 is shown. the evolutionary distances were computed using the maximum composite likelihood method (irinyi et al. 2015) and are in the units of the number of transitional substitutions per site. there was a total of 285 positions in the final dataset. evolutionary analyses were conducted in mega7 software (tamura et al. 2004). 236 veterinaria italiana 2021, 57 (3), 233-237. doi: 10.12834/vetit.1821.9620.3 aureobasidium pullulans infection in a dog waller et al. in five weeks, the lesion recovered, showing that antifungal therapy was not critical in this case. furthermore, some general practices are also important, such as keep clean the body site with physiological solution many times a day, avoid contaminated environment, keep the patient well fed, and avoid any aggravating stressful situation. conclusions this is the first report of subcutaneous phaeohyphomycosis in a dog caused by aureobasidium pullulans. this case report stresses the importance of including a. pullulans as a possible fungal pathogen in dogs when ulcerative lesions in subcutaneous tissue are observed, and, more in general, the importance of considering infections by unusual fungi as opportunistic infections in the diagnosis of cutaneous lesions. acknowledgments the authors would like to thank frederico schmitt kremer for bioinformatic analysis. the authors are grateful to the following brazilian institutes: to conselho nacional de desenvolvimento científico e tecnológico (cnpq), to coordenação de aperfeiçoamento de pessoal de nível superior (capes), to fundação de amparo à pesquisa do estado do rio grande do sul (fapergs) for student and research scholarships. frequentlyand can't be differentiated by morphology. cultures are essential to diagnosis. in our case, the culture allowed to the fungal identification, which was confirmed by the molecular analysis. still, the growth of a. pullulans on the sda on the agar plates and the absence of contaminants confirmed the high technical skill of the operators. furthermore, due to the ability of this species to survive uncommon environments, the reuse of laboratory solutions and buffers for cytology (hofman et al. 2008) must be avoided. all procedures in our case were individually performed with no risk of pathogen transfer to stock solutions, and this laboratorial care should be taken into consideration for all laboratories in any microbial suspicion. the clinical outcome after the sample collection, the veterinarian recommended daily topical applications of rifamycin. this therapy was interrupted after a week, due to the negative result of the bacteriological analysis. once a.  pullulans infection was confirmed, a protocol including oral itraconazole for four weeks was recommended in accordance with what has been described in the literature (joshi et al. 2010, pikazis et al. 2009 chen et al. 2016, de oliveira et al. 2013). however, due to financial matters, the owner decided not to treat the animal. aureobasidium pullulans was repeatedly isolated even if the lesion started to decrease spontaneously in a few days. 237veterinaria italiana 2021, 57 (3), 233-237. doi: 10.12834/vetit.1821.9620.3 waller et al. aureobasidium pullulans 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xila v., velegraki a. & aronit k. 2009. extended fungal skin infection due to aureobasidium pullulans. clin exp dermatol, 34, e892-e894. salkin i.f., gordon m.a. & stone w.b. 1976. cutaneous infections of a porcupine (erethizon dorsatum) by aureobasidium pullulans. sabouraudia, 14, 47-49. tamura k., nei m. & kumar s. 2004. prospects for inferring very large phylogenies by using the neighbor-joining method. proc natl acad sci usa, 101, 11030-11035. 355 department of veterinary sciences, school of agrarian and veterinary sciences, university of trás-os-montes e alto douro (utad), veterinary and animal science center (cecav), p.o. box 1013, 5001-801 vila real, portugal. *corresponding author at: department of veterinary sciences, school of agrarian and veterinary sciences, university of trás-os-montes e alto douro (utad), veterinary and animal science center (cecav), p.o. box 1013, 5001-801 vila real, portugal. e-mail: juangarciadiez@gmail.com. parole chiave analisi della corrispondenza multifattoriale, brucellosi, fattori di rischio, piccoli ruminanti, vaccino rev-1. riassunto tras-os-montes e alto douro è la regione a più alta prevalenza di brucellosi ovi-caprina del portogallo. nel tentativo di ridurre i livelli di prevalenza e di proteggere la salute pubblica, dal 2001 al 2004 è stato condotto un programma di vaccinazione dell'intera popolazione ovi-caprina con brucella melitensis rev-1. sebbene la prevalenza individuale sia scesa dallo 5,6% del 2001 allo 0,4% nel 2007, diversi allevamenti hanno continuato a presentare livelli di prevalenza individuale di oltre il 5,0%. viste le caratteristiche multifattoriali della brucellosi, questo studio ha valutato utilizzando l'analisi della corrispondenza multipla l'influenza esercitata da possibili fattori di rischio e pratiche gestionali negli allevamenti con una prevalenza ≥ 5,0%. i risultati hanno mostrato che il mancato riconoscimento dei sintomi e l’assenza di vaccinazione con rev-1 sono stati i principali fattori che hanno contribuito all'elevata prevalenza individuale della brucellosi nelle greggi. sulla persistenza della malattia hanno contribuito anche altri fattori come il consumo di latte crudo, la presenza di cani negli allevamenti e l'uso di pascoli comuni. la gestione familiare, lo scarso profitto economico, la scarsità di conoscenze, le scadenti capacità manageriali degli allevatori e il mancato supporto veterinario possono spiegare la persistenza degli elevati tassi di prevalenza della brucellosi ovi-caprina negli allevamenti portoghesi. i risultati potrebbero essere utilizzati per migliorare l'efficienza dei programmi di eradicazione della brucellosi. fattori di rischio e pratiche gestionali che influenzano gli elevati tassi di prevalenza di brucellosi ovi-caprina nella regione nord est del portogallo keywords multifactorial correspondence analysis, brucellosis, risk factor, small ruminants, vaccine rev-1. summary the region of tras-os-montes e alto douro in northeast portugal displayed the highest prevalence of brucellosis in small ruminants of the country. a vaccination programme of the whole population with brucella melitensis rev-1 was carried out from 2001 to 2004 in an attempt to reduce prevalence levels and protect public health. although individual prevalence decreased from 5.6% in 2001 to 0.4% in 2007, several flocks continued to present individual prevalence ≥ 5.0%. given the multifactorial characteristics of brucellosis, the current study evaluated farming practices and risk factors in flocks with an individual prevalence over 5% by multifactorial correspondence analysis. results showed that a lack of recognition of the symptoms of brucellosis and lack of rev-1 vaccination were the main factors contributing to the high individual prevalence of brucellosis in flocks. other factors such as the consumption of raw milk, presence of dog commingling with animals and use of communal pastures also contributed to the persistence of the disease. family farms with low economical profit, minimal training/education of farmers, and a scarcity of veterinary support may explain the persistence of factors contributing to the high prevalence of brucellosis. the results of this study highlight several risk factors and farming practices that might have contributed to the maintenance of a high prevalence of brucellosis in flocks with high brucellosis prevalence. these results could be used to adopt new approaches to improve the efficiency of brucellosis eradication programs. adosinda coelho, juan garcía-díez*, joaquim góis, jorge rodrigues and ana cláudia coelho farm practices and risk factors which influence the high prevalence of brucellosis in small ruminant flocks in northeast portugal veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 accepted: 16.02.2017 | available on line: 31.12.2019 356 veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 variables behind small ruminant brucellosis coelho et al. at the farm site, restriction of animal trade and a minimum of 4 serological tests in a 240-day period with negative results. the implementation of the mass vaccination and test-and-slaughter programme (described above), resulted in the decrease of the prevalence of brucellosis in both small ruminants and humans. however, although individual brucellosis prevalence decreased from 5.6% in 2001 to 1.3% at the end of the mass vaccination programme and progressively to 0.4% in 2007 (dgv 2011), individual prevalence of brucellosis in tras-os-montes remains the highest in portugal and some flocks still present prevalence values higher than those observed in 2001. this indicates that control of brucellosis not only depends on test-vaccination-slaughter programmes but also on other factors such as husbandry, grazing, hygiene, veterinary management, training, farmers´ education and implementation of biosecurity measures, among others (garcía-díez et  al. 2013, mainar-jaime et  al. 1999). the prevalence of brucellosis may also vary according to flock size, main animal production, and flock composition (minas 2006, coelho et  al. 2007). moreover, particular characteristics of brucella spp. relating to routes of infection and resistance in the environment make their control in high prevalence areas more difficult (lithg-pereira et  al. 2001). the aim of this study was therefore to evaluate farming practices and risk factors relating to brucellosis in flocks with high brucellosis prevalence values. materials and methods study design a total of 37 registered flocks with animal prevalence of brucellosis ≥ 5% in 2007 was studied. the cut-off value of ≥ 5% was identified as the selection criteria because the brucellosis prevalence rate was 5% at the beginning of the mass vaccination programme in 2001. all flocks belonged to the trás-os-montes e alto douro region (northeast of portugal). information regarding farm characteristics (species, flock size, main animal production, birth date, sex, breed, blood sampling date, rev-1 vaccination date, rbt and cft results and culling date of positive animals) was obtained from interviews with farmers and from the national animal health database (pisa. net®). each farmer participated in an epidemiological survey during their personal interview. interview questions are presented in table i. data analysis farming practices and risk factors for brucellosis were assessed by multifactorial correspondence introduction brucellosis is a contagious zoonotic disease responsible for reproductive failure with important public health significance (seleen et  al. 2010). in small ruminants, brucella melitensis is responsible for heavy economic losses resulting from clinical disease, abortion, neonatal losses, increased births intervals, reduced fertility, commercial restrictions, increased culling rates and the emergency slaughtering of infected animals (radostits et  al. 2000). in portugal, as well as in other european countries, the national veterinary authority is responsible for the control of brucellosis in small ruminant (portugal 2013). tras-os-montes and alto douro, a region of the northeast portugal, has the highest prevalence of brucellosis in small ruminants (portugal 2013) as well as the highest number of cases of brucellosis outbreaks (dgs 2014). due to the public health concern, the national veterinary authority implemented a mass vaccination programme for young and adult small ruminants. the programme, which ran from 2001 until 2004, aimed to decrease high prevalence rates through the use of a commercial, live, freeze-dried vaccine against brucellosis. briefly, all sheep and goats over 3  months were vaccinated with b.  melitensis strain rev-1 (ocurev, shering and plough, us) via conjunctival route. animals were identified with both a tattoo in the left ear and special ear-tags that included the vaccination date. all animal data were recorded in the national animal health software (pisa. net®). blood samples were obtained from a jugular puncture and were collected at the same time as the vaccine was administered. sheep and goats positive for both rose bengal test (rbt) and complement fixation test (cft) were culled (dgv 2011, directive 91/68/eec). after 12 months, blood samples from young-vaccinated sheep and goats were collected and those which were seropositive were culled. in adult-vaccinated animals, a blood sample was collected after 30 months to assess vaccine response. control flocks were sampled 0 days, 1 month, 4  months, and 12 months after vaccination. this allowed us to assess the vaccine response across the seasons. animal replacement was only allowed with young-vaccinated sheep and/or goats in order to prevent brucellosis outbreaks. commercial trade restriction was enforced for 21 days after the rev-1 vaccination, and the national veterinary authority restricted commercial trade of positive flocks (portugal 2000). after the mass vaccination programme, from 2005 to 2007, rev-1 vaccination was used in sheep and goats from 3 to 6 months as previously described. the following special measures were applied to infected flocks. they include the study of the source of infection through an epidemiological survey 357 coelho et al. variables behind small ruminant brucellosis veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 analysis (mca) to the discreet qualitative variables, especially nominal or ordinal ones (matias 2006). this methodology is an exploratory multivariate technique that converts a matrix of non-negative data into a graphical display in which the rows and columns of the matrix are depicted as points. in an mca plot of the data’s attributes, the negative and positive attributes are represented as separate points (torres and van de velden 2007). all multivariate analysis methods work by computing successive axes of decreasing importance called successive axes. mcas reduce the data contained in large tables and depict them in a vectorial plan. the degree of correlation among variables and/or observations is assessed by their relative proximity. thus, the closer they are, the more correlated they are. ten sets of multifactorial correspondence analysis (mca) linked to risk factors detected in the interviews were carried out using andad version 7.1 developed by instituto superior técnico, centro de valorização de recursos minerais, instituto superior técnico of lisboa. the main result of the correspondence analysis was a 2-dimensional plot of the associations between qualitative, explanatory, and outcome variables. it was assumed that the initial data had 2 clouds in 2 multi-dimensional vector spaces: 1 cloud for the columns (the variables studied), which were represented by 4 letters, and 1 cloud for the rows (flocks), which were represented by 2 letters and 2 numbers or 3 letters and 1 number. the mca succeeded in constructing factorial axes that enabled the modalities to be positioned according to their coordinates on the selected factorial map. the interpretation of the quality of a mca consists of the following: a) the contribution of an axis to the total inertia of the cloud is given by the ratio of the corresponding eigenvalue by the sum of all eigenvalues. this percentage is used to select the number of significant axis; b) this partial contributions allows us to determine which points play a major role in the orientation of the factorial axis; and c) the representation of a point in the euclidean space is defined as the difference between the square of the distance of the point of origin and the square of the distance of the point in the profile. three factorial axes whose variables exhibited the greatest behavioural variability were considered in this study. the plot identifies clusters of associated variables. those clusters with greater distance from the intersection have stronger associations. explanatory variables of less than -  0.5 and more than 0.5 in the analysis were considered to show a significant association (high risk) whereas those having variables of less than -  1.0 and more than 1.0 were considered to show a very high significant association (very high risk). in this study, the original data enabled the detection of 3 axes that explained 45.8% of the total variance. the second and third table i. risk factors and farming practices which may influence the prevalence of brucellosis in small ruminant flocks of trás-os-montes e alto douro region (northeast of portugal)*. farmers socio-demographic characteristics age sex education (no education, primary education, higher education) existence/knowledge of brucellosis infection in the family farm characterization farm registration number location flock characterization species at farm (sheep, goat, cattle, others) number of small ruminants in the flock characterization of the flock by sex, age, production (milk or meat) existence of contact with other flocks animal movement/purchase purchase of animals from local farms purchase of animals from fairs purchase of animals from animal sellers purchase from other country existence of official documents of animal movement (entrance/exit) quarantine for purchased animals farm management use of communal pastures existence of cleaning and disinfection (c&d) plan frequency of c&d operations manure removal (frequency and destination of manure) existence of pest control frequency of pest control verifications existence of fly control frequency of fly control verification verification of the of animal cleanliness presence of tap water or spring water milking technique is used in the farm presence of dogs at farm dogs eat or may eat fetal membranes, foetuseses or abortions reproductive management identification of the parturition season existence of a maternity pen technical assistance during reproduction season isolation of females before and after parturition from the flock proper removal of foetuses/abortions/fetal membranes existence of artificial insemination use of own or lent males brucellosis status characterization of brucellosis-positive animals (sex, age, breed, main production) official brucellosis classification of the farm correct identification of brucellosis-positive animals correct identification of brucellosis-positive animals vaccinated with rev-1 permanence (in days) of brucellosis-positive animals at farm before culled kids and lambs are properly vaccinated with rev-1 farmer drinks raw milk farmers eat fresh cheese made from raw milk existence of animal products commerce (milk, cheese) sale of milk sale of cheese made from raw milk (fresh or cured) identification of main clinical signs of the flock abortions metritis orchitis retention of fetal membranes mastitis existence of fertility problems decrease in milk/meat production yield *data obtained from farmers´ interviews and from the national animal health database (pisa.net®) 358 veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 variables behind small ruminant brucellosis coelho et al. comprising both sheep and goats that were used for meat production. multifactorial correspondence analysis the results of the mca of farming practices and risk factors for brucellosis are presented in figures 1 and 2. a significant association between the following variables was observed (figure 1): absence of abortions, animal movement/commercial trade and clinical signs of brucellosis, pest control in farms, dimensions accounted to 22.4% and 12.7% of the data variance, respectively. results farmer socio‑demographic composition and flock characterisation a total of 37 farmers were interviewed. all the respondents were males. over 85% were older than 51 years and reported primary-level education. the combined flocks included over 150 animals, figure 1. plot of multifactorial correspondence analysis related to flock’s characteristics for axis 1 and axis 2. flock isolated (fis); flock not isolated (fnis); owners with other herds (ooh); owners without other herds (onoh); positive animals (pos); negative animals (neg); correct higienization of premises (dsf ); absence of correct higienization of premises (ndsf ); fly control (fc); absence of fly control (afc); pest control (pc); absence of pest control (apc); presence of breeding management (bm); absence of breeding management (abm); animal isolation on birth period (isb); no isolation of animals on birth period (nisb); proper removal of fetal membranes, abortus, etc. (pr) improper removal of fetal membranes, existence of abortions (upr); absence of abortions (ab); tap water (tpw); absence of tap water (atpw); utilization of communal pastures (cop); absence of utilization of communal pastures (ncop); rev-1 vaccination (r1); absence of rev-1 vaccination (nr1); trade of animal and/or its products (tap); absence of trade of animal and/or its products (atap); consumption of raw milk/fresh cheese (crwm); no consumption of raw milk/fresh cheese (acrwm); dogs eat fetal membranes and/or abortions (deat); dogs sometimes eat fetal membranes and/or abortions (dset); dogs never eat fetal membranes and/or abortions (dnet); presence of clinical signs of brucellosis (sig); absence of clinical signs of brucellosis (nsig); human brucellosis (hbr); absence of human brucellosis (nhbr). 359 coelho et al. variables behind small ruminant brucellosis veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 contrarily, the variable “farmers drink raw milk and/or eat fresh cheese made from raw milk” (-  0.52) was presented in the negative dimension (figure 2). at dimension 2, the following variables: correct removal of abortions (foetuses and foetal membranes), absence of dogs that eat foetuses and foetal membranes, not sharing pasture areas, use of tap water, implementation of fly control programme, proper cleaning and disinfection of farm premises, absence of rev-1 vaccination and existence of reproduction/husbandry programme. in decreasing absence of animals vaccinated with rev-1, fly control programme, and the use of tap water. the variables with the greatest partial contributions for the variability were, in decreasing order: absence of abortions (3.15), absence of animal movement/ commercial trade (3.15), absence of clinical signs of brucellosis (2.11), pest control at the farm level (1.82), presence of tap water (0.82), fly control (0.66), absence of animals vaccinated with rev-1 (0.57), absence of dogs in flocks that eat foetuses and foetal membranes (0.57) for the positive dimension. figure 2. plot of multifactorial correspondence analysis related to flock’s characteristics for axis 1 and axis 3. flock isolated (fis); herd not isolated (hnis); owners with other herds (ooh); owners without other herds (onoh); positive animals (pos); negative animals (neg); correct higienization of premises (dsf ); absence of correct higienization of premises (ndsf ); fly control (fc); absence of fly control (afc); pest control (pc); absence of pest control (apc); presence of breeding management (bm); absence of breeding management (abm); animal isolation on birth period (isb); no isolation of animals on birth period (nisb); proper remove of fetal membranes, abortus, etc. (pr) inproper remove of fetal membranes, abortus, etc. (upr); absence of abortion (ab); tap water (tpw); absence of tap water (atpw); utilization of communal pastures (cop); absence of utilization of communal pastures (ncop); rev-1 vaccination (r1); absence of rev-1 vaccination (nr1); trade of animal and/or its products (tap); absence of trade of aninal and/or its products (atap); consumption of raw milk/fresh cheese (crwm); no consumption of raw milk/fresh cheese (acrwm); dogs eat fetal membranes, and/or abortions (deat); dogs sometimes eat fetal membranes and/ or abortions (dset); dogs never eat fetal membranes and/or abortions. (dnet); presence of clinical signs of brucellosis (sig); absence of clinical signs of brucellosis (nsig); human brucellosis (hbr); absence of human brucellosis (nhbr). 360 veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 variables behind small ruminant brucellosis coelho et al. results indicated that flocks with a high prevalence of brucellosis are associated with absence of abortions and absence of clinical signs of brucellosis. in previous studies, coelho and colleagues (coelho et  al. 2007) observed that prevalence of brucellosis was higher in large flocks, with mainly over 150  animals. since flocks in the study area were extensive and required large pasture areas, the presence of abortions and/ or other clinical signs could have been difficult to be observed by farmers. in addition, the association between the lack of abortions and high prevalence of brucellosis may be associated to the lack of communitation of abortions by farmers to the local veterinary authorities, despite this being mandatory (portugal 2006). if farmers notice the existence of abortions to the national veterinary authority, the flock is subjected to several sanitary measures, including restrictions on commercial trade and new blood sample testing. in addition, the flock is restricted to the farm area. animals belonged to large flocks are mainly fed in large pastures (mainly communal pastures). thus, in case of positive results (or even in case of abortion notification), flocks cannot leave the farm to prevent the spread of the disease through pastures. however, sometimes there is no ability to feed them on farms since feed is based on pasture. the trade of live animals (mainly kids and lambs) represents the main economical income for many farmers. thus, farmers may therefore not report abortions in order to avoid any economic losses associated to the restriction measures. this could explain the association between the absence of (reported) abortions and the high prevalence of brucellosis. the low levels of training/education of farmers regarding brucellosis may be considered as a barrier in eradication programmes (litgh-pereira 2001). lack of knowledge about policies related to animals, veterinary management or farm biosecurity could pose a risk for both animals and humans (garcía-díez et al. 2013). this lack of knowledge about the disease and its zoonotic characteristics may explain the relationship observed between flocks with high prevalence of brucellosis and farmers who declared that they drink raw milk or eat cheese made from raw milk (sofian et  al. 2008). most family farms, such as those in the study, are characterized by low economical profit. this fact suggests a lack of veterinary support and may explain the lack of knowledge regarding the association of clinical signs such as abortions, metritis, orchitis or infertility with brucellosis infection (mainar-jaime et al. 1999). the lack of notification of abortions may not only be associated with avoiding restrictive measures, but also with a lack of knowledge about legislation and clinical symptoms related to brucellosis by farmers. order, the following characteristics were observed in the positive dimension 2 (figure  2): dogs eat foetal and abortions products (2.51), not sharing pasture areas (1.10), correct removal of foetuses and foetal membranes following abortion (0.98), use of tap water (0.81) and implementation of fly control programme (0.66). in contrast, the following characteristics were observed in negative dimension 2: existence of reproduction/husbandry programme (-  1.03), absence of cleaning and disinfection programme of farm premises (- 0.67), absence of rev-1 vaccination (- 0.61) and presence of dogs that eats foetuses and foetal membranes (- 0.51). the third dimension (figure 2) contributed 10.7% of data variance. a significant association between farmers who keep dams separate during the parturition period, existence of reproduction/ husbandry program, farmers who had another(s) herd(s) and implementation of fly control program was observed in the negative dimension 2. for the positive dimension 3, the modalities that made the highest partial contributions to variability, in decreasing order, were: dams that are kept away during parturition (1.81), existence of reproduction/ husbandry program (1.56), farmers who had others herds (1.32), cleaning and disinfection program of farm premises (0.68) and farmers who had brucellosis (0.61). in contrast, the following variables were observed in negative dimension 3 (figure  2): absence of dogs that eat foetuses and abortions (-  1.09), absence of farmers who drink raw milk or eat fresh cheese made from raw milk (-  0.82), farmers who remove abortions (foetuses and fetal membranes) (- 0.62) not sharing communal pastures (-  0.59) and deficient cleaning and disinfection of farm premises (- 0.52). discussion eradication of brucellosis in small ruminants is a necessary task to avoid heavy economic losses, reproductive issues (mainly abortions and/or neonatal losses) and the emergency slaughtering of positive animals. moreover, control of brucellosis is also necessary in order to avoid zoonotic outbreaks, protect public health and guarantee the animal trade, especially in areas with high prevalence values (minas 2006). since brucellosis is a multifactorial infectious disease, the characteristics and management of flocks are potential risk factors. the application of a multifactorial correspondence analysis showed, in a graphic form, the association of risk factors and farm practices related to high brucellosis prevalence values. however, the disadvantage of this approach is that it considers all risk factors to have equal importance. 361 coelho et al. variables behind small ruminant brucellosis veterinaria italiana 2019, 55 (4), 355-362. doi: 10.12834/vetit.1162.6419.2 infected reproductive tissues (baek et  al. 2003). dogs infected with brucella spp. can spread organisms into the environment through urine, vaginal secretions, aborted foetuses or faeces. since elimination of infected animals may not necessarily eradicate the disease, they should instead be included in all sanitary measures carried in the flock (i.e. blood sampling). the application of good farm practices (gfp) that also include the existence of a reproductive/husbandry programme was observed to be a protective factor in those farmers that suffered brucellosis. the presence of gfp may be associated with access to knowledge of treatments recommended by medical practitioners to treat zoonotic infection and sanitary recommendations that were made by the national veterinary authority during the compulsory epidemiological survey (gunn et al. 2008). since the control of brucellosis is multifactorial, the characteristics of the flock (i.e. species, size) as well as its management (i.e. feeding, reproductive management) should be considered when determining the potential risk factors. the current study demonstrated that high prevalence values of brucellosis were mainly related to flock size, the absence of clinical signs of brucellosis, including abortions, and the absence of the rev-1 vaccination. familiar farms, the absence of veterinary technical support, and the low levels of training/education of farmers regarding brucellosis may explain the influence of these factors. acknowledgements the authors thank to a. sobral, m.a. armada nunes, l. fonseca, and a.p. figueiras, from the regional veterinary services, and to s. quintans, r. valentim, and a. pina fonseca from the national veterinary authority of portugal. the work was supported by the strategic research project pest-oe/agr/ ui0772/2011 financed by the foundation for science and technology (fct, portugal). the author j. garcía-díez was supported by research grant sfrh/bd/85118/2012 of the foundation for science and technology (fct, portugal) rev-1 vaccination is an essential measure to control brucellosis in areas of high prevalence (blasco 2006). although kids and lambs between 3 to 6 months old must be vaccinated with rev-1, the relationship between the high prevalence of brucellosis and absence of rev-1 vaccination may be compatible with lamb and kid management strategies whereby (unvaccinated) animals originally destined for slaughter are retained in the flock (minas et  al. 2004), increasing the risk of new infections and maintaining the disease within the flock (blasco 1997, blasco 2002). the implementation of a biosecurity plans at the farm level is fundamental to controlling brucellosis (ganter 2008). measures such as the control of animal movement, cleaning and disinfection, pest control, reproductive management or preventive veterinary programmes among others are required. since b.  melitensis has little resistance to most disinfectant agents (cfsph 2009), the correct cleaning and disinfection (c&d) of farm premises (reviriego et  al. 2000) is fundamental to limit its survival. thus, the absence of correct hygienic practices explains the relationship between the high prevalence of brucellosis in flocks without proper cleaning and disinfection procedures (mainar-jaime et al. 1999). infected animals with brucellosis are characterised by the excretion of brucella spp. trough vaginal discharges, mainly during calving, that contaminates the environment. the absence of c&d procedures and proper manure management creates an environment in which insects thrive. these flies could act as mechanical carriers for the dissemination of brucella spp. across the farm environment through, for example, feeders and/or taps facilitating oral infection. the presence of dogs has been described as a risk for brucellosis infection in farm animals (aguiar et al. 2007). dogs represent a potential epidemiological threat in areas in which brucellosis is endemic since they can act as mechanical disseminators by feeding on aborted foetuses, dragging them along and spreading 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ramezani a. 2008. risk factors for human brucellosis in iran: a case-control study. int j infect dis, 12, 157-161. torres a. & van de velden m. 2007. perceptual mapping of multiple variable batteries by plotting supplementary variables in correspondence analysis of rating data. food qual prefer, 18, 21-129. 305 veterinaria italiana 2021, 57 (4), 305-310. doi: 10.12834/vetit.2111.12262.1 accepted: 28.04.2020 | available on line: 31.12.2021 department of microbiology, faculty of veterinary medicine, urmia university, urmia, iran. *corresponding author at: department of microbiology, faculty of veterinary medicine, urmia university, urmia, iran. e-mail: h.dastmalchi@urmia.ac.ir. somayyeh ahmadi, habib dastmalchi saei*, seyed meisam abtahi froush and mohammad zavarshani keywords avian, colibacillosis, escherichia coli, heterophil, pcr, phylogenetic groups, immunological activities, iran. summary avian pathogenic escherichia coli (apec) is a major cause of colibacillosis and is associated with economic losses to the poultry production worldwide. heterophils are the first line of immune defense of the avian host against invasive pathogens. in this study, apec isolates from chickens with colibacillosis were assigned to phylogenetic groups and immunological activities of heterophils against these groups were examined. a total of 92 apec isolates was obtained from 106 samples of diverse organs collected from chickens with colibacillosis from different farms in west azerbaijan province, iran. isolates were assigned to phylogenetic groups based on the clermont triplex pcr method, and immunological activities (including phagocytosis, respiratory burst and bacterial killing) of heterophils against these groups were examined. as results, the frequency of a, b1, b2 and d groups were 35.87, 44.57, 5.43 and 14.13%, respectively. in addition, opsonized escherichia coli isolates belonging to b1 group significantly enhanced the level of respiratory burst (0.52 ± 0.02%) while the killing level of them was significantly lower than the other groups (29.40 ± 5.09%). there was no significant difference in phagocytic activity of heterophils against the phylogenetic groups. in conclusion, incomplete immune responses to b1 phylogenetic group maybe a principal cause of mortality by colibacillosis caused by this group. it is suggested to study heterophilic immune reaction against e. coli phylogenetic group for development of effective prevention strategy. effects of different phylotypes of avian pathogenic escherichia coli isolated from broiler chickens with colibacillosis on heterophils functional activities: an in vitro study are extra-intestinal pathogenic strains (clermont et al. 2000). in poultry, avian pathogenic e. coli (apec) is the main cause of, colibacillosis characterized by fever, depression and respiratory inflammation due to primary penetration of heterophils and monoconjugate phagocytes (ramadan et  al. 2016). it has been reported that young animals are more susceptible than adults to this illness. this transient sensitivity of chickens and turkeys to infection during the first weeks of life is associated with impaired quality of innate and adaptive immune system (hussain et  al. 2017a). heterophils, the counterparts to the mammalian neutrophils, are the first line of defense against any microbial attack and play an indispensable role in the immune response of the avian host (genovese et al. 2013). these cells fight against infectious diseases by phagocytosis and respiratory burst (kogut et  al. 2012). the aims introduction escherichia coli is a facultative gram negative bacterium belonging to the normal flora of the mammals and birds intestine (katouli 2010). this organism is one of the most frequent causes of many common primary and secondary bacterial infections, but it is generally considered to be an opportunistic pathogen that can cause infections following the suppression of the host immune system (hussain et al. 2017b). e. coli causes different clinical manifestations in birds among which the colibacillosis is the most common form causing major economic impact (lutful kabir 2010). based on the clermont triplex pcr method, e.  coli strains can be classified into four phylogenetic groups (a, b1, b2 and d), of which groups a and b1 are normal flora and group b2 and, to a lesser extent, group d 306 in vitro study of effects of apec on heterophils functional activities ahmadi et al. veterinaria italiana 2021, 57 (4), 305-310. doi: 10.12834/vetit.2111.12262.1 avian heterophils preparation the isolation of heterophild, their count and their viability were carried out and assessed as previously described (andreasen and latimer 1989, blattes et al. 2017). in brief, the blood samples from healthy chicken were transferred to the edta tubes and mixed with 1% methylcellulose, in calcium and magnesium-free hank's balanced salt, at a 1:5 ratios. the samples were centrifuged for 20 min at 25 g. the buffy coat was isolated, washed and re-suspended. following the procedure, the samples were centrifuged on a ficoll-hypaque density gradient (400 g, 30 min). the mononuclear cell layer and plasma were removed, and contaminant erythrocytes were deleted by hypotonic lysis. the remaining heterophils were washed twice and counted in a neubauer chamber. the viability of the cells was checked by trypan blue dye exclusion. evaluation of respiratory burst of heterophils by nitroblue tetrazolium reduction (nbt) the suspension of opsonized bacteria and intracellular generation of reactive oxygen species (ros) were measured by nbt reduction as described previously (chen and junger 2012, motlagh et  al. 2015). non-opsonized bacteria were used as control. phagocytosis and microbial killing activities of heterophils two tubes were used as test and control. equal volume (0.5 ml) of heterophil suspension (5  ×  106  cell/ml) and chicken serum were mixed in the test tube in equal volume. in the control tube, 0.5 ml of hbss buffer was mixed with chicken serum instead of heterophil suspension. both tubes were incubated for 5 minutes at 37 °c. then, 0.5 ml of bacterial suspension (5 × 107 cfu/ml in hbss) added to each tube and then at this moment, zero time, 100 μl of the test and control tubes were removed and added to the new tubes. the test and control tubes were incubated at 37 °c for 90 minutes. in this stage we had two kinds of data: zero and 90 data. of the present study were first the phylogenetic typing of e.  coli isolated from poultry and then the evaluation phagocytosis, respiratory burst and bactericidal activity of heterophils against different phylogenetic groups. materials and methods sampling one-hundred and six samples of different organs (liver, heart, lung and spleen) were collected between 2015 and 2016 from broiler chickens with colibacillosis on multiple flocks located in different regions (urmia, naqadeh, and khoy) of west azerbaijan province, iran. all the samples were cultured primarily in macconkey agar at 37 °c for 24 h (mca/105465, merck, darmstadt, germany). pink-colored colonies which were lactose positive, suspected as e.  coli on mca were further streaked on eosin-methylene blue agar (emb/101347, emb, merck, darmstadt, germany) for purification. isolates were then subjected to standard biochemical tests including imvic (indole, methyl red, voges-proskauer and citrate), urease production, h 2 s production and various sugar fermentation tests (li et al. 2010). dna extraction and confirmation of e. coli isolates after identification of the e.  coli isolates by conventional methods, a single colony of each isolate was cultured onto blood agar medium and incubated at 37 °c for 18-24 hours. bacterial dna was extracted by boiling method as described previously (obeng et  al. 2012). purity of extracted dna was determined based on measurement of od 260/280 using a nanodrop 2000c spectrophotometer (thermo fisher scientific, wilmington, de, usa). additionally, the integrity of extracted dna was evaluated by electrophoresis in 1% agarose gel. dna with an od260/od280 ratio of ≥ 1.8 was considered to be pure and used in pcr. pcr amplification of a 662-bp dna fragment size of 23s rrna gene was also conducted to determine any inhibitors remained from the extraction procedure by using eco 2083 (5'-gct tga cac tga aca ttg ag-3') and eco 2745 (5'gca ctt atc tct tcc gca tt-3') primers (riffon et al. 2001). detection of phylogenetic groups phylogenetic group assignment of the isolates was performed using the clermont triplex pcr method (clermont et al. 2000). primer sequences for phylogenetic classification are shown in table i. table i. primer sequences used and size of pcr products. primer primer sequence (5'3') size (bp) chua f: gacgaaccaacggtcaggat 279 r: tgccgccagtaccaaagaca yjaa f: tgaagtgtcaggagacgct 211 r: atggagaatgcgttcctcaac tspe4.c2 f: gagtaatgtcggggcattca 152 r: cgcgccaacaaagtattacg 307 ahmadi et al. in vitro study of effects of apec on heterophils functional activities veterinaria italiana 2021, 57 (4), 305-310. doi: 10.12834/vetit.2111.12262.1 groups were observed in 35.87, 44.57, 5.43 and 14.13% of the strains, respectively. evaluation of respiratory burst, phagocytic and bacterial killing activities of heterophils the viability of heterophils was 94%. results of kruskal-wallis test showed that there were statistically significant differences between different phylogenetic groups in the assessment of respiratory burst and microbial killing of heterophils (p value < 0.05), whereas no significant change in the phagocytic activity of heterophils was observed between the groups (table ii). according to our results, the mean ± standard deviation differences of the optical density in the evaluation of microbial killing activities of heterophils against b1 phylogenetic group were less than other groups (p value < 0.05). there were no significant differences between other groups. however, obtained data showed that the heterophils had the highest potential to kill the group b2 bacteria compared to bacteria in groups a and d. in contrast to microbial killing activity, the results showed that the amount of oxygen free radicals during respiratory burst was significantly higher than other groups against e. coli belonging to group b1 (p value < 0.05). in comparison between bacteria belonging to groups a, b1 and d, the group b2 showed the lowest potential to induce respiratory burst in the heterophils. despite the differences between the groups, they weren’t significant in evaluating the phagocytic activities of heterophils against the different phylogenetic groups of e. coli. albeit, the heterophils showed more potential to uptake the group d bacteria compared to other groups (table iii). discussion avian colibacillosis caused by avian pathogenic e. coli (apec) is one of the important disease of poultry in terms of economic losses throughout the world (guabiraba and schouler 2015). there are evidences suggesting a meaningful relationship between pathogenicity of apec and their phylogenetic zero and 90 data were related to zero time (control tube: c0, testing tube: t0) and 90 min (control tube: c90, testing tube: t90) after incubation, respectively. of note, the viability of heterophili along the experiment was checked during experimental set up. fortunately, no significant change was found. ten ml of sterile distilled water was added into each tube in order to lyse the heterophils. the mixture was stirred a few times slowly until the tube contents were thoroughly mixed. one hundred µl of current mixture were removed and transferred to 96 wells plate. for each dilution, five wells were considered and then 20 μl of mtt solution (5 mg/ml, sigma aldrich, usa) were added to each well. then it was incubated for 20 min at 37 °c. after incubation, 150  μl of dmso solution were added to each well to dissolve formasone crystals. the results of optical densities (od) were read at 492 nm wave lenght (biotek elx800, usa). the bactericidal activity (percentage of killing) was calculated by the formula at the end of process (mehrzad et al. 2009). the test and the control tubes which had been incubated for 90 min were centrifuged at 300 g for 10 min. one hundred μl of supernatant (containing no phagocytize bacteria) were added to the five wells of 96-wells plate. the plate was incubated for 20 min at 37 °c after adding 20 μl of mtt to each well. one hundred fifty μl of dmso were added to each well in the next step and then the data were read at 492 nm wave lenght. percentage of phagocytosis was calculated using the formula (gargan et  al. 1993, fijalkowski et al. 2012, mehrzad et al. 2009). data statistical analysis data analysis was done by spss statistical software, version 18. the results of groups were analyzed statistically by kruskal-wallis test. also, mann-whitney u-test was used to compare differences between two groups. p value less than 0.05 was considered significant. results e. coli detection a total of 92 e.  coli isolates were detected from broiler chicken with colibacillosis by biochemical tests as well as amplification of the 23s rrna gene fragment. phylogenetic classification of e. coli isolates all e.  coli isolates were classified to one of the four main phylogenetic groups, with a, b1, b2 and d table ii. the details of kruskal-wallis test of optical density of respiratory burst, microbial killing and phagocytic activities of heterophils. respiratory burst microbial killing phagocytosis chi-square 22.55 19.64 7.56 df 3 3 3 asymp. sig.* 0.00 0.00 0.06 *p value < 0.05 308 in vitro study of effects of apec on heterophils functional activities ahmadi et al. veterinaria italiana 2021, 57 (4), 305-310. doi: 10.12834/vetit.2111.12262.1 against e.  coli. in that study, e.  coli isolates were categorized based on some special factors such as o, fimbriae and k antigens. finally, they reported that the presence of fimbriae and o78 antigens were effective in protecting e. coli against heterophils and macrophages phagocytosis (mellata et al. 2003). as results, e. coli isolates belonging to group b1 were the most stimulating group in secretion of oxygen radicals by heterophils. this could be attributed to differences in bacterial cell surface antigens among studied phylogenetic groups. study carried out by ondrackova and colleagues showed that o149 strain of enterotoxigenic e. coli (etec) was more efficiently associated with the neutrophils and induced a more intensive respiratory burst compared to the o147 strain of etec. they suggested that this difference might be due to the presence of different types of fimbriae and other factors such as flagella. moreover, induction of higher respiratory burst by b1 phylogenetic group might be a reason for considerable economic losses due to this group which is the most common group in the studied region. it has been shown that the accumulation of inflammatory exudates in the airbag of chicken lung following the excessive release of oxygen radicals by heterophils due to much more respiratory burst is one of the main causes of chick death and as a consequence the high mortality rate of colibacillosis (kemmett et  al. 2014). the strong induction of respiratory burst is probably due to the strong presence of pattern recognition receptors such as toll-like receptors (tlrs) and mannose recognition receptors in b1 group (genovese et  al. 2013). interestingly, despite the strong induction of phagocytosis and respiratory burst in group b1, the bacterial death was defective by heterophils. this may be due to the presence of strong anti-free radical agents in group b1. for example yjaa gene encoding protein has been reported to have an important role against oxygen radicals as an anti-oxidant (bonacorsi et  al. 2000). in previous studies, it has been proven that apec can cut down the antimicrobial activity of heterophils (mellata et al. 2003, qureshi 2003). there was no significant difference in phagocytic activity of heterophils, indicating their high phagocytic ability against different phylogenetic groups of apec likely due to the broad array of phagocytic receptors. avian heterophils have been shown to express various combinations of pattern recognition receptors (prr), including toll-like receptors (tlrs), scavenger receptors, dectin-1, and mannose receptors that allow them to recognize and respond to foreign microbial invaders (genovese et  al. 2013). other speculation could be the lack of difference in strategies used by tested apec isolates to avoid phagocytic engulfment. in this regard the role for k1 capsule, o78 antigen, p fimbriae, and the groups (jaureguy et  al. 2008, moulin-schouleur et  al. 2007). in the current study, phylogenetic analysis revealed that apec isolates segregated mainly in phylogenetic groups b1 (41/92; 44.57%) and a (33/92; 35.87%), indicating that intestinal strains are an important cause of colibacillosis in the west azerbaijan, iran. an high prevalence of phylogenetic groups a and b1 strains has also been reported elsewhere in iran from colibacillosis cases (alizade et  al. 2017, ghanbarpour et  al. 2011) as well as among verotoxigenic e.  coli (vtec) isolates from the faeces of healthy sheep and broiler chicken in northwestern iran (saei and zavarshani 2018). resistance to ciprofloxacin, norfloxacin, co-trimoxazole, nalidixic acid and cefoperazone/ sulbactam is especially prevalent among group b1 isolates and group a. antibiotic selection pressure may explain why most of the drug resistant strains of e.  coli belong to the phylogenetic groups a and b1 (obeng et  al. 2012). similar results have also been reported by kazemnia and colleagues (kazemnia et  al. 2014). other speculation could be the derivation of pathogenic e.  coli strains from commensal strains by the acquisition of virulence factors (duriez et al. 2001). in a study carried out on distribution of virulence genes among e. coli isolates from bovine mastitis in iran, isolates belonging to the phylogenetic group b1 exhibited higher prevalence of various virulence genes in comparison to groups a and d (ghanbarpour and oswald 2010). following the identification of pathogenic agents by heterophils, immune responses are initiated and eventually the pathogen is eliminated. in this regard, it has been shown that heterophils play a significant role in early host defense against bacterial agents such as e. coli in poultry (ariaans et al. 2008, gomis et  al. 2003). in the present study, we found a significant difference in functional activities of heterophils against different phylogenetic groups of apec from broiler chicken with colibacillosis. this could be attributed to differences in genetic backgrounds, virulence factors as well as antigenicity among phylogroups of apec. in a study carried out by mellata and colleagues, phagocytic activities of chicken heterophils were evaluated table iii. comparison of optical density of respiratory burst, microbial killing and phagocytic activities of heterophils (mean ± sd) between two groups. group respiratory burst (optical density) microbial killing (%) phagocytosis (%) a 0.41 ± 0.03 41.10 ± 6.11 58.37 ± 13.80 b1 0.52 ± 0.02a 29.40 ± 5.09a 78.06 ± 25.08 b2 0.40 ± 0.05 43.05 ± 5.84 66.36 ± 19.21 d 0.43 ± 0.03 39.26 ± 4.96 85.54 ± 27.13 asignificant differences (p < 0.05). 309 ahmadi et al. in vitro study of effects of apec on heterophils functional activities veterinaria italiana 2021, 57 (4), 305-310. doi: 10.12834/vetit.2111.12262.1 influenced by bacterial genetic, but also affected by the host genetic. therefore, the identification of the dominant phylogenetic groups of e. coli in a region is crucial in order to develop several strategies such as vaccination against colibacillosis. it is hoped that our results help us and other researchers to choose appropriate ways to prevent and treat colibacillosis by identifying the sensitivity of heterophils to different groups of e. coli. grant support this work was supported by the research fund of urmia university, urmia, iran. 0-min region in initial avoidance of phagocytosis has been demonstrated (mellata et al. 2003). further studies on distribution of various virulence factors among different phylogenetic groups of apec and their interaction with heterophils will provide important insight into some of the mechanisms that they employ to avoid uptake and survive elimination by avian heterophils and cause clinical disease. this study, for the first time, indicated that there were significant differences in microbial killing and respiratory burst activities of heterophils against different phylogenetic groups of e. coli without any significant difference in phagocytosis. however, it should be noted that immune responses are not only alizade h., ghanbarpour r., jajarami m. & askari a. 2017. phylogenetic typing and molecular detection of virulence factors of avian pathogenic escherichia coli 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lehoux b. & fairbrother j.m. 2003. role of avian pathogenic escherichia coli virulence factors in bacterial 111 veterinaria italiana 2021, 57 (2), 111-118. doi: 10.12834/vetit.1998.10744.4 accepted: 11.03.2020 | available on line: 31.12.2021 1department of microbiology and parasitology, faculty of veterinary medicine, university of tripoli, libya. 2department of internal medicine, faculty of veterinary medicine, university of tripoli, libya. 3université de tunis el manar, institut de la recherche vétérinaire de tunisie, 20 rue jebel lakhdhar, bab saadoun, tunis 1006, tunisie. *corresponding author at: department of microbiology and parasitology, faculty of veterinary medicine, university of tripoli, libya. e-mail: a.mo@live.com. hiam r. elnageh1, murad a. hiblu2, mohamed s. abbassi3, yousef m. abouzeed1 and mohamed o. ahmed1* keywords antimicrobial resistance, libya, pets, salmonella, s. kentucky. summary the prevalence of salmonella in dogs and cats was investigated and further characterized with serotyping, antimicrobial susceptibility and risk factor analysis. in total, 151 faecal samples from 103 and 48 healthy and nonhealthy (diarrheic) cat and dogs, respectively were examined. salmonellae were confirmed by laboratory and biomedical characteristics including serotyping and antimicrobial susceptibility tests. risk factors typically associated with salmonellae shedding were identified using fisher’s exact tests. salmonella was detected in 18% (n = 27/151) of pets. most of the positive samples 85% (n = 23/27) were from healthy cats, 7.4% (n = 2/27) from healthy dogs and 7.4% (n = 2/27) from a diarrhoeic cat and diarrhoeic dog. in total, 25 salmonellae (93% of strains) were serotyped as s. thompson mostly originated form healthy cats (n = 23/25). all isolates were resistant to tetracycline and trimethoprim-sulfamethoxazole and expressed an overall intermediate susceptibility patterns to ciprofloxacin. also, multidrug resistant s. kentucky and s. minnesota were identified from a diarrhoeic and a healthy dog, respectively. this is the first isolation report of salmonella from cats and dogs in libya. it indeed represents a public health concern which requires further monitoring. prevalence and antimicrobial resistance of salmonella serotypes isolated from cats and dogs in tripoli, libya et  al. 2016, phu huong lan et  al. 2016). unlike typhoidal salmonellae, which can be carried by humans, non-typhoidal salmonella (nts) are frequently associated with animal carriers. these can be transmitted to humans causing gastroenteritis illnesses and invasive (ints) bacteraemia (uche et al. 2017, lokken et al. 2016, afema et al. 2016). nts can be transferred to humans by various routes such as the consumption of contaminated food products (i.e. eggs, poultry, undercooked meat), or through contact with animals or environment sources (i.e. infecting sewage waste and contaminating vegetables) (nair et  al. 2015, uche et  al. 2017). pet animals are usually fed with discarded raw animal parts and treats which can be frequently contaminated with salmonellae (leonard et al. 2011, cdc 2006, cdc 2008, marks et al. 2011, efsa 2018). non-typhoidal salmonella are among the leading introduction salmonella is one of the most prevalent zoonotic foodborne pathogens colonizing different animal species capable of spreading rapidly and causing significant morbidity and mortality (who 2016, efsa 2018, majowicz et al. 2010). the incidences of human salmonellosis have been reported worldwide; however, a substantial proportion of salmonella infections are either not recognized or sporadically classified as atypical salmonella (dotto et  al. 2017, fisher et  al. 2009, graziani et  al. 2015, who 2018). to date, over 2,500 salmonella have been identified with a variety of serotypes affecting both humans and animals (fernández et  al. 2018, gossner et  al. 2016, who 2018, afema et al. 2016). salmonella enterica serovars are responsible for typhoidal and non-typhoidal infections (lokken 112 veterinaria italiana 2021, 57 (2), 111-118. doi: 10.12834/vetit.1998.10744.4 salmonella serotypes in pet animals from libya elnageh et al. showing signs of any illnesses or being under any medication including antimicrobial drugs at least three months prior to sampling. faecal samples and data collection a fresh faecal sample was taken from the rectum of each animal using a small cotton-tipped swap, and rolling the swab inside the rectum. the sample was then placed in a stuart transport medium and kept in cool place. samples were transported to the laboratory of the department of microbiology, faculty of veterinary medicine, university of tripoli, within four hours. after the collection process, data were obtained on each animal using a simple constructed questionnaire to examine potential risk factors that were frequently associated with salmonella shedding. the chosen variables and recorded data included information on age, sex, purpose of ownership (i.e. caring, security and breeding), type and source of food and diet, history of diarrhoea, and history of antibiotic therapy, particularly at the time of sampling. each faecal sample was recorded as the unit of analysis. identification and serotyping of salmonellae faecal samples were initially mixed in a 10 ml solution of brain heart infusion broth containing 5% glycerol and homogenized using a vortex mixer. a volume of 2  ml from the faecal mixture was transferred into 10  ml of sterile buffered peptone water (bpw) broth and incubated for 48 h at 37  °c. a  loopful from each bpw broth was spread onto xylose lysine decarboxylase (xld) agar and incubated at 37  °c for 24 h. plates were then checked for presumptive colonies of salmonella (i.e. red colonies with black centre) and a single typical colony from each plate was transferred onto a nutrient agar and incubated at 37  °c for 24 h. suspected isolates were initially subjected to gram stain examination and then further confirmed by the api 20e system (bio-mérieux). afterwards, confirmed strains were further serotyped according to the kauffmann-white scheme (bio-rad, marnes-la-coquette, france). antibiotic susceptibility tests the confirmed salmonella serotypes were subjected to antimicrobial susceptibility test using the disc diffusion method following clinical and laboratory standards institute (clsi) guidelines (clsi 2015). isolates were tested against eleven antibiotic classes including ampicillin (10 μg), amoxicillin-clavulanate (20/10  μg), azithromycin (15  μg), gentamicin (10  μg), tetracycline (30  μg), ciprofloxacin (5  μg), trimethoprim-sulfamethoxazole (1.25/23.75  μg), causes of food-borne illnesses in humans particularly in africa affecting mainly children aged under five year old (uche et  al. 2017, nair et  al. 2015, le hello et  al. 2013a, fernández et  al. 2018). nts strains, expressing multidrug resistances, including to or against quinolones and beta-lactamase inhibitors, have been increasingly reported worldwide. this is mainly attributed to inappropriate use of antibiotics in food animals (bangera et  al. 2019). in africa, various salmonellae have been reported from various animal species. among these, salmonella typhimurium is the most commonly identified serovars (al-rifai et  al. 2019, thomas et  al. 2019). in north africa, salmonella is a common foodborne pathogen. serovars enteritidis and typhimurium have been increasingly reported (al-rifai et al. 2019). in libya, salmonella expressing resistance to critically important antibiotics such as fluoroquinolones has been reported in 18% of hospitalized patients mainly children (altayyar et  al. 2016, ghenghesh et al. 2013); the source of these outbreaks or the role played by animals has never been investigated. recent global reports from north africa have documented the role of pet animals as a potential reservoir of drug-resistant salmonellae (e.g. esbl-producers and quinolone resistance isolates) (stull et al. 2015, damborg et al. 2015, srisanga et al. 2017, chipangura et  al. 2017, ahmed et  al. 2017, ezenduka 2019, elnageh et  al. 2017). nevertheless, data or studies estimating pet salmonellosis and the associated risk factors are scarse (giacometti et  al. 2017). no information is available about the occurrence and characteristics of salmonella in animals and their zoonotic role in the spread of amr in libya. in the current study, the prevalence and the antimicrobial susceptibility of salmonella serotypes isolated from faecal samples from cats and dogs collected between march and july 2018 were investigated. materials and methods criteria of selection and sampling this study obtained the approval from the department of microbiology and parasitology, faculty of veterinary medicine, university of tripoli, libya. prior to including pets in this study, owners were informed about its purpose and animals were included on the basis of the availability and acceptance of their owners. samples from nonhealthy pets were collected on admission at three local veterinary clinics. non-healthy pets were involved on the basis of typical gastrointestinal (gi) symptoms (e.g. diarrhoea) and referred to as the gi group throughout the study. healthy pets were included from their households and were not 113veterinaria italiana 2021, 57 (2), 111-118. doi: 10.12834/vetit.1998.10744.4 elnageh et al. salmonella serotypes in pet animals from libya serotyping identified 25 s.  thompson. most of them were from healthy cats (n  =  23/25), one from a diarrhoeic cat and one from healthy dog. the remaining two salmonellae obtained from a healthy and one diarrhoeic dog were serotyped as s.  minnesota and s.  kentucky, respectively (table ii). most of the s.  thompson serotypes expressed intermediate susceptibility to ciprofloxacin and resistance to tetracycline and trimethoprim-sulfamethoxazole. the s.  minnesota serotype showed resistance only to ciprofloxacin and tetracycline whereas the s.  kentucky serotype expressed a multidrug resistant phenotype, including to ciprofloxacin. the mics for the s.  kentucky serotype revealed further resistance to ampicillin, fluoroquinolones (ciprofloxacin and levofloxacin), tetracyclines (doxycycline minocycline, and tigecycline), and aminoglycosides (gentamicin and tobramycin) but susceptible to azithromycin, cephalosporins and carbapenems (table iii). discussion salmonella is a colonizing bacterium of cats and dogs. most of these animals may become asymptomatic carriers but may also show clinical signs associated with opportunistic infections and host immunosuppression (giacometti et  al. 2017, marks et al. 2003, finley et al. 2007). dogs may shed salmonella at a prevalence range between 1% to 44% depending on location, population and the applied laboratory methodology (greene et  al. 1998, finley et  al. 2007, reimschuessel et  al. 2017). risk factors including raw food diet, diarrhoea, home-made diets and antibiotics might influence the shedding of salmonella either in healthy or diarrhoeic dogs (hackett and lappin 2003, marks et al. 2011, westermarck 2016, reimschuessel 2017, leonard et  al. 2011, stavisky et  al. 2011). in africa, recent reports have documented various salmonella serovars in apparently healthy dogs with previous diarrhoea at a prevalence of 12% expressing a high rate of multi drug resistance (mdr) (kiflu et al. 2017). in cats, the prevalence of salmonella is less reported chloramphenicol (30  μg), cefotaxime (30  μg) and ceftazidime (30  μg). plates were incubated at 37  °c under aerobic conditions for 24 hours. resistance was indicted by growth reaching the antibiotic disc within the acceptable range based on clsi guidelines. isolates that expressed resistance to ampicillin based on disc diffusion were further tested against imipenem (10 μg) disks for the initial assessment of carbapanemase producers. isolates expressing multidrug resistance phenotypes (≥  3 antibiotic classes) including ciprofloxacin, ampicillin and imipenem were further subjected to antimicrobial susceptibility dilution method for the determination of the minimum inhibitory concentration (mic) range using designated susceptibility testing plates (thermo-sensititre). the mics was obtained by implementing the microdilution sentitretm gnx2f plate (trek diagnostic systems, thermofisher/gnx2f) according to the manufacturing instructions and interpreted using the 2019 criteria of the european committee on antimicrobial susceptibility testing (eucast). risk factors analysis risk factors analysis was carried out on the collected data information to examine potential factors that are significantly associated with salmonella shedding using the fisher’s exact tests at p ≤ 0.05. results one hundred and fifty one pets, 103 cats (62 healthy and 41 nonhealthy) and 48 dogs (37 healthy and 11  nonhealthy), were enrolled in this study. salmonella was detected in 18% (n = 27/151) of the pet faecal samples. of these, 23% (n = 24/103) were from cat and 6% (n  =  3/48) from dog. healthy cats were the main reservoir of salmonellae representing 22% (n = 23/103) and 85% (n = 23/27) of the cat and the total positive samples, respectively (table i). of the 103 cats, 40% (n = 41) had diarrhoea. of these, only 2.4% were positive for salmonella compared to 37% salmonella-positive non-diarrhoeic cats (p  >  0.05). in addition of the 9 cats that had been fed a raw diet at the time of sampling, 4 (44%) were found positive to salmonella while only 21% (n  =  20) of cats which did not have raw diet food were positive to salmonella. among the cat group, 40% (41/103) had been under antibiotic therapy at the time of sampling. of these, only 2% were salmonella positive comparing to 37% salmonella positive nonantibiotics-intake (abs-) samples. however, these differences as well as those found when analysing the same variables in dogs were not significant (p > 0.05). table i. serotypes of salmonella species from faecal samples of libyan pets. animal species proportion of carriage within each group no. of identified salmonella serotypes within each group cats (n = 103) 23% (n = 24) †s. thompson (n = 23) ‡s. thompson (n = 1) dogs (n = 48) 6% (n = 3) †s. thompson (n = 1) †s. minnesota (n = 1) ‡s. kentucky (n = 1) n = number; † healthy (control) origin; ‡ nonheathy (diarrheic) origin. 114 veterinaria italiana 2021, 57 (2), 111-118. doi: 10.12834/vetit.1998.10744.4 salmonella serotypes in pet animals from libya elnageh et al. population of cats in tripoli. this issue together with the possible source of infection requires further investigations. in the current study, 25 salmonella isolates were characterized as s.  thompson serotype. these isolates expressed intermediate susceptibility to ciprofloxacin and were mostly resistant to tetracycline and trimethoprim-sulfamethoxazole (table ii). however, these serotypes were susceptible to azithromycin which is a recommended treatment antimicrobial class against fluoroquinolone resistant salmonellae (crump et  al. 2015). generally, limited information is available on the epidemiology of s.  thompson worldwide. however, outbreak in humans directly linked to the handling of contaminated dog food and treats have been described (cdc 2006, cdc 2008). in the last decade, this serotype was listed within the most than dogs and the available salmonellosis feline reports refer mainly to serious clinical cases (stiver et al. 2003, reimschuessel 2017). in diarrheic cats, the prevalence of salmonella can range from 0 to 8.6% (marks et  al. 2011, giacometti et  al. 2017) whereas healthy house cats, because of housing hygiene, can harbour salmonella in their faeces at a low prevalence (<  1%) (van immerseel et  al. 2004). the prevalence in non-diarrhoeic cats was also reported and ranged between 0 to 14% (giacometti et  al. 2017). in the current study, salmonella was identified in 23% of the cat samples, a percentage much higher than that found in dogs. most of these cats were healthy. also, no significant differences were found in relation to the studied selected variables for cats and dogs which contradicts other published reports worldwide. this might be attributed to sample size of the current study but also could indicate the high colonization status of salmonella among a healthy table ii. antimicrobial resistance features of the collection of salmonella strains isolated from faecal samples of libyan pets. animal serotype health status antimicrobial susceptibility profiling susceptible intermediate resistance 1 cat thompson diarrhoea amp, amc, gmn, azm, chl cip tet, stx 2 cat thompson healthy amp, amc, gmn, azm, chl cip tet, stx 3 cat thompson healthy amp, amc, stx, azm, chl cip tet, gmn 4 cat thompson healthy amp, amc, stx, gmn, azm, chl cip tet 5 cat thompson healthy amp, amc, gmn, chl cip tet, stx 6 cat thompson healthy amp, amc, stx, gmn, azm, chl cip tet 7 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 8 cat thompson healthy amp, amc, gmn, azm, chl tet, cip stx 9 cat thompson healthy amp, amc, gmn, azm, chl cip tet, stx 10 cat thompson healthy amp, amc, tet, cip, stx, gmn, azm, chl 11 cat thompson healthy amp, amc, stx, gmn, azm, chl cip tet 12 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 13 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 14 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 15 cat thompson healthy amp, amc, tet, cip, stx, gmn, azm, chl 16 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 17 cat thompson healthy amp, amc, gmn, azm, chl tet, cip stx 18 cat thompson healthy amp, amc, cip, stx, gmn, azm, chl tet 19 cat thompson healthy amp, amc, tet, cip, stx, gmn, azm, chl 20 cat thompson healthy amp, amc, stx, gmn, azm, chl cip tet 21 cat thompson healthy amp, amc, stx, gmn, azm, chl tet, cip 22 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 23 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 24 cat thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 25 dog thompson healthy amp, amc, tet, stx, gmn, azm, chl cip 26 dog minnesota healthy amp, amc, stx, gmn, azm, chl tet, cip 27 dog kentucky diarrhoea amc, azm, ctx, tex, caz imp amp, tet, cip, stx, gmn, chl amp = ampicillin; amc = amoxicillin-clavulanate; tet = tetracycline; cip = ciprofloxacin; stx = trimethoprim-sulfamethoxazole; gmn = gentamicin; azm = azithromycin; chl = chloramphenicol; caz = ceftazidime; ctx = cefotaxime; imp = imipenem,. 115veterinaria italiana 2021, 57 (2), 111-118. doi: 10.12834/vetit.1998.10744.4 elnageh et al. salmonella serotypes in pet animals from libya frequently isolated salmonellae responsible for human infections in the united states (cdc 2012). in addition, mdr salmonella serotypes including s.  kentucky, s.  thomson, and s.  enteritidis have been increasingly reported posing significant public health challenges (shah et al. 2017). a recent genomic study of s.  thompson strains isolated from an outbreak of contaminated food products revealed distinct genomic characteristics (parker et al. 2015). pet owners are often unaware and/or underestimate the health risks that their pets may present (chipangura et  al. 2017, damborg et  al. 2015, damborg et  al. 2016, stull et  al. 2015, halsby et  al. 2014). household pets are usually kept for specific purposes and the level of interaction with owners, feeding, caring and health services varies between regions and countries (selmi et  al. 2011). in the low-income regions including libya, cats and dogs are frequently fed on non-commercial food particularly remaining raw animal parts of poultry meat (personal communication). feeding on raw food diets and salmonella-contaminated raw food diets are widely reported to be highly associated with a high prevalence of salmonella shedding in cats and dogs; i.e. up to 23 times greater shedding comparing to commercial diets (giacometti et  al. 2017, finley et al. 2007). also, cats compared to dogs can roam more freely between indoor and outdoor environments, increasing the risk of contacting contaminated foods with salmonella from environmental sources such as rodents and birds (giacometti et  al. 2017). poultry frequently act as carrier of various salmonella serotypes and represent the most important source of human salmonellosis worldwide including africa (thomas et  al. 2019, mshelbwala et al. 2017). recent studies investigating meat and intestinal samples of poultry have reported different nts serovars mainly belonging to the s. kentucky and s. thompson serotypes showing high resistance rates to important drugs including ciprofloxacin (bangera et  al. 2019, carli et  al. 2001). these reports have attributed such isolations and documentations to the introduction of imported feed or new flocks from other countries such as the united states and turkey (carli et al. 2001). this may explain the results of the current study particularly the high prevalence of the s. thompson in cats and also highlights the need to investigate other sources of infection. in the current study, the isolated s. kentucky serotype originated from a diarrheic dog. it expressed a multidrug resistance phenotype (table ii). the determination of mics revealed further resistance to ampicillin, fluoroquinolones, tetracyclines, and aminoglycosides but susceptibility to azithromycin, cephalosporins, and carbapenems (table iii). in contrast, s. kentucky has been recently reported in africa, isolated from apparently healthy dogs representing 9.5% of the collected salmonella isolates but susceptible to ciprofloxacin (kiflu et  al. 2017). this serotype was isolated from human, environmental sources and animals from different african countries expressing very high resistance to antimicrobial agents (afema et  al. 2016, le hello et  al. 2013b). global reports have associated the emergence of multidrug resistance salmonellae to the intensive animal husbandry particularly in poultry (oie 2008) as well as travel history in europe, africa and the middle east (le hello et  al. 2013b, mulvey et al. 2013, rickert-hartman and folster 2014, westrell et al. 2014). for instance, oxa-48-producing table iii. the determination of minimum inhibitory concentration range of s. kentucky using antimicrobial susceptibility dilution method. antibiotic agent dilution (mics) range (mg/l) results (mg/l) interpretation ampicillin 8-16 8 r azithromycin 2-16 2 s ceftazidime/ clavulanic acid 0.12/4-128/4 0.12/4 s cefotaxime/ clavulanic acid 0.12/4-64/4 0.25/4 s ticarcillin/ clavulanic acid 16/2-128/2 32/2 s piperacillin/tazobactam 4/4-64/4 4/4 s cefazolin 8-16 8 na cefoxitin 4-64 8 na cephalothin 8-16 8 na cefotaxime 0.25-64 0.5 s ceftriaxone 1-128 1 s ceftazidime 0.25-128 0.25 s cefepime 1-16 1 s cefpodoxime 0.25-32 0.25 s ciprofloxacin 0.25-2 r levofloxacin 1-8 8 r doxycycline 2-16 16 r minocycline 2-16 8 r tigecycline 0.25-8 0.5 r ertapenem 0.25-4 0.25 s imipenem 0.5-16 0.5 s meropenem 1-8 1 s gentamicin 1-16 4 r tobramycin 1-8 4 r amikacin 4-32 4 s trimethoprim/ sulfamethoxazole 0.5/9.5-4/76 s colistin 0.25-4 1 s polymyxin b 0.25-4 1 s na = not available; = no growth; r = resistance; s = susceptible. 116 veterinaria italiana 2021, 57 (2), 111-118. doi: 10.12834/vetit.1998.10744.4 salmonella serotypes in pet animals from libya elnageh et al. afema j.a., byarugaba d.k., shah d.h., atukwase e., nambi m. & sischo w.m. 2016. potential sources and transmission of salmonella and antimicrobial resistance in kampala, uganda. plos one, 11 (3), e0152130. doi: 10.1371/journal.pone.0152130. ahmed m.o., almshawt n.f. & elnageh h.r. 2017. diarrheagenic escherichia coli o157 from libya: recent perspectives and challenges. j public health afr, 8 (1), 685. al-gallas n., abbassi m.s., gharbi b., manai m., ben fayala m.n., bichihi r., al-gallas a. & ben aissa r. 2013. occurrence of plasmid-mediated quinolone resistance determinants and rmtb gene in salmonella enterica serovar enteritidis and 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sjölund-karlsson m., folster j.p., pecic g., joyce k., medalla f., rickert r. & whichard j.m. 2009. emergence of plasmid-mediated quinolone resistance among non-typhi salmonella enterica isolates from humans 339 1umr virologie, inra, ecole nationale vétérinaire d'alfort, laboratoire de santé animale d'alfort, anses, université paris-est, 94700 maisons-alfort, france. 2ecole nationale vétérinaire de maisons alfort, france. *corresponding author at: anses laboratoire santé animale umr 1161, 14 rue pierre et marie curie, aci-bac-002, 94700 maisons-alfort, france. tel.: + 01.49.77.27.07 / 13.39, e-mail: corinne.sailleau@anses.fr. parole chiave virus della malattia emorragica epizootica, infezione sperimentale, sieri di riferimento, diagnosi sierologica. riassunto scopo di questo studio è stato produrre sieri di riferimento contro i sette sierotipi del virus della malattia emorragica epizootica (ehdv‑1, ehdv‑2, ehdv‑4, ehdv‑5, ehdv‑6, ehdv‑7 e ehdv‑8). in una struttura bsl‑3 sono stati inoculati sette vitelli prim' holstein con i ceppi di ehdv selezionati (1 sierotipo per vitello). campioni di sangue (edta e sangue intero) sono stati prelevati periodicamente il giorno dell'inoculo (d0) fino alla fine dell'esperimento (d31). i sieri sono stati testati con due kit elisa (c‑elisa). il genoma virale è stato rilevato da campioni di sangue edta mediante una in-house real-time rt-pcr. per calibrare i sieri di riferimento, i sieri prelevati al d31 sono stati testati e caratterizzati sia con il test di sieroneutralizzazione (snt) che con il test di virusneutralizzazione (vnt). due giorni dopo l'inoculo 5 animali sono risultati positivi alla rt‑pcr specifica per ehdv. a sette giorni tutti gli animali erano positivi. tutti gli animali hanno sieroconvertito a seconda del sierotipo tra d10 e d23, in base al sierotipo ehdv, tra d10 e d23. i saggi di siero‑ e virusneutralizzazione hanno permesso di determinare i titoli anticorpali neutralizzanti di ciascun siero e le potenziali reazioni crociate tra sierotipi. i due kit c‑elisa utilizzati in questo studio hanno mostrato risultati simili. gli antisieri così prodotti e calibrati sono disponibili e possono essere utilizzati per identificare ceppi di ehdv isolati in tessuto colture o come controlli positivi nel test di sieroneutralizzazione. infezione sperimentale di vitelli con sette sierotipi del virus della malattia emorragica epizootica: produzione e caratterizzazione di sieri di riferimento keywords epizootic hemorrhagic disease virus, experimental infection, reference sera, serological diagnosis. summary the aim of this study was to produce reference sera against the seven serotypes of epizootic hemorrhagic disease virus (ehdv‑1, ehdv‑2, ehdv‑4, ehdv‑5, ehdv‑6, ehdv‑7, and ehdv‑8). in a high containment unit, seven prim ‘holstein calves were inoculated at day 0 (d0) with the selected strains (1 ehdv serotype per calf ). blood samples (edta and whole blood) were periodically taken from d0 until the end of the experiment (d31). sera were tested with two commercially available ehdv competitive elisas (c‑elisa). viral genome was detected from edta blood samples using in‑house real‑time rt‑pcr. sera taken on d31 post infection (pi) were tested and characterized by serum neutralization test (snt) and virus neutralization test (vnt) (for calibration of reference sera). viral rna was first detected at d2 pi in five calves. all infected animals were rt‑pcr positive at d7 pi. seroconversion was observed between d10 and d23 pi depending on the ehdv serotype. snt and vnt have allowed to determine the neutralizing antibody titers of each serum and the potential cross‑reactions between serotypes. the two c‑elisa used in this study showed similar results. the calibrated sera are now available for the serological identification of an ehdv isolated on tissue culture or to be used as positive control in seroneutralization assay. corinne sailleau1*, emmanuel breard1, cyril viarouge1, guillaume belbis2, thomas lilin2, damien vitour1 and stephan zientara1 experimental infection of calves with seven serotypes of epizootic hemorrhagic disease virus: production and characterization of reference sera veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 accepted: 16.12.2019 | available on line: 31.12.2019 340 production and characterization of ehdv’s reference sera sailleau et al. veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 de recherche biomédicale, maisons‑alfort, france ‑ crbm) throughout the experiment. calves were confirmed negative for ehdv using a commercial kit for ehdv antibodies detection (thermofisher, lissieu, france) and a pan ehdv rtrt‑pcr assay previously developed in our lab (viarouge et al. 2015). the serological and virological status regarding btv was also checked. the animals were btv‑free. at day 0 (d0), all calves were inoculated intramuscularly at multiple sites in the shoulder region with 5‑6 ml of inoculums (ehdv culture cell supernatants). edta‑treated whole‑blood and serum samples were collected at regular intervals until the end of the experiments up to d31 post‑infection (pi). hundreds of ml of blood were collected from each animal before euthanasia to produce a reference serum for each serotype. the ehdv‑7 infected calf was euthanized at 17 days because it showed severe clinical signs. introduction epizootic hemorrhagic disease (ehdv) is an arthropod‑borne disease of wild and domestic ruminants caused by viruses (ehdv) belonging to the species ehdv within the genus orbivirus of the reoviridae family (mertens et  al. 2005). seven serotypes are officially recognized (ehdv‑1, ehdv‑2, ehdv‑4, ehdv‑5, ehdv‑6, ehdv‑7, and ehdv‑8) and more recently at least 2 putative new serotypes have been reported (maan et al. 2010, shirafuji et al. 2017, anthony et al. 2009, savini et al. 2011). as the clinical pattern is very close to bluetongue infection, laboratory investigations are essential for the diagnosis of the ehdv infection. in most infected areas, bluetongue and ehd viruses co‑circulate (dommergues et  al. 2019, merrill et  al. 2019, toye et  al. 2013) and mixed infections are reported (sailleau et al. 2012, viarouge et al. 2014). this report highlights the need for efficient, specific and sensitive diagnostic tools to identify the causal agent. the molecular tools for ehdv nucleic acid identification are widely used for the diagnosis. in the last ten years, real‑time rt‑pcr assays (rtrt‑pcr) have been developed for the detection of all seven serotypes (clavijo et  al. 2010, maan et  al. 2017, viarouge et al. 2015, wilson et al. 2009) and the typing of the virus (maan et  al. 2017, viarouge et  al. 2015). serological tools including elisa have been described for the ehdv antibody detection (afshar et  al. 1992, thevasagayam et  al. 1995) and two commercial kits were launched in 2010 and 2017. these serological tools allow a serogroup ehdv antibody detection without identification of ehdv serotype antibodies. these are competitive or blocking elisa using an hrp‑labelled monoclonal against the viral protein  7  (vp7). the serum neutralization test (snt) is usually performed to identify exposure to specific ehdv serotypes (oie 2014). even if molecular diagnosis is widely used, virus isolation must be performed for a reliable diagnosis and, particularly, in the case of an emergence. moreover, this viral isolation is needed to facilitate further pathogen characterization and pathogenesis studies. following the viral isolation, reference sera are used in virus neutralization test (vnt) for the serotype determination (oie 2014, pearson et  al. 1992). the aim of this study was to produce and characterize reference sera against ehdv serotypes. materials and methods experimental infection of calves seven prim'holstein calves (2 to 3 months‑old) were housed in a high containment unit (centre table i. viral strains used to calves inoculation and to vnt. strain origin titre tcid 50 /ml used for ehdv-1 usa1955/01 ref strain 2.5 10e6 inoculation of calf 7706/vnt ehdv-2 can1962/01 ref strain 1.10e7 inoculation of calf 7713/vnt ehdv-4 nig1968/01 ref strain 5.6 10e5 inoculation of calf 7716/vnt ehdv-5 aus1979/06 ref strain 3.2 0e5 inoculation of calf 7724/vnt ehdv-6 aus1981/07 ref strain 2.3 10e6 inoculation of calf 7730/vnt ehdv-7 isr2006/01 israël 2006 5.6 10e5 inoculation of calf 7739/vnt ehdv-8 aus1982/06 ref strain 1.10e4 inoculation of calf 7748/vnt ehdv-1 guy french guyana (2011) 3.2 10e4 vnt ehdv-1 reu réunion island (2011) 1.8 10e4 vnt ehdv-2 aus1979/05 australia (1979) 1.8 10e6 vnt ehdv-2 jap/01 japon 1.8 10e7 vnt ehdv-2 guy french guyana (2014) 1.8 10e4 vnt ehdv-2 guad guadeloupe island (2011) 3.2 10e6 vnt ehdv-6 mor morocco (2007) 3.2 10e6 vnt ehdv-6 reu réunion island (2009) 5.6 10e3 vnt ehdv-6 guy french guyana (2016) 5.6 10e3 vnt ehdv-7 aus1981/05 ref strain 2.5 10e4 vnt ehdv-7 reu réunion island (2014) 1.8 10e6 vnt 341 sailleau et al. production and characterization of ehdv’s reference sera veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 (s/n < 30%: positive; s/p > 40%: negative and s/n between 30 and 40%: doubtful). serum neutralization test (snt) sera sampled at the last day of the study (d17 pi for ehdv‑7 infected calf and d31 pi for the others) were titrated for the presence of serotype specific ehdv antibodies by snt. each serum was tested against all ehdv serotypes. briefly, two‑fold dilutions of sera (in duplicate) were done in dmem glutamax medium (gibco, grand island, new york), starting at ½ (range of dilution: 2‑256). fifty microlitres of dmem, containing 100 tcid 50 of each ehdv serotype, were incubated in microtitre plates, with 50 µl of the diluted sera for 1 hour at 37 °c. then, 20,000 bsr cells were added in each well, with 100 µl of dmem supplemented with 10% fetal calf serum and 2% sodium pyruvate. microtitre plates were then incubated for 6‑7 days at 37 °c. the neutralizing titre of each serum was defined as the highest dilution allowing 50% neutralisation of the 100 tcid 50 . the titers thus defined has been used to determine the four neutralizing unities (nu) required for the vnt. the serum titre gives the value of the one neutralizing unit. for example, if the titer is 128 the dilution to do to obtain 4 nu will be 1/32. virus neutralization test (vnt) each reference serum was evaluated against its homologous ehdv serotype and against those where cross‑reactions were observed in snt. the reference sera were diluted in mem to obtain four nu. vnt was carried out in microtitre plates. briefly, four nu of reference sera (50 µl) were added to a 10‑fold dilution series of ehdv (50 µl). the serum‑virus mixtures were incubated for 1 h at 37  °c. as performed for snt, bsr cells (20,000 cells in 100 µl/well) were then added and the plates were incubated for 6 days at 37 °c (sailleau et al. 2000). a two‑way reduction of at least 2 logs is considered to classify a virus as belonging to the serotype of the antibody which neutralized it. results clinical observations the calves infected with ehdv serotypes 1, 2, 4, 5, 6 and 8 did not show apparent clinical signs until d31. only the ehdv‑7 infected calf was euthanized at 17 days post inoculation because it showed severe clinical signs (apathy, diarrhea, prostration, unable to stand, anorexia). ethic all experimental protocols were reviewed by a state ethics commission and have been approved by the competent authority (reference: 2017051515187349). all adequate measures were taken to minimize pain or discomfort. viruses the seven ehdv strains used for the inoculation of calves (table i) have been titrated and tested with the seven real‑time serotyping rt‑pcr assays (viarouge et al. 2015) in order to control their purity (absence of other serotypes). all strains were passaged on kc cells (culicoides sonorensis cell line) (wechsler et  al. 1989) before the inoculation of calves. as ehdv‑8 failed to grow on kc cells, a bsr passage was performed to produce the ehdv‑8 inoculum. real time pcr total rna was extracted from 100 µl of edta‑blood using the kingfisher 96 robot and the magvet universal isolation kit (thermofisher, cambridge, uk) according to manufacturer’s instructions. finally, the rnas were eluted with 80 µl of ultrapure water and 5 µl were used, after denaturation at 95  °c for 3  minutes, in pan‑ehdv rtrt‑pcr assay (viarouge et al. 2015). serological analyses elisa the detection of ehdv vp7 antibodies was performed using a competitive elisa (thermofisher, lissieu, france). two negative controls (cneg) and two positive controls (cpos) were analysed in duplicate on each plate. the results were expressed as inhibition percentage (%inh) = [optical density (od) of the cneg ‑ od sample/od cneg]*100. the assay was performed according to manufacturer’s instructions (%inh > 60%: positive; %inh < 55%: negative and %inh between 55 and 60%: doubtful). this kit is no longer available. the serum samples were also tested with another celisa (id screen® ehdv competition, idvet, grabels, france) following the manufacturer’s instructions. two negative controls (cneg) and two positive controls (cpos) were analysed in duplicate on each plate. the results were expressed as sample/negative control (s/n) = (optical density (od) of the sample / od cneg)*100. 342 production and characterization of ehdv’s reference sera sailleau et al. veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 a b c 20 25 30 35 40 45 d0 d2 d4 d7 d10 d14 d18 d23 d28 d31 c yc le t h re sh o ld (c t ) days post inoculation 7706 (ehdv-1) 7713 (ehdv-2) 7716 (ehdv-4) 7724 (ehdv-5) 7730 (ehdv-6) 7739 (ehdv-7) 7748 (ehdv-8) -20 0 20 40 60 80 100 d0 d2 d4 d7 d10 d14 d18 d23 d28 d31 p er ce n ta g e in h ib it io n ( p i) days post inoculation 7706 (ehdv1) 7713 (ehdv2) 7716 (ehdv4) 7724 (ehdv5) 7730 (ehdv6) 7739 (ehdv7) 7748 (ehdv8) > 60% pos > 55 < 60% = dtx 0 20 40 60 80 100 120 d0 d2 d4 d7 d10 d14 d18 d23 d28 d31 c o m p et ti o n p er ce n ta g e days post inoculation 7706 (ehdv1) 7713 (ehdv2) 7716 (ehdv4) 7724 (ehdv5) 7730 (ehdv6) 7739 (ehdv7) 7748 (ehdv8) < 30% pos > 30 < 40% = dtx figure 1. follow-up of the biological parameters in calves after experimental infection with different epizootic hemorragic disease virus (ehdv) serotypes. a. viral genome (real time rt-pcr assay); anti ehdv vp7 antibodies. b. lsivet ehdv blocking, thermofisher, france. c. id screen® ehdv competition idvet france. 343 sailleau et al. production and characterization of ehdv’s reference sera veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 elisa results there was a complete agreement between the results obtained with the two elisas used (figures 1b and 1c). animals inoculated with ehdv‑1, ehdv‑2, ehdv‑6, ehdv‑7 and ehdv‑8 seroconverted between d10 and d23 pi. the ehdv‑4 infected calf failed to seroconvert and low level of anti‑vp7 antibodies were detected between d28 and d31 pi in the animal infected with ehdv‑5. the reference sera from the calves inoculated with serotype 4 or 5 were found negative and doubtful, respectively (close to the positivity threshold) when tested with both elisa kits. the reference sera from ehdv‑1, ehdv‑2, ehdv‑6, ehdv‑7, and ehdv‑8 were elisa‑positive. follow‑up of the serological and virological parameters rtrt‑pcr before infection (at d0), all animals were ehdv rtrt‑pcr negative. viral rna was first detected at d2 pi in calves inoculated with ehdv‑1, ehdv‑2, ehdv‑4, ehdv‑6, and ehdv‑7. all animals were rtrt‑pcr positive at d7 pi. five out of 7 animals remained ehdv rtrt‑pcr positive until the end of the experiment. the ehdv‑4 infected calf became rtrt‑pcr negative from d14 pi. the ehdv‑7 infected calf remained positive until d17 pi (date of euthanasia) (figure 1a). table ii. serum neutralization test results obtained with epizootic hemorragic disease virus (ehdv) antisera. the titres are expressed as the reciprocal value of the endpoint serum dilution. shadded cells: titre of sera with their homologous serotype. in bold: titres of sera with heterologous serotypes (cross-reaction). in bracket: the dilution used for each reference serum to obtain 4 neutralizing unit. antisera virus 7706 (ehdv-1) 7713 (ehdv-2) 7716 (ehdv-4) 7724 (ehdv-5) 7730 (ehdv-6) 7739 (ehdv-7) 7748 (ehdv-8) ehdv1 128 (1/32) 8 < 4 < 4 < 4 4 < 4 ehdv2 8 64 (1/16) < 4 < 4 4 32 4 ehdv-4 < 4 < 4 128 (1/32) 8 < 4 < 4 < 4 ehdv-5 < 4 < 4 16 384 (1/96) < 4 < 4 < 4 ehdv-6 < 4 < 4 < 4 < 4 96 (1/24) 4 16 ehdv-7 < 4 8 < 4 < 4 < 4 128 (1/32) < 4 ehdv-8 < 4 < 4 < 4 < 4 < 4 < 4 32 (1/8) table iii. virus neutralization test results obtained with different epizootic hemorragic disease virus (ehdv) strains against homologous and heterologous antisera. values represent the log reduction in cytopathic effect (neutralization). a two-way reduction of at least 2 logs is considered to classify a virus as belonging to the serotype of the antibody which neutralized it. virus antisera ehdv-1 usa1955/01 ehdv-1 guy ehdv-1 reu 7706 (ehdv-1) 2.4 2 2 ehdv-2 can1962/01 ehdv-2 jap/01 ehdv-2 aus1979/05 ehdv-2 guy ehdv-2 guad ehdv-7 aus1981/057 ehdv-7 isr2006/01 ehdv-7 reu 7713 (ehdv-2 2 1.46 4.4 2 2.71 2.7 1.5 1.25 ehdv-4 nig1968/01 ehdv-5 aus1979/06 7716 (ehdv-4) 2.4 1.4 ehdv-5 aus1979/06 ehdv-4 nig1968/01 7724 (ehdv-5) 2.7 1 ehdv-6 aus1981/07 ehdv-6 reu ehdv-6 mor ehdv-8 aus1982/06 7730 (ehdv-6) 3.4 1.9 2 1.7 ehdv-7 aus1981/05 ehdv-7 reu ehdv-7 isr2006/01 ehdv-2 can1962/01 7739 (ehdv-7) > 2.7 3.5 2 1 ehdv-8 aus1982/06 ehdv-6 aus1981/07 ehdv-6 reu ehdv-6 mor 7748 (ehdv-8) > 3 2.39 1.51 2 344 production and characterization of ehdv’s reference sera sailleau et al. veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 could not be carried out, it is not possible to assert that the symptoms observed in the infected calf were partially or totally due to ehdv‑7 infection. similarly to what observed by other authors, also in this study most of the infected animals become ehdv pcr positive from d2 pi (batten et  al. 2011, breard et  al. 2013, eschbaumer et  al. 2012). except for ehdv‑4, all animals remained pcr positive until the end of the experiment. depending on the serotype inoculated, seroconversion (elisa) was observed between d10 (ehdv‑2) and d23 pi (ehdv‑8). the serum from ehdv‑5 inoculated calf gave doubtful results from d28 pi. surprisingly, the ehdv‑4 infected calf remained negative in elisa during the entire experimental period while the snt showed a strong titer (128) at d31 pi. this result could be due to the fact that the anti‑vp7 antibodies produced against ehdv‑4 have a low affinity for the epitope on which the competition with the anti‑vp7 monoclonal (used in both elisa kits) is performed. the same hypothesis can be formulated for the ehdv‑5 serum, which gave doubtful results with both elisas. altogether, the results obtained with the two commercial c‑elisa kits used in this trial were perfectly similar (figures 1b and 1c). more importantly, the four serotypes (1, 2, 6 and 7) mainly circulating in the world were well detected by both elisas. the neutralizing antibody titres obtained for the seven reference sera ranged from 32 to 384 (table ii). the close antigenic relationship between some serotypes has already been reported in previous studies (anthony et al. 2009, maan et al. 2010). both in snt and vnt, strong cross‑reactions were found between ehdv‑2 and ehdv‑7 and ehdv‑6 and ehdv‑8. however, these cross‑reactions were not observed for all isolates: ehdv‑2 reference serum neutralized ehdv‑7 aus1981/05 (log reduction in cpe 2.7) but not other ehdv‑7 strains (ehdv‑7 isr and ehdv‑7 reu) (log reduction in cpe < 2). anthony and colleagues (anthony et al. 2009) have suggested that ehdv‑2 and ehdv‑7, and ehdv‑6 and ehdv‑8 have diverged (or are still diverging) from two different common ancestors. some isolates which were weakly neutralized by their homologous antiserum (e.g. ehdv‑6 reu and ehdv‑2 jap/01) (table iii) probably have a lower antigenic relationship with the isolate used for the production of serum in calf. despite the cross‑reactions described, the use of the seven calibrated antisera in vnt made the serotype identification of an ehdv infected tissue culture possible. to date conventional or real‑time rt‑pcrs are commonly used for fast and reliable identification of ehdv (maan et  al. 2010, maan et  al. 2017, viarouge snt and vnt snt and vnt were performed only with the reference sera. the seven sera were titrated against all serotypes of ehdv in order to detect potential cross‑reactivity (table ii). the titres obtained in the snt performed with homologous serotypes ranged between 32 and 384. weak cross‑reactions (titre = 8) were observed between reference sera of ehdv‑1 and ehdv‑2, ehdv‑2 and ehdv‑1 and ehdv‑7, ehdv‑5 and ehdv‑4. relative strong cross reactions (titers  =  16  or  32) were obtained between sera of ehdv‑4 and ehdv‑5, ehdv‑7 and ehdv‑2, and ehdv‑8 and ehdv‑6. in order to confirm and complete the serological data generated by snt (titres/cross‑reaction), vnt was performed by using several ehdv strains (table iii). each reference serum neutralized all homologous strains except ehdv‑2 antiserum, which neutralized only 4 of 5 ehdv‑2 strains tested. ehdv‑2 jap was only partially neutralized (reduction of 1.46). similarly to snt results, cross‑reactions ehdv‑2 reference serum neutralized ehdv‑7 aus1981/05 but not the other ehdv‑7 strains, while ehdv‑8 reference serum cross‑ neutralized ehdv‑6 aus1981/07 and ehdv‑6 mor. discussion no case of ehdv infection has been so far reported in europe but the risk of introducion is very high. in the last 15 years, ehdv‑1, ehdv‑6 and ehdv‑7 have been detected in the south or east of the mediterranean basin (the maghreb, egypt, israel, turkey, and jordan) (ahmed et al. 2019, ben dhaou et al. 2016, golender et al. 2017, temizel et al. 2009). the availability of reliable and standardized reagents for laboratory diagnosis is crucial in the event of the emergence of this disease. the main purpose of this experimental infection was to produce reference sera to be used in serological assays, either to identify by vnt the ehdv isolated on tissue cultures (unknown serotype against known sera), or as positive controls in snt (unknown sera against known serotypes). of the seven animals infected with different ehdv serotypes, six remained healthy throughout the entire experimental period and no clinical signs of ehd were observed. the calf inoculated with the ehdv‑7 isr2006/01 strain was euthanized at d17 pi. the clinical signs observed (apathy, diarrhea, prostration, unable to stand and anorexia) were different from those described during the 2006 epizootic in israel (yadin et  al. 2008). in a similar experimental infection of holstein cattle using the same strain, no clinical signs were reported (eschbaumer et  al. 2012). as a post mortem examination of the experimentally infected animals 345 sailleau et al. production and characterization of ehdv’s reference sera veterinaria italiana 2019, 55 (4), 339‑346. doi: 10.12834/vetit.2104.11179.1 acknowledgments the authors would like to thank all the veterinary and technical staff of the animal experimental facilities of the crbm for their efficient help in animal handling and samples collection. et al. 2015). however, the genetic variations that may appear over time in the ehdv genome (in region where specific primers/probes for typing assays are designed) can cause the failure of the rt‑pcr. therefore, virus isolation in tissue culture and vnt should be kept as the gold standard for detecting and serotyping ehdv isolates. specific and reliable antisera are therefore essential for this serological test. afshar a., wright p.f., taylor l.a., shapiro j.l. & dulac g.c. 1992. development and evaluation of an enzyme‑linked immunosorbent assay for 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res vet sci, 94 (3), 769‑773. viarouge c., lancelot r., rives g., breard e., miller m., baudrimont x., doceul v., vitour d., zientara s. & sailleau c. 2014. identification of bluetongue virus and epizootic hemorrhagic disease virus serotypes in french guiana in 2011 and 2012. vet microbiol, 174, 78‑85. 143 veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 accepted: 17.03.2020 | available on line: 31.12.2021 1veterinary medicine, department of virology, firat university, turkey. 2department of virology, veterinary medicine, namik kemal university, tekirdag, turkey. *corresponding author at: veterinary medicine, department of virology, firat university, turkey. e-mail: habayli@firat.edu.tr. hasan abayli1 and hakan bulut2 keywords antibody response, bovine ephemeral fever, dna vaccine. summary bovine ephemeral fever (bef) is an arthropod-borne viral disease characterised by a short-term clinical expression that can lead to significant losses in high-yielding cattle and water buffaloes. in this study, we aimed to generate a recombinant plasmid expressing the glycoprotein (g) of the bef virus (befv) and to stimulate a humoral immune response to this protein in balb / c mice immunised with the recombinant plasmid. expression of the encoded protein was demonstrated by western blotting and immunoperoxidase tests. the suitable plasmids were intramuscularly administered to balb/c mice on days 0, 14 and 21. the antibody response in the immunised mice was measured by a plaque reduction neutralization test (prnt) and enzyme-linked immunosorbent assay (elisa). according to befv elisa, only two of the seven animals in these groups exceeded the cut-off value. a significant difference was observed in the mean od values at 450 nm absorbance in the pcdna4-g-immunised group when compared with those in the plasmid control group at 30 days (p < 0.05). according to prnt50 results, a 1:20 (p < 0.05) antibody response was obtained at 30 days in pcdna4-g (100 µg)-immunised mice, whereas this ratio was 1:80 (p < 0.001) in befv-immunised mice (1,000 pfu/0.5 ml). we conclude that the humoral immune response was stimulated in experimental mice immunised with the recombinant plasmid. however, disappointingly, the antibody response was markedly low in pcdna4-g-immunised mice. short-term humoral immune response of the pcdna4-g plasmid expressing the bovine ephemeral fever virus g gene in balb/c mice the only viral protein that stimulates the production of neutralising antibodies that protect cattle from bef (uren et al. 1994). bef is a rare viral disease that benefits from early treatment (e.g. non-steroidal anti-inflammatory administration and calcium supplements) (st george 1988). however, due to the rapid onset of clinical findings, it is likely that late intervention in all animals is possible in breeding sites with high cattle populations. to date, befv has caused endemic outbreaks in africa, the middle east, asia and australia. unusual serious outbreaks exceeding the 10%-20% mortality rate have recently been reported (abayli et al. 2017). preventive measures are therefore recommended to limit yeld losses, animal deaths and commercial restrictions (davis et al. 1984, st george et  al. 1986, st  george 1988, uren 1989, nandi and negi 1999, tonbak et  al. 2013). vaccination is the most important measure for protecting animals against befv. inactive, recombinant, attenuated introduction bovine ephemeral fever (bef) can lead to trade restrictions between countries and significant yield losses in high-yielding cattle and buffaloes. bef is also known as a 3-day sickness because the clinical symptoms persist for 3-4 days. the disease is transmitted among susceptible animals through culicoides species. the causative agent is a bovine ephemeral fever virus (befv), which has a negative-sense, single-stranded (ss) rna genome (st george et al. 1995, hertig et al. 1996, nandi and negi 1999). the befv genome, which is 14,900-nucleotide (nt) long, contains a total of 12 gene regions as well as leader and trailer sequences (walker 2009, walker and klement 2015, bulut and azkur 2016). similar to other rhabdoviruses, the befv virion encodes five structural proteins: matrix protein (m), glycoprotein (g), nucleoprotein (n), polymerase protein (l), and phosphoprotein (p) (walker 2009). glycoprotein is 144 veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 dna vaccines against befv abayli & bulut cells in six-well plates using lipofectamine 2,000 (thermo fisher, usa). fresh medium was supplied 24 hr after transfection, and the expression of glycoprotein was assessed by immuno-peroxidase tests and western blotting at 72 hr after transfection. at the same time, transiently transfected vero cells were treated with zeocin (thermo fisher, usa) (100  µg/ml, the lethal dose tested in vero cells) to obtain a stable cell line. immunoperoxidase test the cells were then treated with 1 ml of 0.1% triton x-100 for 15 min to allow permeabilization at room temperature. blocking was performed at room temperature for 1 hr with 5% skimmed milk powder prepared by dissolving in 0.05% pbst-20 solution [with pbs containing 0.05 % (v/v) tween 20]. once washed with pbs (phosphate-buffered saline), the cells were treated with 3% h 2 o 2 for 1 hr to eliminate the endogenous peroxidase effect. following two washes with pbs, befv g1 specific monoclonal antibody (emai, australia, diluted 1/50 in pbs) was added to each well. western blotting purified proteins from the transfected cell lysate were obtained using the ni-nta spin kit (qiagen, germany) purification system, used for western blot analyses. purified proteins were separated on 10% resolving and 5% stacking sds-page gel in a mini electrophoresis unit (bio-rad, usa) for 120 min at 130 volts. the transfer of proteins in the acrylamide gel to the pvdf membrane (millipore, usa) was carried out using the mini-transblot transfer system (bio-rad, usa) for 60 min at 100 volts. the membrane was blocked with 5% skimmed milk in pbs. following blocking, the membrane was washed several times with wash solution (pbs with 0.05 tween 20) and incubated at room temperature for 2 hr in befv g1 specific monoclonal antibody (diluted 1/100). the membrane was washed again and incubated in hrp (horseradish peroxidase)-labeled anti-mouse igg conjugate (sigma-aldrich, usa, diluted 1/1,000) for 1  hr. after washing, the membrane was incubated for 15 min in the chromogen-substrate solution (0.05 m tris ph 7.2, 1 mg/ml diaminobenzidine, and 0.3% h 2 o 2 ). after washing the membrane in distilled water, the size of the proteins was estimated relative to a peptide molecular weight marker (mw-sds 200, sigma-aldrich, usa). immunization preparation two-three µl of glycerol stocks carrying pcdna4-g recombinant plasmid and control plasmid (pcdna4-lacz) were grown in lb medium (ampicillin, and subunit vaccines have been developed against bef, and some of these are commercially available (walker and klement 2015). one of the most effective befv vaccines is a live attenuated vaccine formulated with the saponin quil a (vanselow et al. 1995). in this study, we investigated the expression of and short-term humoral immune response to the pcdna4-g plasmid in balb/c mice immunised with the attenuated vaccine on days 0, 14 and 21. materials and methods virus and cell culture in this study, we use the befv/ad12/tur strain (genebank: ky012742), which was isolated from the turkish bef outbreak in 2012. the virus was propagated in vero cells, as previously described (abayli et al. 2017). rna isolation and rt‑pcr the viral rna extraction from the virus culture supernatant was performed using a qiaamp® viral rna mini kit (qiagen, germany) according to the manufacturer’s instructions. the primers were designed using dna baser (heracle biosoft s.r.l., romania) based on the complete genome sequences of the befv/ad12/tur strain. reverse transcription polymerase chain reaction (rt-pcr) was performed with the qiagen onestep kit. briefly, the reaction mixture [5× rt-buffer (10  µl), 10 mm dntp mix (2 µl), primers mix (10 pmol) (4  µl), 5’-atgttcaaggtcctaataattacc-3’ and 5’-ttaatgatcaaagaacctatc-3’), rt-pcr enzyme mix (2 µl) and rna (50 ng)] was adjusted to a volume of 50 µl with rnase-free water. after the reverse transcriptase step at 50  °c (30 min), amplifications were carried out at 94  °c for 15 min, followed by 35  cycles at 94  °c for 45 sec, 55  °c for 1 min, and 72 °c for 1 min. construction of pcdna4‑g the full-length glycoprotein gene (befv/ad/12tur, 3060-4931 nucleotide position) amplified by rt-pcr was transferred into the pcdna4/hismax©topo® ta expression vector (thermo fisher, usa) by direct ligation to form pcdna4-g. pcdna4-g was then transformed into top10 competent cells. the orientation of the pcdna-g construct and the absence of unwanted mutations were checked by sequence analysis. expression of recombinant glycoprotein the pcdna4-g plasmid was transfected into vero-e6 145veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 abayli & bulut dna vaccines against befv sera from pcdna4-g-, befv-, pbs-, and pcdna4/ lacz-immunised mice were diluted 1/100. aliquots (100-µl) of these serum samples were transferred into the wells of a elisa-plate, which was incubated for 2 hr at room temperature and shaking at 50 rpm. after incubation, the wells were washed seven times with wash solution. after washing, 100 µl of 1/5,000 diluted streptavidin hrp (thermo scientific, ma, usa) was added to each well. the plate was incubated for 1 hr at room temperature. after washing, a solution of tetramethylbenzidine (tmb, sigma aldrich, ma, usa) substrate was prepared, and 100 µl of tmb substrate was added to all wells. the plate was then incubated in the dark for 10 min. the reaction was stopped with 100 µl of 1m h 2 so 4 . the plate was read at 450 nm absorbance in the elisa reader. plaque reduction neutralization test (prnt) t75 flask-cultured cells were trypsinized and passaged to a 24-well plate (0.02 × 106 cells/well). all mouse serum samples were removed from the freezer (-  20  °c), thawed at room temperature, and then inactivated by incubating at 56 °c for 30 min. aliquots (20 µl) of each serum sample were added to a microcentrifuge tube containing 180  µl of dmem-f12. a 100-µl aliquot of the vortexed mixture was serially diluted with dmem-f12 (sigma-aldrich, ma, usa) (1:10, 1:20, 1:40, and 1:80). inactivated serum samples were studied individually in 24-well cell culture plates, and different plates were used for each experimental group. in addition to the sera from the experimental group, four wells of plasmid control, two wells of virus control, and two wells of vero e6 cells were included on each plate. mouse brain-adapted befv stock was removed from the freezer (- 80 °c) and diluted with dmem-f12 to contain 50 pfu/100 µl of the virus. a 100-µl aliquot of this diluted virus was transferred to serum tubes with serial dilutions. the mixture was vortexed and incubated at 37 °c for 1  hr. after incubation, this mixture (200 µl) was added to the medium-removed cells in a 24-well plate and seeded for 1hr by adsorption. the plates were mixed at 15-min intervals. during the incubation, 0.3% overlay medium (1:1 mixture of 2x dmem-f12 and 0.6% agarose) was prepared. after 1 hr, the inoculum was removed from the plate wells, and the cells were coated with 1 ml of overlay medium. the plates were incubated at 37 °c, 5% co 2 for 4 days. cells were examined under the microscope and scored for cpe (cytopathic effect) foci. calculations and statistical analysis prnt50 (the serum titer required to reduce viral plaques by 50%) was used to calculate the 60 µg/ml, 50 ml). the tubes were incubated overnight in a shaking incubator (37 °c, 180 rpm). the next day, the tubes were centrifuged (4,500 rpm for 15  min) and plasmid purification was performed using the purelink™ expi endotoxin-free maxi plasmid purification kit (thermo fisher, ma, usa). plasmid dna pellets (100 µg) were diluted with 40 µl of sterile pbs. ten microliters of lipofectamine 2,000 were then added to the plasmid-pbs solution. a  50-µl aliquot of the plasmid-pbs-lipofectamine mixture was prepared for injection into the mice assigned to the plasmid-immunised group. the mouse brain adapted befv (1,000 pfu/500 µl) was prepared for injection into the mice assigned to the befv-immunised group. ethical statement all animals used in the study were obtained from the experimental research center of firat university. the firat university animal experiments local ethics committee approved the use of experimental animals (protocol number: 2016/50). experimental study female balb/c mice (4 weeks old) were divided into four groups (n  =  7, each). the experimental mouse groups were designed as two study groups (pcdna4-g-, befv-immunised) and two control groups (pcdna4-laczand pbs-immunised). the mice in the vaccine group were sedated with ketamine (200 mg/kg/ip) and, after about 10 min, the injection site was disinfected with 70% ethanol and injections were performed. all injections were performed on the quadricep muscle (except the befv immunised group given ip) on days 0, 14, and  21. blood was collected from the tail vein on days 0, 13, 20, and 30 from all animals in the experimental groups. the tubes were incubated overnight at 4  °c for centrifugation (4,000 × g for 15 min) and kept in a - 20 °c freezer until use. antibody response elisa in the experimental group, antibody response was determined using a modified version of a commercially available befv elisa (emai, camden park, australia). all wells in the bef elisa plate were first blocked overnight with 5% (w/v) skimmed milk diluted in 0.5% pbst-20. after blocking, the wells were washed twice with wash solution [0.05% (v/v) pbst-20]. non-immune and immune blood 146 veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 dna vaccines against befv abayli & bulut immunoperoxidase test and western blotting the immunoperoxidase test was performed on transiently transfected vero cells using befv g1 specific monoclonal antibody and cells expressing gene with dab chromogen were imaged at 72 hr. in the transfected vero e6 cells, brown staining indicating the presence of g protein of befv was observed after microscopic examination, whereas staining did not occur in negative control cells (figure 1). figure 2 shows western blot analyses of the purified proteins obtained from pcdna4-g transiently transfected vero cells with g1 specific monoclonal antibody of befv as g protein of size 77 kda. elisa and prnt50 the serum samples of the last immunization of pcdna4-g immune mice were studied using befv elisa. when the od (optical density) values of the last sera were examined on an individual basis, it was determined that only mouse #1 and #5 exceeded the cut-off value, while the others were below this value. despite this, higher igg antibody results were obtained in all individuals in the pcdna4-g immune group than in the negative groups. these results show that the humoral response is achieved in all neutralization titers. the critical value in the neutralization test was 1:10. cut-off values for the elisas were calculated by adding three-fold of the standard deviation to the mean of the negative control sera. statistical analyses were performed using ibm spss 22 software, and graphs were generated using the graphpad prism 5 package program (graphpad, san diego). the elisa values were tested for normality, and a one-way anova test was preferred because the values between the compared groups showed a normal distribution. tukey (if homogeneous) and tamhan t2 (if not homogeneous) tests were used according to whether the variances were homogenous in the post-hoc tests. the kruskal wallis h test was preferred for the neutralization test. the mann whitney u test was used to determine which groups were responsible for the difference. results rt‑pcr and sequence analysis the befv g gene was amplified in full length (1871  bp) by rt-pcr. after the sequence analysis, there was no disorientation or undesired stop codon in the pcdna4-g structure. figure 1. immunoperoxidase test using befv g1 spesific monoclonal antibody in pcdna4-g-transfected vero e6 cells. a. vero e6 cells transiently transfected with the pcdna4-g vector. b, c. vero e6 cells stably transfected with the pcdna4-g vector. d. control vero cell (40×). a b c d 147veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 abayli & bulut dna vaccines against befv mice immunised with pcdna4-g. in the evaluation of the test, the cut-off value of the final serum samples was calculated as 0.965 (figure 3). the results were also evaluated on a group basis and group averages of the same day were calculated and compared with negative controls in pairs. accordingly, the mean od value of the sera from the pcdna4-g immune group was significantly different on day 30 when compared with the plasmid control group (p < 0.05) and pbs immune group (p < 0.001). the befv immune group revealed this difference on the 13th day (p < 0.001) (figure 4). table 1 shows the prnt50 results and percentages of neutralizing antibodies inhibiting befv under cell culture conditions. according to these results, in mice #1 and #5 in the pcdna4-g immune group, a 1:20 serum dilution inhibited befv by approximately 70%. therefore, the antibody titer was accepted as 1:20. other mice in the same group exceeded the 50% inhibition value at a 1:10 dilution and remained below this ratio at a 1:20 dilution. therefore, the neutralizing antibody titers of these mice were considered 1:10. according to the prnt50 results, five of the befv-immunised mouse sera were 1:80 and the remaining two inhibited befv by more than 50% at a serum dilution of 1:40. dilution of befv immune sera at 1:160 produced less than 50% inhibition. accordingly, antibody titers were calculated to be 1:80 in five of the befv-immunised mice and 1:40 in the remaining two mice (data not shown). discussion befv vaccines show a protective immune response between 6 months and 1 year. live vaccines in areas with dense mosquito populations might pose a risk if the vaccine strain regains virulence. in addition, contamination during the preparation of live vaccines poses a risk to animal health (tzipori and spradbrow 1973, spradbrow 1975, aziz-boaron et al. 2013). only repeated inoculations of attenuated vaccines induce a protective response against befv. this indicates that the virus loses its antigenicity as well as pathogenicity during attenuation (spradbrow 1975). therefore, some researchers have aimed to increase vaccine efficacy by using adjuvants with figure 2. purified recombinant g protein visualized by western blotting. m = protein ladder; lane 1 = control vero e6 cell lysate; lane 2 = purified g protein. mice number 1 0.0 0.5 1.0 1.5 2.0 2.5 2 pcdna4-g befv cut-o� 3 4 5 6 7 o d v al u e figure 3. individual serum elisa antibody measurements of befvand pcdna4-g-immunised mice. the cut-off value indicates the final serum od mean of all negatives plus three-times the standard deviation, calculated as 0.965. days 0 0.0 0.5 1.0 1.5 2.0 2.5 non-immun 13 14 20 21 30 o d v al u e befvpcdna4-g pcdna4-lacz a ab b c c cb b b bc a a bc a* *** *** *** ab * * *** figure 4. antibody measurements (befv elisa kit) of sera from immunised mice. the graph shows the mean od value of the individuals in each experimental group with standard errors. different letters (a, b, c) for the same day indicate differences between the groups. the significance of the difference is indicated by *. a single star (*) indicates p ≤ 0.05 and three stars (***) indicate p < 0.001. the arrows indicate the days of immunization. 1 2 m 75 kda 55 kda 148 veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 dna vaccines against befv abayli & bulut some researchers have noted that humoral immune response alone is not sufficient to protect against befv and cellular immune response is also important (aziz-boaron 2014, della-porta and snowdon 1979). therefore, researchers have focused on recombinant vaccines (which are both safe and potent) rather than inactive or subunit vaccines (wallace and viljoen 2005, zhang et al. 2017). alternatively, viral vector vaccines expressing the recombinant befv g protein have been studied by some researchers. hertig and colleagues (hertig et  al. 1996) found that a vaccinia vector vaccine expressing the befv g protein protected cattle from experimental infection after the second inoculation; however, the virulence of the virus in the experiment was low. zhang and colleagues (zhang et  al. 2017) reported that a recombinant newcastle disease virus vaccine (la sota strain) expressing the befv g protein produced a 1:64-1:128 neutralizing antibody titre in cattle after double vaccination (days 0 and  21). wallace and colleagues (wallace et al. 2005) reported that a lumpy skin disease virus vaccine expressing the befv g protein did not yield satisfactory results even after four inoculations in cattle. viral vector-based vaccines developed against befv also failed to provide the expected results (wallace et  al. 2005, zhang et  al. 2017). in viral vector-based vaccines, antibodies resulting from pre-existing immunity to the vector virus can prevent vector proliferation. therefore, repeated inoculations of such vaccines can also be ineffective (leitner et al. 1999, griffiths and khader 2014). dna vaccines are considered safe because there is no contamination with infectious agents. after dna vaccination, an immune response develops only against the antigen targeted by the vaccine so that vaccinated animals can be easily distinguished from infected animals. (dhama et al. 2008). after viral infection, viral proteins are produced and processed in the cells. similarly, after the plasmid dna enters the cell, the transcription and translation of the encoded genes occur in the cell. thus, viral proteins having the native structure can be produced in attenuated vaccines (vanselow et al. 1985, vanselow et  al. 1995) although it is not known how much of the virus can remain active after adjuvantization, it is estimated that it is 99.9% aggregated with quil  a saponin (walker and klement 2015). this may adversely affect the proliferation of the vaccine strain in the host. subunit vaccine studies, which initially focused on the protective antibody response of the g  protein separated from the native virus (uren 1994), at present continue with focus on recombinant protein production (hansoongnern et  al. 2019). some researchers have demonstrated that the recombinant befv g protein, which has the potential to be used in vaccines or diagnostic kits, is expressed in  vitro using the baculovirus expression system (johal et al. 2008, kanpipit et al. 2018). hansoongnern and colleagues (hansoongnern et al. 2019) reported that the transmembrane domain-deleted g protein (100 µg) adjuvanted with montanide isa 206 displays a neutralizing antibody response in guinea pigs. in such expression systems, it is critical that the protein produced is conformationally similar to the native virus. in addition, the need for purification and the conformational change of the protein during the purification or adjuvantisation may be a disadvantage for these systems. inactive vaccines are safe. however, the virulent live virus in the inactive vaccines should be completely inactive. further, the adjuvants in these vaccines can lead to cytotoxicity (spradbrow 1975) or undesirable reactions (tzipori and spradbrow 1973). inactive befv vaccines produce a short-term antibody response in cattle, and a positive correlation between the level of antibody response and the protection offered by the vaccine is not always observed (st  george 1988, aziz-boaron et  al. 2014). in a previous study, although an emulsified inactive israeli vaccine stimulated an antibody response at a ratio of 1:256 after the third vaccination, the protective efficacy of the vaccine against natural infection was found to be 50%, which was disappointing (aziz-boaron et al. 2013, aziz-boaron et al. 2014). table i. prnt50 data from sera collected from pcdna4-g-immunised mice on day 30. serum dilution rates neg. control cell culture plate 1 cell culture plate 2 mouse 1 mouse 2 mouse 3 mouse 4 neg. control mouse 5 plaque number plaque number % inhib. plaque number % inhib. plaque number % inhib. plaque number % inhib. plaque number plaque number 01:10 28 0 100 2 92.8* 8 71.4* 4 85* 26 0 01:20 34 10 70.5* 20 41.1 26 23.5 19 44.1 29 8 01:40 32 24 25 34 29 29 27 21 01:80 35 33 32 36 32 30 28 (-) indicates inhibition values below 10%; (*) expresses the highest serum dilution rates that inhibit befv by 50% or more. these ratios were accepted as neutralizing antibody titer. 149veterinaria italiana 2021, 57 (2), 143-150. doi: 10.12834/vetit.2077.12075.1 abayli & bulut dna vaccines against befv hurk et al. 2004). in this study, a chemical liposome (lipofectamine 2000), a plasmid dna transfer system, was used to prevent plasmid dna degradation in the blood. liposomes are synthetic vesicles consisting of a phospholipid bilayer structure and are now one of the most important agents used in the transfer of genetic material into cells. liposomes containing plasmid dna can easily pass through cell membranes after transfer and release of the plasmid dna content following fusion with the endosomal membranes. at the same time, liposomes rapidly escaping from endosomes are protected from degradation in this manner. this increases gene expression and contributes to immunogenicity (pereira et  al. 2014, wang et  al. 2011). although it is not known whether liposomes positively contributed to humoral immune response in this study, their effect is predicted to be beneficial. the number of experimental studies using chemical transfer systems should be increased in the future, and the results should be compared with those of other transfer systems. according to minimum tissue damage and cost-effectiveness analysis, the most suitable system should be preferred. in this study, the cellular response was not determined. further, the fate of the long-term response remains unclear. although the antibody response is low in case of dna vaccines, the cellular response might be superior to that observed in case of subunit or inactive vaccines. future studies investigating the cellular and long-term responses are planned. acknowledgments this article was derived from thesis number 519 087 stored in the council of higher education thesis center, turkey. grant support this work was supported by project number vf1627 by the scientific research projects coordination unit of firat university. the cell (alonso and leong 2013). this feature is important in antigenic regions in the targeted protein. dna vaccines can be rapidly produced, purified and combined with different types of vaccines or different gene regions. in the presence of maternal antibodies, which play an important role in protecting against high-risk diseases, the effect of these vaccines can continue (dhama et al. 2008). in addition, dna vaccines are known to elicit a strong cellular response and may be a good alternative to befv vaccines (beláková et al. 2007). pasandideh and colleagues (pasandideh et al. 2018) showed that anti-befv-neutralising antibodies were induced in mice injected with the pcdna3.1-g1 construct. similarly, we investigated the humoral immune response to the construct expressing the befv g gene in balb/c mice in this study. we found that the humoral immune response was lower and later in pcdna4-g-immunised mice than in befv-immunised mice. although the humoral immune response was expected to occur later in case of dna vaccines, the antibody titres measured on day 30 in the pcdna4-g-immunised group were very low. this may be due to the route of administration of the vaccine. we preferred intramuscular injection because it is widely used and practical. after intramuscular administration, myocytes are often transfected. this region is not equipped with dendritic cells, and myocytes do not have the ability to deliver the antigen to cd4+ t cells via mhc ii; therefore, the cellular response was dominant compared to the antibody response (belakova et al. 2007, dupuis et al. 2000). in addition, the degradation of plasmid dna by serum proteins in intramuscular vaccinations might reduce the expression level, resulting in a delayed antibody response (faurez et  al. 2010). some researchers have preferred intra-lymph node vaccination, electroporation or particle-mediated bombardment to avoid this disadvantage. these techniques can increase transfection and significantly increase immune responses; however, their application is difficult, particularly in large animals, and they have not yet progressed to routine use in animals. 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12.02.2020 | available on line: 12.02.2020 1istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, teramo, italy. 2dipartimento di medicina veterinaria, università degli studi di bari, valenzano (ba), italy. * corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy, tel.: +39 0861 332440, e-mail: a.lorusso@izs.it. alessio lorusso1*, paolo calistri1, antonio petrini1, giovanni savini1 and nicola decaro2 novel coronavirus (sars‑cov‑2) epidemic: a veterinary perspective editorial the current threatening highly pathogenic pneumonia outbreak is caused by a novel coronavirus (cov) named sars-cov-2. this name was given by the coronavirus study group (csg) of the international committee on taxonomy of viruses, which is responsible for developing the official classification of viruses and taxonomy of the coronaviridae family (gorbalenya et al. 2020). instead, the disease that sars-cov-2 causes has been named by the who as covid-19. this novel epidemic emerged in december 2019 in wuhan city, hubei province, china and continues to expand. epidemiological investigations revealed that many initial patients were exposed to wildlife at the huanan seafood wholesale market (south china seafood market), which is the largest seafood market in central china, and where different species of bats, minks, snakes, chinese bamboo rats, but also cats of different breeds, porcupines, dogs, poultry and other farm animals are commonly sold. these markets are known as ‘wet markets’ since they are traditionally places that sold dead and live animals out in the open and where blood and other body fluids originating from different animal species represent an exceptional source for the spread of infectious diseases and the jump of species barriers by pathogens. since 1st of january 2020 the south china seafood market has been closed by the wuhan municipal government. to date (february 11th, 2020) chinese health officials have reported 42,708 cases of infections with sars-cov-2 in china, including 10,980 cases outside of hubei province. a total number of 395 infections with sars-cov-2 also are being reported in other countries (24) internationally (who, https://www.who.int/docs/ default-source/coronaviruse/situation-reports/20200211-sitrep-22-ncov. pdf?sfvrsn=fb6d49b1_2). remarkably, human-to-human transmission has been evidenced. so far, the virus killed 1,017 individuals in china and 1 in the philippines. china responded quickly by informing the world health organization (who) of the outbreak and sharing sequence information with the international community after discovery of the causative agent (www. gisaid.org). this permitted the assessment of several molecular methods for rapid sars-cov-2 diagnosis. sars-cov-2 has been also rapidly isolated in permissive cell-culture. keywords sars-cov-2, novel coronavirus, covid-19, epidemic, veterinary. 6 veterinaria italiana 2020, 56 (1), 5-10. doi: 10.12834/vetit.2173.11599.1 sars-cov-2 and veterinary medicine lorusso et al. covs were not considered to be highly pathogenic to humans until the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in 2002 and 2003 in guangdong province, china, lately spread on a global scale (zhong et al. 2003, drosten et al. 2003, fouchier et al. 2003, ksiazek et al. 2003). ten years after sars-cov, another highly pathogenic coronavirus, middle east respiratory syndrome coronavirus (mers-cov) emerged in saudi arabia and other middle eastern countries (zaki et al. 2012). the evidence that sars-cov and mers-cov were transmitted directly to humans from masked palm civets (paguma larvata) and dromedary camels, respectively, (guan et al. 2003, alagaili et al. 2014, hemida et al. 2013), and that both viruses are thought to have originated in bats (cui et al. 2013, hu et al. 2017, lau et al. 2013) led to the hypothesis that also sars-cov-2 may be of animal origin. covs act as primary actors within the so-called human/animal interface across which a plethora of infectious pathogens has been observed to emerge, jump species barriers and eventually evolve, thus finding new ecological niches and causing new epidemiological phenomena. before the emergence of sars and mers-cov, four human covs (hcovs), namely hcov-nl63, hcov-229e, hcov-oc43 and hcov-hku1 were known to infect humans, mostly causing mild infections (eg common cold) in immunocompetent people. interestingly, low pathogenic covs have also their ancestors in animals. based upon the current available genomic sequences, bats are thought to be the natural hosts for hcov-nl63 and hcov-229e, rodents for hcov-oc43 and hku1. coronaviruses needed intermediate hosts (cattle for hcov-oc43 and alpacas for hcov-229e) before being able to infect humans, as it was the case of sars-cov and mers-cov with masked palm civets and dromedary camels, respectively (cui et al. 2019). covs are enveloped positive-strand rna viruses with exceptional genetic complexity and variety. one major contributing factor to cov diversity is high-frequency rna recombination (banner et al. 1991, lai et al. 1985, makino et  al. 1986). new seroand biotypes have arisen from homologous rna recombination, i.e., the exchange of corresponding sequences among related covs (brian et al. 1997, herrewegh et al. 1998, jia et al. 1995, kottier et al. 1995), while heterologous rna recombination events with non-coronaviral donor rnas have led to the acquisition of novel genes (luytjes et  al. 1988, snijder et  al. 1991, zeng et  al. 2008, huang et  al. 2016). sars-cov is paradigmatic of these evolutionary mechanisms as it emerged through recombination of bat sars-related coronaviruses (sarsr-covs). recombination of sars-cov in the spike (s)-protein gene, which recognizes cell surface receptors, might have mediated the initial cross-species transmission event from bats to other mammals. if, on one hand a direct progenitor of sars-cov was not found in bats, on the other, all its genomic elements were identified in bats present in one single cave of yunnan province, china (reviewed in cui et  al. 2018). sars-cov-2 is genetically close to sars-cov (gorbalenya et al. 2020) and it has been proposed that sars-cov-2 is also a recombinant virus. there is a certain evidence that sars-cov-2 originated from recombination between a bat sars-like cov and a coronavirus of unknown origin. additionally, it 7veterinaria italiana 2020, 56 (1), 5-10. doi: 10.12834/vetit.2173.11599.1 lorusso et al. sars-cov-2 and veterinary medicine has been proposed that snake is the most probable wildlife animal reservoir for the sars-cov-2 based on the virus relative synonymous codon usage (rscu) bias, which is closer to that of snake compared to other animals. truth to be told, neither sars-cov-2-like sequences nor elements of its genome have been evidenced in snakes so far (ji et  al. 2020). researchers in guangzhou, china, have suggested that pangolins (gen. manis, linnaeus 1758), long snouted, ant-eating mammals often used in traditional chinese medicine, are the probable direct animal source of sars-cov-2 for humans. this suggestion originated from the detection of covs closely related to sars-cov-2 in pangolins. however, caution is needed and genetic analyses are currently ongoing (https://www.nature.com/articles/d41586-020-00364-2). importantly, the csg also clearly evidenced that sars-cov-2 clusters with sars-covs within the species severe acute respiratory syndrome-related coronavirus of the genus betacoronavirus (gorbalenya et al. 2020). sars-cov-2 has been assigned to an existing species of hundreds of known viruses largely isolated from humans and bats. these viruses have names derived from sars-cov, but only the viral isolates originating from the 2002-2003 outbreak have been confirmed to cause sars in humans. thus, the reference to sars reflects the phylogenetic grouping rather than linking this virus to a specific disease (i.e., sars) in humans. moreover, it is important to point out that sars-cov-2 is not a descendent of sars-cov (gorbalenya et al. 2020). covs are well-known to veterinary virologists since decades. taxonomically, they are members of the subfamily orthocoronavirinae in the family coronaviridae and the order nidovirales. this subfamily consists of four genera alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. the alphacoronaviruses and betacoronaviruses infect only mammals. the gammacoronaviruses and deltacoronaviruses infect birds, but some of them can also infect mammals (cui et al. 2018). most of the breakthrough studies on strictly veterinary covs of the past century focused on mouse hepatitis virus (mhv), feline infectious peritonitis virus (fipv) and infectious bronchitis virus of poultry (ibv). nowadays, porcine epidemic diarrhea virus (pedv) causes severe gastroenteritis in young piglets, leading to significant morbidity, mortality, and ultimately economic losses (wang et al. 2019). porcine hemagglutinating encephalomyelitis virus (phev) mostly leads to enteric infection but can also infect the central nervous system, causing encephalitis, vomiting and wasting in pigs (mora-diaz et  al. 2019). recently the novel swine acute diarrhea syndrome coronavirus (sads-cov) has been described in pigs (wang et  al. 2019). covs are also a common cause of enteric (canine enteric coronavirus, ccov type i and ii) and respiratory (canine respiratory coronavirus, crcov) disease in dogs (decaro and buonavoglia 2008). the key role of veterinary virologists in the field of discovery, viral evolution, genome manipulation and pathogenesis studies of covs has been highlighted over recent years. it has been indeed proposed that type i ccovs and fcovs evolved from a common ancestral virus, and that the canine and feline type ii lineages arose from multiple recombination events with an unidentified genetic source (lorusso et  al. 2008). ccov-ii has been recognized as the ancestor of transmissible 8 sars-cov-2 and veterinary medicine lorusso et al. veterinaria italiana 2020, 56 (1), 5-10. doi: 10.12834/vetit.2173.11599.1 gastroenteritis virus of swine (tgev) (lorusso et  al. 2008) and, interestingly, back recombinant ccov-iis harboring the s protein 5’-end of tgev have been also described in dogs (decaro et  al. 2009). crcov likely originated from bovine coronavirus (bcov), the direct ancestor of human hcov-oc43 (lorusso et  al. 2009). porcine respiratory coronavirus (prcov) is a deletion mutant of its parental enteric tgev but with respiratory tropism (pensaert et  al. 1986, bernard et al. 1989, rasschaert et al. 1990); genomic sequences highly similar to pedv were detected also in bats and sads-cov is a recent spillover from bats to pigs (zhou et al. 2018). fcov type i and ii cause a mild or asymptomatic enteric infection in young cats, but during persistent infection, specific mutations in the s protein (chang et al. 2012) may transform the virus into a highly virulent strain of fcov (fipv), that leads to the development of a lethal disease known as feline infectious peritonitis (fip) (chang et al. 2011, haijema et al. 2007, pedersen et al. 2009). the emergence of sars-cov-2 is paradigmatic of the strict relationship existing between human and animal health, ecosystem condition and human habits. it is accepted that many viruses have existed in their natural reservoirs for a very long time. the constant spillover of viruses from their natural hosts to humans and other animals is largely due to human activities, including modern agricultural practices and urbanization. therefore, the most effective way to prevent viral zoonosis is to maintain the barriers between natural reservoirs and human society, in mind of the ‘one health’ concept. nevertheless, all recent human health hazards were caused by zoonotic agents. but how worrisome a particular zoonotic disease is? is it possible to prioritize zoonotic diseases? to answer these questions, authorities should take into account the potential of a given disease to cause a pandemic, how severe can be the impact on humans and animals, and whether the disease is listed as a potential agent of bioterrorism. in all cases, the role of veterinarians is crucial. veterinarians operate in the veterinary public health (vph) system, a major part of public health in which human health and well-being are the central tasks. remarkably, veterinary virologists operating in vph coordinate virus surveillance and pathogenesis studies in domestic animals and wildlife, a crucial aspect in trying to understand the etiology of viral zoonoses, their impact on individual health as well as on populations over time, but also for preparedness for human emerging diseases. in this perspective, the combination of innovative novel diagnostic technologies, including next-generation sequencing, and big data management within vph institutes, represents 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coronavirus differs from transmissible 115 1national reference centre for emerging risks in food safety, istituto zooprofilattico sperimentale della lombardia e dell’emilia-romagna ‘b. ubertini’, milan, italy. 2istituto zooprofilattico sperimentale della lombardia e dell’emilia-romagna ‘b. ubertini’, brescia, italy. 3department of veterinary medicine, university of bari “aldo moro”, bari, italy. *corresponding author at: istituto zooprofilattico sperimentale della lombardia e dell’emilia-romagna ‘b. ubertini’, brescia, italy. tel.: +39 030 2290534, fax: +39 030 2425251, e-mail: paolo.daminelli@izsler.it. keywords cow, donkey, lactic acid bacteria, listeria monocytogenes, raw milk, staphylococcus aureus. summary gram-positive foodborne pathogens such as listeria monocytogenes and staphylococcus aureus can grow in a wide variety of foods, including raw milk. the aim of the study was to compare the growth of l. monocytogenes and s. aureus inoculated in donkey and cow samples of raw milk during a storage time of 11 days at 8 °c. moreover, the study aimed to evaluate the influence of lactic acid bacteria (lab) content on the growth of the two microbiological populations considered. lab content was lower in raw donkey milk than in raw cow’s milk during the entire analyses; on the other hand, ph levels were higher in the donkey milk rather than in the cow’s milk, although both values showed a decrease at the day 11. s. aureus showed no significant differences in the two types of milk. from day 0 to 11, l. monocytogenes increased from 3.68 ± 0.02 log cfu/ml to 6.31 ± 0.07 log cfu/ml and from 3.64 ± 0.04 log cfu/ml to 4.59 ± 1.04 log cfu/ml, in donkey milk and in cow’s milk, respectively. our results showed that donkey milk is a more favourable matrix to support the growth of l. monocytogenes than cow’s milk. paolo daminelli1,2*, roberta barrasso3, elena dalzini1, elena cosciani-cunico1,2, giorgio zanardi2 and giancarlo bozzo3 raw donkey milk versus raw cow’s milk. a preliminary study to compare the growth of listeria monocytogenes and staphylococcus aureus veterinaria italiana 2020, 56 (2), 115-121. doi: 10.12834/vetit.2140.13666.1 accepted: 05.10.2020 | available on line: 10.12.2020 is destined for the cosmetics and food industries (brumini et al. 2016, soto del rio et al. 2017). pasteurized dm is usually sold directly from the farms; however, considering its nutritional properties, it can be sold raw, with three days of shelf-life (giacometti et al. 2016). some authors (pilla et  al. 2010) highlighted that foodborne pathogens are generally absent in raw dm and somatic cells and total bacterial count (tbc) are often low, suggesting it could be a safe food, provided that the mammary gland is healthy and the animals are milked in good hygienic conditions. previous studies (carminati et  al. 2014, quigley et  al. 2013) showed that dm has different microbial flora, mainly composed of lactic acid bacteria (lab). these are characterized by bacteriocins production active against some gram-negative bacteria (mottola et  al. 2018, murua et  al. 2013), although the presence of undesirable pathogens responsible of food-borne diseases have introduction milk is a nutritious food product for humans, and it is obtained from a variety of animal sources, such as cows, goats, sheep, donkeys and buffaloes (mehmeti et  al. 2017). since milk contains many important nutrients and provides a suitable physical environment, it represents an ideal growth medium for both non-pathogenic and pathogenic bacteria (quigley et al. 2013, white 2001). donkey milk (dm) is considered the best substitute for human milk in infant nutrition when breast-feeding is not available (monti et al. 2007). so, due to its tolerability (i.e. digestibility, palatability, low allergenicity) and bioactivity (i.e. lysozyme activity), dm could be used as a dietary supplement (souroullas et  al. 2018). nevertheless, dm is still a “niche product” which often can only be retailed in farms for direct consumption, while a smaller part 116 veterinaria italiana 2020, 56 (2), 115-121. doi: 10.12834/vetit.2140.13666.1 raw donkey milk versus raw cow’s milk daminelli et al. milk is produced by the secretion of the mammary gland of farmed animals, it has not been heated to more than 40 °c or undergone any treatment with an equivalent effect. the direct sale of raw milk from farms to consumers is allowed in several european countries, provided that the operation complies with the hygienic criteria in regulation (ec) no. 853/2004 and the general food law [regulation (ec) no. 178/20022]. on the basis of regulation (ec) no. 853/2004, dm is included under the section “other milk producing species,” where the tbc is less than 1.500.000 cfu/ml at 30 °c. in addition, regulation (ec) no. 2073/20053 includes the microbiological “food safety criteria” for listeria monocytogenes in ready-to-eat (rte) foods and the “process hygiene criteria” for coagulase-positive staphylococci (cps). annex i of regulation (ec) no. 2073/2005 sets out the microbiological criteria for foodstuffs, including the criteria for l.  monocytogenes in rte foods (criteria 1.1 to 1.3); in particular, in rte foods able to support the growth of l.  monocytogenes, when food business operator (fbo) is not able to demonstrate that the product will not exceed the limit of 100  cfu/g throughout the shelf-life, the criteria is the absence of the pathogen. annex ii of this regulation specifies that fbos shall conduct, as necessary, studies to evaluate the growth of l. monocytogenes that may be present in the product during the shelf-life under reasonably foreseeable storage conditions. considering that consumers not always respect the advice to boil the raw milk before consuming it (claeys et al. 2013), in the present study we considered the raw milk as a rte product. thus, the aim of the study was to compare the growth of l. monocytogenes and s. aureus inoculated in donkey and cow samples of raw milk during a storage time of 11 days at 8 °c. moreover, we aimed to evaluate the influence of lab content on the growth of the two microbiological populations considered. materials and methods milk contamination and sampling the study was carried out during years 2017 and 2018. two different batches of dm and raw cow’s milk were supplied from local farms, collected into sterilized 1-litre laboratory bottles and transported in coolers to izsler’s laboratories (istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna, brescia, italy) immediately after been described (cavallarin et  al. 2015, efsa biohaz panel 2015). lactoferrin, lysozyme, immunoglobulins and lactoperoxidase carry out an antimicrobial activity in milk (baldi et  al. 2005, yamauchi et  al. 2006) and their content is different among species, breeds and individuals because of genetic or breeding variants (brumini et al. 2016). the low microbial count of dm (aspri et  al. 2017) is related to the excellent natural anatomical position of the udder and its small size (doreau and martin-rosset 2011), as well as the presence of natural antimicrobial components. this antimicrobial activity of dm is mainly attributed to lysozyme and, to a lesser extent, to lactoferrin (uniacke-lowe et al. 2010). salimei and colleagues (salimei et al. 2004) showed that the average concentration of lysozyme in dm is three times higher than in human milk, while this component is absent in the milk of cows, ewes and goats (vincenzetti et  al. 2007). lysozyme in dm ranges from 0.67 to 3.74 g/l and maintains the same high percentage over the total protein during 150 days of lactation (guo et  al. 2007, vincenzetti et  al. 2011, šarić et  al. 2012, šarić et  al. 2014). the interaction between lactoferrin and the lipopolysaccharidic layer (lps) causes disruption of the outer membrane. moreover, this situation promotes the susceptibility of gram-negative bacteria to the lysozyme by increasing the membrane permeability (benkerroum 2008, ellison and giehl 1991, farnaud and evans 2003). because of this mechanism, gram-negative bacteria are less sensible to lysozyme than gram-positive due to their outer layer, which does not allow the entry of lysozyme molecules into the target places in peptidoglycan structure (floris et al. 2003). the abundance of lactose seems to favour the growth and survival of adapted probiotic lactobacilli, although there is a high content of lysozyme (chiavari et  al. 2005, coppola et  al. 2002). studies conducted by zhang and colleagues (zhang et  al. 2008) on the ability of the lab microflora to grow in dm, showed that enterococci could be the major portion of growing bacteria. enterococci, in fact, are more resistant to lysozyme than lactobacilli and, among lactobacilli, sensitivity to lysozyme is species-specific or strain specific (neviani et al. 1991). therefore, the high content of lysozyme in dm is responsible for the presence of only coccus-shaped species (carminati et al. 2014). according to regulation (ec) no. 853/20041, raw 1 e1 european commission (ec) 2004. commission regulation of 29 april 2004 laying down specific hygiene rules on the hygiene of foodstuffs. off j. l139, 05/08/2004, 55. 2 european commission (ec) 2002. commission regulation of 28 january 2002 laying down the general principles and requirements of food law, establishing the european food safety authority and laying down procedures in matters of food safety. off j. l31, 30/09/2002, 1-24. 3 european commission (ec) 2005. commission regulation of 15 november 2005 on microbiological criteria for foodstuffs. off j. l338, 07/12/2005, 1-26. 117veterinaria italiana 2020, 56 (2), 115-121. doi: 10.12834/vetit.2140.13666.1 daminelli et al. raw donkey milk versus raw cow’s milk 6888-1:1999/amd. 1:2003 to enumerate the cps concentration in milk. during the milk storage, on control samples, the enumeration of tbc, enterobacteriaceae (ent), lab and cps (s. aureus and other species) was performed by iso 4833:2003, iso 21528:2017, iso 15214:1998 and iso 6888-1:1999/amd. 1:2003, respectively. the ph was determined using an instrument with automatic temperature compensation (hanna instruments hi 223). on contaminate samples, the enumeration of l.  monocytogenes was performed using the iso 11290-2:2017 while the enumeration of cps s. aureus was carried out using the iso 6888-1:1999/ amd. 1:2003. statistical analysis microbiological results were expressed as log cfu/ ml. for each analysed parameter and for each type of studied milk, the individual means and standard deviations were determined on the basis of the average of the single replicate of two milk batches. three different increasing rates were evaluated starting from the observation of different tendency in lab, l. monocytogenes and s. aureus between the two different types of milk from the day 0 to the day 11, divided by the level found at time 0. results the results were expressed as mean value and standard deviation (sd) of the two samples of dm and the two samples of cow's milk used in the present study during the pre-established time intervals (0, 3, 5, 7 and 11 days). lab concentration was lower in raw dm than in raw cow’s milk during the entire experiment; on the other hand, ph levels milking. for each pathogen considered, a mixture consisting of three different strains was formed: one registered reference strain and two field strains previously isolated from cow's milk and cheese; in particular, atcc® 19115tm (reference strain), lm 273250 and lm 332764 for l.  monocytogenes, and staphylococcus aureus subsp. aureus rosenbach atcc® 25923™ (reference strain), cps 54057 and cps 283463 for s.  aureus. the strains, stored in a freezer at - 80 °c, were individually revitalized in bhi (brain heart infusion) liquid culture medium and incubated at 37 °c for at least 15-18 hours in aerobic conditions. then, each strain was re-suspended in bhi at a lower temperature in order to adapt the microorganism to the storage conditions of 8 °c as suggested by technical guidance document for conducting shelf-life studies on l.  monocytogenes in rte products (eucrl 2017). all strains were separately diluted in physiological solution and then each pathogen was separately mixed in equal volume to obtain a multi-strain cocktail of l.  monocytogenes and a multi-strain cocktail of s. aureus. dm and cow’s milk were divided in 3 groups and inoculated with 1% v/v of physiological solution to obtain control samples or 1% v/v of each multi-strain cocktail to obtain the contaminated samples. samples were incubated at 8 °c for 11 days. the sampling was carried out on single replicates (9 ml each) for each sampling time at 0, 3, 5, 7 and 11 days during the milk storage and the analyses were performed. analysis and test methods the presence/absence of natural contaminations of milk were evaluated on control samples (not contaminated samples) at time 0 by iso 11290-1:2017 to detect the l. monocytogenes presence and by iso table i. values of ph and enumeration of lactic acid bacteria (lab), total bacterial count (tbc), enterobacteriaceae (ent) and coagulase-positive staphylococci (cps) (expressed in log cfu/ml) in raw dm and raw cow’s milk during the storage at 8 °c for 11 days. the results are expressed as mean ± standard deviation (sd). matrix parameter sampling interval (days) 0 3 5 7 11 raw donkey milk ph 7.31 ± 0.05 7.50 ± 0.13 7.46 7.26 ± 0.08 6.95 lab 1.30 ± 0.30 1.65 ± 0.05 1.65 ± 0.05 1.54 ± 0.54 1.81 ± 0.81 tbc 5.66 ± 0.47 6.18 ± 0.93 7.48 ± 0.20 7.90 ± 0.20 8.43 ± 0.07 ent < 1 < 1 < 1 < 1 < 1 s+ 1.26 ± 0.37 1.20 ± 0.28 < 1 1.23 ± 0.33 1.75 ± 1.05 raw cow’s milk ph 6.69 ± 0.02 6.68 ± 0.02 6.43 ± 0.11 6.02 ± 0.62 5.61 ± 0.85 lab 3.48 ± 0.39 4.33 ± 0.74 5.23 ± 1.37 5.78 ± 2.06 6.17 ± 2.16 tbc 5.16 ± 1.31 8.08 ± 0.69 8.45 ± 0.42 8.82 ± 0.47 8.70 ± 1.44 ent 2.26 ± 1.78 4.59 ± 1.73 5.31 ± 1.97 5.97 ± 3.36 2.15 ± 0.98 s+ 1.75 ± 1.05 < 1 < 1 2.08 ± 1.52 1.83 ± 1.17 118 veterinaria italiana 2020, 56 (2), 115-121. doi: 10.12834/vetit.2140.13666.1 raw donkey milk versus raw cow’s milk daminelli et al. matrices resulted more favourable to support the growth of the two bacteria considered. the ph value of both raw dm and raw cow’s milk at time 0 was similar to the values reported in literature by salimei and colleagues (salimei et  al. 2004) and by guo and colleagues (guo et al. 2007). the average ph value (7.31 ± 0.05 at time 0 and 6.95 after the storage of 11 days) of dm was higher than that of cow milk (6.69 ± 0.02 at time 0 and 5.61 ± 0.85 after 11 days). this may be explained by the lower casein  n and phosphate contents in dm than in cow milk (salimei et  al. 2004). moreover, the slight change of the ph values in dm could be associated with the presence of natural concentration of antimicrobial compounds like lactoferrin and lysozyme, which act directly on bacteria (chiavari et al. 2005), maintaining almost unvarying ph values (coppola et al. 2002, zhang et al. 2008). on the basis of regulation (ec) no 853/2004, dm respected the limit of tbc concentration (1,500,000 cfu/ml at 30  °c) until the third day of storage, while in cow’s milk the limit was exceeded earlier. s.  aureus showed no changes in its concentration during the entire period of analysis, both in raw dm and in row cow’s milk. on the other hand, l.  monocytogenes showed a greater increase rate (0.717) in the dm than in the cow’s milk (0.260). the increase rates regarding lab highlighted an inverse trend to l.  monocytogenes, showing a growth of 0.316 in dm and of 0.750 in cow’s milk. the inversely related growth between listeria and lab can be explained considering that lab produced undetermined antimicrobials such as organic acids, hydrogen peroxide, antifungal peptides and bacteriocins that can inhibit the growth of listeria  spp. by competitive exclusion (zhao et al. 2004, 2006). studies conducted by balla and colleagues (balla et  al. 2000) and by gilmore and colleagues (gilmore et  al. 2014) reported that many enterocins from various enterococcal species isolated from many different environments are bactericidal to l.  monocytogenes. these include enterocin q (cintas et  al. 2000), enterocin a (nilsen et  al. 1998), enterocin p (kang and lee 2005), bacterocin 31 (tomita et  al. 1996), bacteriocin 51 were higher in the dm rather than in the cow’s milk, although both values showed a decrease at the day 11 (table i). s.  aureus had no significant differences in the two types of milk considered (table ii); specifically, in the raw cow’s milk s.  aureus showed almost the same value at time 0 (3.53 ± 0.37 log cfu/ml) and at time  11 (3.45 ± 0.78 log cfu/ml), conversely in the raw donkey milk s. aureus decreased from time 0 (3.36 ± 0.35 log cfu/ml) to time 11 (2.95 ± 0.23 log cfu/ml). on the other hand, l.  monocytogenes increased from the value of 3.68 ± 0.02 log cfu/ml at time 0 (the day of the inoculation) to the value of 6.31 ± 0.07 log cfu/ml in the dm and from the value of 3.64 ± 0.04 log cfu/ml at time 0 to the value of 4.59 ± 1.04 log cfu/ml in the cow’s milk (table ii). l. monocytogenes revealed a great variation between the inoculation (day 0) and the day 11 in the dm and a low variation in the cow’s milk (table iii). discussion this preliminary study estimated the growth of l.  monocytogenes and s.  aureus experimentally added to dm and cow’s milk during a storage time of  11 days at 8 °c, to evaluate which of the two table ii. enumeration of staphylococcus aureus and listeria monocytogenes (expressed in log cfu/ml) inoculated in raw dm and row cow’s milk during the storage at 8 °c for 11 days. the results are expressed as mean ± standard deviation (sd). matrix parameter sampling interval (days) 0 3 5 7 11 raw donkey milk staphylococcus aureus 3.36 ± 0.35 3.31 ± 0.32 3.24 ± 0.42 3.15 ± 0.42 2.95 ± 0.23 listeria monocytogenes 3.68 ± 0.02 3.65 ± 0.08 4.41 ± 0.42 5.72 ± 0.27 6.31 ± 0.07 raw cow’s milk staphylococcus aureus 3.53 ± 0.37 3.30 ± 0.27 3.37 ± 0.41 3.34 ± 0.47 3.45 ± 0.78 listeria monocytogenes 3.64 ± 0.04 3.98 ± 0.35 4.16 ± 0.50 4.54 ± 1.50 4.59 ± 1.04 table iii. mean values (m), standard deviation (sd) and standard error (se) of the increase rates among two times (day 0 and day 11). m from time 0 to time 11 expresses the difference between the parameter recorded in the day 11 and in the day 0, divided by the level found at time 0. raw donkey milk raw cow’s milk from day 0 to day 11 lab m 0.316 0.750 sd 0.446 0.425 se 0.316 0.300 listeria monocytogenes m 0.717 0.260 sd 0.029 0.274 se 0.021 0.194 staphylococcus aureus m -0.120 -0.028 sd 0.023 0.117 se 0.017 0.083 119veterinaria italiana 2020, 56 (2), 115-121. doi: 10.12834/vetit.2140.13666.1 daminelli et al. raw donkey milk versus raw cow’s milk + 8 °c, may be able to cause an evident ph reduction, directly through the production of bacteriocins or indirectly through the fermentation activity carried out on the sugars. therefore, the results obtained suggest a particular caution in the consumption of raw dm and confirm the need to give this product a very short shelf life, as correctly established by the order of 10 december 2008 of the ministry of labour, health and social policy, according to which the shelf life of raw milk indicated by the producer may not exceed three days from the date on which it is made available to the consumer. regarding the data obtained from the contamination of milk with s.  aureus, this pathogen does not seem to be particularly influenced by the different concentration of lab and lysozyme. this result suggests the need for further studies to better assess which other enzymatic components can help to ensure a higher level of hygienic and safety in dm compared to raw cow's milk. grant support this work was supported by the department of food microbiology and the primary production department, istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna ‘b. ubertini’, brescia, italy. (yamashita et al. 2011) and several others. similarly, amézquita and brashears (amézquita and brashears 2002) observed that some lab could competitively inhibit l.  monocytogenes in ready-to-eat meats at refrigeration temperature even though the competitive bacteria did not grow. moreover, although some literature sources reported strong antibacterial activity of dm, the majority of these reports are related to its activity toward gram-negative bacteria members of the enterobacteriaceae (šarić et  al. 2012, tidona et  al. 2011, zhang et  al. 2008). indeed, in our study, the enumeration of ent in donkey milk was fewer (1 log cfu/ml) than in cow’s milk during the 11 days of analysis (table i). the results indicate that dm represents a more favourable matrix for support the growth of l. monocytogenes compared to cow's milk. although the data obtained are relative to a limited number of samples, it is possible to state that probably the high concentration of lysozyme in the dm is not able to compensate for the poor concentration of lab. moreover, in a particularly delicate matrix like milk, the concentration of lab is relevant, thanks to the jameson effect, to bio-compete with any pathogens present in the raw material. in fact, the ph profile relative to cow's milk suggests that the lab population present, even at a temperature of 120 veterinaria italiana 2020, 56 (2), 115-121. doi: 10.12834/vetit.2140.13666.1 raw donkey milk versus raw cow’s milk daminelli et al. amézquita a. & brashears m.m. 2002. competitive inhibition of listeria monocytogenes in ready-to-eat meat products by lactic acid bacteria. j food prot, 65 (2), 316-325. aspri m., economou n. & papademas p. 2017. donkey milk: an overview on functionality, technology, and future prospects. food rev int, 33 (3), 316-333. baldi a., politis i., pecorini c., fusi e., chronopoulou r. & dell'orto v. 2005. biological effects of milk proteins and their peptides with emphasis on those related to the gastrointestinal eco system. j dairy res, 72, 66-72. balla e., dicks l.m., du toit m., van der merwe m.j. & holzapfel w.h. 2000. characterization and cloning of the genes encoding enterocin 1071a and enterocin 1071b, two antimicrobial peptides produced by enterococcus faecalis bfe 1071. appl environ microbiol, 66, 1298-1304. benkerroum n. 2008. antimicrobial activity of lysozyme with special relevance to milk. afr j biotechnol, 7, 4856-4867. brumini d., criscione a., salvatore s., vegarud g.e. & marletta d. 2016. whey proteins and their antimicrobial properties in donkey milk: a brief review. dairy sci & technol, 96, 1-14. carminati d., tidona f., fornasari m.e., rossetti l., meucci a. & giraffa g. 2014. biotyping of cultivable lactic acid bacteria isolated from donkey milk. lett appl microbiol, 59 (3), 299-305. cavallarin l., giribaldi m., soto-del rio m.d., valle e., barbarino g., gennero m.s. & civera t. 2015. a survey on the milk chemical and microbiological quality in dairy donkey farms located in north-western italy. food control, 50, 230-235. chiavari c., coloretti f., nanni m., sorrentino e. & grazia l. 2005. use of donkey’s milk for a fermented beverage with lactobacilli. le lait, 85, 481-490. cintas l.m., casaus p., herranz c., hâvarstein l.s., holo h., hernández p.e. & nes i.f. 2000. biochemical and genetic evidence that enterococcus faecium l50 produces enterocins l50a and l50b, the sec-dependent enterocin p, and a novel bacteriocin secreted without an n-terminal extension termed enterocin q. j bacteriol, 182 (23), 6806-6814. claeys w.l., cardoen s., daube g., de block j., dewettinck k., dierick k., de zutter l., huyghebaert a., imberechts h., thiange p., vandenplas y. & herman l. 2013. raw or heated cow milk consumption: review of risks and benefits. food control, 31 (1), 251-262. coppola r., salimei e., succi m., sorrentino e., nanni m., ranieri p., belli blanes r. & grazia l. 2002. behaviour of lactobacillus rhamnosus strains in ass’s milk. ann microbiol, 52, 55-60. doreau m. & martin-rosset w. 2011. dairy animals: horse. in encyclopedia of dairy sciences, 2nd ed. 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food microbiol, 251, 67-72. souroullas k., aspri m. & papademas p. 2018. donkey milk as a supplement in infant formula: benefits and technological challenges. food res int, 109, 416-425. tidona f., sekse c., criscione a., jacobsen m., bordonaro s., marletta d. & vegarud g.e. 2011. antimicrobial effect of donkeys’ milk digested in vitro with human gastrointestinal enzymes. int dairy j, 21, 158-165. tomita h., fujimoto s., tanimoto k. & ike y. 1996. cloning and genetic organization of the bacteriocin 31 determinant encoded on the enterococcus faecalis pheromone-responsive conjugative plasmid pyi17. j bacteriol, 178 (12), 3585-3593. uniacke-lowe t., huppertz t. & fox p.f. 2010. equine 289 #these authors contributed equally to this work. 1faculty of veterinary medicine, university of bari, bari, italy. 2lebanese university, doctoral school of sciences and technology, beirut, lebanon. 3istituto zooprofilattico sperimentale della sicilia “a. mirri”, italy. 4urmite, um 63, cnrs 7278, ird 198, inserm 1095, aix-marseille university, marseille, france. 5lebanese agricultural research institute, fanar, lebanon. 6lebanese university, faculty of agriculture, beirut, lebanon. 7lebanese university, faculty of sciences, section i, hadath, lebanon. 8istituto zooprofilattico sperimentale dell' abruzzo e del molise, teramo, italy. *corresponding author at: faculty of veterinary medicine, university of bari, bari, italy doctoral school of sciences and technology (edst ), rafic hariri university campus, hadath-liban. tel.: +961 (3)959739, fax: +961-470941, e-mail: mayssaa.dabaja@gmail.com. keywords coxiella burnetii, mst, lebanon, ticks, milk. summary this study was carried out to detect and characterize coxiella burnetii in ruminant milk samples and in different tick species from seropositive farms in four lebanese regions. milk and tick samples were screened for c. burnetii presence by quantitative real-time pcr (qpcr) targeting is1111 region followed by multispacer sequence typing (mst). the overall positive percentages of 9.6% (27/282) and 95.45% (84/88) for c. burnetii were recorded in ruminant milk and tick samples, respectively. in detail, the c. burnetii dna was recorded in 52/54 (96.3%) of rhipicephalus annulatus, 20/21 (95.24%) of rhipicephalus turanicus, 6/6 (100%) of hyalomma anatolicum, 5/6 (83.3%) of rhipicephalus sanguineus and 1/1 of rhipicephalus bursa. after genotyping of some is1111-positive samples (17/111), different mst genotypes were identified. out of 15 positive ticks, 10 were infected with mst2 genotype, 4 were infected with mst7 genotype and 1 was infected with mst57. moreover, genotypes mst20 and mst58 were found in one cow and one goat milk samples, respectively. the present study confirmed the high genetic diversity of c. burnetii in lebanon. mayssaa fawaz dabaja1,2,5*#, grazia greco1#, valeria blanda3, maria tempesta1, ali bayan7, alessandra torina3, gesualdo vesco3, rosalia d'agostino3, rossella lelli8, mohamad ezzedine2,7, hussein mortada6, didier raoult4, pierre edouard fournier4 and mohamad mortada2,7 multispacer sequence typing of coxiella burnetii from milk and hard tick samples from ruminant farms in lebanon veterinaria italiana 2020, 56 (4), 289-296. doi: 10.12834/vetit.1799.13290.1 accepted: 17.06.2020 | available on line: 31.12.2020 is considered as potential weapon for bioterrorism (alibek et al. 1999). c.  burnetii reservoirs include many wild and domestic mammals (fernández-aguilar et  al. 2016), with ruminants being the main source for humans (berri et al. 2001, fournier et al. 1998). the bacterium may also occasionally be detected in arthropods including ticks (szymańska-czerwińska et  al. 2013). hard and soft ticks are one of the most important arthropods that are known to be naturally infected with c.  burnetii (cutler et  al. 2007, maurin et  al. 1999, angelakis et  al. 2010). ticks get infected with c.  burnetii during feeding on their animal host and introduction q fever is a zoonotic disease caused by coxiella burnetii worldwide distributed except in new zealand (cutler et  al. 2007). this pathogen is a small gram negative, intracellular bacterium (maurin et al. 1999, péter et al. 1988) that multiplies in the phagolysosomes of eukaryotic host cells (hackstadt et al. 1981, arricau-bouvery et al. 2005). this bacterium evolves in highly infective spore-like forms that are able to survive in the environment for several months (evstigneeva et  al. 2007). c.  burnetii is classified as a category ‘b’ agent by the centers for disease control (atlanta, usa) and 290 coxiella burnetii in lebanon dabaja et al. veterinaria italiana 2020, 56 (4), 289-296. doi: 10.12834/vetit.1799.13290.1 qpcr targeted to the is1111 region (klee et  al. 2006) (table i). additionally, to investigate for c. burnetii genotypic diversity in lebanon and to exclude cross-reactions with coxiella-like endosymbionts (duron et  al. 2015, elsa et  al. 2015), some of is1111 pcr-positive milk (n.  2) and tick (n. 15) samples from c.  burnetii seropositive animals (dabaja et  al. 2017, dabaja et  al. 2019) were investigated by using mst (glazunova et al. 2005). preparation of samples and pcr analysis total dna was extracted from milk and tick samples by using the pure link genomic dna kit (thermo fisher™ applied biosystems™, waltham, ma usa) as described by the manufacturer’s instruction. briefly, in order to extract dna, 200 μl of milk samples and/ or an hemilateral salivary gland of tick were mixed with 180 μl purelink genomic digestion buffer and 20 μl proteinase k, followed by an incubation at 55 °c with occasional vortexing until lysis is complete over 30  minutes for milk sample and from 4 hours until overnight for tick samples . the detection of the is1111 target (klee et al. 2006) of c.  burnetii in milk and tick samples was carried out by using a high sensitive qpcr (biorad cfx96 real time system). the is1111 was selected as a target because it is present in multiple copies in the genome of this bacterium (klee et al. 2006). the forward primer, cox-f (5’-gt ctta agg tgg gct gcg tg) and the reverse primer, cox-r (5’-ccc cga atc tca ttg atc agc) amplifies a 295 bp fragment that was revealed by a taqman probe (fam-agc gaacca ttg gta tcg gac gtt tat gg-tamra). the qpcr reactions were performed in a final volume of 25 µl using a mixture containing: 1x ssoadvanced universal probe supermix (bio-rad), 0.4 µm of each primer, 0.5 µm of probe, 2 µl buffer of amplification internal control 10x (applied biosystems by life technologies), 0.5 µl internal control of dna amplification 50x (applied by life technologies), 10 µl of dna extract, and h 2 o to volume. pcr parameters were as follows: incubation at 50 °c for 2  min, incubation at 95  °c for 5 min, following 45  denaturation cycles at 95  °c for 15  sec then annealing and extension at 60  °c for 1 min. each sample was determined in duplicate. the sample was considered as positive if the ct was < 40. genotyping of c. burnetii dna detected in tick and milk samples a partial number of tickmilkis1111 positive samples were submitted to the genotyping step by using the mst assay (glazunova et al. 2005). the limited sample size used in this step was due to the over 40 tick species can be naturally infected by this bacterium (maurin et  al. 1999). c.  burnetii can multiply to very high titer levels in the mid-gut and stomach cells of the infected ticks, which can excrete bacteria via saliva and feces. infected ticks can transmit c.  burnetii transtadially and transovarially (dorko et al. 2012). the transmission of c. burnetii to mammal hosts might occur via tick bites or by feces contamination of their wool and skin (nasphv and nasaho 2013, marrie et al. 1990). the middle east is the epicenter of the disease, and outbreaks have being reported in jordan (fuad et  al. 1998), syria (bottieau et  al. 2000), turkey (cetinkaya et al. 2000), iraq (faix et al. 2005), cyprus (cantas et  al. 2011) and iran (esmaeili et  al. 2016). lebanon has mediterranean climate that makes it suitable environment for q fever disease (dabaja et  al. 2019) and arthropods (dabaja et  al. 2017). genotyping characterization of c.  burnetii strains detected in animals, humans and in ticks is useful for epidemiological purpose. multispacer sequence typing (mst ) is a suitable tool to genotype c. burnetii strains because of its high discriminatory power (glazunova et al. 2005, walker et al. 2014). in the present study, the detection and the genotyping of c.  burnetii dna in milk and hard tick samples from ruminant farms in lebanon was performed, as no information is available in the area. materials and methods sampling the analyzed samples were collected under the frame of a previous cross-sectional study performed to evaluate both seroprevalence and via milk shedding of c.  burnetii in 1,633 animals from 429  ruminant farms distributed in 7 lebanese provinces (akkar, baalback-elhermel, bekaa, mount lebanon, nabatieh, north lebanon and south-lebanon) in 2014 (dabaja et al. 2019). breafly, in that study 39.86% of farms (95% ci: 35.23-44.56) and 17.27% (95% ci: 15.43-19.1) of ruminants resulted seropositive (dabaja et al. 2019). moreover, 27/282 (14.08%) milk samples from c.  burnetii seropositive animals were positive for the is1111 target of c.  burnetii (dabaja et  al. 2019). furthermore, 219 adult hard ticks from 30 seropositive farms were collected in june 2014, as previously described (dabaja et  al. 2017, dabaja et  al. 2019). collected ticks belonged to the genera rhipicephalus and hyalomma distributed in 5 species r. annulatus, r. turanicus, r. bursa and r. sanguineus and h. anatolicum (dabaja et al. 2017). in the present study, 2 or 3 ticks were selected from each farm. a total of 88 out of the 219 collected ticks were individually investigated by using 291 dabaja et al. coxiella burnetii in lebanon veterinaria italiana 2020, 56 (4), 289-296. doi: 10.12834/vetit.1799.13290.1 table i. prevalence of c. burnetii in ticks collected in june 2014 from ruminants in lebanon detected by real-time pcr. id farm locality town or village of origin province kind of farm id and species of ticks sex of ticks is 1111 cycle threshold: ct 1 barich *33°16'22''n **35°21'9''e ***358 m south lebanon bovine 1 h. a f 33.56 2 r. a f 26.6 3 r. a f 31.62 2 qinarit *33°30'17''n **35°22'44''e ***233 m south lebanon bovine 10 r. a f 25.0 11 r. a f 32.7 12 r. a f 25.87 3 aynedelbe *33°32'40.87''n **35°24'25.834''e ***41 m south lebanon bovine 13 r. a f 34.15 14 r. a f 22.32 15 r. a f 33.23 4 maaroub *33°17'6''n **35°20'49.2''e ***270 m south lebanon ovine 16 r. t f 26.15 17 r. t f 36.26 18 r. t f 23.19 5 zayta *33°30'29''n **35°23'03''e ***300 m south lebanon bovine 49 r. a f 28.08 50 r. a f 32.86 6 bourghlieh *33°18'36''n **35°14'24''e ***19 m south lebanon bovine 56 r. a f 31.76 57 r. a f 37.18 58 r. a f 33.06 7 hasbaya *33°23'n ***35°41'e ***750 m nabatieh bovine 4 r. a f 29.32 5 r. a f 30.66 6 r. a f 25.14 8 el koulayaa *33°19'48''n **35°34'12''e ***650 m nabatieh caprine + ovine 7 r.s f 37.19 8 r.b f 25.32 9 h.a f 34.43 9 zawtar el charkiyi *33°19' 33''n **35°28'34''e ***475 m nabatieh bovine 19 r. a f negative 20 r. a f 21.12 21 r. a f 34.25 10 mayfadoun *33°20'9.6''n **35°27'43.2''e ***470 m nabatieh bovine 22 r. a f 24.26 23 r. a f 30.86 24 r. a f 21.12 11 marjiiyoun *33°30'n **35°30'e ***860 m nabatieh bovine 25 h. a m 30.3 26 h .a m 34.0 27 h. a m 35.0 12 wata el khiyam *33°19'37.8''n **35°36'40''e ***700 m nabatieh caprine 28 h.a m 26.35 29 r.s m 32.49 30 r.s m 27.29 13 ibel el saki *33°12'36''n **35°22'48''e ***800 m nabatieh ovine 31 r. t f 34.5 32 r. s f negative 33 r. t f negative 14 el wazani *33°16'32''n **35°37'22''e ***297 m nabatieh bovine 34 r. a f 31.0 35 r. a f 32.8 36 r. a f 35.4 15 el wazani *33°16'32''n **35°37'22''e ***279 m nabatieh bovine 37 r. a f 30.2 38 r. a f 27.48 39 r. a f 34.49 16 el wazani *33°16'32''n **35°37'22''e ***279 m nabatieh caprine 40 r. t f 28.25 41 r. s f 37.7 42 r. t f 30.5 id farm locality town or village of origin province kind of farm id and species of ticks sex of ticks is 1111 cycle threshold: ct 17 aynebel *33°00'42''n **35°14'24''e ***800 m nabatieh bovine+ ovine 43 r. t f 35.35 44 r. t f 30.32 45 r. t f 34.36 18 kfarkila *33°10'12''n **35°19'48''e ***700 m nabatieh bovine 46 r. a f 33.2 47 r. a f 31.4 48 r. t f 35.0 19 rmeich *33°00'54''n **35°24''e ***690 m nabatieh ovine 59 r. t f 32.82 60 r. t f 28.84 61 r. t m 33.78 20 aynebel *33°00'42''n **35°14'24''e ***800 m nabatieh caprine 62 r. t f 30.85 63 r. t f 35.0 64 r. t f 30.8 21 aynebel *33°00'42''n **35°14'24''e ***800 m nabatieh bovine 65 r. a f 35.86 66 r. a f 34.14 67 r. a f 37.63 22 zahleh *33°50'48''n **35°4'07''e ***963 m bekaa ovine 51 r. t f 39.4 52 r. t f 30.7 23 zahleh *33°50'48''n **35°54'07''e ***963 m bekaa ovine 53 r. t f 33.44 54 r. s f 30.0 55 r. t f 32.54 24 machha *34°32'25''n **36°7'56''e ***349 m bekaa ovine 68 r. a f 30.89 69 r. a f 33.98 70 r. a f 31.6 25 adbel *34°32'6''n **36°57'50.4''e ***300 m akkar bovine 71 r. a f negative 72 r. a f 33.95 73 r. a f 30.93 26 al kantara *34°31'33.078''n **36°00'3.0711''e ***375 m akkar ovine 74 r. a f 36.24 75 r. a f 32.1 76 r. a f 36.55 27 machha *34°32’25’’n **367'56''e ***349 m akkar bovine 77 r. a f 33.0 78 r. a f 35.2 79 r. a f 33.41 28 michmich *34°21'24.0012''n **35°55'51''e ***1,100 m akkar bovine 80 r. a f 36.0 81 r. a f 31.47 82 r. a f 35.33 29 bazbina *34°31' 0'' n **36°12'0''e ***955 m akkar bovine 83 r. a f 32.84 84 r. a f 36.23 85 r. a f 34.65 30 sahel halba *34°33'2'' n **36°4'41''e ***120 m akkar bovine 86 r. a f 34.37 87 r. a f 32.13 88 r. a f 33.41 positivity percentage averall 84/88(+) (95.45%) ct average: 32 *latitude; **longitude ***altitude; f = female; m = male. r. a = rhipicephalus annulatus; h. a = hyalomma anatolicum; r. b = rhipicephalus bursa; r. s = rhipicephalus sanguineus; r. t = rhipicephalus turanicus. 292 coxiella burnetii in lebanon dabaja et al. veterinaria italiana 2020, 56 (4), 289-296. doi: 10.12834/vetit.1799.13290.1 database containing c.  burnetii genotypes from countries in europe and other parts of the world using blast; the new sequences were deposited in the available database on the website: http://ifr48. timone.univ-mrs.fr/mst_coxiella/mst/group_detail. results arthropods is1111 target was detected in 84 of the 88 (95.45%) investigated ticks from c. burnetii seropositive farms with ct value being between 21.12 and 39. positive samples included 52 of 54 (96.3%) r. annulatus, 20  of  21 (95.24%) r. turanicus, 6 of 6 (100%) h. anatolicum, 5 of 6 (83.3%) r. sanguineus and 1 of 1 r. bursa (table iii). milk among 282 milk samples from seropositive ruminants, is1111 dna had been detected in 9 of 86 (10.47%) cattle, in 6 of 93 (6.45%) sheep and in 12  of  103 (11.65%) goat specimens, as indicated in previous study (dabaja et al. 2019). mst because of the low amount of dna, it was possible to perform mst genotyping in 84 is111 positive ticks and 2 is111 positive milk samples only (tables iii and iv). as result of this study, three previously described genotypes and two incomplete ones were identified (tables iii and iv). of the 15 positive ticks, 10 hosted the mst 2 genotype, 4  the mst 7 genotype and 1 the mst 57 incomplete genotype. of the 2 positive milk samples, one was infected with the known mst 20 and the other one with the incompletely characterized mst 58 (tables iii and iv). mst 2 genotype was widely found in different genera and species of ticks from south lebanon and nabatieh; mst 7 was identified in r. annulatum and r. turanicus from bekaa and akkar whereas mst 20 was detected in the cow milk samples in bekaa region (table iii). mst 57 and mst 58 genotypes, incompletely characterized probably due to the low dna concentration, were found in one tick and one goat milk sample from nabatieh and bekaa, respectively  (table iii). discussion almost all the collected ticks (95.45%) from c.  burnetii seropositive ruminant farms in lebanon were positive for is1111 target. since is1111-based low amount dna remaining from each specimen to perform the mst. multi-spacer typing was performed on is1111 positive specimens using a set of primers targeting 10 variable spacers (cox: 2; 5; 18; 20; 22; 37; 51; 56; 57; 61) according to previous study (glazunova et al. 2005). five µl of dna preparation was amplified in a 50 µl reaction mixture containing 0.2 µm of each primer, 0.05 mm (each) datp, dttp, dctp and dgtp; 1.25 u taq polymerase; mgcl 2 2.5 mm and 1x taq buffer. dna from nine mile strain of c.  burnetii was used as positive control. amplifications were carried out using a 2720 thermal cycler (applied biosystems) according to the following conditions: an hot start step of 15 min at 95  °c, followed by 40  cycles of denaturation for 1 min at 95 °c, annealing for 30 sec at 59  °c, elongation for 1 min at 72  °c and final extension for 7 min at 72 °c. pcr amplicons were visualized by electrophoresis of 6 µl of the pcr product with 2 µl of blue loading buffer on 1.5% agarose gel (0.5xtbe) with sybersafe under uv light. pcr products were purified via vacuum filtration through the nucleofast 96 pcr plate (thomas scientific, dueren, germany), as described by the manufacturer. sequencing reactions were carried out using the big dye terminator cycle sequencing kit (applied biosystems). four µl of purified pcr were added to a 10 µl reaction containing 0.5 µl primers, 1.5 µl big dye buffer, and 1 µl big dye. the sequencing reaction was run in a thermal cycler as follows: an initial denaturation step of 1 min at 96  °c followed by 25 cycles of denaturation for 10 sec at 96  °c, annealing for 5 sec at 50 °c and elongation for 5 min at 60 °c, followed by a final step at 15 °c. sequencing reactions were purified using millipore sephadex plates (millipore, billerica, massachusetts) as per the manufacturer’s instructions, and stored at 4 °c until analyzed. sequencing reactions were analyzed on an abi 3130x genetic analyser (applied biosystems) and sequence assembly performed using the multisequence align software chromaspro (v.2.1.1). the obtained sequences were further compared with the sequences included in the mst reference table ii. percentage of ticks positive for is1111 detection by real-time pcr. tick species no. of positive ticks percentage of positive ticks (95%ci) rhipicephalus annulatus 52/54 96.3% (91.1-100) rhipicephalus turanicus 20/21 95.3% (94.88-95.72) rhipicephalus sanguineus 5/6 83 % (53-100) rhipicephalus bursa 1/1 hyalomma anatolicum 6/6 100% ci = confidence interval 293 dabaja et al. coxiella burnetii in lebanon veterinaria italiana 2020, 56 (4), 289-296. doi: 10.12834/vetit.1799.13290.1 2013), in r. annulatus in senegal (mediannikov et al. 2010), in r. sanguineus in iran, cyprus, italy and switzerland (bernasconi et al. 2002, nourollahi fard et al. 2011, satta et al. 2011, spyridaki et al. 2002), in r. turanicus in turkey, italy, switzerland and greece (capin et al. 2013, bernasconi et al. 2002, satta et al. 2011, psaroulaki et  al. 2006), in h. anatolicum in cyprus (spyridaki et  al. 2002), thus supporting the evidence that c.  burnetii seems to be endemic in ticks in more areas. in our study several mst genotypes of c.  burnetii were found in tick and milk samples from ruminant farms in lebanon. the most frequently detected mst 2 genotype was found in r. annulatus, r. bursa, r. turanicus and h. anatolicum ticks from the south lebanon and nabatieh regions. indeed, the same mst 2 genotype had already been detected in blood samples of human beings affected with acute q fever from france, ukraine and kyrgyzstan (glazunova et  al. 2005). mst 7 genotype, detected in both r. annulatus and r. turanicus ticks from the bekaa and the akkar regions, had already been found in france and russia in human blood samples and cardiac valves (glazunova et al. 2005). conversely, the sequence type detected in the cow milk sample from bekaa region was similar to mst  20. this genotype had been found in animal, human and tick samples from germany, france, spain, italy, hungary, the united kingdom, united states and netherlands and central africa. in pcr-assays designed to detect c.  burnetii cross react with coxiella-like bacteria (elsa et  al. 2015), the results of surveys carried out on ticks and based only on is1111 pcr assay should be interpreted with caution since ticks can harbor either c. burnetii or coxiella-like bacteria. in the present study, in order to exclude misinterpretations, some of the is1111-positive samples were genotyped by using mst that is based on the characterization of a set of targets (glazunowa et al. 2005). based on results obtained using this combined approach, different mst genotypes in tick and a few milk samples from different c.  burnetii seropositive ruminant farms of different provinces of lebanon were detected. c.  burnetii infection had already been recorded in r. bursa in turkey (capin et  al. table iii. multispacer sequence typing (mst ) genotypes of c. burnetii in ticks and milk specimens from ruminant farms in lebanon. sample id source & host species town or village of origin: gps coordinates, elevation province date cycle threshold: ct mst genotype 2 r. a (f) bovine barich: *33°16'22''n; **35°21'9''e; ***358 m south lebanon 6/2014 26.6 2 6 r. a (f) bovine hasbaya: *33°23'n;**35°41'e; ***750 m nabatieh 6/2014 25.14 2 8 r. b (f) ovine el koulayaa: *33°19'48''n; **35°34'12''e; ***650 m nabatieh 6/2014 25.32 2 10 r. a (f) bovine qinarit: *33°30'17''n;**35°22'44''e; ***233 m south lebanon 6/2014 25 2 14 r. a (f) bovine ayn eldeleb: *33°32'40.87''n; **35°24'25.834''e; ***41 m south lebanon 6/2014 22.32 2 20 r. a (f) bovine mayfadoun: *33°20'9.6''n; **35°27'43.2''e; ***470 m nabatieh 6/2014 21.12 2 24 r. a (f) bovine zawtarcharkiyi: *33°19'33''n; **35°28'34''e; *** 475 m nabatieh 6/2014 21.12 2 28 ha. a (m) caprine wataelkhiyam: *33°19'37.8''n; **35°36'40''e; ***700 m nabatieh 6/2014 26.35 2 38 r. a (f) bovine el wizani: *33°16'32''n; **35°37'22''e; ***279 m nabatieh 6/2014 27.48 2 40 r. t (f) caprine el wizani: *33°16'32''n; **35°37'22''e; ***279 m nabatieh 6/2014 28.25 2 44 r. t (f) ovine ayn ebel: *33°00'42''n; **35°14'24''e; ***800 m nabatieh 6/2014 30.2 57 52 r. t (f) ovine zahle: *33°50'48''n; **35°54'07''e; ***963 m bekaa 6/2014 30.7 7 53 r. t (f) ovine zahle: *33°50'48''n; **35°54'07''e; ***963 m bekaa 6/2014 33.44 7 70 r. a (f) bovine machha: *34°32'25''n; **36°7'56''e; ***349 m akkar 6/2014 31.6 7 75 r. a (f) ovine alkantara: *34°31'33.078''n; **36°00'3.0711''e; ***375 m akkar 6/2014 32.1 7 19 milk goat rayak: *33°51'3''n; **36°00'42''e; ***929 m bekaa 5/2014 37.74 58 3038 milk bovine alkarak: *33°51'n; **35°55'35''e; ***1000 m bekaa 3/2014 39 20 *latitude; **longitude ***altitude; f = female; m = male. r. a = rhipicephalus annulatus; h. a = hyalomma anatolicum; r. b = rhipicephalus bursa; r. s = rhipicephalus sanguineus; r. t = rhipicephalus turanicus. table iv. genotyping details of the detected strains according to multispacer sequence typing (mst ). m st ge no ty pe co x2 co x5 co x1 8 co x2 0 co x2 2 co x3 7 co x5 1 co x5 6 co x5 7 co x6 1 2 5 6 3 5 x 5 8 1 5 6 7 4 6 3 5 6 5 8 x 5 x 20 3 2 6 x 5 4 x 10 6 5 57 4 6 3 5 5 5 x x 5 6 58 4 8 2 x x x x x x x 294 coxiella burnetii in lebanon dabaja et al. veterinaria italiana 2020, 56 (4), 289-296. doi: 10.12834/vetit.1799.13290.1 conclusions this study provides the first molecular evidence of c.  burnetii as well as a preliminary picture of the genetic diversity of the q fever agent in different tick species and milk samples from ruminant farms in lebanon. although, based on the results of our study, there is no evidence of a role of ticks in the transmission of infection to the ruminants, appropriate biosecurity measures should be taken to prevent zoonotic risk since different genotypes were found in ticks and milk samples. acknowledgments we thank the director general of the lebanese agricultural research institute (lari) dr. michel afram, and our many friends from the ministry of agriculture lebanon who collect us milk and ticks samples from different region of the country. particular, mst 20 was most frequently associated with cattle but rarely with goats. mst 20 was found in a single goat sample in the netherlands (tilburg et  al. 2012), in two milk samples in the united states (pearson et al. 2014), and in a large goat herd with abortion problems in the united kingdom (reichel et  al. 2012). however, in most cases, the genotype mst 20 was associated with cattle and cow’s milk (glazunova et  al. 2005, astobiza et  al. 2012, pearson et  al. 2014, sulyok et  al. 2014, mediannikov et  al. 2010). these findings provide further evidence for the presumed host-specific adaptation of this agent. finally, although incompletely characterized for technical reasons, other two genotypes, mst 57 and mst 58 were detected in a r. 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scotland u.k, 74-217. spyridaki i., psaroulaki a., loukaides f., antoniou m., hadjichristdolou c. & tselentis y. 2002. isolation of coxiella burnetii by a centrifugation shell-vial assay from ticks collected in cyprus: detection by nested polymerase chain reaction (pcr) and by pcrrestriction fragment length polymorphism analyses. am j med hyg, 66 (1), 86-90. sulyok k.m., kreizinger z., hornstra h.m., pearson t., szigeti a., dán á., balla e., keim p.s. & gyuranecz m. 2014. genotyping of coxiella burnetii from domestic ruminants and human in hungary: indication of various genotypes. bmc vet res, 10, 107-112. szymańska-czerwińska m., galińska e.m., niemczuk k. & zasepa m. 2013. prevalence of coxiella infection in foresters and ticks in south-eastern poland and comparison of diagnostic methods. ann agric environ med, 20 (4), 699-704. 61 veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 accepted: 15.04.2021 | available on line: 27.07.2021 1veterinary teaching hospital, university of teramo, loc. piano d’accio snc, teramo, italy. 2department of life, health & environmental sciences, university of l’aquila, piazzale salvatore tommasi 1, l’aquila, italy. 3istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, teramo, italy *corresponding author at: veterinary teaching hospital, university of teramo, loc. piano d’accio snc, teramo, italy. e-mail: sarahsconza@libero.it. sarah sconza1*, giuseppe paradiso galatioto2, nicola d'alterio3, domenico robbe1, fulvio marsilio1 and augusto carluccio1 keywords non-epidemic emergency, earthquake, snowfall, veterinary first respond. summary the ultimate goal of any disaster response, or a natural or a man-made event, is to get the best outcome for the highest number of people. from a veterinary point of view, the best outcome includes either the protection of animals (conventional and unconventional pets) or the safeguarding the wholesomeness of food supplies in the “one health” perspective. the evolution of the italian veterinary role in disaster management has changed across the last 35 years and has grown with the awareness that animals and human beings share the same vulnerability to disasters. the university of teramo, following its experiences in different disaster scenarios, proposes a veterinary presidium to support public authority in responding to catastrophic events in the italian context, in order to rescue small, large and unconventional animals. the proposed veterinary presidium is made up of 3 skilled people certified to react to different population needs. indeed we propose different teams to rescue small, large or non-conventional animal, trained to work together in a stress situation and under coordination of the civil protection function 2. this presidium with its 3 different skilled teams under the supervision of the advanced veterinary medical center (avmc) and by reporting to it will provide the best competences based on the needs of the population and the authorities, in view of the “one health” perspective. proposal for a veterinary presidium to support public authority in responding to catastrophic events in the italian context classified as incident-related risks under the d.lgs. n. 334 of 1999 were registered (d.lgs. 334, 1999). even if in italy nuclear power reactors are no longer operational, there are plants in france, switzerland and slovenia that are less than 200 km away from the national border2. in this environmental context, laws on national risks had already been issued by pre-eu states, especially in relation to seismic risk. specific actions were implemented by the veterinary services after the disasters of seveso, chernobyl and dioxins in the ‘fires land’ (campania), as well as after the floods that struck liguria, piedmont, emilia-romagna and introduction several geographical, demographic and socio-economic variables make italy more exposed than other countries to natural, technological or anthropic disasters. within these scenarios veterinary services might indeed play a fundamental role (table i). according to ministry for environment, land and sea protection, 9.8% of the italian territory has a high hydrogeological risk (ministero dell’ambiente 2013). according to the seismic classification of the italian municipalities updated to 2010, 731 municipalities are in the high-risk zone, 1,909 in the medium risk zone and at least 2 million people live in areas exposed to volcanic risk (vesuvius, campi flegrei, etna, aeolian islands)1. with regard to technological risks, at april 30, 2012, 1,152 plants 1 http://www.protezionecivile.gov.it/jcms/it/classificazione.wp. 2 http://www.protezionecivile.gov.it/jcms/it/attivita_nucleare.wp. 62 veterinary presidium in case of catastrophic events sconza et al. veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 local authorities capable to respond to catastrophic events should be established. this presidium could support national veterinary services in all the disaster phases: in prediction and in prevention, in disaster preparedness, first answer and in the emergency overcoming. veterinary medicine and disaster: guaranteed activities animals and humans share the same vulnerability during disaster. massive deaths of animals due to catastrophic events have led to severe famines in the past centuries. in addition, as a result of natural disasters, an increase in epidemic risks is recognized. given the huge damage resulting from the disasters, since the last decades of the last century western countries have developed and organized a civil protection system. it was based on the coordination and integration of the various bodies involved in disaster recovery and on the principles of humanity, neutrality and impartiality, enshrined in the oslo guidelines (oslo guidelines 2007). the organizational models vary according to the political and administrative characteristics in the different countries. for example, in usa the veterinary response to disasters was mainly developed by veterinary associations, in particular by the american veterinary medical association (avma). thanks to the efforts to create a systematic approach to disasters, it was able, in may 1993, to include veterinary services in the national response plan for disaster recovery, getting them into the national disaster medical system (ndms) (lundin 1994). under the direction of the department of agriculture, in 1994 the veterinary medical assistance teams (vmat) became operative in the control, treatment and eradication of animal disease outbreaks, evolving in 2007 into national veterinary response team (nvrt) (wingfield and palmer 2009). its responsibilities include assessing the veterinary medical needs of the community, caring for medical treatment and stabilization of animals implementing animal and zoonotic disease surveillance. as official responders to a disaster or emergency3, nvrt should also gather information about hazardous substances and provide technical assistance to assure food safety and water quality, hazard mitigation and care and support of animals. in france, the response to emergencies is articulated in different levels: municipal (mayor), departmental (prefect), zonal (interregional) and central (national). at each level, a veterinarian is in charge of coordinating all activities related to animal tuscany, the earthquakes which occurred in abruzzo and emilia romagna and the massive landslides that hit campania. it was, however, after the earthquake of campania and basilicata in november 23, 1980, that it was proved first the importance of the veterinary interventions during emergency. it was the first step towards a modern system of civil protection and the practical application of veterinary emergency management (mantovani et  al. 1998a). this pioneering experience has promoted a cultural movement that has led to an increasing attention and preparation of local veterinary services for the response to non-epidemic emergencies. ministry of health had established the activities during both the preparoty phase and the intervention one (circolare ministero della salute 1992). in 1998 the department of civil protection has drawn up the guidelines about the management of non-epidemic emergencies, that has been updated in 2002 (d.p.c. 1998). the evolution of non-epidemic emergencies management in italy is not linked to scientific acquisitions but to socio-cultural factors, legislative production and to the change of the country's istitutional framework. this paper proposes, through an excursus on the veterinary approaches in the different countries, to identify intervention guidelines for the management of animals during natural catastrophe. in some areas of the italian territory, especially in small isolated areas and in difficult environments, the national veterinary services need support to fully guarantee all the required activities, as in the case of the l'aquila and central italy earthquake, in 2009 and 2016, respectively. from these observations it becomes evident that in the area affected by the disaster a veterinary presidium, coordinated by the health function together with the civil protection system and/or table i. description of the different types of disaster (mantovani et al. 1998b). natural disasters technological disasters earthquake leaks and chemical contamination flood joints of radioactive materials avalanches and landslides large-scale intoxication exceptional snowfall explosions fires massive accidents volcanic eruptions collapse of dams storms and tidal waves massive movements of populations and refugees hurricanes typhoons massive invasions of wild and / or synanthropic animals drought and famine 3 http://eeda.cfsph.iastate.edu/11jan/l_role/vetrole0080. php?frames=frameless. 63 sconza et al. veterinary presidium in case of catastrophic events veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 although the focus of the veterinary disaster response is the veterinary services of local health authorities, other veterinary institutions may be involved in disaster management, i.e. istituti zooprofilattici sperimentali, regional veterinary offices, local practitioneers and groups of experts from other regions or from other public veterinary istitution (i.e. army, university) as well as non-governative organizations (ngo) that, if coordinated by the veterinary services, could give their support in taking care of the rescued animals, as regulated by the memorandum between civil protection department and the ngos (protocollo d’intesa 2018). the veterinary actions that have to be guaranteed during non-epidemic emergencies according to the italian model are illustrated in table ii. veterinary response presidium to catastrophic events: intervention guidelines in 2016, the world organisation for animal health (oie) has developed these guidelines for disaster management and risk reduction in relation to animal health, animal welfare and veterinary public health with the goal of strengthening health and food hygiene, within the organization of relief efforts. most of the operational responses are under the responsibility of the fire department, which includes both a medical and health service (sssm) and well-organized veterinary bodies (vétérinaires sapeur pompier, vps4). the fields of action of the vsps are: prevention and response to technological and biological risks, inspection and control of live animals and foodstuffs of animal origin, veterinary support in recovery operations, training and follow-up of rescue teams with dogs, management of animals and materials for veterinary actions, health and hygiene education, sampling and advice on deceased animals, advice to the fire brigade command about the risks of food and environmental damage during disasters and advice to the fire brigade command about the expected risks and their prevention5. in italy, the veterinary services fall under the remit of the ministry of health. the organization of health services is commissioned to the regional administration and is divided into local health units. in accordance with d.lgs. 502/1992, the veterinary services, together with the services of public hygiene, food hygiene and nutrition and occupational medicine, report to the prevention department (d.lgs. 502 1992). the law 225/1992 established that the italian civil protection (l. 225 1992) and veterinary services, as members of the national health system, are activated in emergency situations by the local civil protection authority (l.  225 1992). nowadays, this law was repealed and replaced by the d.l. 1/2018 in which the civil protection code that gives new definition and purpose of the national civil protection service, was introduced. in particular, this law defines the national civil protection service as the system that exercises the civil protection function consisting of the set of skills and activities aimed at protecting life, physical integrity, property settlements, animals and the environment from damage or the danger of damage resulting from natural disasters or from human activity. the response to disasters is managed by coordination centers at municipal, provincial and national level. in each of these centers, all public and private bodies are represented according to their functions. the veterinary activities are coordinated within function 02 health, social and veterinary assistance, at the head of which a first-aid officer is usually placed. however, the flexibility of the system allows organizational structures to be modulated according to need. 4 https://www.veterinaire.fr/fileadmin/cru-1616430119/user_upload/ documents/profession/code-de-deontologie/1890_code_deonto07-04-bd.pdf; http://www.veterinairespompiers.fr/. 5 http://www.recrut.com/metier/v%c3%a9t%c3%a9rinaire-_sapeurpompier_553. table ii. activities provided by the veterinary services according to the italian organization (dpc 1998, mantovani et al. 1998b). natural disasters immediate actions subsequent actions identification of every possible food resource available and determine if the products are still edible and safe restore normal slaughtering activities, meat inspections, milk collection and storage; organization of treatment or slaughter of injured animals and identification of those still destined for human consumption supply medicines, vaccines, disinfectants and pesticides; destruction or containment of carcasses or animal waste disinfection of areas and companies at risk; providing shelter, food and water to the dispersed animals establish epidemiological surveillance; control of zoonosis evacuate animals from areas at risk (in case of repeatable events) pest control food security technological disasters recognize the origins of contaminants at risk remove or shelter animals from polluted areas prevent animals from feeding on contaminated fodder or pasture monitor contaminants in meat, milk and other animal products ensure toxicological surveillance among the animal population 64 veterinary presidium in case of catastrophic events sconza et al. veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 epidemics or environmental emergencies by monitoring animals, feeding stuff and foodstuffs of animal origin. the mentioned objectives are in line with the concept of italian civil protection and with the government's desire to integrate veterinary services into the public health system (leonardi et al. 2006, d.p.c. 2001). according to some authors, these different phases should not be considered in a strictly chronological order but recovery activities, planning and risk assessment could be developed simultaneously but, for a better quality of the interventions following the disaster, preparation and planning of risks and coordination and recovery activities are necessary. in this regard, the authors suggest the realization of a veterinary presidium for response to catastrophic events that could support the national response system in the preparation and approach to disasters and other emergencies, acting as primary intervention and guaranteeing high quality assistance for all small, non-conventional and large animals with different skilled teams. in order to optimize actions on the disaster scene, the veterinary presidium must consider a basic operative team composed by a veterinary surgeon (qualified either in small, large or unconventional animal), a veterinary technician and an epidemiologist, trained to work together in situations of strong emotional stress, in a non-permissive environment and in highly critical conditions. the recovery and advisory team for either small, large or unconventional animals is included both in the first phase of rapid response and in the deferred one (about 72 hours after the catastrophic event) and, in the latter case, it appears to support the advanced veterinary medical center (avmc). the avmc is a modular self-erecting structure with telescopic opening, easy to identify in the tent field tent and with high internal brightness. the avmc guarantees treatment, hospitalization of the rescued small and unconventional animals and of those of the guests of the tent emergency area, a diagnostic screening by images (ultrasound) and/or laboratory (blood-biochemical, chemical-physical urine tests), behaving as provided for the 2nd level advanced medical position (amp). for large animals, paddocks or shelters should be set up to monitor rescued animals pending a second long-term placement, in cooperation and/or coordination the local health units. this presidium will work in symbiosis with the coordination and control line of medical and veterinary area at the municipal, provincial, regional and national level. this model, which in some respects follows the french model, includes, in addition to the operating teams, also figures of coordination at the level, in the first instance, of the capacity of veterinary services in member countries. the document does not prescribe how veterinary services should act, but leaves it to each oie member country to adapt to local needs based on their context. it identifies inter-sectoral and multi-disciplinary approaches as essential principles in disaster management and stresses that the plans of veterinary services should be included in the national disaster management and risk reduction plans for non-epidemic emergencies, in issues related to climate change (floods, hurricanes, droughts, famines, etc.), earthquakes, nuclear and industrial accidents and accidental or intentional epidemics caused by man (oie, 2016). the different experiences in italy and in other countries suggest that the veterinary services should be active in the different phases of a disaster: • mitigation and prevention; these activities aim at early warning of epidemic and / or environmental risks, monitoring events and defining prevention measures whenever possible: epidemiological surveillance of zoonosis and animal diseases in general; control of the entire chain of meat production, dairy products and other food products of animal origin; environmental epidemiology; • disaster preparedness: “local preparedness plan” organised by the local veterinary services as an integral part of the national response system to a catastrophic event; integration of veterinary services in a general prevention plan at different levels (local, regional, national); training of veterinarians and other figures involved in the civil protection system (firefighters, volunteers, rescue teams with canine support); • first respond: advanced veterinary support in the operating areas to ensure assistance to rescuers and save and search animals; advice on the “health” risk of first responders in relation to animals and food of animal origin; animals as psychological support to the survivor; • recovery-overcoming the emergency: ‘reconstruction’ of routine veterinary assistance and veterinary public health; contribution in verifying the onset of 65 sconza et al. veterinary presidium in case of catastrophic events veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 biphasic consequential response to the needs of health aid (d.m. 2001), that are: 1. a rapid response, given by the territorial organization on the basis of immediately available local resources; 2. a deferred response, which will be articulated in the hours following the event with the contribution from the outside (d.m. 2001). both answers include: • an alarm phase, during which we will try to acquire all those elements that can be useful to size the event both in terms of quality and quantity. this phase can be preceded by the attention and early warning phases (in this succession) when you are faced with a predictable event; • an emergency phase in which all the operations necessary for rescue will be carried out (d.m. 2001). as described above, the avmc is designed to be used in emergencies that go beyond the possibilities of response of local structures (d.lgs. 1/2018). this structure must: • be ready for use as soon as possible after the alarm (3-4 hours); • be able to treat 50 patients with a red-yellow code within 24 hours and for three days; • have 72 h of operational autonomy; • be able to treat at least 50 yellow/red codes per day. in order to be sure of the suitability of drugs and consumables concerning conservation, validity and price, the supply of the 2nd level amp is entrusted to a hospital pharmacy identified from every single region. this choice must take into account the logistic aspects for the good functioning of the amp, and the territorial and forecast elements of the risks (p.c.m. 2003). the presidium pursues the following essential tasks that may slightly change depending on the catastrophic scenario: each team skilled either for small, unconventional or large animals rescues animals using the triage system and in shortest possible time, in respect of the “golden hour” rule. the veterinary triage begins by verifying: • the patient's medical needs • the available medical resources. when compared to the emergency triage of human medicine, evaluations and treatment decisions are different, due to the difference between the two medicines. factors responsible for these differences are: function 2 of the mixed operating center (com). the head of the health department is responsible for human health, veterinary and social assistance. it must therefore have a veterinary referent who, based on the knowledge of the livestock heritage of the area involved in the event, will coordinate the interventions carried out by the teams and will request, if necessary, additional personnel, emergency vehicles and equipment. all these professional figures always act under the guidance of the civil protection system and/or local authorities in the area struck by the disaster and in complete communion of intents. they should: • assess the medical and veterinary needs of the affected community; • provide treatment and stabilization of injured animals; • implement epidemiological surveillance of animal diseases and zoonoses and assess risks to public health; • provide technical assistance to ensure the quality of food and water; • apply measures to reduce of hazards; • provide care and support for animals, guaranteed by a qualified expert in disaster and emergency response. it is a modular presidium in which to channel specialized resources with specific skills, as well as ready-to-use emergency facilities in response to critical and emergency medical situations connected to the veterinary emergency. this disaster veterinary presidium must necessarily meet different needs on the disaster scene and must have: 1. recovery and consulting team for small animals; 2. recovery and consulting team for large animals; 3. recovery and consulting team for unconventional animals. the current international geopolitical conditions have opened up a new emergency front in which the health security of the migrant population coincides with the public health needs of the host population. in this condition of continuous emergency and persistent epidemic risk, it is necessary to have clear guidelines and tested effective operational plans but also health/veterinary presidia that respond to specific needs of ‘self protection’ and ‘force protection’. the activities that are set up in an emergency scenario, are carried out by authorities responsible for the response to the territorial emergency and by the health manager of the function 2 in the coordination centres. however, it should always be considered that every disaster event shows a 66 veterinary presidium in case of catastrophic events sconza et al. veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 table iii. schematic overview of veterinary interventions based on the type of disaster. re co ve ry ev ac ua tio n re sc ue re m ov al sa fe ty eu th an as ia earthquake * * * * * * volcanic eruptions * * * * * * avalanches and landslides * * * * flood * * * * exceptional weather events * * * * fires * * * * nuclear, radiological, biological and/or chemical event * * * * * humanitarian emergencies * * * * * * • identification of the sources of food supply for the animals remaining available; • identification of the facilities that remained active and usable. of particular importance is the availability of places for the shelter of live animals, areas for the collection of carcasses, plants for their destruction (incinerators) and landfills; • identification of mobile shelter structures (prefabricated, tents) and fencing nets, in order to create a shelter for small animals owned by the displaced within the tent camp; • control of synanthropic animals; • control and recovery of unconventional and possibly dangerous animals, e.g. snakes, spiders; • epidemiological surveillance and possible prophylaxis; • epidemiological surveillance of transmissible animal diseases, and in particular of zoonoses; • epidemiological surveillance of toxicity and chemical and radioactive contamination phenomena; • control of vectors of infectious diseases. a schematic overview of veterinary interventions based on the type of disaster is resumed in table iii. to ensure the feasibility of these activities, the recommended equipment of the veterinary teams is as follows: • vehicles: veterinary teams must also be able to make available refrigerated transport vehicles for any requests that may come from legal institutions following catastrophes; • orientation systems: local maps, gps, compass; • material for sampling and field analysis; • the euthanasia option; • the little margin of evaluation between the favourable and prolonged course of animal patients, considering long-term or permanent disabilities or recourse to intensive care; • the difficulty of transport for large numbers of animals and some kind of species, as the non-conventional ones; • limited veterinary medical resources (structures, materials, spare parts, and personnel, varying assistance capacity in the 24 hours); • recognizing that the care given to animals still depends on the owner's disposable income, the “medical resources” do not only include facilities, materials, spare parts, personnel and time but also money. in a catastrophe, all the animals will receive the first courses, independently of the economic possibility of the owner (wingfield and palmer 2009, wingfield et al. 2009). this system requires a color code (red, yellow, green, orange, blue and black) that is well coded internationally to recognize the timeliness of the intervention (wingfield and palmer 2009, wingfield et al. 2009): • admission to the avmc, feeding and eventual treatment of the animals affected by the catastrophe that need it; • medical support for sar k9 dogs, for research and recovery; • medical screening for each animal that arrives or leaves the disaster site; • transfer of animals to a safe area; • control of any euthanasia, in order to guarantee the least possible suffering to the animal; • removal and disposal of carcasses with the organization of collection points that are easily accessible to the vehicles, taking into account the advisability of not creating excessive difficulties for the road traffic of rescue vehicles; • capture and identification of wandering animals and consequent control of stray dogs; • organization of shelters and animal care, as follows: evacuation shelter for animals: animals kept in structures near the reception area of the owners, to facilitate the evacuation and care of them; response shelter: a kennel in which the animals are identified and can be found by their owners; pet friendly shelter: organization in which animals and people are housed together; 67 sconza et al. veterinary presidium in case of catastrophic events veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 of the l’aquila, students, veterinary technicians and voluntaries. the reception area of piazza d'armi has proved to be an aquila example of “pet friendly shelter”, counting the following population: • > 2,500 people; • 46 dogs; • 21 cats; • 1 parrot; • 10 canaries. during its activity, which ended june 8, 2009, the unite tent provided the following services: • veterinary assistance activities in the l'aquila area; • integrated health action in the context of amp; • prevention, control and management of the problems inherent to forced human or animal cohabitation: education of the owners (leash, excrement collection, muzzle); health care; behavioral assistance (dr. gentile aq); zoonosis prevention and control; • census of all the animals present in the field; • control of: ticks; fleas; intestinal parasites; rats (deratting); vaccination (leptospirosis); • reporting and/or checking of ‘dangerous’ or problematic animals within the reception area; • management of ‘problematic’ or problematic dogs of human/animal cohabitation; • preparation of external cages for the sheltering of ‘problematic’ animals; • reporting of wandering animals; • reporting to the coordination of lost/ abandoned animals; • participation in the reunion of animals/owners; • distribution of food and animal support material (collars, leashes, dog beds, cat litters, carriers/cages, plastic bags for collecting excrements); • reporting the presence of rats or mice; • practical training for veterinary medicine students; • distribution of medicines and health consumables for small and large animals • protective gear, clothing and footwear; • writing tools; • work tools; • media and radio communication; • lighting; • disinfection; • documentation through pictures; • tools for the capture and euthanasia of animals; • triage tools, according to law (d.p.c.m. 2007); • first aid set. unite experiences on disaster scenario l’aquila earthquake 2009 (sconza and paradiso galatioto 2015) following the earthquake that struck l'aquila on april 6th 2009 at 03.32 am, the faculty of veterinary medicine of teramo (unite) expressed its willingness to the president of the veterinary order of l'aquila, as early as 7 april, and sent some volunteers to piazza d'armi for veterinary assistance interventions. thanks to the availability and collaboration of the italian red cross, teramo provincial committee, in the figure of the president dr. valentino ferrante, on april 14th, the unite veterinary tent was activated at the base camp of piazza d’armi, clearly visible and integrated into the amp. as proposed in this work for the avma, the tent was equipped with: • examination tables; • ultrasound machine; • surgical basic set; • cage shelters; • infusion pumps; • drug closet; • equipped laboratory for emergencies including: blood cell counter; blood chemical and electrolytes analyser; urine test; centrifuge; • microscope; • material storage area; • food storage area. the tent was active 7 days a week from 8.30 am to 6.00 pm and included the use of veterinary surgeons at unite (22 figures), some practioneers 68 veterinary presidium in case of catastrophic events sconza et al. veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 conclusions from the historical excursus of the veterinary medicine on emergencies and from the unite field experiences it emerges that, as for the human medicine, even veterinary medicine should consider these 4 general principles: 1. mitigation phase: mitigation is the most costefficient method for reducing the impact of hazards. a precursor activity to mitigation is the identification of risk; 2. preparedness phase: this phase is a continuous cycle of planning, organizing, training, equipping, exercising, evaluation, and improvement activities that allows the nation to ensure effective coordination and the enhancement of capabilities to prevent, protect against, respond to, recover from, and mitigate against disaster events. it includes resources identification, development of a local emergency plan, exercises (round tables and practical exercises), database of animals identification and emergency teams at the local level; 3. response phase: it occurs during an emergency and includes the mobilization of the identified emergency staff, including first responders, to a disaster. response procedures should be pre-determined by the university and hospital, and are detailed in disaster plans during the preparedness phase; 4. recovery phase: this phase comprises the actions taken to return to the ‘status quo’. the recovery phase occurs after an emergency is over. typically, this involves rebuilding and making sure that the business have the possibility to be restored. veterinary plans to respond to disasters must be locally organized according to the attitudes and vocations of the territory but must never neglect the basic needs of the animals (wingfield 2015): • rapid assessment of needs; • animal shelter; • animal evacuation and transport; • control and capture of animals; • animal-owner reunification; • research and recovery of animals; • admission to the site and feed the animals; • critical infrastructure support; • veterinary assistance; • animal decontamination; • management of wild animals; • management of unconventional animals; • management of animal mortality. donated by companies in the veterinary sector to veterinary practitioneers of l’aquila; • admission and adoption at the chiareto teaching farm (unite); • ‘psychological’ assistance and support to owners of pets; • guaranteed zoological assistance for: animals owned by people evacuated in the various tent camps; animals left near home with evacuated owners; stray or lost animals; reference center for all other veterinary care facilities. from april 14th to june 8th, the report of the zooiatric activity carried out on small animals counted the assistance of about 1,200 animals, with transport and hospitalization of 78 subjects at unite. this activity was daily reported to civil protection veterinary crisis unit in order to monitor medical and veterinary actions. abruzzo exceptional snowfall 2017 in january 2017, the abruzzo region was hit by several weeks of heavy snowfall and earthquakes. this disaster combination has caused several deaths in people and a lot of damages to livestock and their farms. unite was called to the authorities working table as integral part of the command group and has made available 15 vets and vehicles in order to give practical support to the local public health veterinary service. in particular, these vet teams coordinated by the civil protection authorities and local public health veterinary service was sent to the territory, in order to: • search animal stuck in the rubble; • check their health status and eventually perform euthanasia; • verify and complete damages check lists; • make a detailed census of live animals and of those which died because of snowfall and/or provide a list of the damages caused by the earthquakes to their farmstead; • report local livestock needs to the authorities. in this contest, the unite vets collaborate with other teams to guarantee population support. 69 sconza et al. veterinary presidium in case of catastrophic events veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 intervention must be guaranteed from the first hours, especially as first aid teams, previously organized, prepared and trained. in particular, trained professional figures able to work with unconventional pet must be involved in the disaster support (quarta and leonardi 2015). these first aid teams can contribute to multidisciplinary collaboration (busani et  al. 2006, calicchia et  al. 1992), and to the integration between the various ‘knowledge’ to pursue a single final purpose: the reduction of the damage caused by disasters in view of the uniqueness of public health. the significantly negative impact on the communities affected by disasters due to the absence of a veterinary plan is supported by an extensive bibliography (heath et al. 2001, levy et al. 2011, zottarelli and bronken 2010, hunt et al. 2008, ivers and ryan 2006, ketai et  al. 2006, wang et  al. 2010, garde et al. 2013a, garde et al. 2013b, pasquali et  al. 2006, garde et  al. 2013c), few legislative acts (l.r. emilia-romagna 1 2005) indicate that veterinary activity must be guaranteed within 24  hours of the disaster. from this work, emerges that a first-respond busani l., caprioli a., macrì a., mantovani a., scavia g. & seimenis a. 2006. multidisciplinary collaboration in veterinary public health. ann ist super sanità, 42, 397-400. calicchia m.c., mantovani a. & scorziello m. 1992. intersectoral cooperation training: a tool for health personnel development. ann ist super sanità, 28, 507-510. decreto legislativo (d.lgs). 2018. decreto legislativo 2 gennaio 2018, n. 1. codice della protezione civile (off j, 17, 22/01/2018). decreto legislativo (d.lgs.). 1992. decreto legislativo 30 dicembre 1992, n. 502. riordino della disciplina in materia sanitaria, a norma dell'articolo 1 della legge 23 ottobre 1992, n. 421 (off j s., 305, 30/12/1992). decreto legislativo (d.lgs.). 1999. decreto legislativo 17 agosto 1999, n. 334. attuazione della direttiva 96/82/ ce relativa al controllo dei pericoli di incidenti rilevanti connessi con determinate sostanze pericolose. (off j s., 228, 28/09/1999). decreto ministeriale (d.m.). 2001. decreto ministeriale del 13.02.2001. adozione dei criteri di massima per l’organizzazione dei soccorsi sanitari nelle catastrofi (off j, 81, 06/04/2001). dipartimento della protezione civile (d.p.c.). 1998. linee guida per l’azione veterinaria nelle emergenze non epidemiche. dipartimento protezione civile, roma. dipartimento di protezione civile (d.p.c.). 2001. comunicato relativo al decreto del ministro dell’interno delegato per il coordinamento della protezione civile 13 febbraio 2001, concernente: adozione dei “criteri massima per l’organizzazione dei soccorsi sanitari nelle catastrofi” (off j, 81, 06/04/2001). direttiva del presidente del consiglio dei ministri (d.p.c.m.). 2007. direttiva del presidente del consiglio dei ministri del 13 dicembre 2007. procedure e modulistica del triage sanitario nelle catastrofi (off j, 91, 17/04/2008). garde e., acosta-jamett g. & bronsvoort b.m. 2013a. review of the risks of some canine zoonoses from references free-roaming dogs in the post-disaster setting of latin america. animals, 3, 855-865. garde e.j., pérez g., acosta-jamett g. & bronsvoort b.m. 2013b. characteristics of a canine distemper virus outbreak in dichato, chile following the february 2010 earthquake. animals, 3, 843-854. garde e., pérez g.e., acosta-jamett g. & bronsvoort b.m. 2013c. challenges encountered during the veterinary disaster response: an example from chile. animals, 3, 1073-1085. guidelines on the use of foreign military and civil defence assets in disaster relief. oslo guidelines, revision 1.1 november 2007. www.refworld.org/ pdfid/47da87822.pdf. heath s.e., voeks s.k. & glickman l.t. 2001. epidemiologic features of pet evacuation failure in a rapid onset disaster. javma, 218, 1898-1904. hunt m., al-awadi h. & johnson m. 2008. psychological sequelae of pet loss following hurricane katrina. anthrozoos, 21, 109-121. ivers l.c. & ryan e.t. 2006. infectious diseases of severe weather related and flood related natural disasters. curr opin infect dis, 19, 408-414. ketai l., currie b.j. & alva lopez l.f. 2006. thoracic radiology of infections emerging after natural disasters. j thorac imaging, 21, 265-275. legge (l.). 1992. legge 24 febbraio 1992, n. 225. istituzione del servizio nazionale della protezione civile (off j s., 64, 17/03/1992). legge regionale (l.r.) emilia-romagna. 2005. legge regionale emilia-romagna 7 febbraio 2005, n. 1 che modifica la l.r. 26 luglio 2012, n. 9 . norme in materia di protezione civile e volontariato. istituzione dell’agenzia regionale di protezione civile. leonardi m. & diegoli g. 2013. attività veterinarie nelle emergenze non epidemiche. in gli anmali, l’uomo e l’ambiente: ruolo sociale della sanità pubblica veterinaria (g. battelli, r. baldelli, f. ostanello, s. prosperi, eds). bononia university press, bologna, 250-260. 70 veterinary presidium in case of catastrophic events sconza et al. veterinaria italiana 2021, 57 (1), 61-70. doi: 10.12834/vetit.2115.115013.1 civile e le onlus, 2018. http://www.protezionecivile. gov.it/documents/20182/990040/protocollo+dpc_ animalisti/2bed4b4d-44dc-472b-a33c-fa0da64e6fd7. quarta p. & leonardi m. 2015. disaster planning per detentori di rettili ed altri animali esotici. in la gestione delle emergenze veterinarie. marsilio, aracne, ariccia, 129-146. sconza s. & paradiso galatioto g. 2015. proposta di un dispositivo veterinario di risposta ad eventi catastrofici. in la gestione delle emergenze veterinarie. marsilio, aracne, ariccia, 147-174. wang l.y., wu w.p., li s.z., fu q., wang q., tian t. & yang s.j. 2010. the risk evaluation and response to the spread of hydatid disease after yushu earthquake in qinghai province. chin j parasitol parasit dis, 28, 315-317. wingfield w.e. & palmer s.b. 2009. veterinary disaster response. wiley-blackwell, iowa. wingfield w.e. 2015. issues in veterinary disaster medicine. http://www.dcavm.org/disastermedicine.pdf. wingfield w.e., nash s.l., palmer s.b. & upp j.j. 2009. veterinary disaster medicine: working animals. wiley-blackwell, iowa. world health organization (who). 2002. world health organization study group. future trends in veterinary public health: report of a who study group. who, geneva. world organization for animal health (oie). 2016. guidelines on disaster management and risk reduction in relation to animal health and welfare and veterinary public health. oie, paris. https://www.oie.int/fileadmin/ h o m e / e n g / a n i m a l _ we l f a r e / d o c s / p d f / o t h e r s / disastermanagement-ang.pdf. zottarelli l.k. 2010. broken bond: an exploration of human factors associated with companion animal loss during hurricane katrina. sociological forum, 25, 110-122. leonardi m., borroni r. & di gennaro m. 2006. veterinary medicine in disasters. ann ist super sanità, 42, 417-421. levy j.k., lappin m.r., glaser a.l., birkenheuer a.j., andersen t.c. & edinboro c.e. 2011. prevalence of infectious diseases in cats and dogs rescued following hurricane katrina. javma, 238, 311-317. lundin s.c. 1994. avma emergency preparedness and response guide. american veterinary medical association, schaumburg. mantovani a., caliguri v., leonardi m. & luini m. 1998a. the role of veterinary medicine in non-epidemic emergencies. proc. 1st european congress on emergency medicine. repubblica di san marino, 22-25 aprile, 185-188. mantovani a., leonardi m., luini m. & volpini a. 1998b. veterinary public health in emergencies. eur j emerg med, 5, 108-109. ministero dell’ambiente e della tutela del territorio e del mare. 2013. il rischio idrogeologico in italia, minambiente, roma, 2013. ministero della salute. 1992. circolare del ministero della salute n. 11 del 18 marzo 1992. piano organizzativo ed operativo per attività d’emergenza dei servizi veterinari. pasquali p., agrimi u., borroni r., busani l., graziani c., leonardi m., poglayen g., macrì a. & mantovani a. 2006. capacity building for surveillance and control of zoonotic disease under emergency conditions. fao, rome, italy. presidenza del consiglio dei ministri (p.c.m.). 2003. conferenza unificata seduta del 22.05.2003. criteri di massima sulla dotazione di farmaci e dispositivi medici di un posto medico avanzato di ii livello utilizzabile in caso di catastrofe. protocollo d’intesa tra il dipartimento della protezione 205 1department of veterinary medicine, faculty of agriculture and veterinary, university of prishtina “hasan prishtina”, republic of kosovo. 2department of animal science, university of wyoming, laramie, wy 82071, united states of america. *corresponding author at: department of veterinary medicine, faculty of agriculture and veterinary, university of prishtina “hasan prishtina”, republic of kosovo. e‑mail: driton.sylejmani@uni‑pr.edu. keywords antimicrobial susceptibility, brain, mic, ovine listeriosis, serotyping. summary in 2018, a case of neural disease suspected of listeriosis was reported in a flock of sheep in kosovo with the death of ewes and 5 lambs. samples from the brain of only three dead animals were subjected to histopathological and bacteriological analysis. maldi-tof ms was applied to confirm suspected listeria spp. isolates from culture and multiplex pcr was applied for molecular serotyping. all isolates were tested for antimicrobial susceptibility by microdilution broth method. the histopathological analysis of the brain specimens showed typical changes for listeria monocytogenes. listeria spp. was isolated in brain samples from all three animals, and all the isolates were confirmed using maldi-tof ms and pcr down to the species level (listeria monocytogenes). the molecular characterisation using multiplex pcr revealed all isolates as listeria monocytogenes serotype 4b. all l. monocytogenes isolates were found to be susceptible to penicillin, erythromycin, tetracycline, streptomycin, trimethoprim/ sulfamethosazole, quinupristin/dalfopristin, kanamycin, vancomycin, and gentamicin but resistant to nitrofurantoin and lincomycin. this study shows the emergence of a highly virulent strain in sheep farms in kosovo and a possible threat to public health. afrim hamidi1, bledar bisha2, izedin goga1, baolin wang2, avni robaj1 and driton shefqet sylejmani1* a case report of sporadic ovine listerial meningoencephalitis in kosovo veterinaria italiana 2020, 56 (3), 205-211. doi: 10.12834/vetit.2166.12781.3 accepted: 29.05.2020 | available on line: 31.12.2020 the brain along interneuronal connections both in retroand anteretrograde directions (oevermann et  al. 2010, granier et  al. 2011, henke et  al. 2015). listeriosis associated with encephalitis is more prevalent among ruminants, especially adult sheep, with an attack rate of 10-20% and a mortality rate of 5-10%, respectively. lambs as young as 5 weeks old may develop the septicaemic form of the disease, whereas older feedlot lambs (4-8 months old) develop the encephalitic form (wesley et  al. 2002, lozano et al. 2011). ruminants affected by listerial encephalitis generally show marked neurological symptoms, including ataxia, masticatory problems, failure of jaw closure, drooping of ears, upper eye lids and lips, swallowing problems, tongue palsy, circling, head tilt and leaning to one side, drooling of saliva, and facial paralysis (braun et al. 2002, kumar et  al. 2007, dons et  al. 2007, brugere-picoux 2008, rocha et  al. 2013). once l.  monocytogenes enters the brain in small ruminants, its elimination is introduction listeriosis is an infectious disease caused by listeria monocytogenes, a gram-positive, intracellular, nonsporulating rod, which affects a wide range of animals as well as humans (george 2002, nash et al. 1995). in both humans and livestock (sheep, cattle, goats, and less frequently, poultry), listeriosis can manifest as encephalitis, septicaemia, and abortion (bartt 2000, kumar et  al. 2007, brugere-picoux 2008). the disease usually occurs sporadically, thus outbreaks are only occasionally reported (walland et  al. 2015). in animals, listeriosis outbreaks are primarily described in small ruminants (goats, sheep) and cattle (wagner et al. 2005, budrant et al. 2011, dreyer et  al. 2015). the neurological form, otherwise known as circling disease, is usually observed in farm animals where the bacterium gains entrance to the body through abrasions of the buccal mucosa. it ascends unilaterally along the trigeminal nerve, resulting in encephalitis. l.  monocytogenes can spread intraaxonally within case report 206 listerial meningoencephalitis in kosovo sylejmani et al. veterinaria italiana 2020, 56 (3), 205-211. doi: 10.12834/vetit.2166.12781.3 case report case presentation in march 2018, 10 adult ewes as well as 8 lambs from a flock of 277 sheep in central kosovo displayed symptoms such as depression and anorexia followed by neurological signs. forty-eight hours from the initial onset of the symptoms, the affected ewes and lambs manifested severe clinical signs such as temperature of 40.5 °c, grinding of teeth (crunch empty), circling and tilting of the head, unilateral facial paralysis, drooping ears and lips and supporting the head against the manger and walls. within 7 days after the disease onset, 6 adult sheep and 5 lambs died. samples were obtained from the brainstem of the dead animals. a silage diet had been administered to the flock of sheep. based on the narrative provided by the sheep flock owner, one of the dead lambs (3 month old), was exclusively fed with milk from an ewe. histopathological analysis and findings in order to investigate the cause of the disease and the fatalities, samples from three dead animals (ewes and lamb) were obtained from the brainstem to conduct histopathological analysis. the samples were fixed in 10% buffered formalin, dehydrated, and embedded in paraffin, 5  µm sections were stained with hematoxylin and eosin (h&e), and observed with a light microscope with 10x-40x objectives. examination of the medulla oblongata revealed suppurative encephalitis characterized by microabscesses as well as neuroparenchymal lesions (figure 2 and 3, black arrow) in all affected animals. perivascular cuffing of lymphomononuclear cells was observed in the medulla oblongata with enhanced virchow-robin spaces (figure 1, black arrow). isolation of listeria monocytogenes from brain samples at necropsy of two dead ewes and one lamb, two brain samples from each animal were collected for bacteriological analysis. subsequently, samples were cultured in fraser broth (merck) and incubated aerobically for 24-48 h at 37 °c. after incubation, an aliquot was streaked onto palcam listeria selective agar (merck) and incubated aerobically for 24-48  h at 37  °c. presumptive identification of l.  monocytogenes was based on the observation of typical colony morphologies on palcam agar, gram staining, haemolysis patterns, catalase and oxidase reactions, and tumbling motility. the characteristic gram-positive coccobacilli (short rods) less likely in comparison to cattle (godreuil et  al. 2003, rocha et  al. 2013). sporadic ovine listeriosis cases are mainly attributted to serogroup 1/2a, 1/2b, and 4b (low et  al. 1993, kotzamanidis et  al. 2019). l.  monocytogenes is typically found in soil, vegetables, plants, feces, sewage, genital secretions and the nasal mucous membranes of healthy animals. the disease occurs more frequently in the winter and the early spring and is associated with encephalitis after silage intake (wiedmann et  al. 1994, bertsch et al. 2013). in these cases, mortality rates of 3.1% and 1.3% have been observed in ewes and lambs, respectively (nash et al. 1995, lyon et al. 2008). l.  monocytogenes is commonly present in silage, but it can multiply only in silage which has not been fermented properly (ph 5.0-5.5) (doumith et  al. 2004, kumar et  al. 2007). livestock intended for food production play an important role in the zoonotic transmission of the disease. humans commonly acquire l.  monocytogenes following consumption of products such as contaminated raw milk and, minimally processed foods of plant origin (contamination likely traced back to animal operations); however, rare cases of direct transmission have been reported (heger et  al. 1997, nightingale et  al. 2004). outbreaks of human listeriosis have been mainly linked to consumption of ready-to-eat (rte) foods, including soft cheeses. it should be noted that outbreak investigations and source attribution are often complicated by the typically long incubation time of l.  monocytogenes in conjuction with the ubiquity of this pathogen, which often leads to contamination of foods not likely to be associated with listerial presence (granier et  al. 2011, amato et  al. 2017, buchanan et  al. 2017). further, the occurrence of antibiotic resistance of isolates of l.  monocytogenes from non-human samples has been reported for a number of important antibiotics, including tetracycline, doxycyline, erythromycin and sulfamethoxazole/trimethoprim (vela et  al. 2001, granier et  al. 2011, who 2016, amato et al. 2017 ). well-established breakpoints for use in determination of antimicrobial susceptibility of l.  monocytogenes veterinary clinical isolates are not available, except for select antibiotics such as penicillin g and sulfamethoxazole/trimethoprim, complicating interpretation of antimicrobial susceptibility data and requiring extrapolation based on available information from other closely-related bacteria. the aim of the current study was to provide descriptive analysis of an outbreak in sheep associated with high mortality, determine the cause of fatalities, identify and characterize the etiological agent, and determine the antibiotic susceptibility of the isolates. 207 sylejmani et al. listerial meningoencephalitis in kosovo veterinaria italiana 2020, 56 (3), 205-211. doi: 10.12834/vetit.2166.12781.3 which were catalase positive and oxidase negative, and displayed umbrella shaped growth pattern in motility medium as well as tumbling motility in wet mounts at room temperature were considered as ‘presumptive’ listeria isolates. typical listeria colonies were transferred to cryotubes containing brain heart infusion broth (bd difco) with 20% glycerol and maintained frozen at - 20 °c until used for confirmation. confirmation of isolates of l. monocytogenes downstream confirmation analyses revealed the presence of l. monocytogenes in six brain samples (three different animals) analyzed (table i). the confirmation of isolates of l.  monocytogenes was achieved by matrix-assisted laser desorption/ ionization mass spectrometry (maldi-tof ms)-based identification. maldi-tof ms and biotyping was performed using the brukerultraflex  ii tof/tof in positive ion reflector mode pre-calibrated with bacterial test standard (bruker) according to the manufacturer’s instructions using an ethanol/ formic acid extraction protocol. spectral analyses and sample identifications was performed using the brukerbiotyper software (ver. 3.1.). identifications were only accepted if scored ≥  2.0 by the biotyper algorithm. all isolates were confidently identified by maldi-tof ms as l.  monocytogenes (score higher than 2.4). molecular serotyping of l. monocytogenes l.  monocytogenes strains previously confirmed by maldi-tof ms, including five isolates (b1, b2, b3, b4, and b5) from the first sheep, five isolates (b6, b7, b8, b9, and b10) from the second sheep, and eight isolates (b 11, b12, b13, b14, b15, b16, b17, and b18) from the lamb, were subjected to molecular serotyping by targeting six genes of l.  monocytogenes, lmo0737 (691 bp), lmo1118 (906  bp), orf2819 (471 bp), orf2110 (597 bp), prfa (274 bp), and prs (370 bp) (doumith et  al. 2004, kerouanton et al. 2010). pcr was performed figure 3. perivascular infiltration of lympho mononuclear cells in medulla oblongata (hematoxylin and eosin stain; 40× objec.) figure 2. microabscess in medulla oblongata (hematoxylin and eosin stain; 40× objec.) figure 1. neuroparenchymal lesions from infiltration of lympho mononuclear cells (hematoxylin and eosin stain; 40× objec.). table i. number of samples and l. monocytogenes isolates included in the study. number of samples (brain) strains* serotypes 2 (from sheep 1) b1, b2, b3, b4, b5 4b 2 (from sheep 2) b6, b7, b8, b9, b10 4b 2 (from lamb 1) b11, b12, b13, b14, b15, b16, b17, b18 4b *b = clinical isolates from the brain of sheep and lamb affected of meningoencephalitis. 208 listerial meningoencephalitis in kosovo sylejmani et al. veterinaria italiana 2020, 56 (3), 205-211. doi: 10.12834/vetit.2166.12781.3 quinupristin/dalfopristin, linezolid, nitrofurantoin, kanamycin, ciprofloxacin, vancomycin, lincomycin and gentamicin. the breakpoints used for the determination of the minimum inhibitory concentrations (mic) using the broth microdilution method, were those recommended by clinical & laboratory standards institute (clsi 2014) for closely-related bacteria (staphylococcus spp., enterococcus spp., streptococcus spp.). there was one exception to this pattern, namely for penicillin for which specific listeria breakpoints are defined. staphylococcus aureus atcc 29213 was used as quality control strain, with susceptibility testing results in the expected range. all 18 l.  monocytogenes strains tested were found to be susceptible to penicillin (mic 0.5-1  µg/ ml), erythromycin (mic 0.25  µg/ml), tetracycline (mic 1  µg/ml), ciprofloxacin (mic 1  µg/ml), tylosin tartrate, sulfamethoxazole/trimethoprim, streptomycin, vancomycin, and gentamicin, but with 2  separate groups, one for genes prfa and prs, and the other four genes lmo0737, lmo1118, orf2819, and orf2110. the molecular serotyping using multiplex pcr revealed prfa, prs, orf2819, and orf2110 genes from all isolates, indicating that the isolates belonged to l.  monocytogenes serotype 4b. antimicrobial susceptibility testing of l. monocytogenes isolates all strains were tested for antimicrobial susceptibility by broth microdilution using the sensititre® gpn3f dehydrated panel (trek diagnostic systems, cleveland, oh) following the manufacturer’s instructions. the following antimicrobial agents were used for the in vitro microdilution broth testing protocol: penicillin, erythromycin, sulfamethoxazole/ trimethoprim, chloramphenicol, tetracycline, daptomycin, streptomycin, tylosin tartrate, table ii. mics of 16 antimicrobial agents for l. monocytogenes isolates from ovine meningoencephalitis. antimicrobials number of isolates (n = 18) with mic of (mg/l) 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1024 penicillin 2 (11.1) 14 (77.8) 2 (11.1) erythromycin 18 (100) tetracycline 18 (100) ciprofloxacin 18 (100) chloramphenicol 15 (83.3) 2 (11.1) 1 (5.6) sulfamethoxazole/ trimethoprim 18 (100) daptomycin 11 (61.1) 7 (38.9) vancomycin 2 (11.1) 14 (77.8) 2 (11.1) streptomycin 18 (100) nitrofurantoin 18 (100) tylosin tartrate 16 (88.9) 2 (11.1) gentamicin 18 (100) quinupristin/ dalfopristin 12 (66.6) 5 (27.8) 1 (5.6) lincomycin 18 (100) linezolid 1 (5.6) 2 (11.1) 15 (83.3) kanamycin 17 (94.4) 1 (5.6) 209 sylejmani et al. listerial meningoencephalitis in kosovo veterinaria italiana 2020, 56 (3), 205-211. doi: 10.12834/vetit.2166.12781.3 gentamicin, and vancomycin, are quite similar to those reported for strains of l. monocytogenes from ovine meningoencephalitis in a study by vela and colleagues (vela et al. 2001). while in other authors' studies l.  monocytogenes isolates have shown resistance to tetracycline (vela et  al. 2001, counter et  al. 2007), the isolates in the current study were susceptible to this antimicrobial. based on the in vitro analysis, it would be expected that penicillin would provide an effective treatment during administration of antibiotic therapy in the current clinical settings. furthermore, low-level resistance to a fluoroquinolone such as ciprofloxacin has been associated with the presence of efflux pumps and potentially the use of fluoroquinolones in animal production, especially broiler production (godreuil et  al. 2003). it is not unusual that all isolates displayed resistance to daptomycin, which corroborated findings of other studies which do not recommend the use of daptomycin for treatment of listeriosis cases (noll et  al. 2018, spanjaard and vandenbroucke-grauls 2008). multiple mechanisms, including some that are transferable, have been reported to be involved in conferring resistance to lincosamides in listeria spp. (bertsch et  al. 2013). different l.  monocytogenes isolates originating from the same tissues (i.e. brain) were shown to contain different antimicrobial resistance phenotypes. this could also indicate further differences between the different strains of isolates of l.  monocytogenes 4b, including other virulence factors. furthermore, this study demonstrates the presence of highly pathogenic l.  monocytogenes serotype 4b strains causing a neural form of listeriosis in small ruminants in kosovo, which is being reported for the first time. listeriosis-positive sheep flocks may pose a grave public health risk, as a great number of popular food products derived from these animals are often consumed in the region, greatly increasing opportunities for zoonotic transmission. therefore, in such cases preventive measures have to be undertaken to avoid the transfer of l. monocytogenes to processed milk and other dairy products, although human listeriosis cases are yet to be reported in kosovo. lastly, the results of our study indicate that l.  monocytogenes from ovine listeriosis manifests susceptibility to most commonly utilized antimicrobials; however, several instances of intermediate susceptibility and full resistance identified in the tested isolates suggests a future need for close monitoring and surveillance of antimicrobial resistance in l. monocytogenes strains of animal origin in kosovo. resistant to lincomycin with an observed mic of 8  µg/ml, daptomycin (mic 2-4  µ/ml) and intermediate to nitrofurantoin with an observed mic of 64 µg/ml. furthermore, a number of isolates were classified as sensitive to some other antimicrobials such as kanamycin (17/18), quinupristin/dalfopristin (17/18), chloramphenicol (15/18), and linezolid (3/18); resistant to chloramphenicol (1/18) and kanamycin (1/18); and intermediate to linezolid (15/18), chloramphenicol (2/18), and quinupristin/ dalfopristin (1/18) (table ii). discussion the findings of suspected neurological disease presented in this case study were established through histopathology and horizontal isolation of the etiological agent, l.  monocytogenes from brain tissues. histopathological findings observed within the brainstem of these animals are consistent with those previously described for ruminant encephalitic listeriosis (maxie and youssef 2007, zachary 2012). the finding of a seasonal occurrence of ruminant listeriosis in this study correlates well with the findings reported by wiedmann and colleagues (wiedmann et  al. 1994). that specific study reported isolation of listeria from brains of sheep and goats affected with listerial encephalitis and from silage consumed by the animals in the winter and early spring. we found that only one serotype, serotype 4b, was implicated as the etiological agent in this outbreak. in another similar study conducted in greece, serotype 4b was reported as the predominant strain (81%) isolated in small animals, in which the neural form of the disease was diagnosed (giannati-stefanou et al. 2006). the antimicrobial susceptibility testing results in our study overwhelmingly support the presence of highly  susceptibile l.  monocytogenes isolates (n  =  18). we report the following levels of susceptibility for the isolates tested: penicillin  (100%), erythromycin (100%), tetracycline (100%), ciprofloxacin (100%), tylosin tartrate (100%), sulfamethoxazole/trimethoprim (100%), vancomycin (88.9%), streptomycin (100%), gentamicin (100%), kanamycin (94.4%), quinupristin/dalfopristin (94.4%), and chloramphenicol (83.3%). all isolates (100%) were resistant to lincomycin and daptomycin. resistance was also observed to chloramphenicol and kanamycin (5.6%). on the other hand, our study indicated that the following proportions of l.  monocytogenes isolates could be included in the intermediate category: nitrofurantoin (100%), linezolid (83.3%), chloramphenicol (11.1%), and quinupristin/dalfopristin (5.6%). the mic ranges obtained in this study for penicillin, erythromycin, 210 listerial meningoencephalitis in kosovo sylejmani et al. veterinaria italiana 2020, 56 (3), 205-211. doi: 10.12834/vetit.2166.12781.3 acknowledgements the authors gratefully aknowledge the local veterinarians of the municipality of ferizaj kosovo, where the listeriosis outbreak was identified as well as the regional animal health and veterinary inspectorate for the cooperation and the resources made available to acces the farm and 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grattarola c., zuccon f., iulini b., knabel s.j. & capucchio m.t. 2013. ruminant rhombencephalitis-associated listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains. appl environ microb, 79 (22), 7114-7114. 247 veterinaria italiana 2021, 57 (3), 247-251. doi: 10.12834/vetit.1824.9678.2 accepted: 04.11.2019 | available on line: 31.12.2021 1departement of surgery, anesthesiology & radiology, faculty of veterinary medicine, mansoura university, egypt. 2departement of pathology, faculty of veterinary medicine, mansoura university, egypt. *corresponding author at: departement of surgery, anesthesiology & radiology, faculty of veterinary medicine, mansoura university, egypt. e-mail: sayedelshafaey@yahoo.com. el-sayed ahmed el-shafaey1*, mohamed hamed2 and zeinab dewidar2 keywords odontogenic tumors, odontoameloblastoma, maxilla, histopathology, goat. summary a 6-year-old female ardi goat was presented for evaluation of an expansile firm painless maxillary ovoid mass. clinical and diagnostic imaging examination revealed a well-demarcated extensive mass occupying the left posterior maxilla and altering its anatomical features. surgical excision was not deemed feasible with this apparently extensive infiltrative features of the tumor and the owner elected to euthanize the goat. gross examination of the mass showed a grayish-brown, multiloculated firm and gritty mass measuring 21  x  11  x  19  cm located on the left posterior maxilla. histopathological examination revealed plexiform and follicular arranged, ameloblast-like odontogenic epithelium. follicular epithelium was disintegrated leaving spaces, identical to solid multicystic ameloblastoma, intermingled with primitive myxoid stroma resembling dental papilla, teeth like hard structure as enamel and dentine, and osteodentine matrix. based on these findings, the tumor was diagnosed as odontoameloblastoma (oa) and to our knowledge this is the first report of oa in a goats. odontoameloblastoma in the posterior maxilla of a 6-years-old ardi goat (capra aegagrus hircus) case description a 6-year-old female ardi goat (37 kg) was referred to the veterinary teaching hospital, faculty of veterinary medicine, qassim university, saudi arabia, for investigation of a large firm painless ovoid mass overlying the left maxilla of undetermined duration (figure 1a). the owner reported that the mass had been gradually increased in size during the last 6 months. the appetite, ruminal motility, rectal temperature, heart and respiratory rates of the goat were within normal range, except for difficulty in chewing with excessive drippling of salivation. however, in the last month with the progressive increase of the mass, there was apparent difficulty in mastication and swallowing and the goat starts to loss its appetite without any response to the traditional medicinal treatment. clinical examination oral examination revealed a well-demarcated, hard, sessile and dark red cauliflower-like mass of approximately 21 x 11 x 19 cm. it was located on the posterior maxilla extending from the left first premolar to the last molar, including the left introduction odontoameloblastoma (oa) is an extreme rare matrix-producing mixed odontogenic tumor that has been described in both humans and animals (barnes et  al. 2005, murphy et  al. 2017). it is characterized by a mixed odontogenic epithelium and odontogenic ectomesenchyme with inductive changes leading to the formation of dentin and enamel in parts of the tumor (dive et  al. 2011). although oas are considered to be benign in nature, they might locally grow in expansile aggressive pattern with sequential destruction of adjacent bone and dental structures of the jaw (mosqueda-taylor et al. 2002). oa has been reported in humans (dive et al. 2011), cattle (lepri et al. 2013), horses (murphy et  al. 2017), sheep (dubielzig and griffith 1982), dogs (kok et al. 2018), monkeys (yanai et  al. 1995) and rats (burrough et  al. 2010, wong et al. 2018). while, to the best of our knowledge, oa has not been reported in goat. this report describes the clinical, radiographic, ultrasonographic and histopathological features of oa in a 6-year-old ardi goat. to the best of our knowledge, oa has never been reported in goats. case report 248 veterinaria italiana 2021, 57 (3), 247-251. doi: 10.12834/vetit.1824.9678.2 odontoameloblastoma in a goat el-shafaey et al. maxillary sinuses, pteroid bone, overlying gingiva, left buccal mucosa, left masseter muscle and soft palate. the growth was almost parallel to the tooth and it was protruded in the oral cavity causing difficult prehension and swallowing. surgical excision was not deemed feasible. the apparently extensive infiltration of the mass to the posterior maxilla and adjacent soft tissues indicated that the animal had a bad prognosis and the owner elected to euthanize the goat at this stage. diagnostic imaging evaluation diagnostic imaging evaluation of the maxillary mass using skull radiograph (65 kvp, 2.0 mas and a 60 cm focal film distance) and sector ultrasonography (5 mhz) have been acquired to assess the extent and characters of the maxillary mass. the radiographic picture revealed presence of a soft tissue radiolucent-mass originating from the posterior left maxilla. in addition, there was numerous radiopaque foci with osteolytic changes at the roots of the premolar and molar teeth and in adjoining area of the maxilla (figure 1b). ultrasonographically, the mass was irregular with hyperechoic multiloculated core filled with heterogonous contents (figure 1c). gross examination on gross postmortem examination, the outer surface of the mass from the oral view showed a number of purulent ulcerative nodules filled with a dark red clotted blood and viscid fluid. however, when the mass was viewed from the left fascial side, its outer surface was smooth and lined by a whitish-gray thick capsule (figure 2a). the cut surface of the mass was grayish-brown, firm and gritty with its multiloculated core filled with irregular brownish trabeculae and granular matrix. there was extensive destruction of the posterior maxilla and replacement by a dark red multiloculated tissue including the 1st  premolar and last molar teeth, while the other teeth were completely resorbed. when the mass was viewed from the maxillary sinus side, it was in the sinus causing effacement of the maxillary sinus (figure 2b). no other abnormalities were seen in all other organs. histopathological examination serial sections from the mass, parotid, submandibular and retropharyngeal lymph nodes were taken and fixed in 10% neutral buffered formalin for 48 h, routinely processed, sectioned at figure 1. a. left sided maxillary large ovoid mass in a 6-year-old ardi goat. b. left lateral radiograph of the soft tissue radiolucent-mass (white arrow) of the left maxilla of the aforementioned goat. c. ultrasonographic view of the left maxillary mass showing irregular architecture with hyperechoic multiloculated core filled with heterogonous contents (black arrow). figure 2. gross appearance of the left maxillary mass of ardi goat. a. the lateral aspect of the mass was smooth and lined by a whitish‑gray thick capsule measured approximately 21 x 11 x 19 cm in diameter (arrow). b. grayish‑brown, firm and gritty cut surface of the mass causing extensive destruction of the posterior maxilla and replacement by a dark red multiloculated tissue (arrow). 1 = lower jaw, 2 = maxillary sinus, asterisk (*) = 1st premolar and last molar teeth. 249veterinaria italiana 2021, 57 (3), 247-251. doi: 10.12834/vetit.1824.9678.2 el-shafaey et al. odontoameloblastoma in a goat fibrinous exudate and neutrophils, proliferation of odontogenic epithelium in the form of strands, intermingled with primitive ectomesenchyme was evident (figure 3b). nests of marked pleomorphism neoplastic cells with presence of ghost cells, mature fibrous tissue stroma were recognisable (figure 3c). necrosis, macrophagic and neutrophilic infiltrations, increased mitotic figures of neoplastic cells and squamous metaplasia were also seen (figure  3c). histopathological examination of parotid, submandibular, or retropharyngeal lymph nodes revealed no micro metastasis. based on the clinical behavior, anatomic location, diagnostic imaging, histopathological characteristics and differential diagnosis for the maxillary odontogenic tumors, the mass was diagnosed as oa. discussion odontogenic tumors are histologically classified according to the presence of odontogenic epithelium, odontogenic mesenchyme, or both (head et  al. 2002). in animals, tumors with both components include oa, feline inductive odontogenic tumor, ameloblastic fibro-odontoma, and odontoma (dubielzig 2003). these odontogenic tumors are seen as mass-like lesions interfering with mastication and even swallowing in affected animal (gardner 1996). regarding the frequency of occurrence of odontogenic tumor, the odontogenic tumors comprise 1% of all oral tumors in domestic animals (munday et  al. 2017). among these various dental tissue tumors, oa has been reported in humans, cattle, horses, sheep, dogs, monkeys and rodents (dubielzig and griffith 1982, yanai et  al. 1995, burrough et  al. 2010, dive et  al. 2011, lepri et al. 2013, murphy et al. 2017, kok et al. 2018, wong et  al. 2018). no reference information for incidence of oa in goats is available. the etiopathogenesis of oas, either in humans or animals, is not well known. localized trauma, infection, and genetic predisposition have all been postulated as possible predisposing factors (thompson et al. 1990, mosqueda-taylor et al. 2002). despite the large size of maxillary mass and missing teeth, the goat involved in this report didn’t have any known trauma or infection or known history of congenital inciting of oa. oas have similar microscopic features and often they are asymptomatic in both animal and human patients (barnes et  al. 2005, lepri et  al. 2013). untreated tumors can become quite large, causing dysphagia, pain and destroying the affected jaw (murphy et  al. 2017). in humans, oas are rarely associated with any symptoms. they usually are small and often incidentally identified on radiological examinations because of unerupted or figure 3. a. follicular shaped ameloblastoma, basophilic enamel matrix in the form of crown with peripheral columnar shaped palisaded ameloblasts (arrow), dysplastic eosinophilic matrix (osteodentine) (thick arrow), primitive ectomesenchyme resembling dental papilla. he. bar, 200 µm. b. plexiform ameloblastoma, cystic degeneration in the center filled with eosinophilic fibrin and neutrophils (thick arrow), primitive ectomesenchyme resembling dental papilla (thin arrow). he. bar, 200 µm. c. squamous metaplasia of neoplastic odontogenic epithelium (arrow). he. bar, 100 µm. 5 µm and stained with hematoxylin and eosin (he) for microscopic examination. histopathological examination showed solid ameloblastoma with follicular pattern, peripheral palisading and reversal of polarity, intermingled with primitive odontogenic ectomesenchyme resembling dental papilla, dysplastic eosinophilic matrix of osteodentine (osteoid matrix trapped odontoblasts) and basophilic enamel production in the form of crown with peripherally columnar-shaped palisaded ameloblasts due to inductive odontogenesis (figure 3a). plexiform ameloblastoma with cystic degeneration in the center, filled with eosinophilic 250 veterinaria italiana 2021, 57 (3), 247-251. doi: 10.12834/vetit.1824.9678.2 odontoameloblastoma in a goat el-shafaey et al. and colleagues (burrough et  al. 2010), lepri and colleagues (lepri et  al. 2013) and murphy and colleagues (murphy et al. 2017). the surgical treatment of oa has proven to be a challenge, in ensuring adequate surgical excision of the tumor while maintaining function. the decision whether to surgically enucleate the tumor or to euthanize the affected animal was based on several factors as, tumor appearance, nature, size, invasion degree and the healthy condition of the affected animal (lepri et  al. 2013). improper resection of the highly invasive tumor, resulting in high rates of tumor recurrence with extensive poor prognosis should also be considered (murphy et al. 2017). the oa in the present case invaded the cancellous bone of the posterior maxilla, which would have indicated a higher likelihood of recurrence if surgical excision had been attempted. thus, the surgical intervention was declined. this decision was in accordance with mendez-angulo and colleagues (mendez-angulo et al. 2014) and kok and colleagues (kok et al. 2018). oas have been reported in a wide range of ages with various position in the mandible or maxilla (dubielzig 2003, burrough et  al. 2010). however, most of the reported oa cases usually occur in animals younger than 5 years of age (lepri et  al. 2013), with high frequency mandibular involvement, particularly the incisor region (murphy et  al. 2017, kok et al. 2018). however, in the present case, the oa was located in the posterior maxilla which confirmed by radiographic, gross and histopathological examination. this location strongly suggests that this neoplasm likely originated from the tooth germ tissues. this could be attributed to the relationship between tooth diseases and abnormalities in the animal, which might increase the risk of odontogenic tumors. similar findings were reported by thompson and colleagues (thompson et  al. 1990), dive and colleagues (dive et  al. 2011) and wong and colleagues (wong et al. 2018). to the best of our knowledge, the present report represents the first record of oa in goats. it should be included in the differential diagnosis of mixed odontogenic tumors in animals. missing teeth in the first 2 decades of life (dive et al. 2011). however, in animals, clinical signs associated with oa include mandibular or maxillary swelling, missing teeth, or tooth-like structures erupted into the oral cavity (yanai et al. 1995, murphy et al. 2017, kok et al. 2018). the difficulties in identifying missing teeth early in domestic species may contribute to this disparity, so the lesions in animals may progress further prior to identification and can require euthanasia due to adverse effects on quality of life specially in cattle and horses (lepri et  al. 2013, murphy et  al. 2017). thus, this report describes the significant growth potential of maxillary oa in goat. differential diagnosis of oa from other mixed odontogenic tumors is of a great challenge for human and animal pathologists over the last decades (mosqueda-taylor et  al. 2002). histologically, oas are closely related to the other matrix-producing odontogenic mixed tumors as odontomas and ameloblastic fibro-odontomas (burrough et  al. 2010). odontomas have a limited growth potential (once they are fully formed they don't increase in size) with abundance of dental matrix, which creates the impression of dysplastic denticles (head et  al. 2002, dubielzig 2003). in contrast, the ameloblastic fibromas have the greatest potential of unlimited growth of dental ectomesenchyme, untreated (gardner 1996). oas are primarily differentiated histologically by a relative abundance of irregular cords with follicles of odontogenic epithelium abutting a small cluster of odontoblasts resembling the enamel structure in the developing tooth germ with osteodentin formation (mosqueda-taylor et al. 2002, kok et  al. 2018). the present case showed relative abundance of neoplastic odontogenic epithelium, odontogenic ectomesenchyme, enamel formation, and mineralized dental tissues, including dentin, osteodentin, and bone-like tissue. therefore, the histological characteristics and aggressive nature of the tumor reported here were consistent with the diagnosis of oa, suggesting a critical review of odontogenic tumors in animals that formerly diagnosed as ameloblastic fibro-odontoma. these findings were in agreement with burrough 251veterinaria italiana 2021, 57 (3), 247-251. doi: 10.12834/vetit.1824.9678.2 el-shafaey et al. odontoameloblastoma in a goat barnes l., eveson j., reichart p. & sidransky d. 2005. odontoameloblastoma. in pathology and genetics of head and neck tumors (who classification of tumors series). lyon, france, iarc press, 312. burrough e.r., myers r.k. & whitley e.m. 2010. spontaneous odontoameloblastoma in a female sprague dawley rat. j vet diagn invest, 22, 998-1001. dive a., khandekar s., bodhade a. & dhobley a. 2011. odontoameloblastoma. j oral maxillofac pathol, 15, 60-64. dubielzig r.r. 2003. histologic classification of tumors of odontogenic origin of domestic animals. in histological classification of tumors of the alimentary system of domestic animals, vol. 10, 46-57. washington, dc, armed forces institute of pathology. dubielzig r.r. & griffith j.w. 1982. an odontoameloblastoma in an adult sheep. vet pathol, 19, 318-320. lepri e., avallone g., mandara m.t. & vitellozzi g. 2013. odontoameloblastoma in a calf. j vet dent, 30, 248-250. gardner d.g. 1996. ameloblastic fibromas and related tumors in cattle. j oral pathol med, 25, 119-124. head k.w., else r.w. & dubielzig r.r. 2002. tumors of the alimentary tract. in tumors in domestic animals (meuten d.j., ed). 4th ed. iowa state press, avenue, ames, iowa, 401-481. kok m., chambers j., ushio n., miwa y., nakayama h. & uchida references k. 2018. amyloid-producing odontoameloblastoma in a black-tailed prairie dog (cynomys ludovicianus). j comp path, 159, 26-30. mendez-angulo j.l., tatarniuk d.m., ruiz i. & ernst n. 2014. extensive rostral mandibulectomy for treatment of ameloblastoma in a horse. vet surg, 43, 222-226. mosqueda-taylor a., carlos-bregni r., ramirez-amador v., palma-guzman j.m., esquivel-bonilla d. & hernandez-rojase l.a. 2002. odontoameloblastoma. clinic-pathologic study of three cases and critical review of the literature. oral oncol, 38, 800-805. murphy b., bell c., koehne a., richard r. & dubielzig r. 2017. mandibular odontoameloblastoma in a rat and a horse. j vet diagn invest, 29, 536-540. thompson i.o., phillips v.m., ferreira r. & housego t.g. 1990. odontoameloblastoma: a case report. br j oral maxillofac surg, 28, 347-349. wong h., hedley j., stapleton n., murphy b. & priestnall s. 2018. odontoameloblastoma with extensive chondroid matrix deposition in a guinea pig. j vet diagn invest, 30, 793-797. yanai t., masegi t., tomita a., kudo t., yamazoe k., iwasaki t., kimura n., katou a., kotera s. & ueda k. 1995. odontoameloblastoma in a japanese monkey (macaca fuscata). vet pathol, 32, 57-59. 195 review 1center for vaccines and immunology, university of georgia, athens, ga, usa. 2department of infectious diseases, university of georgia, athens, ga, usa. * corresponding author at: center for vaccines and immunology, university of georgia, athens, ga, usa. e-mail: tedross@uga.edu. parole chiave virus dell’influenza, vaccini universali per l’influenza, sequenza consenso, computationallyoptimized broadly reactive antigen (cobra). riassunto i virus dell'influenza infettano ogni anno milioni di persone con un onere economico e sanitario sostanziale per la nostra società. le epidemie e le pandemie di influenza sono attribuibili alla continua evoluzione dei virus dell'influenza, rispettivamente causate dal “drift” e dallo “shift” antigenico. uno dei motivi che determina la continua circolazione dei virus influenzali nella popolazione umana è la protezione incompleta conferita dai vaccini influenzali stagionali attualmente disponibili contro possibili ceppi influenzali andati incontro a “drift” o “shift” antigenico. di recente, grandi risorse sono state impiegate per lo sviluppo di un vaccino più efficace contro l'influenza, ampiamente protettivo o universale, con l'obiettivo principale di proteggere la popolazione umana non solo dai ceppi virali attualmente in circolazione ma anche da possibili varianti future, senza la necessità di un suo aggiornamento continuo. per raggiungere questo obiettivo, ed ottenere una risposta immunitaria efficace e ampiamente protettiva, sono stati messi a punto diversi approcci: tra questi, al centro di questa review, ci sono quelli profilattici basati sulle sequenze consenso. approcci vaccinali basati sulla sequenza consenso dell'emoagglutinina per contrastare la diversità del virus influenzale keywords influenza virus, universal influenza vaccine, consensus sequence, computationallyoptimized broadly reactive antigen (cobra). summary each year millions of people are infected by influenza viruses, and this causes a substantial economic and health burden on our society. influenza epidemics and pandemics are attributable to the ongoing evolution of influenza viruses through antigenic drift and shift, respectively. one of the reasons for the continuous circulation of influenza viruses in the human population is the incomplete protection conferred by currently available seasonal influenza vaccines against possible arising drifted or shifted influenza strains. recently, tremendous efforts have been focused on the development of a more effective broadly reactive or universal influenza vaccine. the main objective of underdevelopment vaccines is to protect the human population not only from currently circulating virus strains but also from possible future variants without the need for their continuous update. different approaches have been developed to reach this goal and elicit an effective and cross-protective immune response. among these, consensus-based prophylactic approaches to effectively prevent influenza infections are the major focus of this review. giuseppe andrea sautto1 and ted m. ross1,2* hemagglutinin consensus-based prophylactic approaches to overcome influenza virus diversity veterinaria italiana 2019, 55 (3), 195-201. doi: 10.12834/vetit.1944.10352.1 accepted: 03.09.2019 | available on line: 30.09.2019 in 3-5 million severe cases and 300,000-500,000 deaths globally every year. the economic burden of influenza virus-induced disease is close to $100 billion in the u.s. each year (molinari et al. 2007). infection by a seasonal influenza virus results in fever, nasal discharge, coughing, sore throat, headache, myalgia, and nausea. in the case of a severe disease introduction influenza virus infections can be categorized into two epidemiological forms: seasonal and pandemic (paules et al. 2017). seasonal influenza epidemics are caused by influenza a and b viruses in humans, resulting 196 veterinaria italiana 2019, 55 (3), 195-201. doi: 10.12834/vetit.1944.10352.1 cobra and influenza virus sautto & ross have been developed for the different influenza virus subtypes over the years with a particular focus on the computationally-optimized broadly reactive antigen (cobra)-based methodology. consensus-based approaches and design of computationally-optimized broadly reactive antigens (cobra) in order to overcome antigenic drift and mismatch of current influenza vaccines for pandemic and seasonal viruses, different groups have designed and developed over the years different consensus-based approaches for the influenza virus (giles et al. 2012, castelli et  al. 2013, carter et  al. 2016, wong et  al. 2017, wu et  al. 2017, elliott et  al. 2018, stepanova et al. 2018, tsybalova et al. 2018). from a definition point of view, a protein-based consensus sequence encodes the most common amino acid at each position of a protein. thus, in the context of a pathogen-related protein, the generation of a consensus sequence captures a sequence that is more relevant in a current epidemic outbreak. it has been previously demonstrated that a consensus-based approach for the influenza hemagglutinin (ha) protein is effective in protecting from influenza a infection. moreover, consensus-based prophylactic approaches have been also investigated as a strategy for eliciting broadly reactive immune responses against other viruses such as human immunodeficiency virus (hiv), chikungunya virus (chkv) and hepatitis  c virus (hcv) (scholte et al. 2013, meyerhoff et al. 2017, tarr et al. 2018). more in detail, a novel methodology of antigen design using multiple rounds of consensus building to generate candidates termed computationally optimized broadly reactive antigens (cobras), have been described (giles et  al. 2012, carter et  al. 2016, wong et al. 2017). in brief, the cobra methodology starts from inferring the phylogenetic tree from ha amino acid sequences derived from different outbreak groups in a given influenza a subtypes. primary consensus sequences are then generated for each outbreak group. subsequently, secondary consensus sequences are generated for each subclade using the primary consensus sequences as input. the secondary consensus sequences are finally aligned and the resulting consensus sequences, designated cobra, are generated (figure 1) (giles et al. 2012). expression of this novel antigens is then required to assess the breadth of protection of the elicited immune response in vaccinated pre-clinical animal models. and hospitalization, secondary bacterial infections or a disproportionate inflammatory response represent the most common cause (erbelding et al. 2018). differently, influenza pandemics caused by influenza a emerge at unpredictable intervals and they cause significantly increased morbidity and mortality compared to seasonal influenza (paules et al. 2017). in the past century, four pandemic outbreaks have occurred: in 1918, 1957, 1968, and 2009 (palese 2004). furthermore, in the past few decades, animal influenza viruses, such as h5n1 and h7n9 avian influenza, have caused sporadic human infections and deaths. influenza infections with these viruses, termed pre-pandemic viruses, are originally zoonotic and do not demonstrate sustained person-to-person spread. however, viral mutations may occur, allowing efficient transmission among humans and possibly leading to the next influenza pandemic (nicholson et al. 2003). despite the availability of a seasonal vaccine, influenza remains a public health concern. each influenza season, a set of virus strains, representing one h1n1, one h3n2 and 1 or 2 influenza b isolates, are selected for the inclusion in the annual seasonal influenza vaccine. the effectiveness of this standard of care (soc) influenza vaccine ranges between 10-60% (caspard et  al. 2017, flannery et  al. 2018). the occurrence of a possible mismatch between circulating strains and vaccine strains, due to ongoing antigenic changes (also known as drifts) is the main reason of a low prophylactic effectiveness (osterholm et al. 2012). in fact, the influenza strains used in annual vaccine formulations are selected twice annually following the influenza seasons in the northern and southern hemispheres. for this reason, this strategy keeps us at least one year behind compared to the evolving circulating influenza viruses. moreover, virus growth in eggs during vaccine production may allow for additional mutations, further contributing to the vaccine mismatch with the circulating strains (zost et al. 2017). additionally, seasonal influenza vaccines do not provide protection against possible occurring pandemic strains. in fact, the outbreak of a novel influenza virus with a pandemic potential requires the development of a pandemic strain-specific vaccine. for all these reasons, there is an urgent medical need to improve the efficacy of the current influenza vaccine to limit the public health consequences of both seasonal and pandemic influenza infections. to this end, influenza vaccines that are more broadly and durable protective are needed (erbelding et  al. 2018, sautto et al. 2018, sautto et al. 2019). in this review, we describe hemagglutinin (ha) consensus-based prophylactic approaches that 197veterinaria italiana 2019, 55 (3), 195-201. doi: 10.12834/vetit.1944.10352.1 sautto & ross cobra and influenza virus disease instead of a proper protection (sautto et al. 2018). moreover, consensus-based antigen design is inherently influenced by the input sequences used to generate the synthetic molecule and as such is subject to sampling bias. in order to overcome this bias, the cobra methodology was described to generate broadly protective h5n1 vaccine candidates (giles et  al. 2012). in particular, cobra h5 immunogens were designed based upon ha amino acid sequences from clade 2 h5n1 human infections. these proteins were able to bind sialic acid, as well as mediate the viral particle fusion, confirming their retained functional activity (giles et  al. 2011). vaccination of animal models with cobra h5 immunogens, expressed on the surface of viral-like particles (vlp), elicited protective levels of antibodies endowed with hai activity against representative isolates of each h5n1 subclade of clade 2 (giles et  al. 2012). importantly, vaccinated mice and ferrets were completely protected from a lethal challenge with a clade 2.2 h5n1 virus and the same results were also confirmed in vaccinated nonhuman primates (cynomolgus macaques) (giles et al. 2012). it is noteworthy that this breadth of protection can be potentially increased when using an oil-in-water emulsion formulation and can confer protection against challenge as efficiently as the homologous matched vaccine (allen et al. 2017). finally, the immune responses elicited by the cobra ha immunogens were compared to those elicited by a mixture (polyvalent) of different primary h5n1 isolates. notwithstanding cobra and polyvalent h5n1 pandemic events of influenza are caused by the emergence of pathogenic and transmissible new viral strains to which there is a low or absent herd immunity in the population (nicholson et  al. 2003). outbreaks of highly pathogenic avian influenza (hpai) of the h5n1 subtype are of particular concern because of the high mortality rate (~  60%) and pandemic potential, given the immunological naïve status of the human population (cui et  al. 2017). in order to develop a vaccine that elicits broadly reactive antibody responses against emerging h5n1 isolates, the development of consensus-based h5n1 immunogens has been described. classical consensus-based approaches traditionally consist in aligning a population of sequences and selecting the most common residue at each position. these sequences are expected to effectively capture conserved linear epitopes and elicit cross-reactive cellular immune responses, especially for those antigens that are not expressed on the pathogen surface. these approaches have shown promising results, eliciting cross-reactive immune responses against different antigens including ha (elliott et  al. 2018), neuraminidase (na) (moise et  al. 2013) and matrix (m1) proteins (tsybalova et  al. 2018). as an example, consensus-based h5n1 ha vaccines expressed from dna plasmids elicit broad antibody responses (laddy et  al. 2007). however, the role of more conserved proteins in conferring protection, such as in the case of the m1 protein, is debated. in fact, the immune response targeting these antigens is correlated with a modulation of the figure 1. schematic representation of the cobra strategy. a phylogenetic tree is inferred based on included hemagglutinin (ha) amino acid sequences. as intermediate steps, primary and secondary consensus sequences are generated. subsequently, the secondary consensus sequences are aligned and the resulting final consensus sequences, designated cobra, are generated. following their expression, cobra candidates are then tested in animal models and the functional activity of the elicited immune responses is evaluated. cobra #1 ha cobra #2 ha 198 veterinaria italiana 2019, 55 (3), 195-201. doi: 10.12834/vetit.1944.10352.1 cobra and influenza virus sautto & ross protective efficacy compared to h1n1 ha antigens from seasonal and pandemic strains. in this set of experiments, three of the four initial candidates (named p1, x3, and x6) showed the most effective protection in terms of elicited broad hai response and prevention of infection of mice from a pandemic h1n1 challenge. more in detail, vaccinated mice had little or no detectable viral replication (carter et al. 2016). interestingly, these results were also recapitulated when cobra vaccines were expressed using different formulations, such as live influenza viruses, ha-ferritin nanoparticles and split inactivated vaccines, both in ferret and mouse in  vivo models (allen et  al. 2018, darricarrere et  al. 2018, sautto et al. 2018). importantly, the cobra h1n1-elicited antibody response was also evaluated in the context of an influenza pre-immunity (carter et  al. 2017). in fact, evaluating the efficacy of candidate broadly-reactive or universal influenza vaccines in a pre-immune setting is of pivotal importance since humans are ‘universally’ pre-immune to influenza viral antigens due to previous imprinting (kirchenbaum et  al. 2017). in this regard, it has been shown that ferrets previously infected with a 1986 h1n1 virus and vaccinated with a single dose of the cobra ha vlp vaccines, possess antibodies with a broad hai activity against 11-14 of 15 h1n1 viruses isolated between 1934 and 2013. moreover, ferrets primed with a cobra virus infection and subsequently immunized with cobra vlp vaccines showed a broader hai activity compared to those subsequently immunized with wild-type ha proteins (carter et al. 2017). h3n2 contrarily to influenza seasons in which h1n1 circulating strains are predominant, those in which infections from h3n2 viruses are more frequent, tend to be more severe with a greater number of hospital cases and deaths. this is reflected also in the number of elderly people that are infected with h3n2 viruses, leading to enhanced hospitalizations (jhung et al. 2013). using the cobra approach, a set of candidate h3n2 vaccines were tested in ferrets and mice for their ability to elicit antibodies that neutralize virus infection against not only h3n2 historical vaccine strains but also a set of co-circulating variants that circulated between 2004-2007 (wong et  al. 2017). three of the designed h3n2 cobra vaccines, expressed on the surface of vlp, were able to elicit antibodies recognizing and neutralizing all the co-circulating strains during this era. conversely, wild-type vaccine strains were not able to elicit antibodies with hai activity against these vaccines protected vaccinated mice and ferrets from the challenge with highly lethal h5n1 influenza viruses, cobra-vaccinated animals had higher-titer antibodies to a panel of h5n1 ha proteins, a decreased viral replication, less inflammation in the lungs of mice, and reduced virus recovery in ferret nasal washes. furthermore, the fact that the serum from vaccinated mice protected recipient animals more efficiently than the serum obtained from polyvalent-vaccinated mice and that the cell-mediated immune response was comparable in the cobraand polyvalent-vaccinated animals, suggests a predominant role of the humoral response in conferring this breadth of protection (crevar et al. 2015). on the other hand, vaccination of mice with a cocktail of three cobra h5 vlp-based vaccines, elicited broadly-reactive antibodies recognizing h5n1 viruses from 11 h5n1 clades/ subclades isolated over a 12-year span, confirming the versatility and the possibility of further increase the breadth elicited by cobra immunogens (crevar et al. 2015). h1n1 as already mentioned, influenza virus infections from the h1n1 subtype have caused pandemic outbreaks in the past. the most recent, the swine-origin h1n1 pandemic of 2009, reminded the worldwide community of the ever-present menace of a pandemic event. currently, h1n1 strains are circulating in the human population together with h3n2 strains. interestingly, it has been reported that in influenza seasons with more h1n1 influenza virus cases, children and younger adults are typically those more affected (palese 2004). since 2009, pandemic-like h1n1 strains are those predominantly circulating nowadays. however, the accumulation of mutations is making them drifting from the original pandemic strain (van reeth 2018). in order to overcome possible differences occurring between the vaccine strain and circulating strains, consensus-based approaches have been developed also for the h1n1 subtype. in particular, cobra h1n1 were designed using sequences of h1n1 viruses spanning the past 100 years, including modern pandemic h1n1 isolates (carter et  al. 2016). each of the cobra ha antigens was initially expressed on vlp in order to test their immunogenicity and efficacy in a murine model. interestingly, four of the nine h1n1 cobra ha proteins had the broadest hemagglutination inhibition (hai) activity against a panel of seventeen h1n1 viruses, including seasonal and pandemic h1n1 strains. similarly to cobra candidates for h5n1, cobra for h1n1 were used individually, in cocktails or in prime-boost regimens to evaluate the effect on elicited antibodies and 199veterinaria italiana 2019, 55 (3), 195-201. doi: 10.12834/vetit.1944.10352.1 sautto & ross cobra and influenza virus vaccines, aims to precisely characterize influenza immunity and correlates of immune protection. consensus-based approaches to develop universal or broadly reactive influenza vaccines have shown promising results in eliciting a more effective immune response compared to the current soc influenza vaccine. however, the mechanism(s) underlying cobra-elicited antibody breadth remains to be elucidated (sautto et al. 2018). in fact, owing to their design through multi-layer consensus sequence alignments, it can be speculated that cobra ha immunogens could elicit antibody specificities that recognize conserved epitopes. however, it is also reasonable to hypothesize that their design enables cobra ha immunogens to overcome the negative attributes of immunodominance and thus elicit an antibody response targeting multiple epitopes. to this end, the dissection of the humoral immune response elicited by cobra antigens at the single b-cell level is fundamental and would enable the evaluation of their mechanism of elicited breadth of antibody response. moreover, a molecular characterization of cobra ha elicited antibodies would yield additional insights into their effector functions endowed by this kind of immunogens. it is noteworthy that broadly reactive ha antigens for influenza would represent a powerful platform not only from a prophylactic point of view but also for the development of more effective therapeutics (burioni et  al. 2009, burioni et  al. 2010, sautto et  al. 2017). in fact, these novel antigens can be used also for the generation and screening of cross-effective molecules able to recognize and neutralize divergent influenza isolates and to be further developed as possible broad-spectrum antiviral drugs. co-circulating strains (wong et  al. 2017). therefore, the cobra vaccines have the ability to not only elicit protective antibodies against the dominant vaccine strains but also minor circulating strains that can evolve into dominant circulating strains in the future. moreover, similarly to previously described in  vivo pre-immune studies with h1n1 cobra candidates, ferrets imprinted with historical h3n2 viruses and vaccinated with vlp expressing an h3 cobra ha antigen (named t10) elicited sera with higher hai antibody titers than antibodies elicited by vaccines with wild-type ha (allen et  al. 2018). interestingly, sera from pre-immune ferrets subsequently immunized with another candidate h3n2 cobra vaccine (named t11) even if not endowed with hai activity, were able to neutralize antigenically distinct h3n2 influenza viruses, suggesting a different mechanism of neutralization such as antibody-dependent cellular cytotoxicity (adcc) or other antibody fc-mediated effector functions (allen et al. 2018). conclusion to limit the 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for infectious diseases. j immunol res, id 5890962. sautto g.a., kirchenbaum g.a., ecker j.w., bebin-blackwell a.g., pierce s.r. & ross t.m. 2018. elicitation of broadly protective antibodies following infection with influenza viruses expressing h1n1 computationally optimized broadly reactive hemagglutinin antigens. immunohorizons, 2 (7), 226-237. 47 veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 accepted: 09.09.2021 | available on line: 16.11.2022 1college of veterinary medicine, sichuan agricultural university, wenjiang 611130, sichuan province, china 2key laboratory of animal disease and human health of sichuan province, china. *corresponding author at: college of veterinary medicine, sichuan agricultural university, wenjiang 611130, sichuan province, china. e‑mail: yaanwangyin@tom.com. teng tu1,2, changyu liao1,2, pengfei zhang1,2, mingyuan xiang1,2, zexiao yang1,2, xueping yao1,2, yan luo1,2 and yin wang1,2* keywords bioinformatics analysis, isolation, prrsv nadc30-like. summary in this study, lung tissue was collected from nursery piglets suspected of being infected with porcine reproductive and respiratory syndrome virus (prrsv) in a large-scale pig farm in sichuan, china. polymerase chain reaction (pcr) and reverse transcription quantitative-polymerase chain reaction (rt-qpcr) methods were used to ensure that no other pathogens were present. virus isolation was also carried out where the presence of prrsv was determined by indirect immunoinfluscent assay (ifa). compared with the common prrsv strain, the isolate did not produce evident cytopathic effect (cpe) in the early stage of isolation. cpe was found in the late stage, and the titer was 104.17 tcid 50 /0.1 ml. the strain was named cjs01. bioinformatics analysis showed that it was a nadc30-like strain. the virus load was determined by measuring the nucleic acid load during the proliferation of the strain on marc-145 cells. the strain showed good adaptability on cells, and the virus proliferated on cells for 84 hr when the highest nucleic acid load was achieved. by recombinant analysis of orf3~7 genes and prediction of its epitope, it was found that cjs01 strain might interfere with the immunesystem of the infected animals. isolation and bioinformatics analysis of the nadc30-like cjs01 strain of the porcine reproductive and respiratory syndrome virus pigs of all ages have different degrees of susceptibility to prrsv. prrsv can cause damage to multiple organs of the body and seriously affect the health of pigs. it was once called one of the most expensive diseases (rubio et al. 2009). no matter the semen, embryo or animal body, they all can be used as the natural medium of prrs diffusing, and they play a role in the ultra long distance transmission of the virus (xu et al. 2009). prrsv belongs to the rna virus of arteritis virus, with a total length of 15 kb and 10 orfs (yin et al. 1997). as early as 1996, it has been reported that prrsv has the characteristics of code shifting, recombination and mutation (chen et al. 2008). with the passage of time, it has evolved the coexistence of highly pathogenic (hp) prrsv, gm2/qyyz, 1-7-4 rflp, nadc30-like and other virus strains. the nadc30-like showed 131 aa discontinuous deletion (forsberg et al. 2002). comparing with the classical prrsv strain (vr2332, ch-1a), the amino acids of nsp2 protein of nadc30-like isolate were introduction porcine reproductive and respiratory syndrome is caused by porcine reproductive and respiratory syndrome virus (prrsv) (yin et  al. 2016), which mainly causes severe reproductive disorders, growth retardation, respiratory symptoms, and high mortality (gen et  al. 2012). at the end of the 20th century, in the early stage of epidemic, it was an unknown disease at that time, so named as “mysterious disease of pigs” (kappes et al. 2015). as a new virus, there was a serious lack of understanding of the epidemiology and immunology of prrsv (done 1996). prrs has been found for more than 30 years. in 1990s, the pig industry in europe, the united states and other places was in a period of vigorous development (baltag et  al. 2013, li 2015), which provided the necessary conditions for pathogen transmission. in 1996, chinese scholar guo baoqing and other scholars (guo et al. 1996) successfully isolated a strain named ch-1a, and used serological detection to trace prrsv that has been popular in china for a long time. 48 veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 bioinformatics analysis of prrsv nadc30-like cjs01 strain tu et al. virus (tgev), pseudorabies virus (prv), and classic swine fever virus (csfv) were detected by specific polymerase chain reactions (pcr). nadc30-like infected materials were tested by the prrsv nadc30-like sybr green i real-time quantitative pcr (rt-qpcr) as described by xiang and colleagues (xiang et al. 2020). sample processing nadc30-like infected material and other pathogenic (hp-prrsv, pcv, pedv, tgev, prv, csfv) negative materials were aseptically ground, frozen and thawed several times, then they were centrifuged, and the supernatant was collected. after that, a penicillin and streptomycin solution was added. after above operations have been completed, culturing overnight at 37 °c. after the treatment was completed, the homogenate was centrifuged and filtered by using a filter plug. the filtrate was collected and stored at -80 °c for subculturing. virus isolation marc-145 cells were grew in 24-well culture plates at 37 °c in 5% co 2 until they reached 70-80% confluence.confluent monolayers were then inoculated with filtrated homogenates, then added. consequently, cells were incubated in co 2 constant temperature incubator for 2 hours. after incubation, homogenate was discarded, and dulbecco's modified eagle medium (dmem) was added. the cells were then incubated for 4-5 days. if cpe occurs, wait for the pathological area of cells to reach 60%. the suspension was then recovered, freeze-thawed and centrifuged. after that, the supernatant was extracted and purified before subcultur to the plaque. the purified suspension was continuously subcultured up to 17 passages, and the presence nucleic acid of the virus was controlled every 3 passages. if there is no cpe for 10 passages, the eventual presence of nucleic acid was also tested. the sample was considered as negative after 4 negative results in a row. indirect immunofluorescence assay (ifa) cell monoalyers at 70%~80% confluence were fixed with paraformaldehyde fix solution (pfa) at room temperature for 30 minutes. the pfa was washed repeatedly for three times. then triton x-100 was added and kept still for 30 minutes. after that bovine serum albumin (bsa) was added for blocking and at room temperature for 2 hours. then the blocking solution was discarded and anti-n protein monoclonal antibody was added and kept overnight. the cells were washed repeatedly for three times. after that, fluorescein isothiocyanate was added avoiding light absent at 322-432 (111 aa), 481 (1 aa) and 504-522 (19 aa) (li 2015), which is of great significance for prrsv typing. during the evolution of prrsv, some virulent strains, such as prrsv tj, hunan, hn-1/06, nadc30-like recombinant strain jl580 (zhang et al. 2018, giles et al. 2015) were produced. among all subtypes, recombination often occurs in different hot spot genomic regions (kappes et  al. 2015, hopper et  al. 1992, spilman et  al. 2009), such as gp5, nsp2 and other genes. after recovering from infection, the animal is protected from infection with homologous strains or strains with high homology (he 2012, lager et  al. 1997). however, due to the strong heredity and antigenic variation of prrsv, no widely effective and cross protective preparedness measures have been developed (duan 2011). it has been pointed out that gp5 plays an important role in neutralizing antibody production in humoral and cell-mediated immune response after vaccination or field virus infection (vu et al. 2017, hu et al. 2009). materials and methods materials and reagents the lung tissue of piglets suspected to be infected with prrsv were collected from a large-scale pig farm in sichuan province, china. the marc-145 cell line was preserved in the laboratory of sichuan agricultural university (cheng du, china). escherichia coli dh5α was purchased from beijing tiangen corporation (beijing, china). t-vector pmd19 (simple) was purchased from takara corporation (tokyo, japan). dmem medium (high sugar type) was purchased from gibco, california, united states of america. fetal bovine serum was purchased from sangon biotech, shanghai (china). anti-prrsv n protein monoclonal antibody was purchased from genetex, san antonio, united states of america (usa). primescript rt reagent kit and triton x-100 were purchased from takara corporation, tokyo, japan. genomic dna/rna extraction kit, dna molecular weight standard, dna gel recovery and purification kit and plasmid extraction kit were purchased from tiangen corporation, beijing (china). aceq® qpcr sybr® green master mix was purchased from vazyme-innovation in enzyme technology, nanjing, china. ampicillin was purchased from amresco, houston, usa. 1.1×t3 super pcr mix was purchased from tsingke biological technology, chengdu (china). nadc30‑like positive sample specific detection first, porcine circovirus (pcv), porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis 49veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 tu et al. bioinformatics analysis of prrsv nadc30-like cjs01 strain table i. test primer design table of the porcine reproductive and respiratory syndrome virus (prrsv). primer name primersequence (5’-3’) product length (bp) annealing temperature (°c) prrsv-1f aatcagtcgcggtagtcg 1,360 56 prrsv-1r acagtacgtagaattggcactc prrsv-2f cagtacctcaggtcaggtgtt 1,062 58 prrsv-2r caaccactaaggatcgctggg prrsv-3f ttggcaatgtgtcaggcat 1,120 58 prrsv-3r gcaggggccagaatgtacttg prrsv-4f ctccagatgccggttgtgc 749 55 prrsv-4r atttcggccgcatggttc draw the growth curve we calculated the viral nucleic acid load of each time point in the virus solution with the same volume, and the virus proliferation curve was drew with the time in the x-axis and the nucleic acid load in the y-axis. amplification and bioinformatics analysis of some structural genes specific primers were designed for multiple nadc30-like strains (table i), based on the prrsv nadc30 sequence downloaded from genbank (accession number: jn654459). these were then created by the bgi sequencing co., ltd. we amplified the orf3-7 gene fragment of the nacd like virus strain as described in previous studies with some modifications (liao 2019), and sent the amplified product to the bgi sequencing co., ltd for sequencing. sequences were stitched together and used to generate an evolutionary tree for the reference strain (table ii). in addition to the recombination analysis, we also predicted the protein antigen epitope and conducted homology analysis as described in previous studies with some modifications (zhang 2006). all the bioinformatics analysis software used are listed below: • dnaman v6.0 software for assembling the sequence; • mega v5.1 software for generating maximum-likelihood (ml) trees; • simplot v3.5 software for recombination analysis; • dnastar software for predicting the protein antigen epitope. for 20 minutes. after washing and sucking away the superfluous liquid, eclipse ts100 was used to select the appropriate wavelength for fluorescence observation and recording. after washing and sucking away the superfluous liquid, eclipse ts100 was used to select the appropriate wavelength for fluorescence observation and recording. determination of half infection amount of the virus marc-145 cells were cultured adjusting the cell concentration to 1.5 × 105/ml in 96 wells plate. after that, the viral suspension was inoculated when the monolayer was 80% confluent, and incubated at 5% co 2 atmosphere at 37 °c. sixteen wells were selected as the negative control group, and 8 dilution gradients (10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8) were set at the same time. twenty wells were inoculated with each dilution, and the observation and record were made after incubation for 5 days. tcid 50 was calculated by reed-muench method (benfield et al. 1992). cell attack and virus copy number detection marc-145 cells were prepared, the cell concentration was adjusted to 1.5×105/ml in 12 well plate, when the cell density grew to about 80%, inoculate the virus, and diluted virus solution was added into each well. cells were cultured in co 2 incubator. one hole was selected for cell control, and three repeated test groups were set up. the virus was collected at 12 hr intervals (11 groups in total) within 0-120 hr after the exposure, and each group took 300 μl suspension to extract rna. preparation of cdna rna was extracted. complementary dna was prepared, and stored at - 20 °c. table ii. porcine reproductive and respiratory syndrome virus strains used in the study. strain genebank area year jxa1-p80 fjy548853 china 2008 tj eu86024 china 2006 gx1003 jx912249 china 2010 ch-1a ay032626 china 1996 vr2332 u87392 usa 1992 bj-4 af331831 china 2000 mlv af159149 usa 1992 qyyz jq308798 china 2012 gm2 jn662424 china 2012 mn184c mn184c usa 2008 fjy04 kp860910 china 2014 henan-heb kj143621 china 2012 nadc30 jn654459 usa 2008 50 veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 bioinformatics analysis of prrsv nadc30-like cjs01 strain tu et al. titer of the 15th generation of nadc30-like virus was higher than that of the 4th generation. this indicated that the isolate could adapt to marc-145 cell line. determination and comparative analysis of half amount of virus infection the titer of the isolate on marc 145 cell line was 104.17 tcid 50 /0.1 ml. the results showed that the virus titer was slightly lower than that of the normal isolates, and the virus isolation process did not show obvious cpe in the earlier stage, even though the incubation time was longer. drawing of growth curve after inoculation, the virus solution was detected in each period, and the relative nucleic acid load was measured to draw the growth curve. the results showed that the isolates were in the period of concealment from 0 to 40 hr, entered the logarithmic period at 48 hr, and began to enter the plateau period from 40 to 84 hr, and reached the peak at 84 hr (figure 4). the rapid decline in virus volume may be due to the release of rna from the cells into the extracellular environment at 37  °c and the degradation of rna enzymes at ambient temperature for a long time due to rna virus characteristics. the virus was completely inactivated at 37  °c for 48 hr. it can be seen that stability has results sample nadc30‑like sybr green qpcr test result when tested for hp-prrsv, pcv, pedv, tgev, prv, and csfv by pcr, no specific amplified bands were found. using the prrsv nadc30-like sybr green i qpcr detection method, the sample had a specific amplification curve (figure 1). cytopathy observation the prrsv nadc30-like strain positive materials were inoculated in marc-145 and incubated in co 2 incubator at 37 °c for 4-5 days. the nucleic acid of the virus was detected by qpcr every 3 passages. the results showed that the nucleus expanded slightly and became darker from the third to ninth passage, and it was similar to normal cells. cpe began to break off in large area and adhered to the cell surface in the form of clumps or grapes starting from the 10th passage (figure 2). the cpe mainly showed cell aggregation, shedding, irregular shape and strong refraction. indirect immunofluorescence the cultured marc-145 cells were fixed with 5% pfa and stained with anti-n protein mab and fluorescein isothiocyanate, respectively. marc-145 cells that received nadc30-like strain showed specific fluorescence after 4 days of challenge (figure 3). specific green fluorescence around nucleus or aggregation was observed in group a, but no specific fluorescence was observed in negative control group b. detection of fluorescence quantitative method the nadc30-like virus viral nucleic acid was detected from the 4th generation to the 15th generation. the figure 1. qpcr test result of pig samples infected with porcine reproductive and respiratory syndrome virus. 1 = positive control; 2 = sample. 3 = negative control. figure 2. porcine reproductive and respiratory syndrome virus nadc30‑like cjs01 strain growth in marc‑145 cell line cytopathic effect. a. negative control group. b. infected group. figure 3. porcine reproductive and respiratory syndrome virus nadc30‑like cjs01 strain growth in marc‑145 cell line. immunofluorescence test results. a. infected group. b. negative control group. 51veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 tu et al. bioinformatics analysis of prrsv nadc30-like cjs01 strain respectively (table iv). these results indicated that the gm2/qyz type strain was also identified in the strains of other subtypes, which belonged to a type of recombinant strain, mostly hp-prrsv and nadc30-like. using dnaman and simplot software, partial structural gene analysis revealed that the fragment recombination between nadc30-like strain henan-heb and fjy04 (figure 6-e) occurred in cjs01 strain. the orf5 and orf6 genes were mainly located on the orf5 and orf6 genes, i.e. gp5 and m proteins. the orf5 gene was located at 87~294  bp and 507~603 bp, and the accumulative length of the recombinant fragment was about 303 bp, accounting for 50.2% (303/603) of the orf5 gene. the orf6 gene was located at 459~489 bp. the accumulative length of orf6 gene was about 45 bp, accounting for 8.6% (45/525) of the orf6 gene. the recombinant gene accounted for 31.3% (348/1,112) of gp5 and m genes, and 13.6% (348/2,565) of orf3~7 genes. the orf5 gene and orf6 gene of prrsv cjs01 strain were predicted by antigenic epitopes (figure 6-c and figure 6-d). the results revealed that there were potential epitopes at 87~294 (aa29~89), 507~603 (a189~201) and 1~141 (aal~47), 459~489 (a153~163) of orf5 gene. this indicates potential epitopes in the recombination loci. discussion in this study, nadc30-like strain positive samples were grown on marc-145 cells. a prrsv nadc30great influence on it, and it is also important to choose a suitable time for virus harvest. amplification of partial structural genes according to the instructions of rna extraction kit, viral nucleic acids were extracted. the target bands were located at 749 bp, 1,120 bp, 1,062 bp and 1,360  bp, respectively (figure 5). according to the instructions of the gel recovery kit, the target conditions were recovered and ligated. bioinformatics analysis the nucleotide sequence of orf3~7 was obtained by splicing the entire sequence (2,566 bp). two maximum-likelihood (ml) trees (figure 6-a and figure 6-b) of orf5 and orf3-7 gene fragments were constructed by bioinformatics analysis of bj-4, jxa1 and tj strains. the homology of orf3 gene, orf4 gene, orf5 gene, orf6 gene, orf7 gene and orf3~7 gene versus the nadc30 strain (jn654459) were 94.8%, 95.5%, 94.1%, 92%, 98.1% and 94.4%, table iii. porcine reproductive and respiratory syndrome virus nadc30‑like cjs01 strain growth in marc‑145 cell line. number of wells showing cytopathic effect (cpe). dilution of stock solution number of wells with cpe holes number of wells without cpe cumulative percentage of cpe holes number of wells with cpe number of holes without cpe 10-1 20 0 74 0 100% (74/74) 10-2 20 0 54 0 100% (54/54) 10-3 20 0 34 0 100% (34/34) 10-4 12 8 14 8 60% (14/22) 10-5 2 18 2 27 7% (2/29) 10-6 0 20 0 47 0 (0/47) 10-7 0 20 0 67 0 (0/67) 10-8 0 20 0 87 0 (0/87) 700 600 500 400 300 200 100 0 0 20 40 60 80 100 120 140 r fu h figure 4. growth curve of the porcine reproductive and respiratory syndrome virus nadc30‑like cjs01 strain in marc‑145 cell line. h:hours, rfu:relative fluorescence unit. figure 5. rt‑pcr amplification of cjs01 strain structural gene orf3~7 fragment. m = dna marker dl2000; 1~4 = target segment. m 1 2 3 4 2,000 bp 1,000 bp 750 bp 500 bp 250 bp 100 bp 52 veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 bioinformatics analysis of prrsv nadc30-like cjs01 strain tu et al. figure 6. a. porcine reproductive and respiratory syndrome virus nadc30‑like cjs01 strain: orf3~orf7 structural gene phylogenetic tree. b. gp5 structural gene phylogenetic tree. c. gp5 epitope prediction. d. gp6 epitope prediction. e. simplot analysis of orf3~orf7 recombination sites. a b c d e 53veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 tu et al. bioinformatics analysis of prrsv nadc30-like cjs01 strain the recombination between strains, and the number of nucleotides in one mutation is obviously superior to that in gene recombination. the reason may be the joint action of the error correction mechanism of prrsv itself and the external selection pressure. it is worth noted that the aa37-44 of gp5 is an important neutralizing epitope that determines the neutralization reactivity of serum. aa102 and aa104 of gp5 play an important role in virus susceptibility (sun 2016), and aa10 of gp6 has a great influence on immune neutralization. this study shows that the recombinant is mainly located on gp5 and m protein, and the fragment of the recombinant contains the production range of virus neutralizing antibody and neutralizing antibody, which has a direct relation to immune escape (zhang et  al. 2018). there is antibody-dependent enhancement (ade) effect in prrsv, and it has been proved that the amino acid region leading to this effect is also in this range. with the antigen drift of the strain, the ade effect may follow this region into the recombinant strain, and the effect is also related to the parent strain. the previously reported prrsvnadc30-like jl580 strain can produce stronger immunosuppression, which is speculated to be associated with this interval recombination. if the genes carrying stronger immunosuppressive regions, such as drug resistance genes, are transferred between strains, the harm may be more serious. most of the recombination events of prrsvnadc30-like strain occurred with hp-prrsv strain. in tian k’s study (tian 2017), prrsv nadc30-like was compared with other strains (mn184 and sdsu73). pigs infected with nadc30-like had an early febrile and peak at 8 days post infection (dpi). nadc30-infected pigs developed earlier viremia which could be detected as early as 1 dpi. the nadc30-infected pigs developed interstitial pneumonia, but relatively milder as compared with sdsu73 and mn184. the above results suggested prrsv nadc30-like as relatively mild in virulence. in his study, a prrsv nadc30-like strain showed much lower pathogenicity as compared with hp-prrsv. the harm caused by immunosuppression of hp-prrsv strain among many prrsv strains is the most serious. this study suggests that there are two important epitopes of prrsv in this recombination interval, which control the production of immunosuppression and neutralizing antibodies, respectively. it is considered that this phenomenon is one of the causes of clinical immune failure. through the prediction of the antigenic epitopes of the strain, it was revealed that all the segments of the recombination occurred in the presence of potential or confirmed antigenic epitopes. the occurrence of this recombination phenomenon is speculated to be caused by the huge pressure drive caused by the widespread use of prrsv vaccine. besides, improving like strain with good adaptability was obtained. it was named prrsv cjs01 strain. there was no obvious cpe in the early stage of virus passage up to the 10th passage when an obvious cpe was observed. the adaptability of the strain may be related to n protein. n protein, which accounts for about 35% of the total viral constituent proteins, is an important structural protein in virus assembly and is often enriched in nuclei and nucleoli. the marc-145 cell's poly(a)-binding protein (pabp) specifically binds to the n protein. this combination inhibits cell protein synthesis and reduces virus replication, resulting in a decrease in virus titer in the cell. it was observed that the n protein surface gathered in the nucleus and cytoplasm of the infected marc-145 cells showed a granular appearance. therefore, it was speculated that the nucleus and nucleolus of the infected cells might have pathological changes during the early separation process, which could be one of the reasons for the failure of virus isolation. among them, the variation of n gene in nadc30-like prrsv was located in the presence of antigenic epitopes, possibly leading to changes in the corresponding sites of specific recognition and binding of antigenic determinants. the qpcr method was used to detect the nucleic acid load in the process of virus proliferation. it was found that the nucleic acid content was the highest after 84 hr from marc-145 cell infection. to obtain high titers of virus, collecting and passaging in this period of time would contribute to a good efficacy. through the analysis of the biological characteristics of the isolated strain, it was found that the recombination mutation event also occurred in this strain, and the mutation rate of prrsv was obviously accelerated in recent years. hence, it is speculated that the antigen drift may not only be caused by gene mutation, but also caused by table iv. nucleotide homology analysis. strain orf3 orf4 orf5 orf6 orf7 orf3~orf7 nadc30 94.8 95.5 92.9 96.8 98.1 84.5 bj-4 82.2 85.7 84.9 89.7 91.9 86.3 mn184c 81.7 88.8 88.4 89.1 89.8 87 jxa1 81.7 84.5 85.9 89 90.3 93.3 gx1003 81.2 85.3 85.4 89 89.8 85.7 henan-heb 94.4 95.2 91.9 95.6 96 85.5 fjy04 91.4 93.9 91.9 95.2 95.4 94.5 tj 81.4 85.3 85.9 88.8 90.3 85.8 mlv 82.2 86 85.1 89.7 91.9 86.4 ch-1a 81.4 85.7 86.6 88 90.9 85.9 vr2332 82 86 85.1 89.9 92.5 86.4 qyyz 81.4 84.5 86.1 88 90.9 84.6 ch-1r 80.5 85.7 84.1 88.8 87.6 84.4 gm2 80.8 85.1 83.4 89.3 85.8 95.4 54 veterinaria italiana 2022, 58 (1), 47-55. doi: 10.12834/vetit.2182.14564.1 bioinformatics analysis of prrsv nadc30-like cjs01 strain tu et al. acknowledgments this work was funded by the special project of sichuan veterinary drug innovation team of national agricultural industry technology system. the detection methods of prrsvnadc30-like and strengthening the epidemiological investigation of prrsv are of far-reaching significance for the scientific prevention and control of prrsv. alemán f. , nieves cordones m., martínez v. & rubio f. 2009. differential regulation of the genes encoding the high-affinity k + transporters hak5 of thellungiella halophila and arabidopsis thaliana in response to salinity. environmental experimental botany, 65, 263-269. baltag g. & baranov e. 2013. characteristics and strategic development of pork 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[the cloning and bioinformation analysis of orf5 gene of prrsv-scq strain & prokaryotic expression of dorf5 gene]. master’s dissertation, sichuan agricultural university. zhang h.l., tang d.y. & liu c.x. 2018. adaptions of field prrsvs in marc-145 cells were determined by variations in the minor envelope proteins gp2a-gp3. vet microbiol, 222, 46-54. zhang h., xia m., wang w., ju d., cao l., wu b., wang x., wu y., song n., hu j., tian c., zhang s. & wu h. 2018. an attenuated highly pathogenic chinese prrs viral vaccine confers cross protection to pigs against challenge with the emerging prrsv nadc30-like strain. virol sin, 33 (2), 153-161. xiang m.y., liao c.y., jiang d.k., zhang p., wang y., luo y., yang z.x. & yao x.p. 2020. establishment and application of prrsv nadc30-like sybr green i qpcr detection method. biotechnology bulletin, 36 (5), 80-85. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0885. xu g.d. & li z.h. 2009. progress in the prevention and treatment of porcine reproductive and respiratory syndrome in china. journal china animal health, 1, 67-69. yin z. & liu j.h. 1997. animal virology. 2nd edition. scientific publishing house, beijing, china. yin, j., zhang, h. and qi, l. 2016. research advances on effects of porcine reproductive and respiratory syndrome virus on the immune system of pigs. journal of anhui agricultural sciences, 44, 81-82. 327 dept. of animal production, welfare and veterinary sciences, harper adams university, united kingdom. *corresponding author at: dept. of animal production, welfare and veterinary sciences harper adams university, newport, shropshire, united kingdom, tf10 8nb. tel.: +44 (0) 1952 820280, e-mail: probinson@harper-adams.ac.uk. parole chiave resistenza antielmintica, comportamenti e opinioni, allevamento, interviste semi-strutturate, scienze sociali, galles. riassunto nonostante l'importanza della resistenza antielmintica (ra) nel bestiame, mancano in questo settore le ricerche approfondite che esaminano i comportamenti degli utenti finali e dei loro consulenti professionisti. date la crescente importanza dello sviluppo di modelli di resistenza antielmintica nei bovini e la necessità di evitare che la ra si sviluppi nei bovini nella stessa misura che ha avuto negli ovini, obiettivo di questo studio è stato valutare i fattori che influenzano la scelta e l'uso del prodotto antielmintico, e la consapevolezza e i comportamenti degli operatori nei confronti della ra nel galles del nord. sono state condotte interviste frontali con nove allevatori e tre veterinari. scarsi sono risultati la conoscenza e l'importanza della materia in esame; manca la percezione della minaccia, avvertita solo con portata individuale. il costo influisce in maniera determinante sulla scelta dei prodotti antielmintici, ma sono risultate importanti anche le raccomandazioni sui prodotti provenienti da varie associazioni sparse sul territorio e da altre fonti di consulenza non veterinarie. in conclusione, gli autori auspicano una maggiore sensibilizzazione e formazione di allevatori e veterinari sul questo problema emergente al fine di migliorare le profilassi antielmintiche. indagine qualitativa sugli atteggiamenti e sulle pratiche degli agricoltori e dei veterinari riguardo alla resistenza antielmintica nei bovini in galles keywords anthelmintic resistance, attitudes and opinions, cattle, semi-structured interviews, social science, wales. summary despite the importance of stakeholder practices in the potential development of anthelmintic resistance (ar) in livestock, there is a lack of qualitative research examining the attitudes and behaviours of anthelmintic end users and their professional advisors. given the increasing importance of developing anthelmintic resistance patterns in cattle, and the need to avoid ar in cattle developing to the same extent as it has in sheep, the objective of this qualitative study was therefore to assess the factors affecting anthelmintic product choice and usage, and awareness and attitudes towards ar in cattle in north wales. twelve semi-structured face-to-face interviews were conducted with nine cattle farmers and three veterinarians. farmer knowledge and engagement with the issue of ar in cattle in this study was low. a lack of perceived threat was apparent, with only a demonstrable problem at farm level the likely incentive to change future worming protocols and practice. cost had a very prominent influence on anthelmintic product choice, but importance was also given to product recommendations from social farming networks and other non-veterinary advisory sources. a more proactive approach should be taken to raise farmers’ and veterinarians’ awareness of increasing levels of ar in cattle and improve anthelmintic governance. klaudya charlton and philip a. robinson* a qualitative investigation of the attitudes and practices of farmers and veterinarians in wales regarding anthelmintic resistance in cattle veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 accepted: 26.07.2019 | available on line: 31.12.2019 328 qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle charlton & robinson veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 (bellet 2018) used semi-structured interviews to qualitatively investigate dairy farmers’ attitudes and practices in england. semi-structured interviews with farmers, veterinarians and other stakeholders are increasingly being used to investigate animal health across a wide range of issues (e.g. vaarst et al. 2002, adam et  al. 2017, robinson 2017 a, b, clémence et  al. 2018, lomas and robinson 2018). qualitative research methods such as interviewing allow for the interpretation of data unable to be expressed numerically, such as individual attitudes and reported behaviours with their underlying rationale (saunders et al. 2016), and provide insights into the cultural framings that people use to make sense of their experiences (miller and glassner 2016). in a similar vein, this paper reports on qualitative research involving interviews with cattle farmers and livestock veterinarians in north wales that were designed to elucidate their awareness and attitudes regarding ar in terms of the perceived current and future risks, and how this affects pharmaceutical treatment protocols and engagement with professional advice and herd health planning. wales has a cattle population of approximately 1.1 million cattle (welsh government 2018), some 11% of the overall uk cattle population of 9.9 million (defra 2018). farms in north wales are predominantly family farms, with a mix of dairy and beef cattle and sheep. materials and methods the research ethics committee of harper adams university granted ethical approval for this research project (approval no. 1113-201612-staff). purposive, non-probabilistic sampling was chosen to increase the likelihood of providing information-rich and relevant data (curtis et al. 2000, patton 2015). a veterinary practice in north wales acted as a research gatekeeper by providing potential participants with 70 or more cattle in their herd in either a dairy or a beef commercial enterprise that utilised outdoor grazing for at least part of the year. other farmers were sourced through ‘snowball sampling’ (noy 2008) where participants suggested further contacts, allowing others to be found who could otherwise be inaccessible or difficult to identify (saunders et al. 2016). twelve semi-structured face-to-face interviews (including two pilot interviews) were conducted in north wales between december 2016 and march 2017. the interviewees consisted of four dairy farmers, two beef suckler farmers, three who had both dairy and beef enterprises and three livestock veterinarians. the main objective of interviewing the veterinarians was to triangulate the data from the farmer interviews using an alternative data source introduction the frequent usage of relatively low-cost, broad-spectrum anthelmintic drugs, coupled with inappropriate parasite management strategies, has increased selection pressure for resistant populations in ruminants over the past 40 years, especially in sheep (guerden et al. 2015, martínez-valladares et al. 2015). potentially perceived as a minor and relatively new problem by industry stakeholders (george et al. 2017), the issue of anthelmintic resistance (ar) in cattle has been growing in scientific research significance, especially over the past decade. in countries with important cattle industries such as the united kingdom (uk), new zealand, brazil, argentina and the united states of america, the definite trend is towards increasing ar detections in cattle nematodes, and resistance to the three main anthelmintic classes has been confirmed in all major nematodes of cattle (de graef et al. 2013, leathwick and milller 2013, gasbarre 2014, suarez and cristel 2014, ramos et al. 2016, cristel et al. 2017). indeed, a recent systematic review of the issue of ar in cattle has demonstrated that the problem is global (baiak et  al. 2018). hosking and colleagues (hosking et  al. 1996) reported the issue featuring in the literature back to 1986, but warnings about the importance of the emerging problem may have gone largely unheeded in the field. it is imperative to dramatically slow ar development, and conserve the efficacy of the anthelmintic treatments for cattle currently widely available, as has become a priority within the sheep industry (taylor 2012, learmount et al. 2016). an understanding of the decision-making processes of the farmers who use anthelmintics is important to guide future strategy formulation for encouraging sustainable worming practices (charlier et al. 2016). vende velde and colleagues (vande velde et al. 2018a) and morgan and colleagues (morgan et  al. 2019) emphasize the need for interdisciplinary research between veterinary parasitology and other academic disciplines, and call for a better understanding of farmer behaviours and motivations concerning anthelmintic treatments and helminth control. there has been a general lack of published social science research focusing on the attitudes and awareness of stakeholders regarding ar in cattle. this deficit has begun to be addressed within the last five years in belgium and the uk, although there is still much to be investigated. vande  velde and colleagues have conducted questionnaire (vande  velde et  al. 2015) and semi-structured interview studies (vande velde et al. 2018b) with belgian dairy farmers on their use of anthelmintics. easton and colleagues (easton et al. 2016 a, b, easton et al. 2018) have reported on questionnaire studies in the uk with a wide range of industry stakeholders involved in prescribing and administering anthelmintics in cattle, and bellet 329 charlton & robinson qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 pour-on is a lot easier safety-wise – you don’t get hurt.» (int f9, beef farmer) charlier and colleagues (charlier et  al. 2016) cite economic factors as a major influence on decision-making with respect to anthelmintics, therefore it was expected that farmers would regard economic factors to be of importance in the current study, which was the case with most interviewees, as evidenced by these quotes: «farmers have had a really rough two years and you’ve got to pick the cheapest thing. whether that’s the right thing or not remains to be seen.» (int f3, dairy-beef farmer) «it basically came down to whatever was on offer [...] if it’s coming to the end of the line and they’re doing 30% off, [or] the use-by date’s up in a month and you know you’re going to use it by then, yes, we’ll buy it.» (int f6, dairy farmer) throughout the interviews, product cost clearly influenced product choice, with perceived efficacy and the farmer’s own risk assessment of helminth infection on the farm also influencing treatment choice. two farmers (f3 and f9) suggested that cost took precedence over anything else. this view was also confirmed by one of the vets interviewed: «for a lot of farmers, certainly our farmers, cost is the main thing, instead of looking at what they’re getting for it.» (int v3, veterinary surgeon) despite this, other farmers supported the value of product efficacy and quality above cost, but the price of the anthelmintic product was still highly influential: «we’ll get the advice first and work out what we’re going to use, and then we’ll get a price.» (int f1, dairy farmer) «with wormers, you tend to get what you pay for, you tend to pay for quality, and if you’re quite opportunistic you can go to the local supplier and they’ll have offers and that encourages you to buy the best quality stuff.» (int f2, dairy farmer) «with the pour-ons, if there’s an offer on, then i’ll buy that product. but [anthelmintic product] works well for me and it always seems to come out the cheapest. i’d ask the price for that, because i know that’ll do the job.» (int f9, beef farmer) eight of the nine farmers calibrated their dosage apparatus before use, with the majority acquiring new apparatus annually. all of the farmers stated that they deliberately overdosed cattle when administering anthelmintics. however, only three farmers (f1, f6 and f7) possessed weigh tapes or with knowledge and experience of farmer attitudes and practices (flick 2014). all the interviews were arranged in advance by telephone, and then took place face-to-face at a location chosen by the participants. participants were assured that their identities would remain anonymous throughout the data collection, analysis and write-up process, along with their right to withdraw from the study at any time. written informed consent was obtained prior to the commencement of each interview. all interviews were recorded using a digital recorder, before being transcribed verbatim using nvivo software (version 11, qsr international ltd). the data analysis used a grounded theory approach, using both preconceived themes and those emerging during data collection and analysis in an iterative process involving reading and re-reading the interview transcripts and involving both authors (glaser and strauss 2008, saunders et  al. 2016). nvivo software was also used to code the data during the analysis. results the herd sizes of the farms represented in the interview sample ranged from 70 to 650 cattle, with a mean herd size of 335 across the nine farms. the interviews lasted up to 80 minutes for the farmers and up to 51 minutes for the veterinarians. the following sections describe the key findings of the interviews. decision‑making regarding anthelmintic treatment product and protocol all nine farmers’ worming protocols utilised a combination of anthelmintic treatments and pasture management to lower the impact of parasitism in their cattle. there were various combinations of husbandry strategies such as rotational grazing, grazing silage aftermath, using pastures that had been grazed by sheep in the previous season, co-grazing with sheep, and lowering stocking densities of cattle on pasture. the most commonly chosen anthelmintic class and formulation were the pour-on macrolactone (ml) products, heavily influenced by ease of administration: «i like a pour-on rather than a drench [...] ease of use really, ease of application [...] it’s a lot of work if you’ve got to go and drench so many cattle.» (int f3, dairy-beef farmer) «we tend to use the pour-on for cattle because when you get them in a race, you can easily dose them [...] years ago we used to use the bolus. to be honest with you, the 330 qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle charlton & robinson veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 other farmers tended to attribute ar to media or pharmaceutical industry exaggeration or pseudo-science exacerbating the issue beyond its true seriousness, leading farmers to suggest that there would be a wider coverage of ar if there was a significant problem: «where are the actual facts coming from? are they scientific facts? that’s the biggest problem we’ve got.» (int f1, dairy farmer) «if there was a widespread problem, we’d be made far more aware of it before now. the fact that it’s not very well-advertised and there’s no big push on it – if there was a nationwide problem, we’d know about it. the vets and the drug companies would be straight onto it.» (int f9, beef farmer) «i think you’ve got to be careful that the worming companies aren’t driving the issue and making it sound worse than it is. speaking from my own experience, we don’t have a major problem.» (int f8, dairy-beef farmer) most interviewees had more awareness of ar in sheep, and two farmers (f5 and f7) had prior experience of ar occurring in their own sheep flocks, but there was a general lack of awareness and it was perceived as a lower risk for cattle. the farmers in this study tended to prioritise matters with a more obvious economic impact on their farming enterprise: «the last thing you would look at when you buy a heifer or whatever is what the worm burden would be. the biggest worry around here is [bovine] tb.» (int f1, dairy farmer) «worming control’s in there, but it’s not that high up the list. my main concerns are mastitis, lameness, fertility – they’re the biggest three [...] i suppose they’re every dairy farmer’s biggest three.» (int f3, dairy-beef farmer) the veterinarians also agreed that the issue of ar was a low priority amongst their farm clients, and one explained the issue as follows: «perhaps we’re talking to farmers who don’t have a perceived problem and we’re saying, “you need to do this and that and have all this extra work”, for them to ask, “why?” because they’re already pushed as it is time-wise, and they don’t see it [ar] as a problem.» (int v1, veterinarian) another veterinarian (v3) concurred, attributing the farmers’ lack of ar awareness to difficulties in quantifying reduced production parameters such as slower growth, therefore convincing farmers to refine or completely change their worming protocols was challenging. scales to enable accurate estimation of bodyweight, with the majority estimating weights visually. the farmers were generally confident in the suitability of their cattle worming protocol, with many adopting an ‘if it isn’t broken, don’t fix it’ attitude towards their practices. some did not see any need to ask for advice on worming, preferring to follow long-established traditions: «i’ve got a way of doing things that has worked for years and to me, if it’s not broken, why fix it? i would be disgruntled if i was asked to change things if i had spent time perfecting it.» (int f2, dairy farmer) «i’ve got a worming policy that seems to work, so if it’s not broken then i’m not going to fix it.» (int f9, beef farmer) «i bet [that] 75% of farmers’ worming policies are what grandad’s done [in the past] [...] they do x, y and z every year, and that’s it.» (int f3, dairy-beef farmer) diagnosis before treatment, or assessment of product efficacy did not feature highly in the discussions. only one farmer from the sample of nine had conducted helminth diagnostics in their cattle, and the following quote was the response of one of the veterinarians when asked about faecal egg counting: interviewer: «do you find that cattle farmers utilise faecal egg counts much, in your experience?» veterinarian: «i don’t think i’ve done one for cattle. we’ve done horses and the occasional sheep, but not for cattle [...] it’s not something i’ve ever seen anyone do.» (int v2, veterinarian) awareness and concern regarding ar in cattle the farmers’ opinions regarding ar were strongly influenced by the lack of an apparent problem on their own farm, with most of them downplaying the importance of ar for this reason. five of the nine farmers interviewed had some limited knowledge of the issue of ar in cattle, but others were completely unaware. overall, it was deemed to be of low importance for them as individual farmers: «well, i’m not aware [of it] because i haven’t had to be.» (int f5, dairy-beef farmer) «i do try to [rotate anthelmintics] – i suppose they can get immune to the stuff after a while.» (int f2, dairy farmer) «i’m conscious of it, but i wouldn’t say i’m too concerned about it because it hasn’t affected me.» (int f3, dairy-beef farmer) 331 charlton & robinson qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 suitably qualified persons (sqps) and pharmaceutical sales representatives in the uk, an sqp is an animal medicines advisor who has legal authority under the veterinary medicines regulations 2013 to prescribe and/or supply certain categories of veterinary medicines having been trained and professionally qualified to do so. richens and colleagues (richens et  al. 2015) highlight farmers’ willingness to use agricultural merchants for their livestock vaccine purchases as they were perceived to be cheaper and often more convenient than visiting veterinary practices. this finding led to the same expectations of opinions regarding anthelmintic purchase, and this was confirmed by the interviews. the pricing policies, sales trends and advice of sqps working in agricultural stores were very influential factors on product decision-making for these three farmers: «you can guarantee in april time there will be a litre extra for free, so it’s a good incentive really [...] the [sqp] is quite good to be honest. she knows what works and what doesn’t; what are good sellers, and what isn’t.» (int f2, dairy farmer) «a couple of the lads there [at the agricultural merchants] are farmers themselves. they’ll say: “well we’ve sold a lot of this recently”. so they’re there for advice.» (int f9, beef farmer) however, for others there appeared to be mixed views in terms of the quality of sqp advice, with both farmers and vets showing a degree of scepticism regarding the advice given in such a setting: «it’s the same with worming – this is why they’re overused. sometimes [sales] reps are trying to sell a product so they’ll tell you that you need to use it.» (int f7, beef farmer) «at the end of the day, they’re trying to sell you stuff. you’re in their hands a bit [...] that’s why we lean towards the vet a bit more than the others.» (int f1, dairy farmer) «if i go and pick up a wormer to give at home [...] whether because they may know i’m a vet, they (sqps) don’t usually question me on what i’m wanting it for. i’ve seen other people in agricultural merchants and they’ll ask for a product, and they’re not taken aside to have a chat.» (int v3, veterinarian) veterinary advice some of the farmers also stated that other advisory sources, such as veterinarians, could offer tailored advice which was more specific to their farm. a recurring theme in the interviews was that the veterinarian’s familiarity with their farm conferred attitudes towards the perceived value of advisory sources on anthelmintics official guidelines farmers and veterinarians alike noted the importance of education in combating ar, but the farmers appeared to have little interest in engaging with educational resources and knowledge transfer meetings on responsible anthelmintic use: «to be honest, if something came through my door saying that hcc (hybu cig cymru (meat promotion wales) – farmer levy body) are doing a meeting about wormer resistance, i probably wouldn’t go. it’s not really ever stimulated me enough to do anything. if i haven’t got a problem with it, i’m not going to try to do anything about it.» (int f3, dairy-beef farmer) «i’d have to speak to someone about where my resistance is going to come from in my herd. i’m not bothered about anyone else’s.» (int f8, dairy-beef farmer) the farmers compared unfavourably generic official guidelines and online advice to the specificity of veterinarians’ advice for their particular farm circumstances. none of the farmers interviewed used official worming guidelines provided by the farmer levy boards. advice from other farmers several of the farmers took a proactive approach to obtaining animal health advice from their farming peers through attending discussion groups on cattle health and by talking to farming friends in their locale: «friends i regard to do a really good job of farming – not just like bob next door. i’ve got mates in my discussion group from all over the country. they might not be right, but i’d listen to them first [...] i’d regard their opinions quite highly.» (int f3, dairy-beef farmer) «i get that [product] more because i know other people and have had a word of mouth recommendation [...] when you know people who have used the product and they’re pleased with the product, that is more of an incentive than anything in [the farming press].» (int f2, dairy farmer) «the proof is in the pudding, as it were. if you know a guy who’s had a problem with fluke, and he says “well, i used this [anthelmintic] to get rid of it”, why would you not do that yourself?» (int f9, beef farmer) 332 qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle charlton & robinson veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 «i’ve known the vet and the agricultural merchant all my life. i’ve trusted them and their advice had always worked in the past.» (int f8, dairy-beef farmer). «we do have quite a good friend who’s a vet, so i will ask their advice occasionally.» (int f6, dairy farmer). farmers who received routine herd health visits from the vet (f1 and f3) utilised their advice more. despite positive intentions, the veterinarians found it challenging to implement responsible anthelmintic prescription with farmers: «it’s difficult when they have the same [product] every time to say, “why don’t you try this one instead?” so if they come in and want a wormer, they can have whatever they want.» (int v2, veterinarian). «when they’ve had one way of doing things and it’s been working out okay, they would have to have a real problem for them to really want to change.» (int v1, veterinarian). despite this, veterinarians’ attempts to exercise authority in terms of conservative anthelmintic prescription were apparent, with one veterinarian (v2) noting that they try to offer a benzimidazole (bz) anthelmintic where possible, as opposed to utilising the other categories, such as ml. these factors may be affected by the lack of allocated time to discuss anthelmintic control in detail, despite the veterinarians’ intention to do so: «when farmers walk in, nine times out of ten it’s at a really busy time so [although] you almost want to have a consult, [but] that never happens [...] we can’t actually give them the time they need.» (int v1, veterinarian) future prevention of ar in cattle in terms of their beliefs regarding the development of ar, the farmers tended to attribute the problem to a reaction of nature, an overuse of anthelmintics, and generally poor farming practice, as evidenced by these quotes: «nature has a way – doesn’t matter what the disease challenge is, some will always get around it. but it’s not helped by the fact that people have abused wormers. i feel some people aren’t using them properly.» (int f7, beef farmer) «i think it [ar] occurs on single-species farms with heavy stocking rates. that’s more of a risk factor.» (int f5, dairy-beef farmer) the notion of veterinarians not having the same degree of control over anthelmintics as with antibiotics was one which was highlighted during a superior advisory position to others who were legally qualified to give advice on wormers: «if i want to know something i’d rather ask the vet. they’re dealing with these things every day.» (int f7, beef farmer) «the vets may be a bit more specific [...] about the issues and problems that people have got. so, i suppose, deep down, i would value the vet’s opinion a bit more than someone from the agricultural merchants.» (int f2, dairy farmer) nonetheless, despite the value apparently placed on veterinary advice, there were varying degrees of engagement with veterinarians. concurrent with the findings of richens and colleagues (richens et al. 2015), veterinarians tended to be underused by farmers in the current study for animal health advice, undertaking mainly ‘fire-brigade work’ on farms and being approached for advice only when there was a specific problem: «he [the veterinarian] came the other day to a calving and he’s come out about four times in 16 years. ‘fire-brigade work’ (coming out for emergencies only) would be the policy.» (int f6, dairy farmer). the veterinarians interviewed tended to agree, noting the limitation of these types of contact for advisory opportunities regarding ar, and frustrated that they were unable to allocate sufficient time to discuss worming protocols in any detail: «it’s not one i’ve particularly spoken to farmers about. it tends to be the case where they come in, they buy their wormer, and they disappear. we don’t tend to talk to them a huge amount about worms.» (int v2, veterinarian) «speaking about this job, it is one of the limitations [...] that we can’t actually give them the time they need to discuss things.» (int v1, veterinarian) interestingly, all nine farmers stated that they had herd health plans in place, which had been devised with a veterinarian in all but one case. this provides a valuable opportunity to discuss routine herd health with veterinarians, which should include parasite control, but one farmer attributed unfamiliarity with the veterinarians at their local practice as a deterrent from using them for routine advice: «i will often ask one of the vets [for advice], but there’s a big throughput of vets in my practice [...] you tend to cover the same things with another one again.» (int f5, dairy-beef farmer). furthermore, familiarity and trustworthiness tended to be prominent factors involved in seeking advisory sources regarding anthelmintic treatment: 333 charlton & robinson qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 discussion the detection of ar in cattle has become much more common worldwide in recent years, prompting legitimate concerns that the resistance that is much more prevalent in the helminths of sheep is also fast developing in cattle (taylor 2012, baiak et  al. 2018). the data generated in this study provided the opportunity to better understand the factors underpinning on-farm decision-making with respect to anthelmintic use in cattle, and contextualise the results obtained, consistent with an approach to situate animal health knowledge and practices within their wider socioeconomic and sociocultural backdrop (robinson 2017a, chenais and fischer 2018) it is also provides an insight into veterinary practitioner thinking on the issue, and illustrates a potential lack of veterinary engagement. we draw out three particular lessons. first, financial considerations around product retail price appear often to outweigh suitability and efficacy considerations regarding choice of anthelmintic product and protocol, especially when sold by agricultural stores. this was also discussed by bellet (bellet 2018) in her interview-based research with farmers in england, and noted by easton and colleagues (easton et  al. 2016b) in the context of veterinarians in the uk not selling anthelmintics as often as sqp channels such as agricultural merchants. easton and colleagues (easton et  al. 2016b) suggest that because sqps have a statutory requirement for continuing professional development in the subject area, while veterinarians do not, they can be more up-to-date in their knowledge of the subject. in their study, sqps generally performed as well as veterinarians in an online test of knowledge of helminthology and best practice in livestock and horses. this is not therefore an issue of a lack of the capability of sqps to offer advice on worming protocols. however, the evidence presented in our study would suggest that price often easily outweighs other factors regarding anthelmintic product choice, whether or not specific advice is offered by sqps at the time of purchase. the data would suggest that some farmers may go to the retailer with a predetermined mind-set to choose the least expensive or best value-for-money option, regardless of best practice. it appears that, at least on some occasions, retailers may encourage such an attitude through sales promotions and the advocacy of products that are selling in high volumes. although maximising profit is certainly not the only consideration in livestock farming (robinson 2017a), it is an important influence, and as charlier and colleagues (charlier et al. 2015) point out, farmers have the challenge of profit-making in competitive markets, while simultaneously meeting wider societal demands such as the need to slow the interview process by two of the veterinarians interviewed: «the antimicrobials are under the pom-v [veterinarian-only prescription category], so they [farmers] can’t get them. the only way they’d be able to control anthelmintics is to bring them back into line.» (int v3, veterinarian) «i think vets probably need to tighten up their anthelmintic usage a little bit in terms of “no, you can’t have that first”. the problem is, we haven’t got the monopoly in that you have to go through a vet to get them like you do with antibiotics.» (int v2, veterinarian) however, some of the farmers interviewed had an optimistic view and believed that a new anthelmintic drug would become available in the event of a future resistance problem: «i suppose i’d have to change my products wouldn’t i? just go to the next product and it wouldn’t be an issue.» (int f2, dairy farmer) «the easiest thing to do would be to improve the product. find a product which is like [ivermectin] – when that came out, that was marvellous! [...] that’s the quickest solution [to ar], isn’t it?» (int f8, beef farmer) linking ar to antimicrobial resistance, and the issues surrounding limitations of choice of antibiotic, one dairy farmer suggested that there would be a reversal of trends away from newer products towards those previously used in the past, heavily influenced by food retailer pressures: «you can see the same thing happening with wormers, once there’s a resistance they’ll go back to the old ones [...] obviously the supermarkets are putting their input in with antibiotics, and the same thing will happen with wormers.» (int f1, dairy farmer) one farmer thought there was an onus for collective responsibility, and for the whole industry to take urgent action on the problem of ar in cattle, but another felt that veterinarians may not be incentivised to actively engage with the issue: «if it’s going to be a serious problem, we should do something about it now while we can. but it’s not just farmers’ responsibility – it’s the whole industry. it’s vets, farmers, drug companies, merchants – everyone needs to work together on the problem.» (int f3, dairy-beef farmer) «i think that the vets could push this kind of thing (anthelmintic advice) [...] i suppose the only problem with some vets is that there’s nothing in it for them, especially if they don’t sell [...] wormers.» (int f2, dairy farmer) 334 qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle charlton & robinson veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 proactive approach to herd health planning and regular veterinarian contact may be beneficial, as it facilitates the creation of a partnership between both stakeholders in promoting more sustainable anthelmintic use, as has been advocated by veterinary organisations such as the british veterinary association (bva 2017). changing farmers’ attitudes towards ar through focussing on parasite control within an effective overall herd health framework is vitally important, and that would suggest veterinarians, through herd health planning, have a vital role to play in the governance of anthelmintic protocols and practice. however, the challenge remains that there is a possible divergence between the onus on veterinarians to advise farmers on helminth control in the wider context of farm health planning, and their lack of direct influence over their product choice through minimal sales and revenue stream (easton et  al. 2016b). a similar divergence may arise with other disease conditions. for example, veterinarians in the uk are encouraged to engage in active health planning with cattle farmers on bovine tuberculosis control (e.g. glossop 2013, woodroffe 2014). however, veterinarians may lack interest and be disengaged from the topic because of the regulatory rather than therapeutic frameworks within which the disease is situated, and focus their efforts on other health conditions which they are much more interested in. as a result, they may not be valued or used as btb advisors by farmers to the extent envisaged by state authorities (godfray et al. 2018, hamilton et  al. 2019). instead, they may be part of a perpetuating cycle of not being interested in providing farm-specific advice unless being explicitly paid for it, and therefore not being asked to provide it; a similar situation could prevail with anthelmintic advice. there are also fundamental challenges in getting farmers to engage with herd health planning, as has been found in other studies in the uk (bell et al. 2006, hall and wapenaar 2012). while the study is small in that it involved a sample of 12 people, the findings are nonetheless valuable as a pilot study as they are likely to be indicative of the attitudes and behaviours of not just other welsh cattle farmers and veterinarians, but also the wider farming and veterinary population in the uk. the sample size is legitimised by the findings from other qualitative interview-based studies that have demonstrated how a sample of 12 interviewees can provide data saturation and produce reliable results from which to derive theory (e.g. guest et  al. 2006, mcaloon et  al. 2017). nonetheless, a limitation of the study was that the veterinarians all worked in the same veterinary practice, and two thirds of the farmers interviewed were their clients, which may introduce a bias into the sample. future research on the theme would ideally cover a wider sample across the development of drug resistance. for some cattle farmers, the price of the product and sense of acquiring a bargain, may therefore supersede matching worming strategy and product within an overall herd health plan. second, it is also clear, and consistent with previous studies in the uk and belgium (bellet 2018, vande  velde et  al. 2018), that some cattle farmers do not see a need to reconsider their anthelmintic protocols and practice, as they do not see a resistance problem on their farm. several of the farmers in this study were not aware that ar was a problem in cattle at all, despite being aware of the issue in sheep, and generally adopted an ‘it isn’t broken, so don’t fix it’ attitude reinforced by habit and longstanding routines. a novel finding was that several participants thought that the media or pharmaceutical companies were exaggerating the issue. this line of reasoning is consistent with the findings of other studies in the realm of the public understanding of science, where lay framings of animal or human health issues can diverge from those of scientific experts (e.g. suryanarayanan and kleinman 2012, robinson 2017b), and where the media or the pharmaceutical industry can be blamed or villainised as exaggerating or distorting health concerns (aronowitz 1991, wagner-egger et al. 2011). it is difficult to address a problem that is not appreciated by the stakeholders most involved, and complicated by conspiracy theories that may be hard to dispel, as has been seen with vaccination controversies in both human and animal health (leach and fairhead 2007, bva 2018). third, the study would suggest the need for a more proactive governance approach to responsible anthelmintic usage by farmers, with tighter controls over the prescription of anthelmintics to mitigate against their misuse, and conservation of the available licensed products, as is increasingly the case with antimicrobials. in addition to taking professional advice from sqps and veterinarians, the farmers in this study reported that they were influenced in their anthelmintic choices and practices by farming peers through social and discussion group networks. this finding contrasts with vande velde and colleagues (vande velde et al. 2018), where other farmers’ opinions on parasite control were considered untrustworthy. the trust placed in the opinions of other farmers may be reflective of a generally negative attitude towards paying for veterinary advice. both the farmers and the veterinarians in our sample confirmed that farmers in their area tended to utilise a veterinarian for emergency veterinary work rather than preventive herd health management and advice. this was despite farmer references to the perceived specificity and value of veterinarians’ expertise in the interviews. encouraging a more 335 charlton & robinson qualitative investigation of the attitudes and practices on anthelmintic resistance in cattle veterinaria italiana 2019, 55 (4), 327-337. doi: 10.12834/vetit.1848.9845.3 with respect to the issue, and these must be addressed by raising the profile of the issue of ar in cattle as it has been in sheep, but it could also be argued that not all veterinarians are as engaged with the issue as they could be. indeed, there is a need to expand the qualitative investigation of this issue to veterinary and other industry stakeholder opinions and practices beyond farmers. given the apparent complacency that still exists towards ar in cattle, and as mcarthur and reinemeyer (mcarthur and reinemeyer 2014) eloquently argue, there will need to be a paradigm shift in parasite control knowledge, attitudes and behaviours. this must involve the efforts of multiple stakeholders and through multiple approaches if the problem of ar in cattle is to be managed more effectively in the future. acknowledgements the authors wish to gratefully acknowledge the contributions of the farmers and veterinarians who participated in this research. a larger population of farmers and veterinarians in different regions and veterinary practice areas. conclusions this study in north wales illustrates the complexity of farmer decision-making and opinion formation, and emphasizes the need to understand the situated knowledge and contextualised frameworks within which farmers and veterinarians operate in relation to ar (charlier et al. 2015, charlier et al. 2016, bellet 2018). there are multiple hurdles to overcome if the issue of ar in cattle is not to become one 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ramos f., portella l.p., rodrigues f.s., pötter l., cezar a.s., sangioni l.a. & vogel f.s. 2016. anthelmintic resistance in gastrointestinal nematodes of beef cattle in the state of rio grande do sul, brazil. int j parasitol-drug, 6, 93-101. richens i.f., hobson-west p., brennan m.l., lowton r., kaler j. & wapenaar w. 2015. farmers’ perception of the role of veterinary surgeons in vaccination strategies in british dairy farms. vet rec, 177, 465-470. robinson p.a. 2017a. farmers and bovine tuberculosis: 51 short communication cat scratch disease (csd) is a zoonotic disease mainly caused by an aerobic and pleomorphic gram‑negative bacterium, bartonella henselae belonging to α sub‑group of proteobacteria, family bartonellaceae. cat acts as the principal reservoir of b. henselae which is transmitted among cats mainly by the flea ctenocephalides felis (bouhsira et al. 2013, fabbi et al. 2004, greco et al. 2019, iannino et al. 2018, pinna parpaglia et  al. 2007). bartonella clarridgeiae is another agent of csd, though rare (capitta et  al. 2010, greco et al. 2019). humans mainly acquire infection through an infected cat scratch or bite and can exhibit an acute febrile lymphadenopathy. immunosuppressed individuals may show hepatic and/or splenic peliosis and also bacillary angiomatosis (breitschwerdt 2008, breitschwerdt et  al. 2010, capitta et  al. 2010, fabbi et al. 2004a, iannino et al. 2018, pinna parpaglia et al. 2007, zobba et al. 2009). naturally‑infected cats with b.  henselae are usually healthy carrier and can be bacteremic for weeks to months/years, representing an important reservoir for bartonella (breitschwerdt 2008, chomel et  al. 2006, fabbi et  al. 2004b). young cats (<  1  year) can develop a stronger bacteremia than older cats, as well as the street cats compared to pet cats (chomel et al. 2006). thus, infected cats may represent a risk to human health. moreover, positive cats are considered the most important vehicle for the spread of the disease (ebani et al. 2012, fabbi et al. 2004 a, b, pinna parpaglia et al. 2007, zobba et al. 2009). bartonella infections in symptomatic cats should be confirmed by culturing the organism from blood or tissues but some molecular diagnostic approaches have been developed during the last years. in particular, real‑time pcr assays are commonly used to detect bartonella spp. since bartonella genus shows high genetic variability, a universal method is difficult to develop, so different conventional pcr and real‑time pcr methods, targeting different portions of the genome, have been proposed. for example, gene glta is a common genetic target for bartonella detection and is considered a reliable tool for distinguishing genotypes (la scola et  al. 2003, tapp et  al. 2003). however, this test has showed high cross‑reactivity with other bacteria, such as ehrlichia spp. (colborn et al. 2010). the immunofluorescence antibodiy test (ifat) is the most used serological diagnostic tool for detecting bartonella exposure. this study aimed at detecting bartonella infection in cats from abruzzo region. during 2014, a total of fifty‑two samples of whole blood and sera were istituto zooprofilattico sperimentale dell'abruzzo e del molise 'g. caporale', teramo, italy *corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise 'g. caporale', campo boario, 64100 teramo, italy. tel.: +39 0861 332471, e‑mail: a.angioni@izs.it. keywords abruzzo, bartonella henselae, bartonella clarridgeiae, cat, ifat, real‑time pcr. summary cat scratch disease (csd) is a zoonotic disease, caused predominantly by bartonella henselae and transmitted to humans through a scratch or bite of the cat. cat represents the principal reservoir and healthy carrier of bartonella, which is mainly transmitted, among cats, by the flea ctenocephalides felis. during 2014, fifty‑two samples of whole blood and sera were collected randomly from cats in abruzzo region and were examined by real‑time pcr and ifat tests, respectively. seven samples out of fifty‑two (13.5%) resulted positive for bartonella  spp. in both tests, while six specimens (11.5%) resulted real‑time pcr negative but igg positive; thirty‑nine were instead both real‑time pcr and ifat negative (75%). sequence analysis of a fragment of dna identified b. henselae and b.  clarridgeiae in four and in two real‑time pcr positive samples, respectively. salvatora angela angioni*, luigina di gialleonardo, marco di domenico, daniele giansante, manuela tittarelli and cesare cammà survey of bartonella species in cats from abruzzo region, italy veterinaria italiana 2020, 56 (1), 51‑54. doi: 10.12834/vetit.1884.10006.2 accepted: 11.12.2019 | available on line: 24.04.2020 52 veterinaria italiana 2020, 56 (1), 51‑54. doi: 10.12834/vetit.1884.10006.2 detection of bartonella spp. in cats from abruzzo angioni et al. the amount of dna that could be detected by probit analysis with 95% of sensitivity. amplification efficiency was calculated from slope of the standard curve using the following formula: e = (10‑1/slope ‑ 1) x 100 (vaerman et al. 2004). specificity was assessed by testing nucleic acid from closely genetically related bacterial species, or different species that may infect cats including anaplasma phagocytophilum, borrelia afzelii, rickettsia helvetica, rickettsia monacentis, rickettsia slovaca and coxiella burnetii. the intra‑assay variance (repeatability or short‑term precision) was determined by testing three replicates of the 10‑fold serial dna dilutions (equal to 3.074 to 3.072 copy of dna/reaction) in the same run. similarly, the inter‑assay variance (reproducibility or long‑term precision) was determined by running triplicates of the dna dilutions (3.072 to 3.071 copy of dna/reaction) in three different runs and on separate days. these replicates were used to determine the mean and standard deviation of ct values and the coefficient of variation. the reaction mix (20 µl) contained 2 µl of extracted nucleic acid, 10 µl go taq probe qpcr mmix 2x (promega), primers and probe at a final concentration of 800 nm and 250 nm, respectively (table  i). real‑time pcr was performed on quantstudio 7 flex systems istrument (life technologies) with the thermal condition showed in table i. in order to confirm and characterize the results of real‑time pcr a partial region of the 23s gene was sequenced (parra et al. 2016). obtained sequences were aligned using clustal v (dnastar, madison, wi). each sequence was compared to other homologous regions present in genbank by blast search tool (https://blast.ncbi.nlm. nih.gov/blast.cgi). seven blood samples out of fifty‑two (13.5%) tested positive for bartonella spp. ifat revealed igg for bartonella spp. in all real‑time pcr positive cats, while igm were identified in two cats. six specimens were real‑time pcr negative, igm negative and igg positive (11.5%) with values ranging from 1:40 to 1:160, whereas thirty‑nine collected randomly from cats in abruzzo region and were examined at izsam by ifat and real‑time pcr tests. b. henselae houston1 (atcc 49882) was cultivated in brucella broth (bbl microbiology system cockeysville, md, usa) supplemented with haemin (250 µg/ml) and 8% fildes solution, and incubated at 37 °c in 5% co 2 . log ‑phase cultures were centrifuged (4,500 g, 30 min), washed three times in pbs, ph  7.2 and inoculated into a 25‑cm2 flask (corning, ny, usa) containing l929 cells (ecacc 85011425) and dulbecco minimal essential medium (gibco, introvigen, grand island, ny, usa) supplemented with 2 mm l‑glutamine (200  mm) and 10% (v/v) fetal bovine serum (gibco) and maintained at 37 °c with 5% co 2 . l929 cells infected with b. henselae were harvested by centrifugation at 4,500 g for 30 min and washed twice with pbs. the final pellets were resuspended in pbs and 10 µl were added onto each well of 18‑well slides (gsg robotix italy). slides were air‑dried for at least 1 h, fixed with cold acetone for 10  min and used immediately or stored at ‑ 20 °c until use (capitta et al. 2010, zobba et al. 2009). bartonella spp. igm and igg antibodies were determined by ifat: 10 µl of serum diluited (1:40 to 1:640) in pbs was placed on the b.  henselae l929 slide and incubated at 37 °c in a humidified chamber for 30  min. after two washes in pbs (10 min) and one rinse with distilled water the slides were incubated for 30 min at 37 °c with fluorescein isothiocyanate‑labelled goat anti cat igm (bethyl laboratories inc‑ montgomery, texas) and igg immunoglobuline (sigma‑aldrich, st. louis, mo, usa), diluted in pbs 1% evans blue (sigma‑aldrich, st. louis, mo, usa). the slides washed and dried as described above, were examined with a fluorescence microscope. the titre of <  1:40 was considered negative, a titre of ≥  1:40 was considered positive for both classes of antibodies (capitta et  al. 2010, zobba et al. 2009). a quantitative pcr method was developed to detect bartonella spp. dna in blood cat samples. the highly conserved region of nuog gene (andré et  al. 2016, colborn et  al. 2010, diaz et  al. 2012) was selected to design primers and probe by primer express software (thermofisher scientific). total genomic dna was extracted from 300  µl of whole blood samples or from b.  henselae strain by maxwell® 16 instrument using maxwell r 16 blood dna purification kit (promega), according to the manufacturer’s instructions. the limit of detection (lod) of real‑time pcr assay was determined using 10‑fold serial dilutions of dna extracted from b.  henselae (atcc‑49882d‑5). standard curve was generated and used to calculate table i. primers and probe sequences and thermal condition. nuog_60_f 5’-gcggcataattcgcataacc-3’ nuog_60_r 5’-cacttggcagtgctatccgtatt-3’ nuog_p 5’-fam-acgaccccggctat-3’mgb temperature time 95°c 2 min 1 cycle 95°c 15 sec 45 cycles 60°c 1 min 53 angioni et al. detection of bartonella spp. in cats from abruzzo veterinaria italiana 2020, 56 (1), 51‑54. doi: 10.12834/vetit.1884.10006.2 two different bartonella species: four samples were identified as b. henselae and two as b. clarridgeiae. one positive sample was not identified as probably due to the lower concetration of dna. this survey demonstrated correlation between serological and virological results. reasonably, serological positive individuals without evidence of bacterial dna in the bloodstream might be related with the presence of past infections. however non bacteremic cats may still represent a risk for humans given that reinfections might occurr (fabbi et al. 2004a). it is generally accepted that the ifat test should be performed standalone only when the prevalence of bacteric cats is high (40‑60%). however, when the transmission to humans (for example immune‑depressed individuals) is a risk, virological and serological tests should be performed in order to get a complete health status of a cat (fabbi et al. 2004b). in this perspective, the novel molecular method which has been developed in this study, is useful to detect bartonella infected cats and thus for prevalence studies in feline populations. acknowledgments the authors would like to thank alessandra leone and liana teodori for providing the cell line used to prepare the ifat antigen. samples resulted negative by both assays (75%). results are summarized in table ii. the amplification efficiency was 100.4% (slope = ‑ 3.312; r2 = 0.997). the lod, determined as the lowest dilution of dna that could be detected with 95% sensitivity, was equivalent to 0.32 genome copies. the coefficient of variation, determined for intra‑ and inter‑assay repeatability, showed mean values of 0.33% and 2.16%, respectively. no amplification signal was obtained from a panel of different bacterial species tested to evaluate the specificity of the molecular assay. the sequence analysis showed the presence of table ii. serological and real-time pcr results. no. of cats igg igm real-time pcr 1 1/160 neg pos 2 1/320 neg pos 3 1/160 neg pos 4 1/320 neg pos 5 1/80 1/40 pos 6 1/40 1/40 pos 7 1/160 neg pos total positive 7/7 2/7 7/7 pos = positive; neg = negative; no = number. 54 veterinaria italiana 2020, 56 (1), 51‑54. doi: 10.12834/vetit.1884.10006.2 detection of bartonella spp. in cats from abruzzo angioni et al. andré m.r., dumler j.s., herrera h.m., gonçalves l.r., de sousa k.c., scorpio d.g., de santis a.c., domingos i.h., de macedo g.c. & machado r.z. 2016. assessment of a quantitative 5' nuclease real‑time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuog) for bartonella species in domiciled and stray cats in brazil. j feline med surgery, 18 (10), 783‑790. bai y., rizzo m.f., alvarez d., moran d., peruski l.f. & kosoy m. 2015. coexistence of bartonella henselae and b.  clarridgeiae in populations of cats and their fleas in guatemala. j vector ecol, 40 (2), 327‑332. bouhsira e., ferrandez y., liu m., franc m., boulouis h.j. & biville f. 2013. 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cats and dogs from northern sardinia, italy. clin microbiol infect, 15 (suppl. 2), 134‑135. 83 veterinaria italiana 2021, 57 (1), 83-87. doi: 10.12834/vetit.2222.13654.1 accepted: 02.10.2020 | available on line: 27.07.2021 1dipartimento di agricoltura, alimentazione e ambiente (di3a), university of catania, catania, italy. 2dvm freelance. 3istituto zooprofilattico sperimentale of sicily, palermo, italy. *corresponding author at: di3a university of catania, catania, italy. e-mail: alessandrostamilla@gmail.com. alessandro stamilla1*, antonino messina2, roberto puleio3, guido ruggero loria3, francesco antoci3, giuseppe cascone3 and massimiliano lanza1 keywords avian orthoreovirus, broiler, chicken astrovirus, histology, qpcr, quantitative reverse transcription pcr (rt-qpcr), runting stunting syndrome (rss). summary common pathogens of intensive poultry farms, either parasitic or bacterial, such as coccidia or salmonella, are well known and strictly controlled by veterinary management. this case study reports an unusual case of runting stunting syndrome (rss) observed on a sicilian poultry farm of broiler chickens during 2019. the investigation was carried out on five chickens which present delayed in body weight and growth performance. animals showed also difficulty in deambulation and diarrhea. at necropsy, intestinal lesions were detected in three of the five clinical cases. gut samples were collected and analyzed to identify potential pathogens responsible for the rss. presence of viruses was detected by using quantitative reverse transcription pcr (rt-qpcr), while selected tissues were fixed and embedded in paraffin wax according to routine procedures. all histological sections were stained with hematoxylin-eosin. rt-qpcr successfully detected both chicken astrovirus (castv) and avian orthoreovirus (arv). histology evidenced severe specific lesions on the intestinal mucosa in liver and kidneys. chicken astrovirus and avian orthoreovirus rna was also detected in cecal tonsils, kidney and liver, thus implying their possible primary role in inducing the disease. further studies are needed to evaluate the role of other possible factors (low biosecurity measures, e.g.) and, most of all, the consequences in terms of economic losses and animal health impairment. new insights on avian orthoreovirus and chicken astrovirus co-infection in an italian broiler flock: preliminary biomolecular and pathological results the avian orthoreovirus (arv) was firstly isolated from a chicken with arthritis (fahey and crawley 1954). it is one of the most widespread avian viruses causing clinical disease in poultry farming, with subsequent economic losses to total meat production (rosenberger 1989). in poultry flocks, the arv infection causes arthritis and tenosynovitis (heide 1977), immunodepression (kibenge et  al. 1987), enteric symptoms (dutta and pomeroy 1967) and so called “runting-stunting syndrome” (goodwin et  al. 1993). this infection is typically characterized by high morbidity and mortality, poor feed efficiency and delayed growth (dobson and glisson 1992). arv belongs to the reoviridae family and it is constituted by a segmented dsrna genome (spandidos and graham 1976). the chicken astrovirus (castv) is an emerging disease and the most recently identified member introduction the intensive raising process of broiler chickens sometimes can facilitate the spread of opportunistic pathogens, such as bacteria, parasites and viruses, which could increase the mortality rate (fao 2013). most of the pathogens are spread throughout different vectors and may persist in the farm environment for long time (rosenberger 2012). indeed, after a fattening cycle, bacteria and parasites can survive in indoor sheds even after cleaning and disinfection procedures are applied. conversely, viruses are more susceptible to environment condition and chemical disinfection although sometimes, they can survive even for long time after the disinfection (guy 1998, otto et al. 2006). in this report we focused our attention on two viruses linked to modern avian farming: avian orthoreovirus (arv) and chicken astrovirus (castv). case report 84 avian orthoreovirus and chicken astrovirus co-infection in broilers stamilla et al. veterinaria italiana 2021, 57 (1), 83-87. doi: 10.12834/vetit.2222.13654.1 of kidney and liver, almost double in size, in one of the three chicks compared to the organs of the others two birds. loss of weight was clearly visible in the muscular breast which were significantly underweight. moreover, in the carcasses were evident the hemorrhagic lesions with necrotic areas in liver and signs of hemorrhagic enteritis in intestinal tracts (figure 1). representative portion of organs (intestine, liver, kidney and cecal tonsils), fecal content, tracheal and cloacal swabs were collected for laboratory investigation. at the end of this cycle, the mortality rate reached 3%. a peak of 1.7% was reached in the first two weeks of raising cycle. there was no visible changes in litter and differences in feed/water intake as the performance were measured only at the end of the cycle. microbiological screening was performed on the cloacal swabs and from liver and kidney. samples were directly sowed on columbia agar containing 5% sheep blood (oxoid limited, hampshire, uk), mcconkey agar (oxoid limited, hampshire, uk) and inoculated aerobically and anaerobically at 37  °c up to 48 h; subsequently, suspected colonies were purified and identified by vitek 2 (biomerieux, france). at the same time, 10 grams of intestine were homogenized through stomacher in 90 ml of tryptone soya broth and incubated aerobically for 24 h at 37 °c. after incubation, 1 ml of enriched broth was transferred to modified semi-solid rappaport-vassiliadis agar and incubated for 48  h at 40 °c. presumptive salmonella colonies were transferred on xylose lysine desoxychloate agar and brilliant green agar1. for all other bacteria, including fastidious species, samples of duodenum and caecum were inoculated directly into tsa medium, modified brilliant green medium and columbia iii w/5% sb medium (bd diagnostic, diagnostic systems, sparks, md, usa) and incubated at 37 °c for 72 h. microbiological investigation did not evidence the presence of pathogenic bacteria. a parasitological examination has been carried out by direct mount technique through preparation of smears obtained either from each fecal sample (collected from the cloaca) or from intestinal material sampled from three sections of the of avian astroviruses. castv belongs to the astroviridae family and is characterized by a ssrna genome. like other astroviruses, it is a small round shape, non-enveloped virus (méndez et  al. 2013). astroviruses, generally, cause enteric infection and occur in many animal species including humans (smyth 2017). historically, these viruses have been named according to the target species e.g. turkey astrovirus (tastv), but cross infections among different host have been identified, such as castv isolated in turkey (pantin-jackwood et  al. 2006). according to the international committee on taxonomy, it’s possible to distinguish three species, namely avastrovirus 1, 2 and 3 (smyth 2017). castv is an enteric pathogen that often infects birds, either through horizontal transmission by fecal-oral route and also by vertical transmission from naïve in-lay parent bird. therefore, chicks may hatch shedding high levels of virus. in comparison to other viruses, the lack of an envelope confers castv a higher resistance to lipid solvents, and its maintenance in the environment could be also related to arthropoda which act as vectors (rosenberger 2010). previous studies suggest that concomitant enteric viral infections may increase the severity of clinical signs in turkeys and in broiler chickens (spackman et  al. 2010, reynolds et  al. 1987, bon-sang et  al. 2013). however, viral enteric outbreaks, especially those caused by castv and arv in broilers, have never been reported in southern italy so far. case report in a commercial farm, a total of five male chickens ross 308, 46 days old, were selected during the last day of fattening cycle, in order to measure growth performance. in a large shed, previously disinfected and kept empty for 15 day after the previous fattening cycle, a total of 8,000 chickens from the same hatching were reared from the beginning of march to the end of april. chicks were vaccinated against infectious bursal disease virus (ibvd), infectious bronchitis (ib) (793b, h120) and marek’s diseases in the hatchery. a coccidiostat (nicarbazin, 40 ppm, and narasin, 50 ppm) was added to the feeds. no antibiotics were added to diets and to water during raising process. three out of five showed delay in growth, immature appearance if related with their age, and typical clinical signs of suffering such as reluctancy to move, fatigue, traces of diarrhea in the cloacal region and swelling of the infraorbital sinuses. the body weight of these three animals was significantly lower (about 60% less) if compared to weight of the other two chickens (table i). carcasses inspection showed hemorrhagic enteritis, hepatic necrosis and swelling of infraorbital sinuses, furthermore an abnormal size table i. final body weight (bw) and presence of chicken astrovirus (castv), avian orthoreovirus (arv) and avian metapneumovirus (ampv). chick bw (kg) ampv castv arv 1 3.439 x x x 2 1.243 x v v 3 1.350 x v v 4 1.268 x v v 5 3.502 x x x x = absence; v = presence. 85 stamilla et al. avian orthoreovirus and chicken astrovirus co-infection in broilers veterinaria italiana 2021, 57 (1), 83-87. doi: 10.12834/vetit.2222.13654.1 lesions collected from liver, spleen, kidneys and intestinal tract of duodenum, jejunum and ileum, were fixed in 10% buffered formalin, embedded in paraffine vax and routinely processed. sections with a thickness of 2.5 microns were obtained and stained with hematoxylin and eosin (he) for morphological examination. histology examination showed an inflammatory condition of the liver with focal areas of monocyte infiltration and focal areas of degeneration; in the kidney (figure 2) multifocal lympho-granulocyte infiltrates located in the interstitium associated to tubular necrosis were observed, while in the intestinal mucosa diffuse lymphocyte infiltrates with fusion of the villi were also detected (figure 3). all the lesions could be associated to castv and arv infection, which may cause a degeneration of the internal structure of tissues reducing the absorption of nutrient at the intestine level and contemporary renal failure and reduced filtering capacity in kidney. conclusions this study confirms the importance of intestinal inflammation (enteritis) during castv and arv coinfection, both considered responsible for the runting stunting syndrome (rss). historically, castv and arv have been associated with malabsorption syndrome of broiler chickens, but our study proves that these viruses, well known as opportunistic pathogen, are responsible for more aggressive syndromes. it’s clear that clinically infected chicks show hatches small in size and white-colored, sometimes showing a delay in growth, and a general appearance resembling similar to younger chick with immature feathering, yellow color and reduced size of the beak (rosenberger 2012). common symptoms also include enteritis and diarrhea, leg weakness and irregular feathering (kouwenhoven et al. 1978). while castv is predominantly an enteric virus, it is also recognized as infecting organs through the intestine (duodenum, jejunum and caecum). fecal material collected from the intestine were then processed through a saturated sugar (density 1.27 g/ ml) flotation technique, by plating the supernatant phase on smears and observed by direct light microscopy at 100x up to 400x magnification in order to identify eggs or eventually other stages of cestode, nematoda or coccidia according to quinn and colleagues (quinn et al. 1980). cecal tonsils from the suspected clinical cases were used for molecular investigation in order to detect: chicken astrovirus (castv), and avian orthoreovirus (arv) rna. tracheal swabs were also screened for avian metapneumovirus (ampv). total rna and dna were extracted directly from cecal tonsils, liver and kidney tissues and tracheal swabs using a dna/rna purification kit (kylt, germany) according to the manufacturer’s instruction. for the pcr, commercial kits were used: ampv a&b, chicken astrovirus rt-qpcr, avian reovirus rt-qpcr (kylt, germany). each reaction consisted in 4 µl of total rna, 10 µl of 2x rt-qpcr-mix and 6  µl of detection-mix, as reported in the manufacture’s instruction. the multiplex rrt-pcr was performed on a linegene 9600 (bioer, hangzhou, china) according to the following conditions: 1 cycle at 50  °c for 10 min; 1 cycle of 95  °c for 1 min and 42 cycles at 95  °c for 10 sec and 60 °c for 1 minutes. molecular screening confirmed the suspicion of viral mixed infection which affected the three broilers (number 2, 3 and 4). all the tissues belonged from the five chickens resulted positive for arv and castv while all five animals were negative for avian metapneumovirus (ampv a&b) (table i). figure 1. necropsy of a broiler infected by astrovirus (a = hemorragic enteritis; b = liver necrosis). figure 2. broiler: interstitial nephritis, haematoxylin & eosin, 20x. a b 1 iso 6579-1. 2017. microbiology of the food chain. horizontal method for the detection, enumeration and serotyping of salmonella. available online https://www.iso.org/standard/56712.html (accessed on 22 may 2019). 86 avian orthoreovirus and chicken astrovirus co-infection in broilers stamilla et al. veterinaria italiana 2021, 57 (1), 83-87. doi: 10.12834/vetit.2222.13654.1 syndromes are strictly connected with intestinal disease, sometimes associated with tenosynovitis, that was not observed in this outbreak. intestinal lesions reported in this study characterized by severe villous atrophy and crypt hyperplasia, appear to be significantly more severe if compared to previous reports and related to the direct damage of castv, without any involvement of bacterial secondary infection (guy 1998). this difference is related to a concomitant infection with arv that induces more severe clinical signs as reported by spackman and colleagues (spackman et  al. 2010) and bog-san koo and colleagues (bog-san koo et al. 2013). concomitant infection of both enteric viruses may booster the pathogenicity of the syndromes and, in this case, it also causes lesions on kidney and liver, uncommon findings for infections with these kinds of viruses. when singularly detected, castv and arv are considered as opportunistic pathogens which may affect the curve of growth of chicks and their body weight (hauck et al. 2016), but according to this report we have to consider the risk of mixed infection and the related risk of an enhancement of pathogenicity which may imply significant economical losses if the percentage of infected chicks increase significantly. the difference in live weight between sick and healthy broilers was relevant, even three time less than normal body weight at equal age. the loss of production could have a negative impact on farm incomes, therefore, as for all the other poultry pathogens, it is really important to avoid circulation of these viruses in the farm environment, improving all of those procedures to maintain high level of biosafety. this preliminary data may offer new perspectives for industrial poultry farming in sicily, underlining the importance of applying “ad hoc” biosafety programs and represent an original report on lesions caused by these viruses and their extra intestinal spread to other organs. enteric tract, including the liver and kidneys (bulbule et  al. 2013). in our case, the presence virus dna copies and infiltrate in liver and in the kidney showed that the two viruses, with a predominantly enteric tropism, may colonize and induce imbalances and so far pathological lesions in different body organs; in the kidney they affect the filtering function of the glomeruli; in the liver they cause malfunction due to diffuse tissue degeneration. in the intestine the worst damage was reported, in terms of a disorganized structure of villi that appeared confluent/fused with a diffuse lymphocytic infiltrate following the infection (kang et  al. 2018). these findings are consistent with a report that described enteritis simultaneously occurring with astrovirus infection (koci et  al. 2003). the initial suspicion of a viral infection was confirmed by the biomolecular examination, while the investigation for the other typical poultry pathogens (bacteria or parasites) were negative. histopathological examination confirmed a severe and diffuse enteritis and massive monocyte infiltration in liver and kidney. these figure 3. broiler: monocytic enteritis, haematoxylin & eosin, 20x. 87 stamilla et al. avian orthoreovirus and chicken astrovirus co-infection in broilers veterinaria italiana 2021, 57 (1), 83-87. doi: 10.12834/vetit.2222.13654.1 bon-sang k., hae rim l., eun-ok. j., hye-sun j., moo-sung h. & in-pil m. 2013. an unusual case of concomitant infection with chicken astrovirus and group a avian rotavirus in broilers with a history of severe clinical signs. j vet sci, 14 (2), 231-233. bulbule n.r., mandakhalikar k., kapgate s., deshmukh v.v., schat k. & chawak m. 2013. role of chicken astrovirus as a causative agent of gout in commercial broilers in india. avian pathol, 42 (5), 464-473. dobson k.n. & glisson j.r. 1992. economic impact of a documented case of reovirus infection in broiler breeders. avian dis, 36, 788-791. dutta s.k. & pomeroy b.s. 1967. isolation and characterization of an enterovirus from baby chicks having an enteric infection ii. physical and chemical characteristics and ultrastructure. avian dis, 11, 9-15. fahey j.c. & crawley j.f. 1954. studies on chronic respiratory disease of chickens ii. isolation of a virus. comp med vet sci, 18 (1), 13-21. food and agriculture organization of the united nations (fao). 2013. poultry development review. www.fao. org/docrep/019/i3531e/i3531e00.htm. guy j.s. 1998. virus infections of the gastrointestinal tract of poultry. poultry sci, 77, 1166-1175. goodwin m.a., davis j.f., stewart mcnulty m., brown j. & craig player e. 1993. enteritis (so-called runting stunting syndrome) in georgia broiler chicks. avian dis, 37 (2), 451-458. kang k., linnemann e., icard a.h., durairaj v., mundt e. & sellers h.s. 2018. chicken astrovirus as an aetiological agent of runting-stunting syndrome in broiler chickens. j gen virol, 99, 4, 512-524. heide v.d. 1977. viral arthritis/tenosynovitis: a review. avian pathol, 6, 271-284. kang k., el-gazzar m., sellers h.s., dorea f., williams s.m., kim t., collett s. & mundt e. 2012. investigation into the aetiology of runting and stunting syndrome in chickens. avian path, 41, 41-50. kouwenhoven b., vertommen m. & van eck j.h.h. 1978. runting and leg weakness in broilers; involvement of infectious factors. vet sci comm, 2 (1), 253-259. koci m.d., moser l.a., kelley l.a., larsen d., brown c.c. & schultz-cherry s. 2003. astrovirus induces diarrhea in the absence of inflammation and cell death. j virol, 77, 11798-11808. references kibenge f.s.b., jones r.c. & savage c.e. 1985. effects of experimental immunosuppression on reovirus-induced tenosynovitis in light-hybrid chickens. avian pathol, 16, 73-92. méndez e., murillo a., velázquez r., burnham a. & arias c.f. 2013. replication cycle of astroviruses. in astrovirus research: essential ideas, everyday impacts, future directions (s. schultz-cherry, ed.). springer, new york, 19-45. otto p., liebler-tenorio e.m., elschner m., reetz j., löhren u. & diller r. 2006. detection of rotaviruses and intestinal lesions in broiler chicks from flocks with runting and stunting syndrome (rss). avian dis, 50, 411-418. pantin-jackwood m.j., spackman e. & woolcock p.r. 2006. molecular characterization and typing of chicken and turkey astroviruses circulating in the united states: implications for diagnostics. avian dis, 50, 397-404. quinn r., smith h.v., bruce r.g. & girdwood r.w. 1980. studies on the incidence of toxocara and toxascaris spp. ova in the environment. 1. a comparison of flotation procedures for recovering toxocara spp. ova from soil. j hyg (lond), 84 (1), 83-89. reynolds d.l., saif y.m. & theil kw. 1987. enteric viral infections of turkey poults: incidence of infection. avian dis, 31, 272-276. rosenberger j.k., sterner f.j., botts s., lee k.p. & margolin a. 1989. in vitro and in vivo characterization of avian reoviruses. i. pathogenicity and antigenic relatedness of several avian reovirus isolates. avian dis, 33, 535-544. rosenberger j. 2010. darkling beetles as vectors for bacterial and viral pathogens found in poultry litter. proceedings of the 45th national meeting on poultry health and processing. ocean city, md, usa. rosenberger j. 2012. update on the runting-stunting syndrome. ceva egg program online, 3, 1-8. http:// f s 1 . 5 m p u b l i s h i n g . c o m / i m a g e s / c e v a / e p o _ no3-may2012.pdf (accessed on 24 june 2021). smyth v.j. 2017. a review of the strain diversity and pathogenesis of chicken astrovirus. viruses, 9 (2), 29. spackman e., day j.m. & pantin-jackwood m.j. 2010. astrovirus, reovirus, and rotavirus concomitant infection causes decreased weight gain in broad-breasted white poults. avian dis, 54, 16-21. spandidos d.a. & graham a.f. 1976. physical and chemical characterization of an avian reovirus. j virol, 19 (3), 968-976. 89 veterinaria italiana 2021, 57 (1), 89-92. doi: 10.12834/vetit.2110.12149.1 accepted: 07.05.2020 | available on line: 27.07.2021 department of veterinary medicine, university of milan, lodi, italy *corresponding author at: department of veterinary medicine, university of milan, lodi, italy. e-mail: jari.zambarbieri@unimi.it. jari zambarbieri*, guido grilli, tiziana vitiello and paola scarpa keywords atypical pathogens, brevundimonas, dog, moellerella, urinary tract infection. summary bacterial urinary tract infection (uti) is a common condition affecting dogs. urine culture and antimicrobial susceptibility test, associated with the identification of underlying cause, are of primary importance in order to select a correct treatment, especially in presence of comorbidities. two cases of immunecompromised dogs affected by urinary tract infection (uti) have been described: the first, probably immunosuppressed due to old age, was in poor body condition, with severe odontolithiasis and periodontitis; the second was affected by chronic kidney disease in advanced stage. urine cultures isolated two rare and atypical pathogens, moellerella wisconsensis and brevundimonas vesicularis, both showing sensitivity versus floroquinolones which were selected for the treatment. after a 4 weeks treatment, a second culture demonstrated the resolution of infection in both cases, in absence of clinical signs.to date neither of the two bacteria have been reported as cause of uti in dog. urinary tract infection by atypical uropathogens in dogs multocida and anaerobic bacteria are rarely recognized in dog (ball et al. 2008). case description case 1 a 14-year-old male poodle was examined as suffering asthenia, anorexia and ataxia since few days. the clinical examination revealed a poor body condition score, dehydration, muscle atrophy, bilateral congiuntivitis and otitis, severe periodontitis and odontolithiasis. the ultrasound examination showed signs of chronic nephropathy, bilateral pyelonephritis, and prostatic hyperplasia. a bilateral hip luxation was identified by x-ray. serum creatinine was increased (1.9 mg/dl) and urinalysis evidenced a significant proteinuria [urinary protein  :  creatinine ratio (upc) 1.40], leukocyturia (10/hpf ) and bacteriuria. sample for urine culture was inoculated on blood agar as well as macconkey agar and incubated at 37 °c for 24 hours or 48 hours in negative cases. growth of a single organism with a count of ≥  105 colony-forming units (cfu)/ml was considered to represent the infection and the bacteria was identified using appropriate routine identification introduction bacterial urinary tract infection (uti) is a common condition affecting dogs presented to first opinion and specialist practitioners. utis occur in approximately 14% (5-27%) of dogs in their lifetime with variable age of onset, especially in presence of predisposing factors and/or comorbidities (wong et  al. 2015). utis are a major reason for improper antibiotic prescription in small animal practice and the responsible bacterial populations have evolved with increasing resistances to many antimicrobials. international society for companion animal infectious diseases (iscaid) published guidelines to promote a cautious and reasoned use of antibiotics in cases of utis pointing out the importance of microbiological culture and the identification of underlying causes in order to select a correct treatment (weese et al. 2019). escherichia coli is the most commonly isolated pathogen, followed by other species such as staphylococcus spp., enterococcus  spp., streptococcus  spp., proteus  spp., pseudomonas  spp. and klebsiella spp. (wong et al. 2015, hall et al. 2013). other minor species, including acinetobacter  spp., bacillus spp., bacteroides spp., citrobacter  spp., clostridium  spp., corynebacterium  spp., lactobacillus  spp., morganella morganii, pasteurella case report 90 urinary tract infection in dogs zambarbieri et al. veterinaria italiana 2021, 57 (1), 89-92. doi: 10.12834/vetit.2110.12149.1 creatinine 4.3 mg/dl), normotensive; the substaging based on proteinuria (upc 1.06) was dubious due to the presence of an ongoing infection. a moderate anaemia (red blood cells 3.93  x  106/μl, haemoglobin 9.7 g/dl haematocrit 27.5%) and mild hyperphosphataemia (serum phosphate 4.9  mg/dl) were thought to be the consequences of ckd, associated with the presence of anaemia of chronic disease. urinalysis showed a low urine specific gravity (usg 1,007), proteinuria (upc 1.06), leukocyturia (100/hpf ) and bacteriuria. the procedures for bacterial identification and evaluation of antimicrobial susceptibility were the same reported in case 1. the bacterium identified with api 20ne (bio-mèrieux sa, marcy l’etoile, france) was brevundimonas vesicularis (api cod. 0441004, 91.5% identification). antimicrobial agents tested were amikacin (ak), ampicillin (amp), amoxicillin clavulanate (amc), cephalexin (cl), cefoperazone (cfp), cefovecin (cvn), cefuroxime (cxm), doxycycline (do), enrofloxacin (enr), gentamicin (cn), marbofloxacin (mar), oxacillin (ox), trimethoprim/ sulfamethoxazole (sxt). the isolated bacterium was sensitive to ak, cfp, enr, cn, mar and sxt. the dog was treated with marbofloxacin (2 mg/kg for 4 weeks) according to antimicrobial sensitivity test and two weeks later urinalysis and culture showed the resolution of infection with the persistence of low usg (1,010) and proteinuria (upc 1.19), probably due to underlying ckd. discussion moellerella wisconsensis is a rare gram-negative bacillus belonging to the family of enterobacteriaceae, named definitively in 1984. its pathogenic rule remains nowadays unclear due to two factors: moellerella is rarely identified in clinical methods including colony morphology, gram-stain and biochemical characteristics of isolates. the bacterium identified with api 20e (bio-mèrieux sa, marcy l’etoile, france) was moellerella wisconsensis (api cod. 1244060, 99.9% identification). antimicrobial susceptibility of m.  wisconsensis was tested by the disk diffusion method using the mueller-hinton agar (kirby-bauer method). antimicrobial agents tested were amikacin (ak), ampicillin (amp), amoxicillin clavulanate (amc), cephalexin (cl), cefoperazone (cfp), cefovecin (cvn), cefuroxime (cxm), doxycycline (do), enrofloxacin (enr), gentamicin (cn), nitrofurantoin (f), oxacillin (ox), trimethoprim/ sulfamethoxazole (sxt). the isolated bacterium was sensitive to cfp, enr and cn. after a few days of antibiotic treatment (enrofloxacin 5 mg/kg sid for 3 days, prosecuted at home for 4 weeks) and supportive care, clinical conditions improved and the dog was discharged as normoazotemic. case 2 a 7-year-old female labrador retriever was referred for a second consultation after a diagnosis of chronic kidney disease (ckd). at clinical examination the dog was in good general condition with pale mucous membranes. the diagnostic investigations allowed to stage ckd in stage 3  vs  4 (serum table i. results of the complete urinalysis of case 1. parameter result rv color yellow clarity subclear usg 1,016 > 1,030 ph 5.5 5.5-7 protein ++ glucose negative ketones negative bilirubin + blood +++ hemoglobin +++ nitrite negative rbc occasional < 5/hpf wbc 10/hpf < 5/hpf epithelial cells absent casts absent bacteria rods crystals absent upc 1.40 < 0.5 rv = reference value; usg = urine specific gravity; rbc = red blood cells; wbc = white blood cells; upc = urine protein : creatinine ratio; hpf = high power field. table ii. result of the antimicrobial susceptibility test of case 1. antibiotic result amikacin i amoxicillin clavulanate r ampicillin r cefovecin r cephalexin r ceftriaxone i doxycycline i enrofloxacin s gentamicin s nitrofurantoin r s = susceptible, standard dosage regimen; i = susceptible, increased exposure; r = resistant. 91 zambarbieri et al. urinary tract infection in dogs veterinaria italiana 2021, 57 (1), 89-92. doi: 10.12834/vetit.2110.12149.1 to the best of our knowledge, this is the first isolation of moellerella wisconsensis in a clinical case of uti. brevundimonas vesicularis, named definitively in 1994, is an aerobic gram-negative rod belonging to the caulobacteraceae family and is the main representative of genus brevundimonas, currently composed by 25 species. it is considered an opportunistic pathogen able to cause severe and invasive infections in presence of underline predisposing conditions and/or coinfections. brevundimonas vesicularis is the most isolated pathogen in human medicine and it has been found primarily in patients with bacteraemia but also in eye, urine, wounds, central nervous system, heart, joints, liver, cervical specimens and also in the lung sputum of a cystic fibrosis patient (ryan and pembroke 2018). to date, only few cases have been described in animals (carnevia et  al. 2013, suchodolski et al. 2010). the treatment of brevundimonas spp. infections is frequently difficult because these bacteria can be resistant to many antibiotics including β-lactams and fluoroquinolones; however, in our case, the isolation showed sensitivity versus some cephalosphorines and fluoroquinolones and the antibiotic treatment was effective (carnevia et al. 2013). to the best of our knowledge, this is the first isolation of brevundimonas vesicularis in a clinical case of uti. the reported cases reinforce the importance of the execution of performing urine culture and antimicrobial susceptibility in all cases of suspected uti. samples and only a few numbers of case report in humans and animals are available; belonging to the group of enterobacteriaceae is probably a natural inhabitant of gastrointestinal tract and the pathogenic role is evident only if predisposing factors are present (cardentey-reyes et  al. 2009). in humans, moellerella has been identified in cases of diarrhoea, cholecystitis, in a bronchial aspirate and in a blood culture; in animals it has been reported in faecal samples of captive raptors and in the oral cavity of a wild raccoon in usa, in a lung of a goat and in poultry meat in italy, in liver and kidney from a cow in portugal (cardentey-reyes et  al. 2009, bangert et al. 1998, sandfort et al. 2002, casalinuovo and musarella 2009, anastácio and leão 2016). moreover, a case reported a moellerella infection in a dog with a clinically relevant chronic vaginal discharge in poland (zielińska et al. 2015). moellerella is generally susceptible to most antibiotics that are active against gram-negative bacteria; however, in the case reported in dog the bacteria showed significant resistances versus amoxicillin clavulanate, ampicillin, sulphonamides and trimethoprim and resulted sensible to amikacin, gentamicin and fluoroquinolones (cardentey-reyes et al. 2009, zielińska et al. 2015). interestingly, these results are similar those observed in our case.. table iii. results of the complete urinalysis of case 2. parameter result rv color pale yellow clarity clear usg 1,007 > 1,030 ph 5.5 5.5-7 protein + glucose negative ketones negative bilirubin negative blood ++ hemoglobin negative nitrite negative rbc occasional < 5/hpf wbc 100/hpf < 5/hpf epithelial cells transitional casts absent bacteria rods crystals absent upc 1.06 < 0.5 rv = reference value; usg = urine specific gravity; rbc = red blood cells; wbc = white blood cells; upc = urine protein : creatinine ratio; hpf = high power field. table iv. result of the antimicrobial susceptibility test of case 2. antibiotic result amikacin s amoxicillin clavulanate r ampicillin r cefadroxil r cefovecin i cephalexin r ceftriaxone s doxycycline i enrofloxacin s gentamicin s marbofloxacin s nitrofurantoin s trimethoprim-sulphonamide s s = susceptible, standard dosage regimen; i = susceptible, increased exposure; r = resistant. 92 urinary tract infection in dogs zambarbieri et al. veterinaria italiana 2021, 57 (1), 89-92. doi: 10.12834/vetit.2110.12149.1 anastácio s. & leão h. 2016. moellerella wisconsensis: what’s its role in cattle disease. experimental pathology and health sciences, 8, 35-36. ball k.r., rubin j.e., chirino-trejo m. & dowling p.m. 2008. antimicrobial resistance and prevalence of canine uropathogens at the western college of veterinary medicine veterinary teaching hospital 2002-2007. can vet j, 49, 985-990. bangert r.l., ward a.c., stauber e.h., cho b.r. & widders p.r. 1998. a survey of the aerobic bacteria in the feces of captive raptors. avian dis, 32, 53-62. cardentey-reyes a., jacobs f., struelens m.j. & rodriguez-villalobos h. 2009. first case of bacteremia caused by moellerella wisconsensis: case report and a review of the literature. infection, 37 (6), 544-546. carnevia d., letamendía m. & perretta a. 2013. pathogenic gram-negative bacteria isolated from ornamental fish in uruguay: characterization and antibiotic resistance. bull eur assoc fish pathol, 33 (6), 181-186. casalinuovo f. & musarella r. 2009. isolation of moellerella wisconsensis from the lung of a goat. vet microbiol, 138, 401-402. hall j.l., holmes m.a. & baines s.j. 2013. prevalence and antimicrobial resistance of canine urinary tract pathogens. vet rec, 173 (22), 549-556. references ryan m.p. & pembroke j.t. 2018. brevundimonas spp.: emerging global opportunistic pathogens. virulence, 9, 480-493. sandfort r.f., murray w. & janda j.m. 2002. moellerella wisconsensis isolated from the oral cavity of a wild raccoon (procyon lotor). vector borne zoonotic dis, 2, 197-199. suchodolski j.s., xenolius p.g., paddock c.g., steiner j.m. & jergens a.e. 2010. molecular analysis of the bacterial microbiota in duodenal biopsies from dogs with idiopathic inflammatory bowel disease. vet microbiol, 14, 394-400. weese s.j., blondeau j., boothe d., guardabassi l.g., gumley n., papich m., jessen l.r., lappin m., rankin s., westropp j.l. & sykes j. 2019. international society for companion animal infectious disease (iscaid) guidelines for the diagnosis and management of bacterial urinary tract infections in dogs and cats. vet j, 247, 8-25. wong c., epstein s.e. & westropp j.l. 2015. antimicrobial susceptibility patterns in urinary tract infections in dogs (2010-2013). j vet intern med, 29, 1045-1052. zielińska s., woynarowki a., łoś j.m., milewska k. & łoś m. 2015. isolation of moellerella wisconsensis from a chronic vaginal discharge in a crossbred dog. aust vet pract, 45, 37-38. 11 rapid communication veterinaria italiana 2020, 56 (1), 11-12. doi: 10.12834/vetit.2246.12523.1 accepted: 21.04.2020 | available on line: 24.04.2020 keywords covid-19, severe acute respiratory syndrome coronavirus 2, pandemics, coronavirus infections, zoonoses. summary sars-cov-2 is a zoonotic virus that has achieved community spread among humans and become a pandemic. transmission from humans to dogs, domestic cats, tigers, and lions has occurred. pigs, cats, ferrets, and primates have been identified as good candidates for susceptibility to sars-cov-2. the potential implications indicate the need for one health surveillance, intervention, and management strategies to mitigate the effects on animal populations and prevent a second preparedness failure during this health emergency. one health center of excellence, university of florida, gainesville, florida, united states *corresponding author at: one health center of excellence, university of florida, 1604 mccarty drive, room g047, gainesville, fl 32603, united states. tel.: +1 352 294 8465, e-mail: icapua@ufl.edu. rania gollakner and ilaria capua* is covid-19 the first pandemic that evolves into a panzootic? sars-cov-2 is the most recent example of an emerging zoonotic infectious virus that has converted ‘pandemic potential’ to reality. whilst the origin of this virus has not yet been confirmed, the most likely candidate is the bat, the pangolin or a combination of both (andersen et al. 2020). having successfully crossed the species barrier to the human population and achieved intra and inter-community spread, the world now fights to mitigate the human health consequences and survive the socio-economic ramifications. but is this pandemic only a pandemic or can sars-cov-2 extend its spread to that of a panzootic? are we risking multiple spillover episodes in animal populations which may result in sars-cov-2 becoming endemic in multiple animal species and populations? following the extensive and well documented nature of its spread in humans, as of april 12th 2020, two cases of sars-cov-2 transmission to dogs, 2  cases of transmission to domestic cats, 4 cases of transmission to tigers, and 3 cases of transmission to lions have been reported (government of the hong kong 2020 a, b, c, chini 2020, wcs newsroom 2020). although the dogs did not develop clinical signs, one domestic cat presented with vomiting, diarrhea, and breathing difficulties, and the tigers and lions presented with dry coughs and wheezing. in addition, preliminary studies have demonstrated cat to cat spread of sars-cov-2 and the production of specific neutralizing antibodies against sars-cov-2 in this species (shi et al. 2020, zhang et al. 2020). these observations may be statistical outliers (perhaps a consequence of close contact of domesticated mammals with very high infectious virus loads from the human population), or as the tipping point from which to acquire the transmission characteristics necessary to achieve intra and inter community spread in the new target species. proactive consideration of the potential implications of a ‘reverse’ zoonosis is appropriate to create management strategies that mitigate the potential for adverse effects on the respective animal populations whilst also seeking to control any future recirculation of adapted animal viruses back into humans. pigs, cats, ferrets and non-human primates have similar or identical sars cellular receptors to those found in humans (wan et  al. 2020). this potentially provides sars-cov-2 with a related cellular entrance mechanism to infect a varied series of hosts without requiring further significant genetic changes. genetic changes randomly acquired when the virus replicates could lead to it developing the ability to become endemic in some animal populations, including domestic pets. the sars-cov-2 pandemic and subsequent panzootic potential highlight the need for a one health approach. it is important that harmonized guidelines for surveillance and intervention in wild, captive, and companion animals are developed to facilitate a better understanding of viral spread in novel host populations. the proposed interventions should include quarantine and care packages for infected animals. whilst potentially at lower risk, food 12 veterinaria italiana 2020, 56 (1), 11-12. doi: 10.12834/vetit.2246.12523.1 event would represent a second major preparedness failure during the same public health emergency. acknowledgments olga munoz, dvm, msc, for her edits and thoughtful revisions. animals may still be considered in future guidelines as the cellular receptor mechanisms mentioned previously render the target species jump possible even though the risk of close contact with humans is lessened. with the current information available it is not possible to predict if sars-cov-2 will cause a panzootic. however, not being prepared for such an covid-19: from pandemic to panzootic gollakner & capua andersen k.g., rambaut a., lipkin w.i., holmes e.c. & garry r.f. 2020. the proximal origin of sars-cov-2. nat med [internet], 89 (1), 44-48. https://www.nature.com/ articles/s41591-020-0820-9.pdf. the government of the hong kong special administrative region press releases. 2020a. pet dog tests positive for covid-19 virus. [cited 2020 apr  1]. https://www.info. gov.hk/gia/general/202003/19/p2020031900606.htm. the government of the hong kong special administrative region press releases. 2020b. pet dog further tests positive for antibodies for covid-19 virus. [cited 2020 apr 1]. https://www.info.gov.hk/gia/ general/202003/26/p2020032600756.htm. the government of the hong kong special administrative region press releases. 2020c. pet cat tests positive for covid-19. [cited 2020 feb 4]. https://www.info.gov.hk/ gia/general/202003/31/p2020033100717.htm. chini m. 2020. coronavirus: belgian woman infected her cat [internet]. the brussels times. 2020 [cited 2020 apr 1]. https://www.brusselstimes. c o m / a l l n e w s / b e l g i u m a l l n e w s / 1 0 3 0 0 3 / coronavirus-belgian-woman-infected-her-cat/. wcs newsroom. a tiger at bronx zoo tests positive references for covid-19; the tiger and the zoo’s other cats are doing well at this time [internet]. [cited 2020 april 5]. https://newsroom.wcs.org/news-releases/articletype/ articleview/articleid/14010/a-tiger-at-bronx-zoo-test s-positive-for-covid-19-the-tiger-and-the-zoos-othe r-cats-are-doing-well-at-this-time.aspx. shi j., wen z., zhong g., yang h., wang c., huang b., liu r., he x., shuai l., sun z., zhao y., liu p., liang l., cui p., wang j., zhang x., guan y., tan w., wu g., chen h. & bu z. 2020. susceptibility of ferrets, cats, dogs, and different domesticated animals to sars-coronavirus-2. science, apr 8. pii: eabb7015. doi: 10.1126/science. abb7015. zhang q., zhang h., huang k., yang y., hui x., gao j., he x., li c., gong w., zhang y., peng c., gao x., chen h., zou z., shi z. & jin m. 2020. sars-cov-2 neutralizing serum antibodies in cats: a serological investigation [internet]. wuhan, 2020. https://www.biorxiv.org/content/10.110 1/2020.04.01.021196v1.full.pdf. wan y., shang j., graham r., baric r.s. & li f. 2020. receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars. j virol, 94 (7). pii: e00127-20. doi: 10.1128/ jvi.00127-20. 297 veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 accepted: 11.03.2020 | available on line: 31.12.2021 istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. *corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. e-mail: lo.sacchini@izs.it. lorena sacchini, lucilla ricci, katiuscia zilli, romina romantini, guido di donato, diana neri, tiziana persiani and elisabetta di giannatale* keywords s. typhimurium, monophasic s. typhimurium, antimicrobial resistance, mlva. summary salmonellosis is currently the second most common zoonosis in european union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease trend in the yearly number of infections caused by salmonella. in italy, s. typhimurium and monophasic s.  typhimurium represent the most prevalent serotypes. in this paper, we investigated these two serovars isolated from 2012 to 2017 in abruzzo and molise regions. a set of 345 strains isolated from human sporadic cases, surface water, food and animals were collected and analyzed. monophasic s. typhimurium strains were found to be resistant to streptomycin, sulfisoxazole, ampicillin, tetracycline and nalidixic acid, while s. typhimurium isolates showed high levels of resistance to tetracycline, sulfisoxazole and ampicillin. the 5-loci multilocus variable-number tandem repeat analysis (mlva) identified 88 genotypes (gt), six of which were common for the two serovars. several mlva profiles were shared by human and non-human isolates. mlva had sufficient typing resolution for epidemiological studies of s. typhimurium but demonstrated poor discriminatory in trace-back study of monophasic s. typhimurium. characterization of salmonella typhimurium and monophasic salmonella typhimurium isolated in abruzzo and molise regions, italy, from 2012 to 2017 in the eu, a total of 91,857 human salmonellosis cases were reported; salmonella enteritidis and salmonella typhimurium were the most common serovars, followed by monophasic s.  typhimurium, s.  infantis and s.  newport (efsa and ecdc 2018). in 2018, the number of reported confirmed human cases and the eu notification rate were at the same level as in 2017 (efsa and ecdc 2019, efsa and ecdc 2018). in italy, s.  typhimurium and monophasic s.  typhimurium are two of the most commonly serovars isolated from humans and animals. a similar situation has also been reported in other european countries (molbak et  al. 2014, mueller-doblies et  al. 2018). in italy, over last few decades, various trends have been observed in the prevalence of different salmonella serotypes. between 1980 and 1988 s.  typhimurium was the serotype most commonly isolated from humans, and in the 90’s the highest number of cases were caused by s. enteritidis. since 2000, and until 2012, s.  typhimurium was once again the most prevalent serotype reported. the introduction salmonella spp. is one of the most common foodborne pathogens and salmonellosis is the second most frequently reported zoonosis in europe (efsa and ecdc 2019, efsa and ecdc 2018). monophasic variants of salmonella typhimurium-like strains, lacking the fljb-encoded second phase h antigen appear to be of increasing importance in many european union (eu) member states and a rapid emergence and dissemination of such strains in livestock animals, companion animals and humans has been observed (efsa j 2010). while the number of salmonella-positive samples collected annually is generally decreasing, the monophasic s. typhimurium is one of the few serovars for which the opposite trend has been described both in humans and in animals and it is currently considered an emerging epidemic serovar. on the basis of genetic similarity, the monophasic strains with formula 4,[5],12:i:are regarded as variants deriving from s.  typhimurium that display similar virulence and antimicrobial resistance characteristics. in 2017 298 characterization of salmonella typhimurium in italy sacchini et al. veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 italia surveillance network. the enternet italia is coordinated by the istituto superiore di sanità and relies on the collaboration of peripheral laboratories. it is incorporated into the european surveillance system for foodand waterborne diseases (fwd) and coordinated by the ecdc. the strains isolated from animals were collected in farms. isolates from food were obtained from the producers and from retail stores during official veterinary controls. all the strains were serotyped according to the kauffmann-white-le minor schema by the slide agglutination method (grimont et  al. 2007) using commercial antisera (statens serum institute, copenhagen, denmark). to confirm the strains serotyped as s. typhimurum or monophasic variant, a pcr assay was also performed, as previously described (barco et al. 2011). antimicrobial susceptibility testing the susceptibility to sixteen antibiotics was performed by the disc diffusion method (kirby-bauer) in accordance with the clinical laboratory standards institute guidelines (patel et  al. 2017) using the following bd sensi-disctm (becton and dickinson, berkshire, u.k): ampicillin (a) 10  μg, amoxicillin-clavulanic acid (amc) 20/10 μg, cefazolin (cz) 30 μg, gentamycin (g) 10 μg, kanamycin (k) 30 μg, enrofloxacin (en) 5 μg, trimethoprim/sulfamethoxazole (sxt) 1.25/23.75 μg, tetracycline (t ) 30 μg, ceftazidime (caz) 30 μg, colistine (cl) 10  μg, sufisoxazole (su) 300 μg, nalidixic acid (na) 30  μg, streptomycin (s) 10 μg, chloramphenicol (c) 30  μg, cephalothin (cf ) 30 μg and ciprofloxacin (cip) 5  μg. escherichia coli atcc 25922 was used as a control. multilocus variable‑number tandem repeat analysis (mlva) the 345 strains were typed using 5 locus mlva (sttr9-sttr5-sttr6-sttr10-sttr3) according to the protocol previously described by ecdc (takkinen et  al. 2011) using multiplex pcr and capillary electrophoresis on an abi 3500 instrument with pop 7 polymer (thermo fisher scientific, ma, usa). the mlva profiles were expressed according to the nomenclature published by larsson and colleagues (larsson et al. 2009). the genetic diversity of each locus was determined using the simpson’s diversity index (sdi) calculated for a dataset of s.  typhimurium and monophasic s.  typhimurium, comprising of only one strain per mlva genotype and using the online tool available at the comparing partitions website (http://www. comparingpartitions.info/?link=tool). mlva clustering was performed using the monophasic s. typhimurium was isolated for the first time in italy from a patient in 2003 and the number of cases has increased in the last years (graziani et al. 2013, dionisi et al. 2009). s.  typhimurium was the predominant serovar isolated from veterinary samples between 2002 and 2011, however in 2012, the numbers of monophasic s.  typhimurium-positive samples exceeded the numbers of s.  typhimurium and a steady rise in the prevalence of the monophasic variant has been observed in the following years (https://www.izsvenezie.it/temi/malattie-patogeni/ salmonella/enter-vet/) (accessed on 22 october 2019). the emergence of a clonal group of serovar s.  typhimurium and monophasic s.  typhimurium r-type assut (resistante to ampicillin, streptomycin, sulphonamides and tetracycline) strains in italy, denmark and the united kingdom has also been described (lucarelli et al. 2010). multilocus variable-number tandem repeat analysis (mlva) is a highly discriminative typing method (efsa 2013) that has emerged as a powerful tool for subtyping of food-borne bacterial pathogens (nadon et al. 2013) and it is used for s. typhimurium subtyping, especially in outbreak investigations (torpdahl et  al. 2007, petersen et  al. 2011, ross et al. 2011). the main aims of this study were to investigate the epidemiology of s.  typhimurium and monophasic s.  typhimirium (4,[5],12:i:-) in abruzzo and molise regions, to elucidate the characteristics of the resistotypes and genotypes circulating in the environment and in animal and human populations between 2012 and 2017. materials and methods strains collection a set of 345 strains including 65 isolates of s.  typhimurium and 280 isolates of monophasic s.  typhimurium were collected in abruzzo and molise regions of italy between january 2012 and december 2017. the strains of s. typhimurium were isolated from fecal samples of chickens (n = 2), pigs (n  =  9), wild birds (n  =  6), humans (n  =  13), pork meat (n  =  6), and surface water samples (n  =  31). the strains of monophasic s.  typhimurium were isolated from fecal samples (from 15 pigs, 2 chickens and from 145 humans), from food (21 from pork, 2 from mixed beef-pork minced meat, 1 from lamb, 1 from turkey, 8 from bivalve molluscs) and from 85 surface water samples. the strains isolated from human feces and surface water were sent to our laboratory by regional hospitals and environmental protection agency, respectively, within the enternet 299 sacchini et al. characterization of salmonella typhimurium in italy veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 results the antimicrobial resistance traits of the two salmonella serovars are shown in table ii. more than a half of tested isolates were resistant to tetracycline (61.35% for s. typhimurium and 66.39% for monophasic s.  typhimurium). moreover, a large number of monophasic s. typhimurium strains were resistant to streptomycin (81.89%), sulfisoxazole (58.49%), ampicillin (52.08%) and nalidixic acid (48.30%), while s.  typhimurium isolates showed high levels of resistance to sulfisoxazole (38.64%) and ampicillin (36.36%). fewer monophasic s.  typhimurium isolates were resistant to cefazolin, kanamycin, ceftazidime, colistin, cephalotin and ciprofloxacin, while s.  typhimurium isolates were fully susceptible to these antimicrobials (table ii). more than 30% of monophasic s.  typhimurium belonged to the assut r-type (95 strains) and only the 5.6% to the acssut type (16 strains) while for s.  typhimurium the assut and acssut profiles accounted for 6.87% (3 strains for each serovars). the mlva analysis classified the 345 strains (figure 1) into 88 gts. six of the mlva profiles were common for both serovars, while 33 were specific for s. typhimurium, and 49 for monophasic s. typhimurium. the six common genotypes between the two serotypes were gts 25 (3-11-9-na-211 mlva profile), 27 (3-12-10-na-211), 61 (3-15-11-na-211), 64 (3-15-12-na-211), 70 (3-17-11-na-211) and 75 (3-8-10-na-211). bionumerics software package version 7.6. (applied-maths, sint-martens-latem, belgium) with mst (minimum spanning tree) method. clonal complexes (ccs) were retrieved with the most stringent (conservative) definition (spratt et al. 2004) where all members assigned to the same cc differed only by one locus with the nearest member included in the same cc. table i shows gts-mlva patterns conversion. table i. gts-mlva patterns convertion. salmonella typhimurium monofasic salmonella typhimurium mlva pattern gts mlva pattern gts mlva pattern gts 2-10-6-6-112 2 1-9-na-na-210 1 3-14-9-na-210 60 2-13-na-na-311 3 2-19-na-na-211 10 3-15-11-na-211 61 2-16-6-12-212 4 3-10-10-na-211 15 3-15-12-na-210 63 2-16-6-14-112 5 3-10-11-na-211 16 3-15-12-na-211 64 2-17-na-na-105 6 3-10-12-na-211 17 3-15-7-na-211 65 2-17-na-na-211 7 3-10-15-na-211 18 3-15-8-na-na 66 2-17-5-7-212 8 3-10-9-na-112 19 3-15-8-na-211 67 2-17-7-14-112 9 3-11-10-na-211 21 3-15-9-na-211 68 2-20-na-na-211 11 3-11-11-na-211 22 3-17-12-na-311 70 2-22-9-14-112 12 3-11-15-na-211 23 3-9-12-na-211 75 2-6-4-9-212 13 3-11-8-na-211 24 3-9-5-na-211 76 2-8-5-9-212 14 3-11-9-na-211 25 3-10-9-na-210 81 3-11-9-na-211 25 3-12-na-na-211 26 3-13-na-na-211 82 3-12-10-na-211 27 3-12-10-na-211 27 3-11-na-na-211 83 3-12-12-12-211 31 3-12-11-na-211 28 3-13-14-na-211 43 3-12-11-18-na 29 3-13-7-na-211 44 3-12-12-na-211 30 3-14-na-21-311 49 3-12-13-na-211 32 3-14-12-7-211 55 3-12-3-na-211 33 3-14-16-17-311 57 3-12-7-na-211 34 3-15-11-na-211 61 3-12-8-na-211 35 3-15-11-na-311 62 3-12-9-na-na 36 3-15-12-na-211 64 3-12-9-na-211 37 3-17-12-na-211 69 3-13-na-7-211 38 3-17-12-na-311 70 3-13-10-na-211 39 3-19-12-na-311 71 3-13-11-na-211 40 3-19-na-na-311 72 3-13-12-na-211 41 3-8-10-na-211 73 3-13-13-na-211 42 3-9-na-na-210 74 3-13-7-7-211 45 3-9-12-na-211 75 3-13-8-na-211 46 4-12-9-7-211 77 3-13-9-na-210 47 4-13-9-22-311 78 3-13-9-na-211 48 6-24-15-22-11 79 3-14-10-na-211 50 6-9-13-9-211 80 3-14-11-na-211 51 6-19-15-18-na 84 3-14-11-9-211 53 2-19-6-14-112 85 3-14-12-na-211 54 2-17-na-na-210 86 3-14-13-na-211 56 3-10-na-na-210 87 3-14-3-na-211 58 2-20-5-13-112 89 3-14-8-na-211 59 table ii. antibiotic resistance rates (%) of s. typhimurium and s. 4,[5],12:i:isolated between 2012 and 2017 in abruzzo and molise regions. antibiotic % resistant s. typhimurium s. 4,[5],12:i:ampicillin (a) 10μg 36.36 52.08 amoxicillin/clavulanic acid (amc) 20/10 μg 11.36 20.38 cefazolin (cz) 30 μg 0,00 3.02 gentamicyn (g) 10 μg 4.55 3.02 kanamycin (k) 30 μg 0,00 5.66 enrofloxacin (en) 5 μg 6.82 12.08 trimethoprim/sulfamethoxazole (sxt) 1.25/23.75 μg 4.55 9.81 tetracycline (t) 30 μg 61.36 66.79 ceftazidime (caz) 30 μg 0,00 0.01 colistine (cl) 10 μg 0,00 1.89 sufisoxazole (su) 300 μg 38.64 58.49 nalidixic acid (na) 30 μg 9,09 48.30 streptomycin (s) 10 μg 22.73 81.89 chloramphenicol (c) 30 μg 6.82 6.04 cephalothin (cf ) 30 μg 0.00 6.79 ciprofloxacin (cip) 5 μg 0.00 0.00 300 characterization of salmonella typhimurium in italy sacchini et al. veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 (3-15-11-na-311) included human and pig samples, gt72 (3-19-na_na-311) and gt61 (3-15-11-na-211) contained strains isolated from humans and the surface water. gt27 (3-12-10-na-211) included s.  typhimurium isolated from humans and surface water and belonged to cc2 that contained isolates from pig. cc5 included only isolates from surface water, while cc3, cc4 and cc7 comprised of strains from pig and surface water. gt85 (2-19-6-14-112) group included only poultry isolates and clustered closely with gt5 (2-16-6-14-112) samples from surface water in cc9. we identified four genotypes that were shared between the human and environmental or animal samples. the genotype that contained the higher number of isolates was g62 (mlva profile 3-15-11-na-311) that was identical for seven pigs and 1 human isolate. gt61, with a mlva pattern 3-15-11-na-211, was assigned to isolates from four surface water samples and from one human sample. genotype 27 (3-12-10-na-211) was found in one water, two wild birds and 1 human isolates, and g72 (3-19-12-na-311) was shared between one strain collected from surface water and two strains isolated from humans. for monothasic s.  typhimurium, clustering analysis retrieved only one major cc which grouped almost all the mlva genotypes (45 out of 55) (figure 3). several genotypes, i.e. gt28 (3-12-11-na-211) and gt37 (3-12-9-na-211) included, beside human isolates, samples from more than one source (pig, turkey, poultry, water, etc.). three mlva profiles, 3-11-10-na-211 (gt21), 3-12-3-na-211 (gt33) and 3-11-15-na-211 (gt23), were shared between humans and bivalve molluscs; while, 3-11-11-na-211 (gt22) included monophasic variant strains isolated from humans and sheep. fifteen genotypes were retrieved from human isolates only. gt51 (mlva profile 3-14-11-na-211) was the most common genotype identified in monophasic s.  typhimurium (85 human feces and 25 surface water strains), followed by gt41 (3-11-10-na-211) assigned to 14 surface water and 3 human isolates. other frequently identified mlva pattern included the degree of polymorphism of mlva genotypes associated with the two serovars was quantified by calculating the diversity index of each locus (table  iii). for s.  typhimurium, sttr9 showed the lowest number of alleles (n = 4) with a sdi of 0.622, sttr5 resulted as the most variable locus (15 allele) with sdi of 0.934. for monophasic s.  typhimurium only sttr5 and sttr6 were highly discriminating, other loci showed an sdi below to 0.325 (table iii). ten ccs were assigned using mst analysis of s.  typhimurium based on mlva typing results (figure 2). the major clonal complex (cc1) included 8 genotypes, three of which were associated with human and non-human isolates. gt62 figure 1. minimum spanning tree (mst) of 65 s. typhimurium and 280 s. 4,[5],12:i:analyzed between 2012 and 2017 in abruzzo and molise regions. the node labels represent the unique genotype (gt). the diameter of each node is proportional to the number of isolates. the branch length is proportional to the loci differences. table iii. number of partition, simpson’s diversity index (sdi) and confidence interval (ci 95 %) for the five loci of the mlva panel for s. typhimurium and s. 4,[5],12:i:-. the calculation of confidence intervals for simpson's index was performed by use of the large sample approximation, thereby improving the objective assessment of the discriminatory power of typing techniques. locus s. typhimurium s. 4,[5],12:i:# partitions sdi ci (95%)* # partitions sdi ci (95%)* sttr9 4 0.622 (0.536-0.708) 3 0.072 (1.000-0.168) sttr5 15 0.934 (0.903-0.964) 9 0.868 (0.843-0.893) sttr6 13 0.899 (0.853-0.944) 11 0.894 (0.868-0.921) sttr10 11 0.725 (0.580-0.869) 4 0.173 (0.038-0.308) sttr3 8 0.777 (0.684-0.871) 5 0.325 (0.168-0.481) #number of different repeats present at this locus; *precision of the diversity index, expressed as 95% upper and lower boundaries. 301 sacchini et al. characterization of salmonella typhimurium in italy veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 possible implication of environmental sources was suspected (cito et al. 2016). the mlva protocol designed for salmonella typhimurium routine typing (takkinen et  al. 2011) is also commonly used for the characterization of monophasic s.  typhimurium and it has facilitated the successful detection and investigation of multiple food-borne disease outbreaks (cito et  al. 2016, barco et  al. 2014, raguanaud et  al. 2012). for routine surveillance of salmonella, mlva seems to have notable advantages over pulsed field gel electrophoresis (pfge) method. the protocol is fast and can be completely automated and the generated data are easily analyzed and compared between laboratories. moreover, mlva has been shown to have a higher discriminatory power than pfge (torpdahl et  al. 2007, hopkins et  al. 2007, best et  al. 2009). in our study, the analysis of the mlva profiles of s.  typhimurium and s.  4,[5],12:i: isolates showed that, in spite of the high similarity and close relationship between the two serovars (switt et  al. 2009), only six mlva profiles were in common between the two serovars. moreover, the genetic resolution power of three mlva loci was much lower for monophasic s.  typhimurium than for s.  typhimurium. cluster analysis performed for monophasic variant retrieved only one major clonal complex that grouped almost all the mlva profiles, 3-11-10-na-211 (g21) assigned to humans (n  =  4), pigs (n = 4), surface water (n = 3) and bivalve molluscs (n = 3). 3-12-11-na-211 mlva profile (gt28) found in human (n = 1), surface river (n = 1), pigs (n = 1), cattle (n = 1) and chikens (n = 2) strains and 3-12-3-na-211 (gt33) assigned to human (n = 4),pigs (n = 3),surface river (n  =  2) and strains isolated from bivalve molluscs (n = 2). discussion in italy, s.  typhimurium and monophasic s.  typhimurium are two of the most commonly reported serovars isolated from human and veterinary samples. while, in general, the total number of yearly salmonella isolation have been decreasing, the opposite trend has been observed for a few serovars including monophasic s.  typhimurium (efsa and ecdc 2019). our data confirmed this observation as were reported four times greater number of isolated monophasic variant than s.  typhimurium strains. the high dissimilarity of number of sampling between the two serogroups was partly due to an epidemic outbreak (mlva 3-14-11-na-211) of monophasic s. typhimurium observed in abruzzo region during 2013 and 2014 (67.80% of the strains) where a figure 2. minimum spanning tree (mst) of 65 strains of s. typhimurium isolated in abruzzo and molise regions between 2012 and 2017. the node labels represent the unique genotype (gt) and are colored according to the source of isolation. the diameter of each node is proportional to the number of isolates. the branch length is proportional to number of different loci. clonal complexes (ccs) are shaded in grey. figure 3. minimum spanning tree (mst) of strains of 280 monophasic s. typhimurium:isolated in abruzzo and molise regions between 2012 and 2017. the node labels represent the unique genotype (gt) and are colored according to source of isolation. the diameter of each node is proportional to the number of isolates. the branch length is proportional to number of different loci. clonal complexes (cc) are shaded in grey. 302 characterization of salmonella typhimurium in italy sacchini et al. veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 2017 approximately 40% of human s. typhimurium isolates, and 80% of monophasic s.  typhimurium were resistant to multiple antimicrobials. in pigs, multi-drug resistance (mdr) was observed in approximately 60% of s.  typhimurium and 77% of the monophasic variant isolates (efsa and ecdc 2019). in recent years, there has been an overall decline in the level of antimicrobial resistance in serovar s. typhimurium in several european countries related to a decrease in the number of isolates of multiresistent dt104 (meakins et al. 2008). to some extent this reduction has been counterbalance by the increase in prevalence of monophasic s.  typhimurium that expresses resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (mueller-doblies et  al. 2018, lucarelli et  al. 2010). in our study, we found that more than 30% of s.  typhimurium were resistant to four antibiotics (assut resistance type) and only 5.6% were additionally resistant to chloramphenicol (acssut resistance type). overall, the majority of monophasic s.  typhimurium isolates appeared to show a multidrug resistance phenotype while the majority of s.  typhimurium isolates were resistant to only a few antimicrobial drugs (data not showed). in conclusion, based on the characterization of human and non-human s.  typhimurium and monophasic s.  typhimurium strains obtained over a 6-year surveillance at the regional level, we found antimicrobial resistance results comparable to those reported by the last efsa report from 2017. the mlva 5-loci scheme resulted suitable for s.  typhimurium typing but the discriminatory power observed for monophasic s.  typhimurium was insufficient when used as an exclusive typing method in epidemiological studies. use of powerful discrimination typing method based on whole genome sequencing (wgs) such as snps analysis or core genome mlst are recommended to identify epidemiological relationship of monophasic s. typhimurium isolates. confirming the lack of discrimination of sttr3, sttr10 and sttr9 loci, as previously demonstrated by barco and colleagues (barco et  al. 2015) on the contrary, for s.  typhimurium, the diversity of mlva profiles was conferred by all the five loci (sdi > 0.6). hopkins and colleagues (hopkins et  al. 2007) reported that since mlva is a highly discriminatory method, some minor changes in targeted loci can be found in outbreak-related strains. therefore, for salmonella the authors proposed a difference of no more than two repeats at one single locus to identify isolates that are part of the same outbreak. as the mlva protocol for typing of monophasic s.  typhimurium includes three out of five highly stable loci, the correct interpretation of mlva profiles can be critical especially for large scale epidemiological investigations and for source attribution studies. in our work, mlva analysis of s.  typhimurium assigned only four genotypes in common between human and non-human isolates (pig or surface water), while, for monophasic s.  typhimurium about half of genotypes were shared by human isolates and the isolates from other sources (pig, turkey, poultry, water, etc.). these findings support the conclusion of barco and colleagues (barco et al. 2015) that 5-loci mlva scheme applied to monophasic s.  typhimurium may not provide a sufficient resolution for source attribution studies. in the definition of an outbreak caused by monophasic s.  typhimurium, cito and colleagues (cito et  al. 2016) reported the pfge profile and the r-type. another study confirmed the insufficient resolution of mlva scheme in the characterization of s.  4,[5],12:i:isolates, given that isolates with considerable diversity in terms of single-nucleotide polymorphisms (snps) were indistinguishable by their mlva profiles (petrin et al. 2019). the efsa-ecdc report from 2017 (efsa and ecdc 2018) describing the prevalence of antimicrobial resistance in eu included data on salmonella isolated from humans, fattening pigs and calves. in 303 sacchini et al. characterization of salmonella typhimurium in italy veterinaria italiana 2021, 57 (4), 297-304. doi: 10.12834/vetit.2060.12083.1 barco l., barrucci f., cortini e., ramon e., olsen j.e., luzzi i., lettini a.a. & ricci a. 2015. ascertaining the relationship between salmonella typhimurium and salmonella 4,[5],12:i:by mlva and inferring the sources of human 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salmonella typhimurium 4,5,12:i:in schools associated with imported beef in poitiers, france, october 2010. euro surveill, 17, 20289. ross i.l., davos d.e., mwanri l., raupach j. & heuzenroeder m.w. 2011. mlva and phage typing as complementary 297 1faculty of veterinary medicine, benha university, moshtohor, toukh 13736, egypt. 2animal health research institute, p.o. box 264-giza, cairo 12618, egypt. 3friedrich-loeffler-institut, institute of bacterial infections and zoonoses, naumburger str. 96a, 07743 jena, germany. *corresponding author at: friedrich-loeffler-institut, institute of bacterial infections and zoonoses, naumburger str. 96a, 07743 jena, germany. e-mail: gamalwareth@hotmail.com. keywords brucella spp., maldi, amos-pcr, cattle and buffaloes, egypt. summary brucellosis is a widespread disease in egypt which cause huge economic losses in the dairy industry. the present study aims at isolating and identifying brucella (b.) spp. circulating in bovine and buffalo dairy herds kept at farmers houses in four districts of the delta region of egypt. one hundred and five  tissue specimens were collected from seropositive cattle and buffaloes. the samples included 10 vaginal swabs, 3 placentas, 3 uteri and 86 supra-mammary lymph nodes from dams, as well as 3 stomach contents from aborted fetuses. matrix-assisted laser desorption ionization (maldi) and the conventional biotyping techniques were used for preliminary identification of isolates into the genus level. amos-pcr was applied to differentiate brucella isolates into species level. nineteen brucella strains have been identified, four b. abortus strains were recovered from cattle and 15 b. melitensis strains were isolated from both cattle (n  =  8) and buffaloes (n  =  7). the predominant occurrence of b. melitensis in bovines raises the fact that b. melitensis clone can cross species barriers and can establish a permanent reservoir in cattle and buffaloes. presence of culture-positive animals at householders represent a high-risk factor for human infection. this knowledge is of significant importance in the control of brucellosis in bovines. ashraf abd eltawab1, fatma el-hofy1, mahmoud hamdy2, shawky moustafa1, enas soliman1, wedad ahmed1, mohamed ramadan1 and gamal wareth1,3* isolation and molecular identification of brucella spp. in bovine herds kept at householders in the delta region of egypt by maldi-tof and amos-pcr veterinaria italiana 2020, 56 (4), 297-300. doi: 10.12834/vetit.1980.10596.3 accepted: 23.01.2020 | available on line: 31.12.2020 of the others are not proved yet. regarding their capability of causing disease in humans, b. melitensis is considered the most virulence species followed by b. suis, and b.  abortus which, amongst the three species, is considered the mildest one (galinska and zagorski 2013). in egypt, brucella was reported for the first time in 1939 (refai 2002). in the 1980ies, attention was directed to the animal diseases when an open-door policy was applied and friesian cows were imported to establish governmental farms. in that period, the incidence of brucellosis in some farms reached 38% (refai 2002). recent national serological investigations gave indirect proof of the presence of brucella in cattle, buffalo, sheep, and goat herds nationwide (wareth et  al. 2014a). in egypt only few laboratories are capable of isolating brucella. despite the implementation of the control program, brucellosis is an infectious zoonotic disease worldwide caused by brucella (b.) species. the disease might have serious public and animal health impact as infections are often associated to chronic debilitation in humans and reproduction failure in animals (godfroid et  al. 2011). in the sexually mature female animals, the disease provokes metritis, abortion, stillbirth, retention of the placenta and drop in milk production, in males the disease can cause orchitis and epididymitis. infertility may be seen in both sexes (ducrotoy et al. 2014). up-to-date, the genus brucella comprises of 12 accepted phenotypically recognized species; b.  abortus, b. suis, b. melitensis, b.  neotomae, b. ovis, b. canis, b. inopinata, b. microti, b. pinnepedails, b.  ceti, b. papponi, and b.  vulpex. only b. melitensis, b. suis except bv 2, b.  abortus and to some extent b. canis are well-known pathogens for humans (chiliveru et  al. 2015), while the zoonotic potential short communication 298 brucellosis in egyptian dairy herds eltawab et al. veterinaria italiana 2020, 56 (4), 297-300. doi: 10.12834/vetit.1980.10596.3 performed in 25 µl of a reaction mixture containing final concentrations of 10 pmol/µl for each primer with 0.2 µl of 5  u/µl of taq dna polymerase (promega), and 10  mm/1µl of deoxynucleotide triphosphates (dntp) (eppendorf ). one μl dna template was added to each reaction mixture. the pcr mixtures were overlaid with 2.5 µl of pcr-puffer (genaxxon) and complete with hplc until 25  µl. the amplification was performed at a denaturation temperature of 93  °c for 5 min. this was followed by 35 cycles at 90  °c for 60 sec, 60  °c for 60 sec, 72  °c for 60 sec and 1 final extension at 72  °c for 5 minutes. positive control (b. abortus 544 dna) and negative control (hplc water) were included in each reaction. after amplification, all reaction mixtures were analyzed by electrophoresis in a 1.5% agarose gel, stained with ethidium bromide, and photographed. visible bands of appropriate sizes of (223 bp) for brucella spp. were considered positive reactions. to confirm results of maldi-tof and secure identification of brucella species, the amos-pcr (b.  abortus, b. melitensis, b. ovis and b. suis-pcr) was done according to bricker and halling and matope and colleagues (bricker and halling 1994, matope et  al. 2009) with few modifications. briefly, the pcr was performed in a final volume of 25 µl consisting of a reaction mixture containing 10 × pcr buffer (genaxxon) without mgcl. 0.2 μm each of b. abortus, b. melitensis, b. ovis, b. suis, and is711-specific primer added to the reaction (table i). 10  mm of dntps (eppendorf ), 0.2 µl of 5 u/µl of taq dna polymerase (eppendorf ) were added and complete with hplc water until 25  µl. 1 μl dna template was added to the 24 μl reaction mixture. the cycling conditions utilized were initial denaturation of 95 °c for 5 min; followed by 30 cycles of amplification using the following parameters: at 95 °c for 60 sec, annealing for 120 sec at 58  °c, extension for 120  sec at 72  °c, and a final elongation step of 72  °c for 5  min. the pcr products were separated by electrophoresis the disease is still endemic among livestock and humans. buffaloes and cattle are a valuable and economic component of egyptian rural household and number of buffaloes is higher than in any other neighboring country in the near east region. control programs have been instituted by the general organization of veterinary services (govs) to prevent the spread of brucellosis in the country particularly in large dairy herds (wareth et al. 2014a). more than 70% of total livestock populations are owned by smallholders. few cattle and buffaloes are kept in the household to produce milk, meat, and dairy products for home consumption or to sell often unpasteurized products in the local markets. the current study was aimed to identify the brucella species circulating in small buffalo and cattle dairy herds in different localities of delta region in egypt using matrix-assisted laser desorption ionization (maldi), conventional and amos pcrs. a total of 105 seropositive animals (90 cattle, 15  buffaloes) were used in the study. all animals were serologically positive for brucella by rose bengal test (rbt) and elisa within the surveillance and eradication program instituted by the govs, egypt. the animals originated from qalyubia, gharbia, dakahliya, and menofia. from each animal one sample was collected at abattoirs. different tissue specimens have been taken: 86 lymph nodes, 3 uteri, 3 placenta and 10 vaginal swabs from dams, as well as 3 stomach contents from aborted fetuses. the specimens were collected aseptically and were kept in an icebox and transferred directly to the laboratory for isolation. the organ surface was burned and the internal parts were smeared by the swab. the swabs were plated on brucella agar for primary isolation and subsequent identification of brucella species was done according to colony morphology, gram stain, motility, co 2 requirement, h 2 s production, phage lysis, and the biochemical tests e.g. catalase, oxidase and urease tests (alton et  al. 1988). a single colony was selected from the brucella selective agar media and sub-cultured again on blood agar media to obtain uncontaminated colonies. after 48 hours of incubation, one colony was picked and submitted to matrix-assisted laser desorption/ionization (maldi-tof) for genus brucella identification as described previously (murugaiyan et  al. 2014). the score values between 2.0 and 2.29 considered ‘secure genus identification and probable species identification’. genomic dna was extracted with the high pure template preparation kit (dna hp kit, roche applied sciences, mannheim, germany) according to the manufacturer’s instructions. two pairs of primers amplifying different regions of the brucella genome were used, the primer b4 and b5 that amplify a 223  bp fragment of the 31-kda outer membrane protein. pcr assay conditions were table i. primers used in conventional and amos-pcr for identification of brucella in samples collected from seropositive cattle and buffaloes in the delta region of egypt. conventional pcr primer nucleotide sequence 5'-3 ' amplicon size b4 primer tgg ctc ggt tgc caa tat caa 223 bp b5 primer cgc gct tgc ctt tca ggt ctg amos-pcr b. abortus gac-gaa-cgg-aat-ttt-tcc-aat-ccc 498 bp b. melitensis aaa-tcg-cgt-cct-tgc-tgg-tct-ga 731 bp b. ovis cgg-gtt-ctg-gca-cca-tcg-tcg 976 bp b. suis gcg-cgg-ttt-tct-gaa-ggt-tca-gg 285 bp is711 tgc-cga-tca-ctt-aag-ggc-ctt-cat 299 eltawab et al. brucellosis in egyptian dairy herds veterinaria italiana 2020, 56 (4), 297-300. doi: 10.12834/vetit.1980.10596.3 19  isolates, twelve strains were isolated from cattle and seven from buffaloes. failure of isolation from the other cases may be due to a low number of viable brucella organisms in specimens and/or due to massive contamination with other bacteria (wareth et  al. 2014b). b. abortus was isolated from cattle, while b. melitensis was isolated from both cattle and buffaloes. previously it was assumed that brucellosis in cattle was mainly caused by b. abortus, less frequently by b. melitensis and rarely by b. suis (radostits et  al. 2000). in recent years, b. melitensis has been described as cause of great outbreaks in cattle and now is becoming a worldwide emerging problem and difficult to be controlled. this is likely due to the ineffective vaccination programs and lack of knowledge regarding the causative agents in different hosts (alvarez et  al. 2011). the major limitation of the control program in egypt is the difficulty to detect all infected animals, especially carriers hosts i.e. dogs, cats, rats (wareth et al. 2017). seronegative/culture-positive animals within the herds also play a significant role in re-emerging and dissemination of the infection (el-diasty et  al. 2018). in egypt, b. melitensis bv3 and b. abortus bv1 are the predominant source of brucellosis. b. melitensis have been isolated from sheep, goat, cattle, buffaloes, rats and nile catfish (wareth et  al. 2014a), while b.  abortus has been isolated from cattle, buffaloes and once from dog and cat (wareth et al. 2017). isolation of b. melitensis from cattle and buffaloes, the non-original hosts, can be attributed to raise sheep and goats together with cattle and buffaloes. the current results are in accordance with previous studies that highlighted the fact that b. melitensis clone can cross species barriers and establish a reservoir in cattle and buffaloes (abd-el halim et  al. 2017, hosein et  al. 2018, wareth et  al. 2014a). different molecular typing methods such as pcr techniques can differentiate brucella at the species and the biovars level (al  dahouk et  al. 2005, bricker and halling 1994). these methods require little expertise, easy to perform and provide valuable alternatives to biochemical typing (fretin et  al. 2008). application of maldi-tof ms was used successfully for microbial identification into the subspecies level, demonstrating that this technology is also a potentially effective tool for detection of microorganisms (croxatto et  al. 2012). combination of amos-pcr and maldi-tof were able to identify brucella spp. from a single colony in a short time and are promising techniques for fast and accurate identification. the results obtained in the current study should be considered during control and eradication program of brucellosis. all strains were recovered from small dairy farms which should be included in the control programs. using 1.5% agarose gel (w/v). visible bands were considered positive reactions at appropriate sizes of 498 bp for b. abortus, 731 bp for b. melitensis, 976 bp for b. ovis and 285 bp for b. suis. as shown in table ii, the bacterial examination of the collected samples from seropositive cattle and buffaloes revealed 19 brucella strains, 12/90 strains were recovered from cattle and 7/15 were recovered from buffaloes. all recovered isolates showed the characteristic colonial morphology of brucella on brucella agar, brucella selective media, and blood agar. the colonies were convex, round, 1-2 mm. in diameter, with smooth margins, round edges, translucent and of golden color (pale honey-colored). all isolates were gram-negative, non-motile, catalase and oxidase-positive. they required co 2 for growth and produced h 2 s. conventional pcr and maldi-tof confirmed the classical bacteriology results on the 19 strains as brucella with clear bands and score values >  2.3, respectively. the score >  2 is considered positive for genus brucella, while the score values between 2.3 and 3.0 indicate ‘highly probable species identification’. madi-tof identified four strains as b. abortus and 15 strains as b. melitensis. amos-pcr differentiated four b.  abortus strains at appropriate band size of 498 bp, while a total of 15 strains were identified as b. melitensis at appropriate band size of 731 bp. all b. abortus strains identified in the current study were recovered from cattle samples, while b. melitensis strains were isolated from cattle (n = 8) and buffaloes (n = 7). brucellosis is endemic and prevalent in the middle east, in the mediterranean basin, in some countries of africa and asia, and in some areas of latin america. however, still the epidemiological situation is not clear in some of those regions. the present work was planned to isolate brucella spp. from cattle and buffaloes in different governorates of the delta region in egypt. bacteriological examination of 105 collected samples produced table ii. number of collected samples and number of brucella isolates obtained from seropositive cattle and buffaloes in the delta region of egypt. source of samples cattle buffaloes no. of samples no. of isolates % no. of samples no. of isolates % lymph node 74 8 10.8% 12 5 41.6% uterus 3 1 33.3% 0 placenta 3 2 66.6% 0 stomach content 3 1 33.3% 0 vaginal swabs 7 0 3 2 66.4% total 90 12 13.3% 15 7 46.6% 300 brucellosis in egyptian dairy herds eltawab et al. veterinaria italiana 2020, 56 (4), 297-300. doi: 10.12834/vetit.1980.10596.3 acknowledgments we would like to thank the academy of scientific research and technology (asrt) egypt for financial support. the work belonging to brucmednet project (id: 698) funded by the arimnet2. abd-el halim m., mohamed a. & shalaby n. 2017. prevalence of brucellosis in buffaloes and its control measures. journal of veterinary medical research, 24, 161-170. al dahouk s., tomaso h., 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n., sayour a. & neubauer h. 2017. isolation of brucella abortus from a dog and a cat confirms their biological role in re-emergence and dissemination of bovine brucellosis on dairy farms. transbound emerg dis, 64, e27-e30. wareth g., melzer f., elschner m.c., neubauer h. & roesler u. 2014b. detection of brucella melitensis in bovine milk and milk products from apparently healthy animals in egypt by real-time pcr. j infect dev ctries, 8, 1339-1343. 347 veterinaria italiana 2022, 58 (3), 347-351. doi: 10.12834/vetit.2239.15416.2 accepted: 04.08.2021 | available on line: 31.12.2022 1department of veterinary medicine, federal university of espírito santo (ufes), campus alegre, 29.500-000 alegre, espírito santo, brazil. 2federal university of espírito santo (ufes), campus alegre, alegre, espírito santo, brazil. 3veterinary consultant,  29122-720 vila velha, espírito santo, brazil. 4incaper, 29590-00 divino são lourenço, espírito santo, brazil. 5department of biosystems engineering, federal university of são joão del-rei, 36301-160 são joão del-rei, minas gerais, brazil. *corresponding author at: department of veterinary medicine, federal university of espírito santo (ufes), campus alegre, 29.500-000 alegre, espírito santo, brazil. tel.: +55 28 35528916, e-mail: marcos.zanini@ufes.br. marcos santos zanini1*, larissa ataíde siqueira2, yuri vieira almeida2, laisa savergnini poleze1, dante gnecco zanini3, roberto ramos sobreira4 and ana paula madureira5 keywords american cutaneous leishmaniasis, dogs, nitrofurans, therapy. summary euthanasia of animals is not accepted as a control for cutaneous leishmaniasis caused by leishmania (viannia) braziliensis and drugs used in humans for the treatment of leishmaniasis are not allowed for animals in brazil. miltefosine was authorized for dogs infected by leishmania infantum with variable results for l. braziliensis. thus, nine dogs infected with leishmania (v.) braziliensis were treated by a combination of furazolidone and β-cyclodextrin. the nine dogs were mongrels, weighing between 4-17 kg and 3-10 years old. these dogs had ulcerous lesions in different regions such as scrotal tissue, auricular pavilion and nostrils. serological, molecular and protozoal culture techniques were used for laboratory diagnosis. the treatment used furazolidone + β-cyclodextrin complex (1: 2) at a concentration of 60  mg/ml given orally at a dose of 15 mg/kg every 12 hours. the re-epithelialization of lesions occurred between 35 and 41 days of treatment. during fourteen months the animals were monitored and there was no reactivation of lesions or growth of the protozoan in a culture medium of the biopsies. this study demonstrated that treatment with fzd and cd is effective in reducing the cutaneous lesions caused by l. braziliensis in dogs. treatment of canine cutaneous leishmaniasis by leishmania (viannia) braziliensis in dogs with furazolidone and β-cyclodextrin: case report. maintaining 9 endemic peri-urban areas. dogs can increase the potential for transmission by virtue of 10 representing an untreated reservoir for the parasite. in human, the drug traditionally used 11 is meglumine antimoniate, but its use is not allowed in animals . drugs like pentamidine 12 isethionate, miltefosine, amphotericin b and liposomal amphotericin b can be used 13 (paho 2020). in brazil, cl occurs throughout the country and the causative agent responsible for the largest number of cases in both humans and animals is leishmania (viannia) braziliensis. euthanasia of wild and domestic animals is not accepted as a control measure for cl and although there is treatment for infected humans, infected animals are not treated with the same drugs in order to prevent the development of parasite introduction cutaneous leishmaniasis (cl) is an infectious, non-contagious disease caused by 2 different species of protozoa of the genus leishmania, which affects the skin and mucous 3 membranes. cl is a zoonosis, which affects animals and secondarily humans. infection 4 invariably starts with a bite of the infected sandfly vector taking a blood meal followed 5 by sequestration of the leishmania parasites in the macrophages of the host. the earliest 6 clinical symptoms, a cutaneous lesion or a fever, have no diagnostic or prognostic value 7 (de bruijn & barker 1992). domestic animals are potential vectors of different types of 8 leishmaniasis, and pets play an important role as new opportunistic hosts, case report 348 veterinaria italiana 2022, 58 (3), 347-351. doi: 10.12834/vetit.2239.15416.2 treatment of canine cutaneous leishmaniasis with furazolidone/β-cyclodextrin santos zanini et al. which had ulcers suggestive of cl were examined. the dogs were males between 3-10 years of age, mongrels, weighing between 4 to 17 kg, residing in rural areas of different municipalities in the region. the nine animals had ulcerative lesions in different places such as scrotal epidermis, pinna and nostrils. the laboratory diagnostic protocol was carried out in two stages, first serological diagnosis followed by molecular diagnosis. for serological diagnosis, samples of venous blood were collected to perform the indirect enzyme linked immunosorbent assay (i-elisa) (ribeiro et al. 2007) and western blot (zanini et  al. 2010). for the i-elisa, soluble antigen was extracted from the l.  (v.)  braziliensis seed sample (mhoh/br/1975/m2903) provided by the oswaldo cruz foundation according to authentication certificate n. 034/2012. all dogs were seropositive by the i-elisa with a cut-off point of 3 standard deviations (3sds) of negative controls using polyclonal antiserum igg and soluble antigen of l.  braziliensis. this cut-off of 3sds allows to differentiate l.  braziliensis from sporotrichosis (ribeiro et  al. 2007). in the western blot test, the sera of the nine dogs reacted with fractions of the protein soluble antigen distributed between 54, 66, 70, 80 and 97 kda extracted from a seed sample of l. (v.) braziliensis (mhoh/br/1975/ m2903) as already observed (zanini et al. 2010) using secondary polyclonal igg antibody for symptomatic and asymptomatic dogs in a serological study for cl. for molecular diagnosis, the animals were sedated with dissociative drugs (10% ketamine 15 mg/ kg/im and 20% xylazine 0.2 mg/kg/im) and were anesthetized locally in the region where the procedure was performed, using lidocaine 0.2% (1:10 diluted). after sedation, tissue samples from the edge of the lesions were collected for smear, polymerase chain reaction (pcr) and protozoan culture. the following lesion samples were collected: dog 1, pinna; dog 2, pinna; dog 3, pinna; dog 4, scrotal epidermis; dog 5, pinna and nostril; dog  6, pinna and scrotal tissue; dog 7, pinna; dog 8, nostril; dog 9, pinna. the smears of the lesions were stained with leishman stain and suggestive forms of intracellular phagocyte amastigotes were observed in the sample collected from the ear of dog 5, scrotal epidermis sample from dog 6 and the ear of dog 9. for pcr, primers b1 and b2 (de bruijn and barker 1992) were used since the southern region of the state of espírito santo is endemic only for cl, and these primers amplify a sequence of 750 base pairs of dna from the sub-genus leishmania (viannia). all collected samples, except for the nostril lesion of dog 5, were positive to pcr. plots of the nine biopsy fragments were inoculated in nnn – novy, macneal, and nicolle (modified blood agar) and lit (liver infusion tryptose) media after resistance for human treatment (brasil 2017). however, after 2016 the control of leishmaniasis in dogs has taken a new approach in brazil (brasil 2016) following the approval of a product based on miltefosine for the treatment of visceral canine leishmaniasis. this commercial product offered in the brazilian veterinary products market has no therapeutic recommendation inserted in its package for the treatment of cutaneous leishmaniasis caused by l.  braziliensis. a variable action of this drug on l.  braziliensis was observed by espada and colleagues (espada et al. 2017). in humans, the drug traditionally used is meglumine antimoniate, but its use is not allowed in animals. among several drugs used experimentally, furazolidone (fzd) has shown promising results as a leishmanicidal agent (tempone et al. 2010, passos et al. 2014). the pharmacological activity of fzd is due to its ability to rupture the parasitic nuclear and mitochondrial membrane, causing cell organelles to leak (hunder 1987). tempone and colleagues administered fzd in liposomal and free form (50 mg/kg/day) to animals infected with leishmania (leishmania) chagasi and observed that both forms of the drug were effective in reducing the number of parasites (tempone et  al. 2010). passos and colleagues treated eight dogs infected with l.  (v.)  braziliensis with fzd (35 mg/kg/day) for 21 days followed by domperidone (2 mg/kg/day) for 10 days and observed that the therapy was effective for regression of the lesions, which were epithelialisation without subsequent recurrence and without changes in biochemical and haematological patterns (passos et al. 2014). also new associations of fzd complexed to β-cyclodextrin (cd) for the treatment of leishmaniasis have been proposed to a lower dosage of fzd in relation to the dosage already used in the literature for the treatment of cl (carvalho et al. 2018, carvalho et al. 2020). it should also be noted that the use of fzd orally is recommended by the brazilian society of gastroenterology in human therapy, particularly for the treatment of helicobacter pylori infection (coelho et  al. 2013). thus, this report aims to describe the treatment of cutaneous leishmaniasis by l.  braziliensis in nine dogs using a reduced dose of furazolidone complexed to β-cyclodextrin. case report the microbiology and zoonoses laboratory – ufes (campus alegre, es, brazil) has an extension project with the municipality of íuna, state of espírito santo, brazil for diagnosis and control of dogs with leishmaniasis. all experimental protocols were reviewed by a state ethics commission and have been approved by the competent authority (ceua ufes 13/2018). specifically in this study, nine dogs from the southern region of the state of espírito santo 349veterinaria italiana 2022, 58 (3), 347-351. doi: 10.12834/vetit.2239.15416.2 santos zanini et al. treatment of canine cutaneous leishmaniasis with furazolidone/β-cyclodextrin biopsy of the tissue for recovery of the lesions of the pinna and scrotal epidermis were then performed after regeneration (biopsy of the region of the regenerated nostril was not performed), elisa, pcr and culture tests were repeated. all animals remained positive i-elisa whereas four ear biopsies (dogs 1, 3, 5 and 6) and one scrotal epidermis (dog 6) resulted positive to pcr amplifying fragments of 750  bp. when culturing in nnn-lit medium, four tissues showed secondary contamination by different microorganisms whereas the others did not show growth of promastigote forms and all cultures were negative to pcr. discussion the treatment for cutaneous leishmaniasis with fzd and domperidone in dogs (passos et  al. 2014) at a dosage of 35 mg/kg/day has been used and regression of lesions were consistently observed after 15  days of continuous use of fzd. however, especially when dogs are in good nutritional status and have weight over 10 kg (unpublished data), nervous manifestations such as motor incoordination and vomiting were frequently observed because fzd gradually accumulates in the fatty tissue causing an overdose after the second week of treatment. the application of this experimental therapy (35 mg/kg/day) requires constant monitoring by the veterinarian to manage possible intoxication. the fzd is limited in the clinic due to its potential side effects, such as hepatotoxicity and to the incidence of observed severe side effects when the dose was increased (deng et  al. 2015). thus, immunomodulation contributes to the treatment of leishmaniasis. this can be achieved by the use of domperidone, a gastric prokinetic with antiemetic activity. it is a dopamine d2 receptor antagonist and hyper pro-lactinaemic drug that causes the release of serotonin, which in turn stimulates prolactin production. prolactin appears to have a central role in the specific cellular immune response to leishmaniasis (sabaté et  al. 2014). the association of fzd + cd causes an increase in the solubility of fzd and consequently treatment with penicillin-streptomycin solution for the decontamination of prokaryotes. these cultures of tissue were incubated in a bod (biochemical oxygen demand) incubator at 24 ± 1 °c for 15 days (passos et  al. 2014), before examining for the presence promastigote forms of the parasite were researched. promastigote forms were confirmed as l.  braziliensis by pcr in four samples of auricular lesions (dogs 1, 2, 3, 5 and 9) while the other cultures were contaminated. all animals were subjected to a standardized health treatment, wormed with a commercial product composed of the association of praziquantel, pyrantel pamoate and febantel and started to be fed with commercial food with 21% crude protein. the nine dogs were also evaluated by complete blood test with comprehensive metabolic panel-cmp (aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase and hepatic bilirubin) and basic metabolic panel-bmp (urea, creatinine and albumin). the sizes of the ulcers of dogs caused by l.  braziliensis were monitored using a pachymeter (mitutoyo, taipei, taiwan), and expressed in mm2 according to table i. after laboratory confirmation of the infection with l.  (v.)  braziliensis, it was decided to medicate the animals with the fzd associated with cd in the proportion 1:2 manipulated according to literature (carvalho et  al. 2018, carvalho et  al. 2020). the complex of the fzd + cd (sigma aldrich, darmstadt, germany) manipulated had a concentration of 60 mg/ml. the animals which remained in their houses, were weighed and treated orally at a dosage of 15 mg/kg (5 mg/kg of active fzd) 12/12 hours x 50 days. every 15 days, the animals were evaluated for lesion size, metabolic panel-bmp) and metabolic panel-cpm (vidal et  al. 2009). after 20 days of treatment, the lesions started to regress (table  i) and, at 35  days, there was a re-epithelialization of the pinna and scrotal tissues. the lesions of the mucosa of the nostrils had re-epithelialization at 41 days. after these observations, the animals were monitored for fourteen months, with no observed reactivation of lesions. a complete blood test and table i. chronology of lesion regression (mm2) in dogs 1 to 9 treated with furazolidone and β-cyclodextrin. days of treatment dog 1 2 3 4 5 6 7 8 9 pan pan pan ee pan na pan ee pan na pan 00 576 289 630 900 328 130 414 382 271 372 402 10 505 292 598 910 340 110 410 386 265 180 367 20 100 112 182 332 92 77 224 102 192 66 162 30 00 16 00 87 00 48 38 00 88 41 27 40 00 00 00 00 00 02 00 00 00 00 00 pan = pinna; na = nostril; ee = scrotal epidermis. 350 veterinaria italiana 2022, 58 (3), 347-351. doi: 10.12834/vetit.2239.15416.2 treatment of canine cutaneous leishmaniasis with furazolidone/β-cyclodextrin santos zanini et al. lesion were not performed since several authors reported that the presence of parasite nucleic acids in regenerated cl lesions is a constant presence and the clinical cure of cl is rarely associated with sterile healing (schubach et  al. 1998, mendonça et  al. 2004, passos et  al. 2014). thus, further studies with quantitative pcr (qpcr) in cl lesions should be carried out to determine the threshold between asymptomatic and symptomatic patients, as well as the level of residual parasitic infection that can lead to a new outbreak of leishmaniasis and relapse (wu  et  al. 2020). culture and conventional pcr are extremely sensitive for qualitative diagnosis of cl (mesa et al. 2020) while the use of quantitative pcr (qpcr) to assess parasite load in tissue biopsy would be a reference of confusion. the use of miltefosine to treat cl has shown the need of different concentrations to inhibit the growth amastigote forms of the isolates of l.  braziliensis from different regions of brazil (espada et al. 2019). in addition, the use of miltefosine for the treatment of canine visceral leishmaniasis has been consolidated when associated with continuous use of allopurinol (daza gonzález et al. 2019) but the allopurinol is not effective in inhibiting l.  braziliensis (minodier and parola 2007). this study presents an applicable form for the treatment of canine cutaneous leishmaniasis caused by l. braziliensis. a reduction of the toxicity, particularly when using high doses of the complex. these results stimulated the production of a dense liquid formulation containing 60 mg/ml of the fzd + cd complex (1:2) where the active ingredient fzd has a concentration of 20 mg/ml. to facilitate the dog acceptance, a meat flavouring was added to the product that was administered with the aid of a disposable syringe. for humans, the literature mentions the use of fzd in the treatment of giardia and h. pylori (coelho et  al. 2013) using concentrations of fzd up to 400 mg/day for persons over 12 years old (+/-  50 kg) which indicates a dosage of 8 mg/kg/day. a dark yellowish colour was observed in the urine resulting from renal excretion of fzd but there was no change in the values of the complete blood test, metabolic panel-bmp and metabolic panel-cpm repeating the finding already observed (passos et al. 2014, vidal et  al. 2009) for l.  braziliensis. despite the clinical healing and absence of reactivation or development of lesions, the pcr was positive for six of the seven lesions evaluated after fourteen months, however similar findings were observed (schubach et al. 1998, wu et al. 2020) when treating cl with a pentavalent antimonial. it was observed that the inactivated parasites 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levels of gamma-glutamyl transferase in dogs naturally infected or not by leishmania (leishmania) chagasi. arq bras med vet zootec, 61, 749-751. wu y., tian x., song n., huang m., wu z., li s., waterfield n.r., zhan b., wang l. & yang g. 2020. application of quantitative pcr in the diagnosis and evaluating treatment efficacy of leishmaniasis. front cell infect microbiol, 10, 581639. zanini m.s., viana k.f., reis a.b., campos d.r., mussi j.m., zanini s. & lemos e.m. 2010. leishmania (viannia) braziliensis: immunoblotting analysis for the detection of igg subclasses in the diagnosis of symptomatic and asymptomatic dogs. vet parasitol, 173, 143-146. 23 introduction campylobacter jejuni and coli are the most important cause of bacterial gastroenteritis worldwide infecting humans mostly through consumption of contaminated poultry (dingle et  al. 2002, food et  al. 2014). campylobacter species colonise the gastrointestinal tract of domestic and wild animals and their prevalence in food producing animals, such as cattle, swine and poultry, can exceed the 80% (mughini gras et  al. 2012, efsa 2017). campylobacter has a broad host range and has been detected everywhere, from farm and urban environments to slaughter plants, in wild birds and mammals, companion animals and farm production animals (whiley et  al. 2013, alter et  al. 2005, pearce et  al. 2003). campylobacter species are highly adapted to asymptomatically colonise the intestinal tract of most avian species, reaching high numbers (up to 1010 cfu/g caeca content) in chickens and turkeys (newell et  al. 2008). once campylobacter is introduced into a flock, it spreads quickly. indeed, it can reach a within‑flock prevalence ranging from 60% to 100% (barrios et al. 2006). higher prevalence of infection has been observed in many countries in warmer months, suggesting a seasonal pattern in the colonization of poultry flocks (horroks et  al. 2009). the reason behind this seasonal effect is largely unknown, although a possible role of migratory birds or insects has been suggested (jacobs‑reitsma 1997). campylobacter infections in colonized flocks could be transmitted horizontally within the farm via a variety of routes and vehicles. the possible primary infection sources and transmission routes of campylobacter for poultry flocks have been investigated in numerous studies, but no definitive factors have been identified so far that explain the high levels of prevalence observed in commercial poultry flocks. risk factors associated with the introduction and dissemination of campylobacter within the flocks may include lack of biosecurity istituto zooprofilattico sperimentale dell'abruzzo e del molise 'g. caporale', campo boario, 64100 teramo, italy *corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise 'g. caporale', campo boario, 64100 teramo, italy. e‑mail: s.iannetti@izs.it keywords broiler carcasses, campylobacter, contamination levels, production chain, pfge. summary a research was carried out in italy with the aim of assessing campylobacter contamination in broilers from breeding to slaughter, of defining the genetic diversity of isolates and their antibiotic resistance. sampling was carried out in a slaughterhouse, and in farms representative of the most common broiler production in italy. at farm, the 78.8% (95% c.i.: 74.5%‑82.5%) of cloacal samples tested positive for campylobacter spp. c. jejuni showed higher prevalence in winter than in spring and summer (p < 0.00001, χ2 = 32.9), while c. coli showed an opposite trend (p < 0.00001, χ2= 41.1). at slaughterhouse, the 32.3% (95% c.i.: 30.2%‑35.2%) and the 23.9% (95% c.i.: 21.7%‑26.3%) of skin samples tested positive for c.  jejuni for c. coli, respectively. c. coli showed higher prevalence than c. jejuni at washing (p < 0.05, χ2 = 11.11) and at chilling (p < 0.05, χ2 = 9.26). pfge revealed high heterogeneity among isolates. some clones were identified within the same farm in more than one season, suggesting environmental conditions able to support their persistence; other clones resulted to be spatially distant, suggestive of cross‑contamination. both campylobacter species showed high resistance to nalidixic acid and ciprofloxacin, while resistance to erythromycin was more frequent in c. coli than c. jejuni (p < 0.05; χ2 test). simona iannetti*, paolo calistri, gabriella di serafino, francesca marotta, alessandra alessiani, salvatore antoci, diana neri, margherita perilli, giorgio iannitto, luigi iannetti, giacomo migliorati and elisabetta di giannatale campylobacter jejuni and campylobacter coli: prevalence, contamination levels, genetic diversity and antibiotic resistance in italy veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 accepted: 11.11.2019 | available on line: 24.04.2020 24 veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 campylobacter coli and jejuni in italy iannetti et al. carcasses contamination during slaughtering and campylobacter colonisation at primary production level (efsa 2010c). the aim of this research was to study the prevalence of c.  jejuni and c.  coli infection in chickens at farm and the contamination levels of carcasses in a typical italian chicken production and slaughtering chain. moreover, this study aimed at evaluating the antibiotic susceptibility profiles, the survival and genetic diversity of campylobacter isolated at farm and at different stages of the slaughtering process using pfge. materials and methods sample collection the sampling activities were carried out in one slaughterhouse (slaughter capacity about 110,000 chickens per day) and in three farms located in abruzzi region (central italy). the farms (a, b, c) selected belonged to the same company and were representative of the most common intensive broiler farms in italy, on the basis of the breeding management system, the animal housing system, feeding programs and sanitary protocols. in such farms, broilers are usually grown as mixed‑sex flocks in large sheds under intensive conditions; they are reared on ground on deep litter consisting in chopped straw and fed ad libitum with different feed formulas, depending on different stages of the animals’ growth. the temperatures (24‑33  °c), relative humidity (80‑100%) with natural daylight of 12 h and artificial lighting during 12 h of darkness, are differently settled in relation of animals’ age. sampling was carried out twice for each season (with the exception of august and march for logistic reasons) to take into consideration possible seasonal variations in the infection rates: • winter: from december to february; • spring: from april to may; • summer: from june to july; • autumn: from september to november. at farm, sampling activities were performed during four breeding cycles in farms a, b and c. in particular, samples were collected at different stages of the breeding cycle: • fifteen days before day‑old chicks restocking (water trough, feed trough, fan blades, ground and insects of the species alphitobius diaperinus, litter beetles commonly found in poultry houses); • during day‑old chicks restocking (shipping containers swabs, starter feed, manure); measures, contaminated water or feed, contacts with other infected animal species (wild birds, pets, mice, etc.) and mechanical transmission via insects (barrios et al. 2006, horroks et al. 2009). some authors have suggested that campylobacter can spread from the parent flocks to the progeny (cox et  al. 2002, petersen et  al. 2001b, sahin et  al. 2003, shanker et  al. 1986) although some observations indicate that vertical transmission plays a minor role in campylobacter flock colonization (petersen et  al. 2001a, callicott et  al. 2006). in the european union (eu), enteric infections caused by campylobacter are the most frequently reported zoonosis in humans; in 2016, 246,307 cases of campylobacteriosis have been reported in the eu, with an increase of 6.1% compared with 2015. (efsa/ecdc 2017). the majority of campylobacter infections in humans originate from the consumption and handling of raw or undercooked poultry meat products. in italy, a recent study aimed at investigating an outbreak of campylobacteriosis, showed that in the 70% of cases of c.  jejuni infection, the mlst profiles associated with human disease were most similar to those associated with chicken source (di giannatale et  al. 2016). this result is in line with many other european countries, even if the percentage of human campylobacteriosis due to the chicken source vary among the studies (wilson et al. 2008, mullner et al. 2009, sheppard et al. 2009). therefore, the control of campylobacter in poultry flocks and the reduction of poultry meat contamination are the cornerstones for any public health strategy aiming at reducing the incidence of campylobacteriosis in humans (efsa 2008). in the eu, during 2008 a harmonised and standardised baseline survey on the prevalence of campylobacter in broiler flocks and broiler carcasses was carried out, which assessed a prevalence of campylobacter contamination in broiler carcasses in italy equal to 49.6% (95% c.i. 39.5%‑59.7%) (efsa 2010b). the results of this survey showed that a campylobacter‑colonised broiler batch was about 30 times more likely to have the sampled carcass contaminated with campylobacter, compared to a non‑colonised batch, and that the risk of carcasses contamination increases from july to september (efsa 2010c). according to relevant studies, faecal contamination of carcasses during slaughtering represents the main source of campylobacter in fresh poultry meat (mahler et  al. 2011, guerin et  al. 2011). however, the main factors responsible for campylobacter presence on carcasses have not been identified yet. moreover, previous studies comparing c.  jejuni and c.  coli concentrations at farm and along the slaughtering chain are few (schets fm et  al. 2017). given the results of the baseline survey, the european food safety authority (efsa) recommended the eu member states to identify more clearly the risk factors of 25veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 iannetti et al. campylobacter coli and jejuni in italy italy). colonies were cultured on columbia agar for 48 hours in micro‑aerobic atmosphere, inoculated in mueller hinton broth supplemented with blood, and dispensed into eucamp microtiter plates (trek diagnostic systems, biomedical service, italy). the plates contained known scalar concentrations of the following antimicrobial substances: gentamicin (gm) (0.12‑16 µg/ml), streptomycin (s) (1‑16 µg/ml), ciprofloxacin (cip) (0.06‑4 µg/ml), tetracycline (te) (0.25‑16 µg/ml), erythromycin (ery) (0‑5‑32 µg/ml), nalidixic acid (na) (2‑64 µg/ml), and chloramphenic (cpl) (2‑32  µg/ml). the plates were then incubated at 42  °c in micro‑aerobic atmosphere for 48  hours. c.  jejuni strain nctc 11351 was included for the quality control of the minimal inhibitory concentration (mic) test. antimicrobial resistance was interpreted according to clsi breakpoints (clsi clinical and laboratory standards institute). pfge pulsed‑field gel electrophoresis (pfge) was performed according to the instructions of the 2013 u.s. pulse net protocol for campylobacter (pulse net international 2013). strains of c. jejuni and c. coli were sub cultured on columbia agar at 42 °c for 2 days in micro‑aerobic atmosphere and embedded in agarose blocks (seakem gold agarose, lonza, rockland, usa). the blocks were then lysed, washed and digested with smai enzyme (promega, italy), 25  u at 25  °c for 4 hours. salmonella serovar branderup h9812 was used as standard molecular weight size. pfge was performed using a chef mapper xa (biorad laboratories) with the following parameters: initial switch time of 6.75 s, final switch time of 35.38 s for 18 hours at 6 v and 14 °c in 0.5 x tbe buffer (sigma). after electrophoresis, the gel was stained with sybr safe dna gel stain (invitrogen, usa) and photographed at transilluminator (alpha innotech, usa). bionumerics v. 6.6 software (applied maths, belgium) was used for the analysis of pfge fingerprinting profiles. level of similarity were calculated with the dice correlation coefficient (position tolerance was set at 1%) and unweighted pair group mathematical average upgma clustering algorithm was used for cluster analysis of the pfge pattern. pfge‑clusters were defined at 100% similarity between macro restriction patterns (grotheus et  al. 1991). untypeable isolates were not included in the analysis. data collection and analysis sampling information was collected using specific sampling cards and recorded into microsoft® access database (ms‑access 2010) for further analyses. microsoft® excel (ms‑excel 2010) and xlstat©‑pro (version 7.5) were used for descriptive and statistical analysis of the data. • thirty days after restocking (water trough, feed trough, fan blades, growth feed and the water from the lake located inside the farm and used for chickens). one day before slaughtering, fifty broilers for each batch were identified before leaving the farm with numbered leg‑rings, and cloacal swabs were collected from each identified animal: the number of broilers identified was increased to take into account the possible lack of the leg‑ring during the slaughtering operations. the identification of the broilers allowed preserving the link between the animal sampled before and during the slaughtering process. at slaughterhouse, samples were collected from the neck skin of each identified carcass by excision after bleeding, defeathering, evisceration, washing, chilling. caeca samples were also collected from the same carcasses after the evisceration stage. culture conditions and pcr assays campylobacter strains were recovered from skin after the enrichment according to iso 10272‑1:2006 and to iso 10272‑2:2006 methods. caeca contents were directly plated on mccd agar and incubated at 42 °c for 48 h under micro‑aerobic conditions. for enumeration, 1 ml of caeca contents was added to 9 ml of peptone water ph 7.0 (1:10 g/ml), log‑dilution were performed (until 10‑9 dilution), and the plates were incubated at same conditions. the suspected colonies were then examined according to iso 10272 method and confirmed by multiplex pcr (wang et al. 2002). campylobacter isolates were cultured on columbia blood agar in microaerophilic conditions at 42  °c for 48 hours. species identification was performed using a multiplex pcr (wang et al. 2002). dna from campylobacter strains was extracted using the maxwell 16 tissue dna purification kit (promega corporation, madison, wi) according to the manufacturer's instructions. all isolates were stored at ‑ 80 °c. one thousand ml of water lake was filtered using membrane filter (pore size 0.45 micron, millipore). after filtration, membrane filter was placed in bolton enrichment broth for 48 h at 42  °c in microaerobic condition, later streaked on mccd agar plate for isolation, and then incubated again for 48 h at 42 °c in microaerobic condition. the suspected colonies were identified by multiplex pcr (wang et al. 2002). antimicrobial susceptibility the susceptibility of campylobacter isolates to seven antimicrobials was evaluated with a micro broth dilution method using the ‘sensititre’ automated system (trek diagnostic systems, biomedical service, 26 veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 campylobacter coli and jejuni in italy iannetti et al. multiple comparisons showed a significantly higher prevalence of contamination in winter than in spring and summer (p < 0.00001, χ2 = 32.9; multiple chi‑squared test) for c.  jejuni, while c.  coli showed a higher prevalence level in spring than in summer and autumn (p < 0.00001, χ2 = 41.1; multiple chi‑squared test). the environmental samples, the feed and insects tested all negative for campylobacter  spp. by the detection method. the water collected from the lake located inside the farm 30 days after placement of the day‑old chicks in the shed, and used for chickens, tested positive for campylobacter  spp. by filtration method and campylobacter coli was identified by multiplex pcr. at slaughterhouse, 230 broilers out of 400 (57.5%) were sampled, for 1,333 samples (table i). the rest of the broilers was not sampled due to the loss of the leg‑ring during transport. the 32.3% (95% c.i.: 30.2%‑35.2%) of skin samples tested positive for c. jejuni and the 23.9% (95% c.i.: 21.7%‑26.3) of samples tested positive for c.  coli. the 48.9% (95% c.i.: 42.9‑55.8) of caeca samples tested positive for c.  jejuni and the 28.9% (95% c.i.: 23.4‑35.1) tested positive for c.  coli. figure  2 compares the prevalence of c.  coli and c.  jejuni contamination in samples taken at slaughterhouse. c.  jejuni showed higher prevalence levels than c.  coli in caeca samples, at bleeding, defeathering and evisceration, while after the evisceration stage multiple chi‑squared test was used to verify if in samples taken at slaughterhouse there were significant differences in prevalence levels among sampling seasons and sampling stages. chi‑squared test was used to verify whether the prevalence levels observed for c.  jejuni were significantly different from prevalence levels observed for c. coli in samples taken at slaughterhouse. contamination levels were checked for a normal distribution with the kolmogorov‑smirnov test, and then non‑parametric tests have been used because of non‑normality of the data. kruskal‑wallis test was used to verify significant differences among contamination levels of sampling seasons and significant differences among contamination levels of sampling stages in samples taken at slaughterhouse. multiple comparisons among contamination levels of c. coli and of c. jejuni in skin and caeca samples were performed with dunn test. friedman test was used to verify if there were significant differences among contamination levels of skin samples taken from the same animal after bleeding, defeathering, evisceration, washing and chilling and multiple comparisons were performed with nemenyi test. fisher f‑test was also used to verify whether the contamination levels observed for c.  jejuni were significantly different from contamination levels observed for c.  coli in samples taken at slaughterhouse. results campylobacter prevalence and contamination levels at farm, out of 400 cloacal samples, 315 tested positive for campylobacter spp. (prevalence = 78.8%, 95% c.i.: 74.5%‑82.5%). c.  jejuni showed higher prevalence levels than c. coli during each sampling season. a lower prevalence level was observed in spring than in the other seasons. c.  coli, showed a different seasonal pattern with higher prevalence levels in spring than in the other seasons (figure 1). table i. number of samples taken at slaughterhouse. month of sampling season n. of animals n. of caeca n. of samples taken after the slaughtering stages total n. of samplesbleeding defeathering evisceration washing chilling dec-feb winter 46 46 46 46 46 46 45 275 apr-may spring 60 55 55 56 55 59 55 335 jun-july summer 68 68 68 63 63 63 62 387 sep-nov autumn 56 56 56 56 56 56 56 336 total 230 225 225 221 220 224 218 1,333 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% winter spring summer autumn p re va le n ce (% ) sampling season figure 1. c. coli (blue dots) and c. jejuni (orange dots): prevalence (± c.l. 95%) of contamination of samples taken at farm (cloacal swabs). 27veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 iannetti et al. campylobacter coli and jejuni in italy wallis test). contamination levels of c.  jejuni were significantly higher at bleeding [mean: 4.08 log (cfu)/g; sd: 1.05] than at the other slaughtering stages (p  <  0.0001; kruskal wallis test). details on c.  coli and c.  jejuni concentrations in samples of carcasses collected after the slaughter operations and on the results of the multiple comparisons performed are shown in table ii and table iii. as regards seasonality, concentration of c.  jejuni in skin samples was found significantly higher in summer [mean: 3.89 log (cfu)/g; sd: 1.20] than in other sampling seasons (p < 0.0001; kruskal wallis test), while no statistically significant difference was found for c. coli. in caeca samples no statistically significant difference was observed among contamination levels of seasons for c.  coli, while a significant lower contamination c.  coli showed significantly higher prevalence levels than of c.  jejuni at washing (p  <  0.05, χ2 = 11.11) and at chilling (p  <  0.05, χ2 =  9.26). figure 3 shows contamination levels of c.  jejuni and c.  coli in samples taken at slaughterhouse at different slaughtering stages. concentrations of c.  coli and c. jejuni on neck skin samples of carcasses collected after slaughter operations are shown in table  ii and table  iii, respectively. statistically significant differences were found in campylobacter concentration among the slaughtering stages (p  <  0.0001; kruskal wallis test). results of multiple comparisons performed with dunn test highlighted that contamination levels of c.  coli were significantly lower at defeathering [mean: 1.54 log (cfu)/g; sd: 0.91] and significantly higher at bleeding [mean: 4.17 log (cfu)/g; sd: 0.69] than at the other slaughtering stages (p < 0.0001; kruskal 0% 10% 20% 30% 40% 50% 60% caeca bleeding defeathering evisceration washing chilling figure 2. c. coli (blue dots) and c. jejuni (red dots): prevalence (± c.l. 95%) of contamination of samples taken at slaughterhouse. 0 1 2 3 4 5 6 7 8 caeca bleeding defeathering evisceration washing chilling lo g (u fc )/ g figure 3. c. coli (green dots) and c. jejuni (purple dots): levels of contamination (± c.l. 95%) of samples taken at slaughterhouse. table ii. the concentration of c. coli on samples of chicken carcasses collected after the slaughter operations. c. coli caeca bleeding* defeathering# evisceration washing chilling mean 6.81505115 4.170834346 1.540735312 2.919966832 2.574238328 2.664002739 median 7.173475592 4.170915028 0.995635195 3.146128036 2.579783597 2.676091259 sd 1.112598967 0.694096517 0.906040819 0.793316118 0.296979432 0.630023145 95° perc 7.830002887 5.313414732 3.151232096 4.097512676 3.009558145 3.491361694 5° perc 4.044136728 3.090273684 0.995635195 0.995635195 2.301029996 2 *contamination levels significantly higher (p < 0.0001); #contamination levels significantly lower (p < 0.0001). table iii. the concentration of c. jejuni on samples of chicken carcasses collected after the slaughter operations. c. jejuni caeca bleeding* defeathering# evisceration# washing# chilling# mean 5.374044013 4.078751274 1.831685688 2.787108632 2.545574554 2.76650184 median 5.627636253 3.954215699 0.995635195 2.903089987 2.544068044 3 sd 1.613333167 1.047516471 1.014431325 0.778875511 0.278445066 0.757715035 95° perc 7.661623156 5.946502352 3.538718726 3.678379134 3.004967555 3.614852317 5° perc 2.903089987 2.618264014 0.995635195 0.995635195 2.176091259 0.995635195 *contamination levels significantly higher (p < 0.0001); #contamination levels significantly lower (p < 0.0001). 28 veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 campylobacter coli and jejuni in italy iannetti et al. test). campylobacter concentration was significantly lower at defeathering than at evisceration, washing and chilling, and significantly lower also at washing than at chilling stages (p  <  0.0001; nemenyi test). figure 4 shows mean and median contamination levels found in skin samples of the same animal along the slaughtering chain. pfge pfge analysis with smai restriction enzyme of 367 c.  jejuni isolates in caeca and in the phases of the slaughter process resulted in a total of 103 pulsotypes (with 100% similarity). pfge types of c.  jejuni strains isolated at the various stages of the slaughter process vary from 1 to 6 pulsotypes per cycle. three pfge types (31, 35 and 75) were always present in different product of all batches and farms, while six pfge types (41, 53, 75, 82, 85 and 86) were present in all slaughtering stages (table iv). for each slaughter batch, up to four different c.  jejuni pulsotypes were found at slaughterhouse. cluster analysis of 273 c.  coli isolates showed 70  distinct pulsotypes (with 100% similarity) with none of the pfge types overlapping between the two farms. in particular, pfge types of c. coli strains isolated at the different stages of the slaughter line (p < 0.05; kruskal wallis test) was detected in spring for c.  jejuni [mean: 4.30 log (cfu)/g; sd: 0.77]. significant differences between contamination levels of c.  coli and c.  jejuni along the slaughtering stages were found only for caeca samples with higher level of c. jejuni (p < 0.05; fisher f‑test). as regards campylobacter concentration in the same animal during the slaughtering chain, campylobacter concentration was found significantly different among each sampling point (p  <  0.0001; friedman 2.745 1.214 2.322 1.916 2.330 3.079 0.996 2.653 2.301 2.602 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 bleeding defeathering evisceration washing chilling lo g (c fu )/ g mean median figure 4. contamination levels of skin samples taken at slaughterhouse from the same animal. table iv. pfge pulsotypes of c. jejuni strains isolated at different stages of the slaughter process and at farm and sampling season. major pulsotypes (100% similarity) isolates (no.) source farm sampling season 26 4 washing, evisceration b autumn 29 14 defeathering, evisceration, washing, chilling b autumn 30 6 evisceration, washing, chilling b autumn 31 15 defeathering, evisceration, washing, chilling b autumn washing c spring 35 14 chilling b autumn washing c winter slaughtering bleeding, evisceration, washing c spring 41 10 slaughtering bleeding, washing, chilling b summer 43 4 slaughtering bleeding, evisceration c spring 53 25 slaughtering bleeding, evisceration, chilling a spring 68 10 slaughtering bleeding, evisceration, washing, caeca a winter 72 2 slaughtering bleeding, evisceration b summer 75 24 defeathering a summer slaughtering bleeding, evisceration, washing a winter slaughtering bleeding, evisceration, washing, chilling b summer 77 2 chilling, defeathering a summer 82 16 slaughtering bleeding, evisceration, washing, chilling b summer 85 10 slaughtering bleeding, defeathering, evisceration, washing chilling c winter 86 32 slaughtering bleeding, defeathering, chilling c winter 87 10 defeathering, evisceration c winter 93 2 slaughtering bleeding, defeathering a summer 103 9 slaughtering bleeding, defeathering b autumn 29veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 iannetti et al. campylobacter coli and jejuni in italy for the reduction of campylobacteriosis in humans. to date, the mechanisms underlying campylobacter colonization of farmed broiler flocks and contamination of carcasses during slaughtering have not been fully clarified yet. investments in research are fundamental to improve the knowledge of the physiology, ecology, metabolism and colonisation mechanisms of c.  jejuni and c.  coli in poultry and their surviving capacity in the environment. in addition, although the role of c. jejuni contamination in broiler meat has been extensively studied in many european countries, the importance of c. coli in these food products has been not fully investigated yet. in particular, while the contribution of c.  jejuni to the burden of human illness, through the consumption of raw or undercooked broiler meat, is well known, the same cannot be stated for c. coli. currently, in the eu it is generally considered that, given food regulations (eu 2017) precluding the use of antimicrobial treatments on carcasses (such as hyper chlorination), the most effective intervention strategy is to prevent or reduce flock colonisation at the farm level. the results obtained at farm confirm the high level of prevalence already detected by other previous studies (allen et  al. 2007, di giannatale et  al. 2010, hadžiabdić et  al. 2013, henry et  al. 2011, hue et  al. 2010, rosenquist et  al. 2006, thakur et  al. 2013). regarding environmental samples, feed, water and pests, our results, however, are not in line with the findings of other studies (evans et  al. 2000, bull et  al. 2006) in which the isolation of campylobacter was frequently obtained from feed, water in the drinkers and litter samples. in our research, the environmental samples and those taken from the feed, water and pests resulted all negative for campylobacter detection, with the exception of one sample of water lake, which could suggest a possible introduction of contamination through the use of this water source for animal drinking. vary from one to three pulsotypes for each batch and only three pfge types (18, 44 and 70) were isolated at all slaughtering stages (table v). besides, for c. coli slaughter batches showed a multiple number of pfge types, up to three, with the exception of four carcasses from farm c having a single pulsotype identified in all slaughtering stages. antimicrobial resistance the results of mic and antimicrobial resistance revealed that 92.0% and 93.8% of the isolates from caeca were resistant to quinolones and fluoroquinolones (nal and cip), respectively. the 39.3% of the strains showed resistance to tetracycline, the 13.4% to erythromycin, and few strains resulted resistant to other antimicrobials such as chloramphenicol (1.8%) and streptomycin (0.9%). none of the isolates tested was resistant or sensitive to gentamicin. moreover, resistance to erythromycin was more frequent in c.  coli (27.8%) compared to c. jejuni (6.6%) isolates (p < 0.05; χ2 test), whereas these differences were not observed for the remaining antimicrobial substances. the highest level of resistance was observed to nal and cip, for campylobacter isolates from carcasses. in detail, the 90.0% and 90.6% of the strains were resistant to fluoroquinolones and quinolones, the 64.7% were resistant to tetracycline, and the 31.9% were resistant to erythromycin. the 99% of strains were susceptible to chloramphenicol, streptomycin gentamicin antimicrobials. also for the strains isolated from carcasses, resistance to erythromycin was more frequent in c.  coli isolates (44.0%) compared to c. jejuni (13.2%) (p < 0.05; χ2 test). discussion the prevention and control of campylobacter colonisation in broiler flocks is an important goal table v. pfge pulsotypes of c. coli strains isolated at different stages of the slaughter process and at farm and sampling season. major pulsotypes (100% similarity) isolates (no.) source farm sampling season 9 6 defeathering, washing, chilling a spring 10 2 chilling, evisceration a spring 15 3 defeathering, washing a autumn 17 7 evisceration, chilling a autumn 18 18 slaughtering bleeding, washing, chilling a autumn 24 18 defeathering, evisceration, washing, chilling a winter 33 4 evisceration, washing, chilling c spring 41 6 evisceration, washing a summer 43 3 evisceration, washing a summer 44 36 slaughtering bleeding, evisceration, washing, chilling a summer 70 42 slaughtering bleeding, defeathering, evisceration, washing, chilling a spring 30 veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 campylobacter coli and jejuni in italy iannetti et al. genomic relationships among bacterial isolates, with the ability to correlate isolated microorganisms from different sites and samples (frasao et  al. 2017). typing of campylobacter strains isolated from pigs, poultry, turkey, sheep, and lambs by pfge has been widely described (silva et  al. 2016, lahti et  al. 2017). in particular, silva and colleagues found that campylobacter clones belong to poultry flocks to indicate endemic strains with horizontal transmission among birds, and that the genetic profile associated with different farms suggested different sources of contamination (silva et al. 2016). pfge results showed a high genetic heterogeneity of campylobacter population: the same flocks were colonized by more genotypes. some clones recovered at the early stages of the production chain were not recovered at the later stages, while other clones were predominant in individual breeding cycles and were present in all production chain stages (pulsotype 85 for c. jejuni or pulsotype 70 for c.  coli), confirming the traceability of flock specific strains along the entire processing chain. every season would seem characterised by different genetic sub‑populations of strains of campylobacter. however, other clones were identified within the same farm in more than one season (pulsotype 35 or 75 for c.  jejuni), suggesting the persistence of these genotypes in the environment. these results are consistent with a study of peyrat and colleagues showing the existence of c.  jejuni and c.  coli clones particularly able to adapt and survive overnight on the surfaces of slaughterhouse equipment after cleaning and disinfection (peyrat et al. 2009). on the contrary, the presence of the same clones of c. jejuni and c. coli in different herds (pulsotypes 31 for c.  jejuni), spatially distant, might suggest a cross‑contamination linked to the operators or the means used, favouring the recirculation of these strains among farms. cross‑contamination of carcasses from poultry coming from different flocks but slaughtered at same slaughterhouse seems, therefore, to be unavoidable. the prevalence and contamination levels observed in carcasses confirm the deep influence of slaughtering operations to the final contamination levels. after a clear decrease of prevalence and contamination levels between bleeding and defeathering, a significant increase was observed after the evisceration stage. this trend may be related to the contamination occurred in case of intestine ruptures at the evisceration stage and supported by cross‑contamination due to the strict contact among the carcasses during the slaughtering chain. the reduction of prevalence and contamination levels in skin samples taken after defeathering may be due to the effect of heat treatment after scalding (around 55  °c) (bolder 2002), and to the abrasive many studies are available on the different behaviour of campylobacter species in farms and environment suggesting also a possible contribution of broiler farms to the aquatic environmental campylobacter load (schets et al. 2017). in our study, the comparison among c.  jejuni and c.  coli seems suggesting a different seasonal behaviour of c. jejuni with respect to c. coli in chickens, as shown in figure 1. moreover, it is noteworthy that whereas a clear seasonality of campylobacter spp. contamination, with a highest risk from july to september, was observed in many eu  member countries, this pattern was not recognised in italy (efsa 2010c). the increase of campylobacter spp. contamination in broiler flocks during summer was frequently associated with the possible role of flies in spreading the infection within and between farms, especially in northern countries (hald 2004). at southern latitudes, like in italy, the temperatures conditions could be favourable for the presence of flies and other insects all around the year, thus lacking a clear seasonality in campylobacter spp. contamination. a recent report comparing different types of samples from broiler flocks at farm, showed a greater level of genetic diversity in strains isolated from neck skin and caeca samples than in chicken meat (ugarte et  al. 2015). other studies investigating the genetic diversity of campylobacter concluded that campylobacter concentration increases from farm to slaughter, suggesting also that the full diversity of campylobacter genotypes found at slaughter could be also the result of cross‑contamination during the slaughtering process (colles et al. 2010). commercial broiler farms are an important ecological niche for a wide variety of campylobacter genotypes, thus confirming the complexity of the population structure of these organisms in broiler production and in the chicken food chain. therefore, it is very important to improve sampling strategies with the aim of investigating campylobacter structure population in broiler production (vidal et al. 2016). to our knowledge, this is the first extended study in which the sources of cross‑contamination in a poultry slaughterhouse were studied by pfge in italy: we used pfge to check the traceability of flock specific strains along the entire processing chain. with regard to species difficult to quantify, isolate, or distinguish, such as campylobacter spp., pfge is an important technique enabling research on the entire food supply chain as well as the tracing and estimation of the same strain responsible for contamination, from the raising of the animal to the foodborne illness in a human (frasao et  al. 2017). even if pfge is not the method of choice for molecular typing of campylobacter, this technique is widely used for obtaining a clear comparison of 31veterinaria italiana 2020, 56 (1), 23‑34. doi: 10.12834/vetit.1819.9596.2 iannetti et al. campylobacter coli and jejuni in italy nalidixic acid and tetracycline has been observed in this study. erythromycin resistance is significantly more frequent in campylobacter coli (27.8%) compared to campylobacter jejuni (6.6%) in caeca isolates (p < 0.05; χ2 test), and in campylobacter coli isolates from chicken carcasses (44.0%) compared to campylobacter jejuni (13.2%) (p < 0.05; χ2  test). according to the other studies, high levels of resistance to tetracycline and ciprofloxacin are frequently reported in both the species (ge et  al. 2013, rozynek et  al. 2008). c.  coli is usually more resistant to erythromycin than c.  jejuni, although resistance in c.  jejuni has been increasing (wang et al. 2013). our study demonstrates the significant diffusion of campylobacter infection in broilers in a typical breeding and slaughtering production context and the high genetic variability of the bacterium. to reduce cross contamination of campylobacter flocks with persistent clones during the slaughtering process, efficient hygiene measures are needed. the results of this study provide more information for the definition of proper control options at slaughterhouses and new insight about the possible different behaviour of c.  coli in comparison of c. jejuni. grant this project was funded by the italian ministry of health in the framework of the national research program and carried out by the istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ (izsam), which is the national reference laboratory for campylobacter. action caused by machines that remove microbial slide on the chicken skin. the favourable effect in reducing the level of contamination by the exposure to low temperatures and the dehydration of the carcasses surface during the transit through the chilling tunnel was not confirmed in our study unlike other researches (guerin et al. 2011). the comparison among c.  jejuni and c.  coli concentrations in skin samples taken at slaughterhouse during seasons suggested a different behaviour of these organisms: c.  jejuni seems to be significantly higher in summer, while c.  coli concentration was not significantly different among seasons. an opposite prevalence trend among c.  jejuni and c.  coli was found after evisceration stage, as shown (figure 2). c. coli showed significantly higher prevalence of contamination than of c.  jejuni at washing and at chilling. this finding could suggest a greater resistance of c.  coli than c.  jejuni at lower temperatures, although no statistically significant difference was found among contamination levels of c.  coli and of c.  jejuni at washing and at chilling (figure 3). in general, it is rather difficult to interpret these apparent differences, especially 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economically important transboundary animal disease (tad) of sheep and goats caused by the small ruminants morbillivirus (srmv), formerly known as ppr virus, a member of the genus morbillivirus of the family paramyxoviridae (http:// ictvonline.org/virustaxonomy.asp). it is an acute and highly contagious viral diseases of primarily sheep and goats and is currently emerging to cause infection in camels (balamurugan et al. 2014b) and clinically manifested by high fever, discharges from eyes and nasal orifices, oral necrotizing and erosive ulcers, stomatitis, gastroenteritis, diarrhea and bronchopneumonia (balamurugan et al. 2014b). this transboundary nature of the disease is one of the foremost constraints in enhancing the productivity of the small ruminants in enzootic countries in africa, the arabian peninsula, the middle east, and central and south‑east asia. ppr has a massive potential to cause huge economic losses and it significantly impacts the livestock sector economy and food security of the enzootic countries (govindaraj et al. 2016). after the eradication of rinderpest, a global agreement was stretched on the requirement to eradicate ppr, with the adoption of a ppr global control and eradication strategy (gces) to make the world free from ppr by 2030 (oie and fao 2015a) due to huge socio‑economic impacts of ppr. the food and agriculture organization and oie conjointly initiated an international strategic design for control and eradication with a goal of gathering all stakeholders behind the ppr‑ global eradication programme (ppr‑gep) and mobilizing the supplementary support required for the eradication (oie and fao 2015a) and launched ppr‑gep for the period 2017‑2021, into action with the adoption of a ppr gces. in india, sheep and goats play a pivotal role in the socio‑economic development of rural households and are generally referred for ‘any time money (atm)’ to rural farmers. these animals are constituting an important productive asset of settlers, landless, marginal, and small landholders and it generates a flow of income and employment indian council of agricultural research, national institute of veterinary epidemiology and disease informatics (icar-nivedi), yelahanka, bengaluru-560064, karnataka, india *corresponding author at: indian council of agricultural research, national institute of veterinary epidemiology and disease informatics (icar-nivedi), yelahanka, bengaluru-560064, karnataka, india. e-mail: balavirol@gmail.com. keywords peste des petits ruminants, sheep and goats, seroprevalence, cross‑sectional study, north eastern region, india. summary a seroprevalence study of the peste des petits ruminants (ppr) in small ruminants was carried out in the different states (assam, manipur, meghalaya, mizoram, nagaland, and tripura) in the north eastern region (ner) of india using serum samples collected from april 2017 to march 2018. a total number of 4,163 sera [sheep (n = 508) and goats (n = 3,655)] collected from 345 epidemiological units/villages covering 176 tehsils/blocks in ner were screened by competitive elisa kit for the detection of ppr virus antibodies. the results revealed that the seroprevalence of ppr in small ruminants in assam, manipur, meghalaya, mizoram, nagaland, and tripura was 34.3%, 10.3%, 4.7%, 15.7%, 14.7%, and 5.5%, respectively with an overall 14.5% prevalence.association between the presence of antibodies and goats has been showed to be significant (p < 0.01) at the ner level and within every single state. this manuscript highlights the need for continuous monitoring of this important disease as for the severe economic impact ppr may have in the affected countries. vinayagamurthy balamurugan*, bibitha varghese, d. muthuchelvan, s. sowjanya kumari, k. vinod kumar, r. dheeraj, g. govindaraj, k.p. suresh, d. hemadri, and parimal roy seroprevalence of peste des petits ruminants in small ruminants in the north eastern region of india veterinaria italiana 2020, 56 (1), 55‑60. doi: 10.12834/vetit.1985.10631.2 accepted: 17.01.2020 | available on line: 24.04.2020 56 veterinaria italiana 2020, 56 (1), 55‑60. doi: 10.12834/vetit.1985.10631.2 seroprevalence of ppr in sheep and goats in the ner of india balamurugan et al. political‑administrative section of the country and it comprises arunachal pradesh, assam, manipur, meghalaya, mizoram, nagaland, sikkim, and tripura states of india. this region was purposively selected, as the ppr has been reported from this region since 2010 (balamurugan et  al. 2014a, muthuchelvan et  al. 2014). further, some of the ner states did not implement the national ppr control program. however, arunachal pradesh and sikkim states were excluded in the study due to the ecological niche of hilly terrain and valley, geographically isolated nature of states, the topology of the region. the states under analysis in this study, in ner, have 7.44 and 0.56 million sheep and goat individuals, respectively, as per basic animal husbandry statistics (bahs), 2012 (http://www.dahd.nic.in/). a cross‑sectional prevalence study was conducted between april 2017 and march 2018 to ascertain the prevalence status of ppr virus antibodies in small ruminants population in the epidemiological units (epi‑units) of different states in ner. the initial hypothesis is that ppr antibodies are homogeneous or independent in the populations of the epi‑units in the study region. the rural societies live in villages consisting of clusters of households that follow similar animal husbandry and socio‑economic activities. hence, the village is considered as a distinct epi‑unit in this study as described earlier (balamurugan et al. 2009). accordingly, a list of villages in various blocks/ tehsils in different districts in the state and their sheep and goat populations in each of the state was prepared and in order to have a sizeable population, the villages (epi‑units) having more than 200 sheep and goats (with inclusion and exclusion criteria) were shortlisted for the sampling frame. the sample size for the present study was determined as per cochran, (cochran 1963) formula n  =  z2  [p  (1  ‑  p/e2] by using epitools, where n  =  sample size, z  =  95% confidence level, p  =  30% proportion [animal unit‑level prevalence of 30% was considered as per gces (oie and fao 2015b) as well as based on the prevalence of ppr before the implementation of vaccination in india (singh et  al. 2004a)], e is the precision level (5%). based on these inputs a total sample size of 323, were determined (http://epitools.ausvet.com.au/ content.php?page=1proportion). however, after considering the attrition rate of 10%, the total sample size was 356. the multistage stratified random sampling procedure was adopted for collecting serum samples. in the first stage, the states were stratified and in each stratum (state), 60  sampling primary epi‑units (villages) were equally allocated randomly using r software (r_core_team 2014). in the next stage in each of the selected villages, the number of secondary units (animals) were throughout the year for their livelihood. the disease is enzootic and a number of outbreaks are occurring regularly, in different parts of india throughout the year (balamurugan et  al. 2014b). ppr is a major constraint in small ruminant production causing huge economic losses in terms of morbidity, mortality, productivity losses with trade restriction (balamurugan et  al. 2014b, singh et  al. 2004a). further, the molecular characterization of ppr virus (pprv) and its phylogenetic analyses revealed that the asian lienage iv as the sole circulating in india (shaila et  al. 1996, dhar et  al. 2002, balamurugan et al. 2010, muthuchelvan et al. 2014). for the control of disease, india adopted focused vaccination campaigns (within a radius of 3‑10  km to contain the disease in outbreak situation) in some of the states since 2002 (singh et  al. 2009) and in strategic vaccination mode through implementation of a national control program on ppr (ppr‑cp) in some of the states even before the global framework was planned (balamurugan et  al. 2016, govindaraj et  al. 2019). nevertheless, neither a surveillance plan nor a systematic post vaccination monitoring or evaluation was initiated to assess the effectiveness of the vaccination program. in india, several outbreaks go unrecorded due to under‑reporting or non‑reporting (balamurugan et al. 2011) and further, in spite of vaccination, disease outbreaks are being reported regularly in some of the states or sporadically in regularly vaccinated or geographically restricted regions of india. even though ppr is enzootic in india, some states in the north eastern region (ner) are either free from disease or have a few sporadic outbreaks reports. ppr is of increasing importance and likely to extend its geographic distribution especially in ne states, as ppr outbreaks have been reported in assam (balamurugan et  al. 2014a, chaudhary et  al. 2013, majumder 1997) and in tripura since 2010 (muthuchelvan et al. 2014). nevertheless, systematic epidemiological surveys have not been conducted except for a few isolated studies (balamurugan et  al. 2019). earlier studies on the seroprevalence, generating the baseline data on ppr seroprevalence in goats of ner (balamurugan et  al. 2014a, begum et al. 2016, de et al. 2016, devi et al. 2016, karam et al. 2018), have generally indicated that transboundary migration of animals is related to ppr outbreaks in naïve areas. moreover, studying the prevalence of antibodies would help in the implementation of proper disease control strategies such as vaccination (balamurugan et  al. 2014c). therefore, a cross‑sectional seroprevalence survey is being employed in this study to determine the prevalence of pprv antibodies in ner of india. north eastern region (ner) is the easternmost region of india representing both a geographic and 57veterinaria italiana 2020, 56 (1), 55‑60. doi: 10.12834/vetit.1985.10631.2 balamurugan et al. seroprevalence of ppr in sheep and goats in the ner of india the seroprevalence was estimated by the number of positive animals versus numbers of tested animals as per the method described by thrushfield (2005). the chi‑squared test (χ2) was carried out in ms‑office excel 2016 as per the method described by snedecor and cochran (snedecor and cochran 1967) to understand the association of pprv antibodies in sheep and goats across states and districts within every single state in the ner of india. this cross‑sectional prevalence study assessed the status of the pprv antibodies in sheep and goats in ner from april 2017 to march 2018. the observed percentage seroprevalence of ppr in small ruminants in the studied states of the ner of india were, 34.3%, 10.3%, 4.7%, 15.7%, 14.7%, and 5.5%, in assam, manipur, meghalaya, mizoram, nagaland, and tripura, respectively with the overall prevalence of 14.5%. further, the results of the chi‑square analysis revealed that the association (not independent) of ppr virus antibodies in goats (χ2  =  137.3, p < 0.01) and in small ruminants (χ2  =  330.6, p < 0.01) across the different states in the studied ner of india. it is important to point out that most of the states do not adopt vaccination strategies. state‑wise seroprevalence of pprv calculated by the hypergeometric distribution as per gces guidelines by considering an animal unit‑level prevalence of 30% (oie fao 2015b) in small ruminants. the maximum level of 11 samples to be collected was determined based on the sheep and goat populations in each epi‑units. thus, a maximum of 1,320 secondary animal units [660 for each target (sheep or goats) species] was established using epi‑calculator https://www. nivedi.res.in/nadres_v2/epical/stratified/random_ sampling.php.) for this study. in each epi‑unit/village, sera were collected randomly through all india coordinated research project on animal disease monitoring and surveillance (aicrp on admas), a collaborating center of icar‑nivedi, in the respective states. in the epi‑unit, where only either sheep or goats where present, a maximum of 11  samples of either species was collected. the sample epi‑units under examination are in gis map (figure 1) using qgis software 2.18.6 version. the collected sera were labeled and transported in an ice‑cool shipment container to the laboratory. samples were stored at ‑ 20 °c until further use. all the sera were tested by using competitive elisa kit (singh et al. 2004b). figure 1. the epi-units (villages) location are depicted (as • a dot) in the gis map of the studied states in the north-eastern region (ner) of india. north eastern region of india ner assam meghalaya tripura nagaland manipur mizoram 58 veterinaria italiana 2020, 56 (1), 55‑60. doi: 10.12834/vetit.1985.10631.2 seroprevalence of ppr in sheep and goats in the ner of india balamurugan et al. table i. serum samples screened for each state and prevalence of ppr antibodies in small ruminants in the ner of india. name of the states no. of the tehsil/ block in each state no. of the village / epi-unit in each state no. of the serum samples screened no. of the samples positive in elisa prevalence of ppr virus antibodies (ci value at 95%) seroprevalence % status in epi-units (no.) total sheep goats total sheep goats total sheep goats < 30 30-70 > 70 assam 43 48 724 260 464 248 138 110 34.25 (31-38) 53.08 (47-59) 23.71 (20-28) 28 12 8 manipur 24 59 758 132 626 78 13 65 10.29 (8-13) 9.85 (6-16) 10.38 (8-13) 55 3 1 meghalaya 25 60 655 655 31 31 4.73 (3-7) 4.73 (3-7) 56 3 1 mizoram 18 60 682 40 642 107 0 107 15.69 (13-19) 0 (0-9) 16.67 (14-20) 47 12 1 nagaland 41 59 723 76 647 106 7 99 14.66 (12-18) 9.21 (5-18) 15.3 (13-18) 51 7 1 tripura 25 59 621 621 34 34 5.48 (6-10) 5.48 (6-10) 55 4 0 total 176 345 4163 508 3655 604 158 446 14.51 (13-16) 31.10 (27-35) 12.2 (11-13) 292 41 12 chi square value: goats (χ2 = 137.3, p < 0.01); states (χ2 = 330.6, p < 0.01) of the epi‑units had < 30% seroprevalence, which necessitates of a comprehensive intensive active surveillance or vaccination programs. this information would be very useful in the formulation of effective disease management strategies as well as in the implementation of a national ppr vaccination program in the ner states of india. more systematic, intensive and comprehensive serological active surveillance program in small ruminants along with measurement of clinical prevalence or monitoring of the occurrence of sporadic outbreaks in different clinical forms of ppr in the enzootic areas must be undertaken in order to develop effective control measures/strategies for ppr to make disease‑free ner of india. acknowledgments authors wish to thank the indian council of agricultural research (icar), new delhi, india for financial support of the institute project (ixx14475), and the icar‑nivedi for the constant support and encouragement. the authors also thank all the aicrp on admas of ner states, collaborating center of icar‑nivedi, for sending the serum samples for regular surveys to monitor the status of livestock diseases in the country as well as for providing information about the status of ppr‑cp implementation for this study. the authors are thankful to all the ner states animal husbandry and veterinary services departments and their officials, field veterinarians, para‑veterinarians for their assistance during samples /data collection in the survey and for their kind help and co‑operation. antibodies is presented in table i. results of single states are available upon request. the results here provided indicate that the disease incidence is sporadic in the states of ner and in some places the animals are naïve for pprv infection (balamurugan et al. 2014a, de et al. 2016, singh et al. 2004a). generally, variation in seroprevalence could be due to differences in sample size, prevailing management practices, humidity or season as reported earlier (singh et  al. 2004a). however, in the present study, as per the sampling plan, a cross‑sectional study was conducted to estimate prevalence with a specified level of confidence and desired precision with the maximum statistical sample size for the finite or large population representing the target population from different epidemiological units of the studied region. sporadic nature of the disease in the ne region, especially in assam and tripura states, will cause an increase in the number of outbreaks, as there is no vaccination of small ruminants is undertaken at present in the ner, which in turn favors the spread of disease to other parts. furthermore, most of the selected epi‑units of ner had neither seroprevalence of > 30% (n  =  292) nor protective population immunity of > 70% (n  =  333), which implies that goat population is free from pprv antibodies, as there were neither ppr outbreaks reported nor vaccination strategies practiced in ner except focused vaccination in selected outbreaks places. 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vet ital, 45, 449‑462. singh r.p., saravanan p., sreenivasa b.p., singh r.k. & 404 not found 135 veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 accepted: 05.10.2020 | available on line: 31.12.2021 icar-indian veterinary research institute, hebbal, bengaluru-560024, karnataka, india. *corresponding author at: icar-indian veterinary research institute, hebbal, bengaluru-560024, karnataka, india. e-mail: venkat1953@gmail.com. r.p. tamil selvan, b.p. sreenivasa, m. hosamani, suresh h. basagoudanavar, saravanan p. and r. venkataramanan* keywords foot and mouth disease virus, immune response, linear regression, liquid phase blocking elisa, virus neutralization test, alternative to challenge. summary virus neutralization test (vnt ) and liquid phase blocking elisa (lpbe) are accepted tests for screening and as in vitro alternative to challenge in fmd vaccine potency testing. to replace vnt by lpbe for the screening of cattle, the optimized tests need to be first evaluated for their diagnostic performances. to replace it with lpbe in the absence of protection data, the interrelationship between vnt and lpbe have to be established to find out lpbe cut-off titer corresponding to the currently used vnt titers. accordingly, vnt and lpbe were carried out using known negative (n = 306) and positive samples [serotype o (n = 43), a (n = 14) and asia1 (n = 11)], for the initial screening. the cut-off of < 1.5 log 10 lpbe was comparable with that of < 1.2 log 10 vnt titer for screening. lpbe was comparable to vnt in terms of specificity, sensitivity as shown by roc curve and least varying (coefficient of variation 7.73% in lpbe vs 24.19% in vnt ). based on linear regression model using 471 bovine sera, the predicted lpbe titers corresponding to the currently used log 10 vnt titers of 1.65, 1.5 and 1.5, were 2.24, 1.87 and 2.00 for o, a and asia1, respectively. these lpbe titers hence can be used as cut-off titers for classifying cattle as protected or not protected until correlation based on in vivo challenge between protection and antibody titer is established. performance characteristics of virus neutralization test (vnt) and liquid phase blocking elisa (lpbe) and their relationship in the cattle immunized with trivalent foot and mouth disease vaccine vaccination using inactivated oil-adjuvanted vaccine containing all three representative strains of fmdv, typeo, a and asia1 is indeed the mainstay tool for reducing the spread of the disease. in india, vaccination is included in the national fmd control programme (fmd-cp) (dadf annual report 17-18 2018). with the increase in coverage of fmd-cp, the quantity of vaccine required has significantly increased as well as the requirements for quality control of the vaccine. among the various quality parameters, the potency of fmd vaccine assumes major importance as it has a direct influence on the herd immunity. as per indian pharmacopeia, 2018 (ipc 2018), and world organization for animal health (oie 2018), potency testing of fmd vaccine requires vaccination of fmdv seronegative cattle. hence, it is essential to have a validated serological test to detect fmd free animals for vaccine testing. the basis for the serological tests is that the fmdv exposure or vaccination induces detectable levels introduction foot and mouth disease (fmd) is one of the highly contagious virus infections of cloven-hoofed animals (grubman and baxt 2004). it adversely affects the productivity and profitability of food animal industry and the endemic countries are banned for trading animals or its products. presently, the disease is globally widespread and highly prevalent in asia and africa (king and henstock 2015). the causative agent of the disease, fmd virus (fmdv), belongs to the genus aphthovirus of the picornaviridae family. it has seven genetically and immunologically distinguishable serotypes named as o, a, asia1, c and sat1, 2, and 3. in india, serotype o, a, and asia1 are prevalent and the current fmd vaccine targets these serotypes (pdfmd annual report, 11-16 2016). even if fmd in an endemic region could be limited through controlling the movement of animals and animal products from outbreak areas, mass 136 veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 vnt and lpbe in assessing the immune response to fmd vaccine tamil selvan et al. (gmem) containing 10% each of tryptose phosphate broth and fetal calf serum were used to propagate the vaccine strains of fmdv (o/ind/r2/1975, a/ ind/40/2000, and asia1/ind/63/1972). the virus was harvested about 18 h post-infection when the cytopathic effect was maximum. the virus was clarified by centrifugation at 200 g for 10 min and stored at - 80°c use. for lpbe, inactivation of the virus was done by exposing it to 3 mm binary ethyleneimine for 24 h at 37 °c for two successive cycles. the inactive virus was aliquoted and stored at 80°c. working control serum high virus neutralizing (vn) titer, medium vn titer and fmdv negative sera having mean antibody titer (log 10 vnt titer) of 2.30, 1.88 and 0.9 for o, 2.60, 1.96 and 0.9 for a, 2.60, 2.19 and 0.9 for asia1, respectively, were simultaneously tested with test samples to monitor the repeatability and authenticate the validity of results of the vnt and lpbe. the high and medium sn titer sera were collected from cattle vaccinated with inactivated trivalent fmd vaccine on day 28 post-vaccination (dpv). fmdv negative sera were collected from an apparently normal hallikar breed of the bull calf of < 6 months with no history of either vaccination or infection. the fmd free status was confirmed by a negative result in 3abc elisa (hosamani et al. 2015). reference negative serum sera from normal male cattle (n = 306) of hallikar breed aged 6-9 months with no history of infection or vaccination and tested negative in the 3abc indirect elisa served as fmd negative samples to calculate diagnostic specificity. reference positive serum monovalent sera for the fmdv serotype o (n = 43), a (n = 14) and asia1 (n = 11) were collected from cattle immunized with inactivated oil adjuvant vaccine containing fmdv o/ind/r2/1975, a/ind/40/2000 and asia1/ind/63/1972 strains, respectively. the sera were collected between 14-130 dpv to serve as reference reagents for the optimization and monitoring of the diagnostic performance of the vnt and lpbe. trivalent vaccinate serum sera from the cattle (n = 471) vaccinated with inactivated oil adjuvanted/vectored fmdv vaccine expressing capsid protein of o, a and asia1 vaccine strains were used as test samples to determine the functional equivalence between vnt and lpbe titers. of antibody response. with regard to screening of cattle, a positive/negative cut-off of 1.2 log 10 in virus neutralization test (vnt) is recommended (oie 2018) for certification of individual cattle. since the results of serological tests vary between laboratories and strains of the virus, it needs to be established at each laboratory with a set of known positive/ negative samples (oie 2018). to date, the diagnostic performance of vnt has not been evaluated using indian vaccine strains of fmdv. in trivalent vaccine potency testing, animals are challenged with one serotype and clinical protection of 75% is set as a cut-off. for the other two serotypes, predicted vnt titers are used to assess the protection status. in india, the mandated vnt antibody titers for o, a and asia1 are 1.68, 1.50 and 1.50 log 10 , respectively for use in fmd-cp. despite the fact that the vnt serves a gold standard for assessing the protection, it has many limitations including live virus handling, time-consuming, less robust, demands cell culture facility and expertise. to overcome the limitations of vnt, various forms of elisa (hamblin et  al. 1986, mccullough et  al. 1992) are evaluated. of these, liquid phase blocking elisa (lpbe) (hamblin et  al. 1986) has been successfully employed and is considered as a reference methodology (oie 2018). the test uses a constant amount of antigen that binds to fmdv antibodies in serially diluted sera and the leftover antigen is trapped in a sandwich elisa. for screening of animals, an lpbe cut-off titer of 1.60 log 10 is recommended by oie. however, as with vnt, the lpbe cut-off titer for screening needs to be established for individual laboratories for each strain of fmd virus. at present, the lpbe cut-off titer for either screening or potency testing is not available for indian vaccine strains. determining the protective titer of lpbe demands a large number of challenge experiments. as an alternative, if a functional relationship between vnt and lpbe titers can be established, the lpbe protective titers can be deduced for the currently used vnt cut-off titers. in the present report, we first evaluated the diagnostic characteristics of vnt and lpbe for using it as a screening test. second, the quantitative relationship between the titers of vnt and lpbe was determined by linear regression to find the possibility of replacing vnt with lpbe for prediction of protection in fmd vaccine potency testing. materials and methods cells and viruses baby hamster kidney-21 (bhk-21) cells clone #13 maintained in glasgow modified eagle medium 137veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 tamil selvan et al. vnt and lpbe in assessing the immune response to fmd vaccine per 20 ml of 0.1 m citrate-phosphate buffer ph 5.0) was added to each well. after 15 min of incubation at 37 °c, the reaction was stopped by adding 1 m sulphuric acid. the optical density (od) of each well was read at 492 nm using a spectrophotometer (tecan infinite-f50 absorbance microplate reader, männedorf, switzerland), and the percent age of inhibition (pi) was calculated. the endpoint was defined as log 10 of that dilution at which half of the wells showed 50% inhibition compared to the 0% inhibition observed in the antigen control. statistical analysis receiver operating characteristic (roc) curve was constructed using the reference sera (positive and negative) in proc package (robin et  al. 2011) and ggplot2 package (wickham 2009) of r  programming language (version 3.1.1-“sock it to me”) (r development core team 2014). the cut-off titer for vnt and lpbe was determined based on the youden index (fluss et  al. 2005) to find out the diagnostic sensitivity and specificity of vnt and lpbe. the area under the roc curve was analyzed by the z test. significance was set at 95%. a simple linear regression model was fit by regressing the titer of lpbe (y) on vnt (x) of test sera. results performance characteristics and repeatability of vnt and lpbe the performance characteristics of vnt and lpbe for the three serotypes of fmdv are presented in the form of roc curve (figure 1a to c). no significant differences were found in the area under the curves between vnt and lpbe for any of the serotypes [serotype o (p = 0.937), a (p  =  0.37) and asia1 (p = 0.733)]. youden index based cut-off estimation revealed that < 1.2 and < 1.5 log 10 titer in vnt and lpbe, respectively, was suitable across all 3  tested serotypes with high sensitivity and specificity (table i). the vnt and lpbe using monovalent vaccinate serum against homologous and two other heterologous vaccine virus serotypes indicated that they were serotype specific and titers against heterologous serotypes were at least 2 fold less than that of the homologous serotype (table ii). the cross-reactivity was least for vnt and minimum for lpbe. the vnt titer results were valid as virus titration and back titration results of fmdv vaccine strains were within 0.5 log 10 differences on either side of 2.0 log 10 tcid 50 . the repeatability of vnt and lpbe as assessed with high titer, medium titer, and negative working control sera indicated that the inter-day variation did not exceed 2 standard virus neutralization test (vnt) the virus neutralization test was performed as described (oie 2018) using bhk-21 cells. briefly, 100 tcid 50 /50 μl of fmdv o, a and asia 1, were incubated with 50 μl of 2 fold serially diluted heat inactivated test sera (0.9 to 3.1 log 10 ) in 96 well tissue culture plates (n = 2 wells/dilution) for 1 h at 37  °c. then, 50  μl of bhk-21 cells (106 cells/ml) in gmem containing 10% fetal calf serum was added to each well. after incubation for 48 h at 37 °c in 5% co 2 incubator, wells were examined for cytopathic effect. antibody titers were expressed as log 10 of the reciprocal of highest serum dilution required for neutralization of 100 tcid 50 of the virus in 50% of the wells. when a sample had a vnt value that was beyond the lowest (< 0.9 log 10 ) and highest dilutions (> 3.1 log 10 ), respective limits of detection (0.9 and 3.1 log 10 ) were considered as the vnt value. liquid phase blocking elisa (lpbe) liquid phase blocking elisa is modified form of sandwich elisa in which the residual antigen, after blocking by antibody in the liquid phase, is captured and detected by serotype-specific anti-140s fmdv polyclonal serum (hamblin et  al. 1986, oie 2018). briefly, separate 96-well elisa plates (nunc-maxisorp, denmark) were coated with serotype-specific (o, a, and asia1) polyclonal anti-140s fmdv rabbit serum (50 μl/well) at a predetermined dilution in carbonate/bicarbonate buffer (ph 9.6) and incubated overnight at 4  °c. in perspex plates, test sera were serially diluted starting from 1/4 to 1/512. at each test, three working control sera as described in ‘working control serum’ section, were included to monitor the performance of the assay. from the perspex plate, serum was transferred to separate fmdv-o, a and asia1 non-binding deep well plates and equal quantity of respective antigen were added (which lead to final serum dilution as 1/8 to 1/1024), and incubated at 4 °c overnight. next day morning, the coated elisa plates were washed three times with washing buffer containing tween-20 0.05% in phosphate-buffered saline and incubated with the antigen-antibody mixture from deep well plates (50 μl /well in duplicates for each serum dilution). the plates were then incubated for 1 h at 37 °c. the plates were washed as before and detector antibody (anti-140s fmdv guinea-pig antibody) in predetermined dilution in blocking buffer was added to the type-specific plates. after 1  h incubation at 37 °c, plates were washed again and 50 μl/well of rabbit anti-guinea pig immunoglobulin-hrpo conjugate (dako, catalogue # p0141, denmark) were added to the wells. the plates were washed after 1 h of incubation and substrate solution (10 mg of orthophenylene diamine and 8 μl of 0.05% h 2 o 2 138 veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 vnt and lpbe in assessing the immune response to fmd vaccine tamil selvan et al. deviations (sd) from the mean titre on 95% of the tested occasions. this extent of less variation around mean titer indicates that the results are reliably reproducible (table iii). relationship between vnt and lpbe titers comparison of lpbe titers with that of vnt indicated that the lpbe titers were higher with a significant (p < 0.0001) positive correlation (spearman’s rho ρ = 0.71, 0.76 and 0.76, for fmdv o, a, and asia1, respectively). a simple linear regression model was fitted using vnt titers as the independent variable and lpbe titers as a dependent variable to predict lpbe titers from vnt titers. model coefficients indicate that the vnt titers are highly significant (p  <  0.001) for serotype o, a and asia1 (figure 2a, b, and c) as compared to intercept only model. the lpbe titers corresponding to the currently used cut-off vnt titers of 1.65 and 1.51 was predicted to be 2.24, 1.87 and 2.00 and for o and a and asia1, respectively (table iv) from the parameter estimates. though the confidence interval was narrow, the predictive interval (figure 2a, b, and c) was wider with a coefficient of determination ranging from a. roc curve serotype o se n si ti vi ty (% ) 100 80 60 40 20 0 100 80 60 40 20 snt lpbe 0 speci�city (%) p-value = 0.937 b. roc curve serotype a se n si ti vi ty (% ) 100 80 60 40 20 0 100 80 60 40 20 snt lpbe 0 speci�city (%) p-value = 0.37 c. roc curve serotype asia1 se n si ti vi ty (% ) 100 80 60 40 20 0 100 80 60 40 20 snt lpbe 0 speci�city (%) p-value = 0.733 figure 1. receiver operating characteristic (roc) curves for virus neutralization test and liquid phase blocking elisa for detecting foot and mouth disease virus antibodies. a. serotype o b. serotype a and c. serotype asia1. x axis indicates the false positive rate while y axis indicates the true positive rate. the arrow indicates the cut-off titers. table i. diagnostic sensitivity, specificity, and accuracy of virus neutralization test (vnt) and liquid phase blocking elisa (lpbe) for the three serotypes of foot and mouth disease virus. test cut-off titer (log 10 ) diagnostic sensitivity (%) diagnostic specificity (%) accuracy (%) o a asia1 o a asia1 o a asia1 vnt < 1.2 95.4 92.9 90.9 97.7 100.0 99.4 97.4 99.7 99.1 lpbe < 1.5 93.3 92.9 90.9 99.0 100.0 99.0 98.9 99.7 98.7 table ii. serotype specificity of virus neutralization test (vnt) and liquid phase blocking elisa (lpbe) using monovalent post‑vaccinate reference sera raised against indian foot and mouth disease vaccine strains. monovalent serum animal id test fmd virus o/ind/ r2/1975 a/ind/ 40/2000 asia1/ind/ 63/1972 o/ind/ r2/1975 bc 889 21 dpv vnt 1.50 < 0.9 < 0.9 lpbe 1.65 < 0.9 1.34 bc 927 21 dpv vnt 1.95 < 0.9 < 0.9 lpbe 2.26 1.20 1.34 a/ind/ 40/2000 bc 830 28 dpv vnt < 0.9 1.95 < 0.9 lpbe 1.65 1.95 1.04 asia1/ ind/63/1972 bc 809 21 dpv vnt < 0.9 < 0.9 1.50 lpbe < 0.9 < 0.9 1.95 bc 914 21 dpv vnt 1.04 < 0.9 1.65 lpbe < 0.9 < 0.9 1.65 figures in bold indicate the serotype specific reactivity. 139veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 tamil selvan et al. vnt and lpbe in assessing the immune response to fmd vaccine 0.44 to 0.57 indicating moderate precision of the regression model. discussion to assess the immune response to fmd, a robust reproducible test with high accuracy is essential. hence, before regressing lpbe titers on vnt titers, we investigated the diagnostic performance characteristics of the test. the cut-off of <  1.5  log 10 titer in lpbe was comparable with that of < 1.2 log 10 titer of vnt for detecting animals with fmdv antibodies (figure 1a to c). the accuracy (%) of lpbe was 98.9, 99.7 and 98.7 for o, a and asia1, respectively (table i). similar cut-off, sensitivity, and specificity values were obtained for diagnostic lpbe, when the test was standardized for estimating the herd immunity in large scale surveillance programme in south america and europe (periolo et  al. 1993, smitsaart et  al. 1998, van maanen and terpstra 1989). the optimized lpbe was more sensitive in detecting fmd positive animals as the test also measures non-neutralizing antibodies. repeatability of lpbe was superior to that of vnt as indicated by the coefficient of variation (max. 7.73% in lpbe vs 24.19% in vnt; table iii), which is below the acceptable limit of 10% (jacobson 1998). this confirms the previous findings that lpbe is reproducible (hamblin et  al. 1986, maradei et  al. 2008) and can be used for screening. a significant moderate positive correlation [spearman correlation coefficient ρ  =  0.71, 0.76 and 0.76, (p < 0.0001)] was found between lpbe and vnt titers for fmdv serotypes o, a and asia1,  respectively. the results agree with the findings on lower correlation coefficient (0.75, 0.78, 0.67 and 0.81) between lpbe and vnt titers, when serum from cattle vaccinated with oil-adjuvanted polyvalent vaccine containing o1/caseros, a/ arg/79, a/arg/87, c3/arg/85 (smitsaart et  al. 1998) viruses was used. in contrast, higher pearson’s correlation coefficients were reported by two different laboratories for monovalent o1/bfs, a10/ holland (0.91) (van maanen and terpstra 1989) and a. vnt o vs lpbe o lo g 10 l p b e 5 0 4 3 2 1 1.0 1.5 2.0 2.5 3.0 log 10 sn 50 o-lpbe 50 = 0.946 + (0.785*o-sn 50 ); r2 = 0.44 fit 95% con�dence interval 95% prediction interval b. vnt a vs lpbe a lo g 10 l p b e 5 0 4 3 2 1 1.0 1.5 2.0 2.5 3.0 log 10 sn 50 a-lpbe 50 = 0.469 + (0.927*a-sn 50 ); r2 = 0.54 fit 95% con�dence interval 95% prediction interval c. vnt asia1 vs lpbe asia1 lo g 10 l p b e 5 0 4 3 2 1 1.0 1.5 2.0 2.5 3.0 log 10 sn 50 asia1-lpbe 50 = 0.678 + (0.873*asia1-sn 50 ); r2 = 0.57 fit 95% con�dence interval 95% prediction interval figure 2. regression of lpbe titer on virus neutralization test (vnt) titers for the foot and mouth disease virus serotypes o, a and asia 1 (a-c): model co‑efficient and coefficient of determination (r2) are given for each serotype. table iii. repeatability of liquid phase blocking elisa (lpbe) (n = 25) in comparison with virus neutralization test (vnt) (n = 20). serum id test serotype o serotype a serotype asia1 mean s.d. cv (%) mean s.d. cv (%) mean s.d. cv (%) bc # 41* vnt 2.31 0.35 15.15 2.60 0.20 7.69 2.61 0.34 13.03 lpbe 2.90 0.08 2.76 2.90 0.08 2.76 2.99 0.05 1.67 bc # 55** vnt 1.91 0.32 16.75 1.90 0.26 13.68 2.15 0.52 24.19 lpbe 2.39 0.15 6.28 2.20 0.17 7.73 2.47 0.18 7.29 bc # 66*** vnt 0.90 0.00 0.00 0.90 0.00 0.00 0.90 0.00 0.00 lpbe 0.92 0.06 6.52 0.93 0.08 8.60 0.96 0.10 10.42 *high vnt titer; ** medium vnt titer; ***negative working control serum; s.d. = standard deviation; cv (%) = co-efficient of variation. 140 veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 vnt and lpbe in assessing the immune response to fmd vaccine tamil selvan et al. vs non-neutralizing antibodies between serotypes and individual variation in immune response might be hypothesised. our results indicate that it is not possible to set a common cut-off for all the three serotypes of fmdv to replace the challenge based potency testing and assessing the herd immunity. for serotype o, the predicted lpbe titers for a mandated cut-off of 1.65 log 10 vnt titer was 2.24 log 10 lpbe. however, a lower lpbe titer of 1.65 and 1.95 was set as the cut-off for serotype o in fgbi-arriah and coda-cerva, respectively (willems et al. 2012). in our study, the lpbe cut-off was deduced to be 1.87 and 2.00 for serotype a and asia 1, respectively. an lpbe cut-off titer of 2.20 (cevan) and 2.11 (var) has been reported for serotype a (mattion et  al. 2009). this variation could be attributed to the methodological differences between the laboratories while optimizing the lpbe and statistical models to determine the cut-off. setting a higher cut-off in lpbe especially for fmdv serotype o and a is also favorable to maximize the probability of protection. these viruses, in fact, give rise to frequent generation of antigenically and phylogenetically different variants as a result of continuous circulation in endemic countries like india. it is concluded that vnt and lpbe with a cut-off of <  1.2 and <  1.5, respectively, can be used for screening of the cattle for fmd antibodies. further, a positive relationship exists between the titers of vnt and lpbe for the fmd serotypes o, a and asia1. a cut-off in the range of 2.24,1.87 and 2.00 log 10 lpbe titer corresponds to the protective vnt titer of 1.65 and 1.51 log 10 for o and a and asia1, respectively, despite the fact that the predictive precision is moderate. this cut-off could be used for classifying the cattle as protected and not-protected till the logistic regression based correlation of protection with antibody titer is established and validated. acknowledgments the authors thank, the director, icar-ivri, bareilly and the joint director, icar-ivri, bengaluru campus for providing necessary facilities to carry out this work. we great fully acknowledge the contribution of dr. aniket sanyal, joint director, and dr. k. narayanan, principal scientist, icar-ivri, bengaluru for corrections and discussion in the manuscript. we also thank all the laboratory staff, icar-ivri, bengaluru for their assistance in carrying out this work. o1/bfs, a5(fra1/68) (>  0.83) (hamblin et  al. 1986). despite the differences between the laboratories, it is clear that higher the vnt titers, higher the value of observed lpbe titers. a possible reason behind the moderate correlation in our study might be due to the presence of more samples in lower vnt titer classes (0.9-1.34), which showed increased variation in lpbe. examination of predicted lpbe titers against serotype o for different classes of vnt titer showed that the initial lpbe titers were 5 times higher than vnt titers that ranged from 0.9-1.04. for the vnt titer classes 1.2-1.65 and 1.81-2.56, lpbe titers were 4 and 3 times higher, respectively (table iv). in the case of serotype asia1, the predicted lpbe titers are 3 times higher in lower and medium vnt titers. however, in the case of serotype a, all predicted lpbe titers were within 2 times of vnt titers. predicted lpbe titers were within the limit of 2 times of vnt titer above 2.71, irrespective of serotype analyzed, which may be due to saturation of antigen by antibodies. though exact reasons for differences in fold increase in lpbe titers are not known, the relatively high antigenic mass of serotype o virus in fmdv trivalent vaccines and/or differences in the relative abundance of neutralizing table iv. predicted log 10 lpbe 50 titer 50 (95% confidence interval) for each log 10 sn 50 titers based on linear regression$. log 10 sn 50 log 10 lpbe 50 titer o a asia1 0.90 1.65 (1.58-1.72) 1.30 (1.24-1.37) 1.46 (1.40-1.53) 1.04 1.76 (1.70-1.83) 1.43 (1.37-1.49) 1.59 (1.53-1.64) 1.20 1.89 (1.83-1.95) 1.58 (1.53-1.64) 1.73 (1.68-1.78) 1.34 2.00 (1.94-2.05) 1.71 (1.66-1.76) 1.85 (1.80-1.90) 1.51 2.13 (2.08-2.18) 1.87 (1.82-1.92) 2.00 (1.95-2.04) 1.65 2.24 (2.19-2.30) 2.00 (1.95-2.05) 2.12 (2.07-2.17) 1.81 2.37 (2.31-2.43) 2.15 (2.09-2.20) 2.26 (2.21-2.31) 1.95 2.48 (2.41-2.54) 2.28 (2.22-2.33) 2.38 (2.33-2.44) 2.11 2.60 (2.53-2.67) 2.43 (2.36-2.49) 2.52 (2.46-2.58) 2.26 2.72 (2.64-2.80) 2.56 (2.49-2.64) 2.65 (2.58-2.72) 2.41 2.84 (2.75-2.93) 2.70 (2.62-2.77) 2.78 (2.71-2.86) 2.56 2.96 (2.86-3.06) 2.84 (2.75-2.94) 2.91 (2.83-3.00) 2.71 3.07 (2.96-3.18) 2.98 (2.88-3.08) 3.05 (2.95-3.14) 2.86 3.19 (3.07-3.31) 3.12 (3.01-3.23) 3.18 (3.07-3.28) 3.01 3.31 (3.18-3.44) 3.26 (3.14-3.38) 3.31 (3.20-3.42) $regression model: y= a+bx; where, y = log 10 lpbe 50 titer; a = intercept; b = slope; x= log 10 sn 50 . 141veterinaria italiana 2021, 57 (2), 135-141. doi: 10.12834/vetit.1907.12234.1 tamil selvan et al. vnt and lpbe in assessing the immune response to fmd vaccine department of animal husbandy, dairing & fisheries (dadf). 2018. annual report 2017-2018. ministry of agriculture and farmers welfare, government of india. fluss r., faraggi d. & reiser b. 2005. estimation of the youden index and its associated cutoff point. biometrical j, 47, 458-472. doi:10.1002/bimj.200410135. grubman m.j. & baxt b. 2004. foot-and-mouth disease. clin microbiol rev, 17, 465-493. hamblin c., barnett i.t. & crowther j.r. 1986. a new enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against foot-and-mouth disease virus. ii. application. j immunol methods, 93, 123-129. hamblin c., barnett i.t.r. & hedger r.s. 1986. a new enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against 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serum neutralization test to evaluate immunity in potency tests of foot-and-mouth disease vaccines. j immunol methods, 124, 111-119. doi:10.1016/0022-1759(89)90192-0. wickham h. 2009. ggplot2: elegant graphics for data analysis. 2 ed. springer-verlag, new york. willems t., lefebvre d.j., goris n., diev v.i., kremenchugskaya s.r., paul g., haas b. & de clercq k. 2012. characteristics of serology-based vaccine potency models for foot-and-mouth disease virus. vaccine, 30, 5849-5855. doi:10.1016/j.vaccine.2012.07.015. world organisation for animal health ( oie). 2018. foot and mouth disease. version adopted by the world assembly of delegates of the oie in may 2017. https:// www.oie.int/app/uploads/2021/03/a-reso-2018.pdf. 221 #these authors contribute equally to this work. 1diagnostic virology laboratory, department of animal health, istituto zooprofilattico sperimentale delle venezie (izsve), viale dell’università 10, 35020 legnaro (pd), italy. 2oie/fao, national reference laboratory for avian influenza and newcastle disease (izsve), viale dell’università 10, 35020 legnaro (pd), italy. 3iga technology services, via linussio 51, 33100 udine, italy. 4veterinary consultant, suivet, italy. *corresponding author at: diagnostic virology laboratory, department of animal health, istituto zooprofilattico sperimentale delle venezie (izsve), viale dell’università 10, 35020 legnaro (pd), italy. tel.: +39 049 8084461, e-mail: msbeato@izsvenezie.it. parole chiave astrovirus suino, metagenomica, intero genoma. riassunto gli astrovirus sono virus identificati nel tratto gastrointestinale dei suini con e senza manifestazioni cliniche. la loro presenza nella popolazione suina è segnalata a livello globale. il loro ruolo come agenti causali di patologia gastroenterica è tuttora poco chiaro. nel presente lavoro si descrive, per la prima volta in italia, l’identificazione e la caratterizzazione genetica, attraverso sequenziamento dell’intero genoma, di un porcine astrovirus tipo 2 (poastv2) in veneto nel 2015. l’analisi filogenetica dell’orf2 evidenzia una alta similarità del virus con altri poastv circolanti lo stesso anno in giappone. poche sono le sequenze complete di astrovirus, e il presente dato rappresenta un importante contributo verso una migliore comprensione delle caratteristiche dei poastv. prima caratterizzazione dell’intero genoma di porcine astrovirus individuato nelle feci di un suino in italia keywords porcine astroviruses, metagenomics, whole genome. summary porcine astroviruses (poastv) are found in the gastrointestinal tract of healthy and diseased pigs worldwide. however, their role in causing enteric disease in pigs and other animals has not been elucidated. in the present report, we describe for the first time in italy, the identification and genetic characterization, through whole genome sequencing, of a poastv2 in pigs in northeast italy in 2015. this instance is the first detection of poastv2 in pigs in italy. the phylogenetic analysis of the complete orf2 segment highlights the high similarity of this virus to those circulating that same year in japan. there are very few full astrovirus genomes available, and the present data represent an important contribution towards a better understanding of the characteristics and evolution of these viruses. luca tassoni1#, gianpiero zamperin2#, eliana schiavon1, vera vendramin3, lara cavicchio1, monica mion1, francesco tonon4, isabella monne2 and maria serena beato1* first whole genome characterization of porcine astrovirus detected in swine faeces in italy veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 accepted: 15.07.2019 | available on line: 30.09.2019 genomes of astvs range in size from 6.4 to 7.7 kb, are polyadenylated at their 3’ end and contain three orfs, namely, orf1a, orf1b and orf2 (cortez et  al. 2017, boujon et  al. 2017). orf1a encodes the nonstructural polyprotein 1a, whilst the entire orf1 sequence encodes both a protease and an rna-dependent rna polymerase, with a ribosomal introduction astroviruses (astvs) are emerging viruses in the family astroviridae that infect a wide range of mammalian and avian species. they are non-enveloped viruses with a radius of 28-30 nm and a single-stranded positive sense rna genome (cortez et al. 2017, boujon et al. 2017). the 222 veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 whole genome sequence of poastv2 in italy tassoni et al. from faeces collected from piglets suffering from acute gastroenteritis in italy. the paucity of whole genome data renders the full characterization and comparison with extant strains difficult and, importantly, limits the analysis studies upon the origin and evolution of these viruses. materials and methods sample collection in november 2015, an outbreak of acute gastroenteritis was observed on a pig farm located in the province of treviso in the veneto region of northeastern italy. this was an open cycle farm with approximately 460 sows and 5,000 piglets. eighty percent of the piglets presented diarrhoea during the early post-weaning phase along with a reduction in food intake; morbidity was high, but mortality was low (approximately 10%). faecal samples were collected from three diseased piglets by a field veterinarian as part of the routine veterinary care animals; in addition, one succumbed piglet was submitted for necropsy. the faeces and the piglet were submitted to the diagnostic laboratory at istituto zooprofilattico sperimentale delle venezie (izsve). microscopic test for parasites was conducted on the intestinal tract, bacteriology tests were performed on the faeces, intestine and liver and virological tests on faeces. virus detection as first screening method, all faecal samples, including one from the dead piglet, were analysed by negative staining electron microscopy (em) for detection of virus particles. briefly, faeces were 10-fold diluted in phosphate buffered saline (pbs), repeatedly frozen and thawed and clarified by a two-step centrifugation (2,500 x g) at 4 °c for 30 min and 7,000 x g at 4  °c for 30 min. an aliquot of 85 µl of the supernatant was ultracentrifuged for 15 min in a beckman airfuge, using an a-100 rotor, at 20 psi (125,000 x g). the grids were stained using a 2% sodium phosphotungstate solution in distilled water (ph 6.8) for at least 3 min. the dried grids were observed using a tem philips operating at 80 kv, at a magnification of 19,000-45,000. morphometric measurements were performed on astrovirus-like positive samples. a minimum of 20 viral particles were measured at a magnification of 36,000 and statistically analysed with soft-imaging software analysis 2.1 (gmbh_ 1996). presence of porcine epidemic diarrhea virus (pedv) was excluded by real time rt-pcr (idexx realpcr pedv/pdcov multiplex rna test). in addition a pan-mamastrovirus rt-pcr analysis was performed. one gram of faeces was frame shift at the orf1a/1b (orf1ab) junction (de benedictis et  al. 2011). orf2 encodes the viral capsid structural polyprotein required for virion formation (de benedictis et al. 2011). based on the host, two genera have been identified: mamastrovirus and avastrovirus (boujon et al. 2017). thirty-three species of mamastrovirus and 7 species of avastrovirus have been identified based on the genetic differences in the complete orf2 amino acid sequence (guiz et al. 2013). astroviruses have been detected in the intestines and faeces of animals and are responsible for gastrointestinal disorders mainly in young individuals (cortez et al. 2017, boujon et al. 2017, de benedictis et al. 2011). however, astrovirus infections have been described in diseased animals with extra-intestinal manifestations, including respiratory and neurological signs (cortez et  al. 2017, boujon et al. 2017, de benedictis et al. 2011). additionally, astroviruses have been detected in clinically healthy mammalian and avian hosts, suggesting that infections in the absence of clinical signs are likely (cortez et  al. 2017, de benedictis et al. 2011). porcine astroviruses (poastvs) belong to the mamastrovirus genus and are distributed worldwide. five genotypes of poastvs have been identified (xiao et al. 2013); these genotypes belong to seven mamastrovirus clades recognized by the international committee on taxonomy of viruses (ictv), namely, clades 3, 22, 24, 26, 27, 31 and 32 (boujon et  al. 2017). this classification possibly reflects the different origins of poastvs along with interspecies transmission and recombination events, some of which presumably occurred with human strains (cortez et al. 2017). poastv was first detected in the faeces of diarrheal pigs in 1980 using electron microscopy (bridger 1980, shirai et al. 1985). poastv1 was the first lineage to be characterized, and it has been identified in us (xiao et al. 2013), canada (luo et al. 2011), the czech republic (indik et al. 2006) and colombia (ulloa and gutierrez 2010). the other genotypes have been identified in the usa, canada, china, and hungary at varying frequencies and have been detected in healthy and diseased animals (laurin et  al. 2011, shan et al. 2012, reuter et al. 2011, reuter et al. 2012, lan et  al. 2011, shan et  al. 2011, chen et  al. 2018, karlsson et al. 2016, zhang et al. 2014). intriguingly, poastv has also been shown to be associated with respiratory disease (padmanabhan and hause 2016), and it was detected in the brains of piglets suffering from congenital tremors (blomstrom et  al. 2014). pigs infected with poastv may or may not present clinical signs. therefore, the economic impact of swine astrovirus infections is poorly understood. here, we applied a metagenomics approach to sequence and analyse the whole genome of poastv first author et al. nick title 223veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 the faecal samples were deposited in the izsve biobank (izsve vir sct3 lvd 15). sequencing and data preprocessing the cdna library was processed with the illumina cbot for cluster generation on the flow cell following the manufacturer’s instructions and was sequenced in paired-end mode on a hiseq 2500 (illumina, san diego, ca). the casava 1.8.2 version of the illumina pipeline was used to convert the sequencing data from .bcl to fastq format. the illumina read quality was assessed using fastqc v0.11.2 [andrews s. fastqc, a quality control tool for high throughput sequence data (http://www. bioinformatics.babraham.ac.uk/projects/fastqc. 2014)]. the raw data were filtered by removing the following reads: a) reads with more than 10% undetermined (‘n’) bases, b) reads with more than 100 bases with a q score below 7, and c) duplicated paired-end reads. the remaining reads were clipped from the illumina truseq adapters with scythe v0.991 [(https://github.com/vsbuffalo/scythe) and trimmed with sickle v1.33 (https://github.com/ najoshi/sickle)]. reads shorter than 80 bases or reads that remained unpaired after the previous filtering were discarded. the high-quality reads were further filtered to remove host contamination by alignment with the sus scrofa transcriptome (ftp://ftp.ncbi.nlm.nih.gov/ genomes/all/gcf/000/003/025/gcf_000003025.5_ sscrofa10.2) using bwa v0.7.12-r1039 (li and durbin 2010), and the mapped reads were discarded. metagenomic analysis taxonomic assignment of the high-quality host-free reads was carried out using blastn 2.6.0+ (altschul et  al. 1990) for alignment with the integrated nt database (version 8th february 2017) and diamond v0.8.36 (buchfink et al. 2015) for alignment with the integrated nr database (version 8th february 2017). alignment hits with e-values larger than 1  ×  10-3 were discarded; this threshold value was selected because it was more conservative than the default threshold (1 × 10-2) used in megan. the taxonomic classification of each read was determined by the lowest common ancestor (lca)-based algorithm that was implemented in megan v6.7.0 (huson et al. 2016). de novo assembly for the reconstruction of the astrovirus consensus sequence, reads taxonomically classified as belonging to the family astroviridae were selected. these reads were assembled de novo diluted 1:5 (w/v) in 4 ml of phosphate-buffered saline solution (pbs). each sample was mixed by vortexing and centrifuged at 14,000 × g for 5 minutes. the obtained supernatant was used for rna extraction. the qiaamp viral rna mini kit (qiagen, hilden, germany) was used according to the manufacturer’s instructions, and the rna was stored at < -70 °c until use. the superscript iii one-step rt-pcr system with platinum taq (invitrogen, 12574-026) was used for the pan-mamastrovirus rt-pcr analysis. the primer set (mamastrof: 5’ ggmatgtgggtnaarcctg 3’; mamastror: 5’ twtggaggggmggaccaaa 3’) was designed to amplify orf2, generating a 355  bp amplicon. the pcr conditions were as follows: 55 °c for 30 minutes (min); 94 °c for 2 min, followed by 35  cycles at 94  °c for 30 seconds (sec), 55  °c for 30  sec, and 68  °c for 20 sec; and a final elongation step at 68 °c for 5 min. virus isolation was attempted in newborn swine kidney (nsk) and porcine kidney-15 (pk15) cell lines using trypsin for three blind passages. results of virus isolation were confirmed by the observation of cytopathic effect (cpe) and by pan-mamastrovirus rt-pcr analysis of the cell culture after the third passage. the faecal samples were stored and maintained at < 70  °c throughout the period of the study. nucleic acid extraction and library preparation sequencing was conducted for one of the three faecal samples with a sufficient amount of dna target. the faecal sample was thawed and mixed by vortexing, and approximately 250  mg of previously defrosted and mixed faeces was sampled using a sterile spatula in a 0.1  mm glass bead tube and immediately suspended in 650 µl of warmed pm1 buffer containing β-mercaptoethanol following the manufacturer’s instructions for the powermicrobiome rna isolation kit (mobio qiagen, carlsbad, ca, usa). the glass bead tube was quickly vortexed and loaded in a tissuelyser instrument (qiagen, carlsbad, ca, usa) for 2  min of homogenization at 30 beats/sec. the rna was extracted following the manufacturer’s instructions. the integrity and quantity of the extracted rna was checked on a caliper gx (perkinelmer, waltham, ma). fifty nanograms of rna was used as input for library preparation with the truseq stranded totalrna sample prep kit (illumina, san diego, ca) directly from the ‘synthesize first strand cdna’ step following the manufacturer’s instructions. the final library was quantified using a qubit 2.0 fluorometer (invitrogen, carlsbad, ca), and the dna quality was tested with the agilent 2100 bioanalyzer high sensitivity dna assay (agilent technologies, santa clara, ca). 224 veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 whole genome sequence of poastv2 in italy tassoni et al. results at necropsy the dead piglet showed peritonitis, oedema of the colon and hyperaemia of the pyloric region of the stomach, and severe catarrhal enteritis with foam and loss of tone of the intestinal tract was observed. the presence of the foam resembled the typical presentation of enteric disease caused by astrovirus in turkeys (toffan et al. 2012). microscopic examination of intestinal tract did not show presence of parasites. bacteriology test highlighted the presence of escherichia coli not belonging to neither o138, o139, o147, o149, o8 serogroups nor o141k85ab and o141k85ac serotypes in intestine and faeces. the presence of salmonella  spp. and clostridium perfringens was excluded. the em examination evidenced only astroviridae virus particles. all faecal samples tested positive for astroviridae by em and by pan-mamastrovirus rt-pcr (figures 1, 2). virus isolation attempts were not successful as cpe was not observed for any samples and absence of astrovirus particles was confirmed by em and rt-pcr. following library preparation and illumina sequencing, we generated 53,586,949 paired-end reads (2 × 125 bp), which allowed the recovery of 15,299 high-quality host-free reads truly belonging to the family astroviridae (a ratio of 0.15% poastv2 dna to exogenous dna). no other reads belonging to other virus families were identified. the poastv2 reads represented a 590-fold sequence coverage of base pairs and a 1165-fold physical coverage of using idba-ud  v1.1.1 (peng et  al. 2012) with the multi-kmer approach using a minimum value of 24, a maximum value of 124 and an inner increment of 10. an assembled sequence of a length comparable to that of the astrovirus genome (6-8 kb) was selected as the astrovirus consensus sequence. to assure that the selected sequence was truly representative of the astrovirus genome present in the sample, we aligned all reads classified as belonging to the family astroviridae against the astrovirus consensus sequence with bwa. a visual inspection of the alignment was performed with tablet v1.14.10.21 (milne et  al. 2010), and the consensus sequence was manually revised based on this alignment. we also checked whether all positions in the astrovirus consensus sequence were filled with the consensus nucleotide for that position by calling variants with lofreq v2.1.2 (wilm et al. 2012). according to the recommendations for lofreq use, the alignment was first processed with picard-tools v2.1.0 (http://picard.sourceforge.net) and gatk v3.5 (mckenna et al. 2010) [the genome analysis toolkit: a mapreduce framework for analysing next-generation dna sequencing data (depristo et  al. 2011, van der auwera et  al. 2013)] in order to correct potential errors, realign reads around indels and recalibrate base quality. lofreq was then run with fixed alignment with the option ‘call-indels’ to produce a vcf file containing both snps and indels. variant indels and snps with a frequency lower than 50% were discarded from the final set. if needed, the remaining variants were used to change the astrovirus consensus sequence accordingly. phylogenetic analysis to classify the virus correctly, the orf2 amino acid sequence was deduced from the nucleotide sequence, and phylogenetic analysis was performed. sequences representative of each poastv clade were included in the dataset along with target sequences obtained from a blastp search (tamura et al. 2013). moreover, a phylogenetic analysis of the orf1ab and orf1a amino acid sequences was conducted on only the poastv2 samples to identify the differences between the phylogenetic tree topologies. the best substitution models were identified for each dataset using modelfinder in mega version 6 (altschulz et al. 1997), and the selected models were applied in the phylogenetic analyses performed using the maximum likelihood method implemented in phyml 3.0 (guindon et  al. 2010). the branch support of the tree was measured by non-parametric bootstrap analysis with 100 replicates. figure 1. electron microscopy picture of astrovirus (larger particles of 18-20 nm) and parvovirus (smaller particles of 27-30 nm) observed directly in faecal sample of puppy no. 6 (36,000 kx). the characteristic ‘star-like appearance’ of astrovirus particles is evident compared to the round shaped morphology of parvovirus. negative staining was obtained with 2% sodium phosphotungstate solution. 225veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 tassoni et al. whole genome sequence of poastv2 in italy figure 2. acrilammide gel of the positive sample submitted to metagenomics analysis. mv ntc c+ mammalian astrovirus 15diapd 5469-10 the poastv2 genome, which allowed us to perform a successful de novo assembly of the detected astrovirus. the phylogenetic tree obtained from the deduced orf2 amino acid sequence, which includes the five genotypes of poastv, allows the classification of the virus poast/italy/diapd5469-10/2015 as poastv2 (figure 3). according to a recent classification, two poastv2 lineages, namely, lineages 1 and 2, were clearly delineated, with the tree supported by high bootstrap values (ito et al. 2017). lineage 1 is characterized by poastv2 sequences originating primarily in asia, with the exception of two poastv2 sequences originating in belgium, and one bovine astrovirus (boastv) (figure  3). lineage figure 3. phylogenetic tree based on deduced amino acid sequences of orf2 of poastv, including poastv of 5 different genotypes. the tree was obtained using the maximum likelihood method implemented in phyml3.0. a non-parametric bootstrap analysis with 100 replicates was conducted to obtain branch supports. the substitution model used was lg+g+i+f, and it was selected using mega6 model finder. *poastv for which only the orf2 sequence is available. 226 veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 whole genome sequence of poastv2 in italy tassoni et al. discussion this study reports for the first time the full-length genome of a poastv in italy belonging to genotype 2. only 303 whole-genome sequences of mammalian astrovirus are publicly available, 79 sequences from swine. in recent years, the number of poastv whole genome sequences has increased; however, the majority of the available mammalian astrovirus whole genome sequences are from humans (90) (data retrieved from genbank, 20 february 2019). a recent study conducted in japan (ito et al. 2017) has contributed in the increase of poastv sequences, but data from europe are limited. the lack of adequate of genome-level data for poastv hampers the determination of a correct classification of poastv and limits our ability to monitor the diversity and evolution of poastv. with reference to data available on poastv in italy, one study investigated the prevalence of poastv in italian swine herds (monini et al. 2015). faecal samples were collected from 2012 to 2014 in the northern, central and southern regions of italy. nineteen poastvs were detected and all were classified by using the orf1b-orf2 sequence as poastv4 (monini et  al. 2015). in this study, no significant association was identified between astrovirus infection and the presence of diarrhoea (monini et  al. 2015). outside italy, poastv2 was detected in healthy pigs in croatia in 2013 (brnic et  al. 2013), with a high prevalence 2 is characterized by higher heterogeneity than lineage 1 in terms of country and species of origin. phylogenetic analysis including the sequences of poastvs 1-5, revealed that the italian poastv2 clusters within lineage 1 and shows the highest similarity (%) with strains from japan and china detected in 2015 (monini et  al. 2015). the paucity of poastv sequences from europe may bias such findings. the different topologies observed in the orf2, orf1ab and orf1a phylogenetic trees (figures  4,  5,  6) confirm the results previously reported in the literature (ito et  al. 2017). color coding allows a simple comparison to be made between the topology obtained for the orf2 (figures 3,  4), orf1ab and orf1a phylogenetic trees (figures 5,  6). recombination events in the evolutionary history of these viruses might explain the differences observed. in addition, the orf1ab and orf1a trees are characterized by lower branch supports and smaller distances between taxa in comparison with the orf2 tree (figures 4, 5, 6). the deduced amino acid sequence of poast2/italy/ diapd5469-10/2015 shows higher similarity with that of bel12r021/2012 (73.15%) for orf2 and with that of bel15v01/2015 for orf1ab and orf1a (96.6% and 97.82%, respectively). figure 4. detail of orf2 phylogenetic tree showing poastv2 only. this figure is an enlargement of the data illustrated in figure 1, focusing on poastv2. each colour identifies further genetic groups within poastv2. only bootstrap values ≥ 60 are shown. lineage 1 samples are coloured in green while lineage 2 are in light blue. whole genome was not available for sequences marked by an asterisk. *poastv for which only the orf2 sequence is available. figure 5. phylogenetic tree of the deduced amino acid sequences of orf1ab of poastv2 only. the tree was obtained using ml method (lg + g) implemented in phyml3.0, performing a 100 replicates non parametric bootstrap analysis for the branch supports. only bootstrap values ≥ 60 are shown in the figure. lineage 1 viruses are coloured in green while lineage 2 are in light blue. 227veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 tassoni et al. whole genome sequence of poastv2 in italy and role. poastv is not considered taken into consideration in differential diagnosis with other pathogens responsible for gastroenteric disorders in pigs; accordingly, its prevalence might be underestimated and other genotypes are probably circulating in italy and europe. the absence of a cell culture systems and animal models hinder the opportunity to further characterize and study the pathogenic role of poastv (cortez et al. 2017). in fact, it remains difficult to associate poastv infections to disease. in this context, the piece of information provided in this study, represent an achievement in this field. this complete poastv sequence can facilitate porcine astrovirus diagnostics and further molecular epidemiology analysis of poastv in italy. notwithstanding the potential for interspecies transmission of astroviruses, resources are still lacking for improve knowledge upon animal astrovirus infections. in addition, further efforts are warranted in order to understand the evolution of astroviruses, with emphasis on their ability to recombine and to cross the species barrier. accession number(s) the raw miseq data were submitted to the ncbi sequence read archive (sra; http://www.ncbi. nlm.nih.gov/traces/sra/) under accession number srr6297822. the astrovirus consensus sequences were submitted to genbank under the accession number mg930777. grant support we thank the italian ministry of health for supporting the work of g. zamperin through the rc izs ve 05/14 project: “sviluppo ed applicazione di strategie di sequenziamento di nuova generazione (ngs) per la caratterizzazione del viroma in volatili e conigli”; project leader: isabella monne. compared to that of other poastv lineages (brnic et al. 2014), and in clinically healthy pigs in the czech republic between 2010 and 2011 (dufkova et  al. 2013). outside europe, poastv2 has been detected in healthy young piglets in china and japan (ito et al. 2017, li et al. 2015, cai et al. 2016), in the usa (xiao et al. 2013) and for the first time in east africa by amimo and colleagues (amimo et  al. 2014). unfortunately, isolation attempts in continuous cell lines failed, confirming the difficulties to replicate astroviruses in vitro, as already reported in the literature (de benedictis et al. 2011). regarding the role of poastv in multifactorial enteric disorders of pigs only scarce information data is available. better surveillance and diagnosis in the field regarding the involvement of poastv during enteric disorders in pigs may shed light on its prevalence figure 6. phylogenetic tree of the deduced amino acid sequences of orf1a of poastv2 only. the tree was obtained using ml method (lg + g) implemented in phyml3.0, performing a 100 replicates non parametric bootstrap analysis for the branch supports. only bootstrap values above 60 are shown in the figure. lineage 1 samples are coloured in green while lineage 2 are in light blue. 228 veterinaria italiana 2019, 55 (3), 221-229. doi: 10.12834/vetit.1873.9956.1 whole genome sequence of poastv2 in italy tassoni et al. amimo j.o., okoth e., junga j.o., ogara w.o., njahira m.n., wang q., vlasova a.n., saif l.j. & djikeng a. molecular detection and genetic characterization of kobuviruses and astroviruses in asymptomatic local pigs in east africa. arch virol, 159, 1313-1319. altschul s.f., gish w., miller w., myers e.w. & lipman d.j. 1990. basic local 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parole chiave bluetongue virus sierotipo 2, cellule di culicoides, feto, pecora, trasmissione transplacentare. riassunto per verificare se il ceppo italiano del sierotipo 2 del virus della bluetongue (btv‑2) è in grado di attraversare la barriera placentare ed infettare i feti ovini, due gruppi di 5 pecore gravide sono stati infettati, rispettivamente, con il ceppo di btv‑2 di campo (gruppo a) e con lo stesso ceppo passato una volta su cellule di culicoides (kc) (gruppo b). dopo l'infezione, a tutti gli animali sono stati effettuati settimanalmente prelievi di sangue in provette con e senza edta (siero) su cui rilevare, rispettivamente, la presenza di rna virale e virus infettante, e anticorpi anti‑btv‑2. al parto, si è proceduto al prelievo di sangue e siero dagli agnelli prima dell’assunzione del colostro. i campioni prelevati sono stati analizzati come descritto per le pecore. in concomitanza con la madre, anche gli agnelli sono stati sottoposti a prelievo. anticorpi anti‑btv‑2 sono stati evidenziati in tutte le pecore a partire dal 12° giorno dall'infezione e per tutto il periodo di studio (68 giorni). negli animali del gruppo a, btv‑2 è stato rilevato dal 7° al 12° giorno dall’infezione mentre in quelli del gruppo b dal 5° al 12° giorno. dei 14 agnelli nati, nessuno presentava anticorpi pre‑colostrali; tre agnelli nati da due pecore del gruppo b erano viremici alla nascita e virus infettante è stato isolato dal sangue fino all’11° giorno di vita. il presente studio ha dimostrato per la prima volta che il ceppo italiano di btv‑2 è in grado di attraversare la barriera placentare di pecore dopo un solo passaggio su cellule kc. infezione transplacentare del ceppo italiano del sierotipo 2 del virus della bluetongue nelle pecore keywords bluetongue virus serotype 2, culicoides cell line, ewe, fetus, transplacental transmission. summary in order to study the capability of a bluetongue virus serotype 2 (btv‑2) field isolate to cross the placental barrier, 2  groups of 5 pregnant ewes were infected with a field btv‑2 italian strain (group a) or with the same strain passaged once in culicoides cells (kc) (group b). following infection, edta‑blood and serum samples were collected weekly and tested for the presence of btv rna/infectious virus and anti‑btv‑2 antibodies, respectively. at lambing, precolostral edta‑blood and serum samples were collected from lambs and tested as before. the lambs were then sampled as scheduled for the dams. all sheep seroconverted on day 12 post‑infection (pi) and remained seropositive throughout the sampling period (day 68 pi). btv was isolated from day 7 pi to day 14 pi in animals of group a and from day 5 pi to day 12 pi in animals of group b. none of the 14 lambs born had pre‑colostral antibodies. three lambs born from two ewes of group b were viraemic at birth and in one lamb infectious virus was isolated from blood up to 11 days of age. this study proved for the first time that a single passage of btv‑2 field strain in kc cells is able to give to btv the ability to cross the placenta barrier and infect foetal tissues. transplacental transmission of the italian bluetongue virus serotype 2 in sheep veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 accepted: 11.06.2019 | available on line: 30.06.2019 massimo spedicato1, irene carmine1, liana teodori1, alessandra leone1, claudia casaccia1, annapia di gennaro1, gabriella di francesco1, giuseppe marruchella2, ottavio portanti1, valeria marini1, maura pisciella1, alessio lorusso1 and giovanni savini1* 132 veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 chicken eggs (ece). even if field isolates are capable of causing abortion in pregnant animals, they have never been detected in foetal tissues (van der slujiis et al. 2011). it is for this reason that btv field isolates have been supposed not able to cross the placental barrier and that abortion to likely be the result of the disease in the dam. however, no experimental trials have ever been designed and performed to support or refute this supposition. from what just said, it can also be deduced that passages in an artificial culture system ‑like ece or cell culture may favour phenotypic changes like changes in virulence, tissue tropism and ability to cross the placenta. it has been demonstrated that one passage on embryonated chicken eggs (ece) and two passages on mammalian cells, were capable of establishing an infection in ovine foetuses (van der slujiis et  al. 2011). recently, it was also demonstrated that two passages, one on kc cells (a cell line derived from c.  sonorensis), and one on cpt‑tert cells (ovine choroid plexus cells) were enough to give to the btv‑2 italian strain the capability of crossing the placental barrier and infect ovine fetuses (rasmussen et al. 2013). concerning btv vertical transmission, much has therefore been achieved since 1955 but much has still to be done. improving our knowledge on the mechanism(s) behind the vertical transmission of btv is of paramount importance in order to better understand the biology and epidemiology of the virus. in this trial, the capability to cross the ewe placental barrier of two btv‑2 strains, one never passaged into cell lines and one passaged once into kc cells, has been evaluated. materials and methods ethics all procedures on animals were accomplished according to the italian decreto legislativo 4 march 2014, no. 26 on the protection of animals used for scientific purposes, and were approved by the italian ministry of health. animals twenty‑nine healthy bergamasca ewes were housed in the facilities of the istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ (izsam). the animals were uniquely identified by ear tag numbers. the ewes were treated with deltamethrin (butox pour on, msd animal health, milan, italy), dewormed and confirmed negative for the presence of btv rna by real time rt‑pcr and anti‑btv antibodies by competitive elisa (celisa) and virus neutralisation (vn). introduction bluetongue (bt) is a viral non‑contagious disease of wild and domestic ruminants caused by bluetongue virus (btv). within the reoviridae family, it represents the type species of the genus orbivirus (mertens and diprose 2004). in the last few years next to the classical 24 serotypes, new serotypes have been described (hofmann et  al. 2008, lefevre et  al. 2008, maan et  al. 2011, zientara et  al. 2014, schulz et  al. 2016, sun et  al. 2016, bumbarov et  al. 2016, savini et al. 2017, marcacci et al. 2018, lorusso et al. 2018). sheep is the most susceptible species, but overt disease has also been reported in cattle, goats and wild ruminants (elbers et  al. 2008 a, b, lefevre et  al. 2008, dal pozzo et al. 2009, maclachlan et al. 2009). even though btv transmission mainly occurs through culicoides biting midges, for some strains sporadic cases of oral (ingestion of infected placenta or colostrum) and vertical (transplacental) transmission have been reported (menzies et  al. 2008, backx et  al. 2009, mayo et  al. 2010, van der slujis et al. 2013, batten et al. 2013, rasmussen et al. 2013). according to the stage of pregnancy and strain involved, infection of fetuses with btv can lead to embryonic death and return to service, abortion, congenital defects and stillbirth (wouda et  al. 2009, santman‑berends et  al. 2010, saegermann et  al. 2011). all these events might have serious economic repecussions. vertical transmission of btv in animals could also play an important role in the epidemiology of natural btv infections (maclachlan et al. 2009, verwoerd and erasmus 2004, saegerman et al. 2011, van der sluijs et al. 2016). some of these animals can be born infected, and may introduce the virus to new areas if the dam is transported, and allow btv to overwinter (van der slujis et  al. 2011, efsa scientific opinion on bluetongue serotype 8 2011, van der sluijs et al. 2016). the first report of transplacental transmission of btv dates back to 1955, when shultz and delay found that vaccination of pregnant ewes with an in  ovo‑attenuated btv vaccine caused the birth of weak and malformed lambs. since then, studies on mother‑to‑foetus transmission of btv in ruminants were conducted under various experimental settings using either cell‑ or in ovo‑adapted strains. the interest on this issue peaked after the north‑european btv‑8 outbreak, that was responsible for abortions and teratogenesis in cattle (barnard and pienaar 1976, gibbs et  al. 1979, stott et  al. 1982, richardson et  al. 1985, parsonson et  al. 1987, maclachlan et  al. 2000, de clerq et  al. 2008 a, b, menzies et  al. 2008, darpel et al. 2009, van der slujis et al. 2011, van der slujis et al. 2013). it was the first time that a field isolate was able to cross the placental barrier. before btv‑8 incursion, vertical transmission was proven only for strains adapted on cells or passaged into embryonated btv‑2 transplacental transmission spedicato et al. 133veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 spedicato et al. btv‑2 transplacental transmission at approximately 120 days of gestation, animals of group  a were inoculated subcutaneously with 10 ml of btv‑2 blood while the 5 ewes of group b with 2 ml of btv‑2 kc . each group was located in the same insect‑proof stable but in different pens. two not‑pregnant, virologically and serologically negative sheep were added to the infected groups (one per group) and used to monitor eventual btv circulation. rectal temperatures and clinical signs of all animals were monitored daily for 21 days. a rectal temperature ≥ 40 °c was considered fever. edta‑blood and serum samples were collected from all animals three times a week and once a week for 68  days, respectively. blood was tested for the presence of btv rna by real time rt‑pcr and for the presence of infectious virus by virus isolation  (vi); serum was tested against btv antibodies by celisa and by vn assay. after lambing, precolostral edta‑blood and serum from lambs as well as colostrum from ewes were collected, and tested serologically and virologically as previously described. subsequently, lambs were sampled as scheduled for the ewes. whenever possible, placentas were collected and screened for the presence of viral rna and infectious virus. all samples were kept at 4 °c until analysis (for a maximum of 2 days). necropsy and tissue collections in case of death, animals underwent post‑mortem examination and gross lesions were recorded. tissue samples from brain, heart, spleen, lungs, ileus, kidney and lymph nodes (submandibular, mesenteric, mediastinic, precrural and popliteal) were removed and splitted into two aliquots: one was fixed in 10% buffered formaldehyde for histology and immunohistochemistry (ihc) and one homogenised in pbs (1:10). once clarified by centrifugation at 5,000 rpm for 15 minutes, homogenates were stored at 4  °c until analysis (for maximum of 2 days). histology and immunohistochemistry tissue samples were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin wax. subsequently, tissue blocks were sectioned at a thickness of 5 µm, stained with haematoxylin and eosin (he) and examined microscopically. sections of collected tissues were also processed for ihc: briefly, tissue sections were heat‑treated for antigen retrieval (121 °c x 8 min in 0.01 m citrate buffer, ph 6.0) and incubated overnight with an the oestrus was synchronized by means of intravaginal sponges soaked with flugestone acetate (crono‑gest sponge, msd animal health, milan, italy) kept in situ for 15 days, followed by the administration of 400 iu of equine chorionic gonadotropin (crono‑gest pmsg, msd animal health, milan, italy) at removal. two days later, four rams were introduced in the flock for three weeks. ten ewes were subsequently diagnosed pregnant by ultrasound examination and enrolled in the study. two weeks before inoculation, ewes were randomly divided into 2 groups and housed in insect‑proof barns. btv‑free status of the animals was rechecked as described above. automatic temporized dispenser of pyrethroids operated during the entire experiment and ewes were treated monthly with deltamethrin. one blacklight trap operated between the first door and the entrance of the barns and further two traps operated within the barns. challenge virus the btv‑2 used for the challenge derived from the spleen of a sheep naturally infected during the 2001 btv outbreak in sardinia, italy (internal reference number 80306/2001). it is worth to note that the outbreak occurred before the beginning of the first btv vaccination campaign which started in 2002 and that the btv‑2 strain used in this trial is genetically distinct from the btv‑2 vaccine strain (elia et al. 2008, savini, unpublished results). to increase the volume of the inoculum and evaluate the vitality of the strain, two sheep were injected. briefly, approximately one gram of the spleen was homogenized and diluted 1:10 in phosphate buffered saline (pbs) added with antibiotics and antimycotics. the homogenate was centrifuged at 5,000 rpm for 10 minutes and the supernatant inoculated subcutaneously to a btv‑free sheep (internal id number 84372). a btv‑free ram (internal id number 84359) was used as second animal. it was further inoculated subcutaneously with 30  ml of pcr‑positive blood collected from the first sheep. blood samples were collected daily from the ram by jugular venepuncture, and btv rna‑positive samples were stored at ‑ 80 °c (virus stock btv‑2 blood ). btv‑2 blood was also propagated in kc cells for 10 days, and the infected tissue culture was harvested, titrated and stored at ‑  80  °c until use (virus stock btv‑2 kc ; titer 5.97 log 10 tcid 50 /ml) . experimental design the 10 pregnant ewes were divided into 2 groups of 5 animals each, namely group  a and group  b. 134 veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 btv‑2 transplacental transmission spedicato et al. the first door and the entrance of the barns. it was negative when tested for the presence of btv rna. clinical signs most animals had fever from day 6 to day 11 post‑infection (pi). on day 14 pi, ewe no. 4 (group  a) died before parturition. clinical signs initially included nasal discharge, salivation and conjunctivitis, and later mild coronitis, severe facial oedema and finally dyspnoea. between days 20 and 32 pi, the remaining 9 ewes gave birth to 14 lambs: six were born from group a and 8 from group b. lamb no. 12 from ewe no. 1 (group a) was born dead. apart from this event, no abortions were reported during the trial, however 6 lambs, 1 from group  a and 5 from group b died within the first 10 days of age. the neonatal mortality rate was 43%. on day 37 pi, an ewe of group  b (no. 10) was euthanized due to severe neurological signs (inability to stand, hyperextension of front and hind limbs and opisthotonus) unresponsive to antibiotic therapy and supportive care. gross pathology, histology and immunohistochemistry of ewes two ewes, one per group, died during the study. at necropsy, the carcass of ewe no. 4 (group  a) showed a generalized yellowish gelatinous subcutaneous oedema, particularly of the head, with sparse subcutaneous petechiae and hemorrhagic suffusions; petechiae and suffusions were also evident on oesophageal mucosa and adventitia, spleen and pleura. other gross lesions included hydropericardium and haemorrhages at the base of the pulmonary artery, lymphadenomegaly, and pulmonary oedema with presence of foamy fluid in trachea. histologically, perivasal oedema and congestion were detected in the brain. heart was affected by an interstitial myocarditis with necrosis (figure 1a) and spleen by an inflammatory process characterized by lymphocytic karyorrhexis (figure 1b). lungs showed a catharral alveolitis with congestion and secondary contamination by bacteria. all organs tested pcr‑positive, but virus isolation was inconclusive. the size of the foetus was normal for its gestational age. macroscopic lesions included renal hyperaemia and splenic petechiae. all sampled organs tested pcr‑negative. at necropsy, the carcass of the ewe (no. 10) of group  b had erosions of the oral mucosa, diffuse anti‑btv vp7 monoclonal antibody (ab22070, abcam, uk) diluted 1:1000. immune reactions were revealed using a polymer‑based peroxidase technique (envision plus kit, denmark). positive and negative controls were included in all ihc reactions. entomological surveillance insects were collected once a week and placed in ethanol 70%. culicoides midges were identified according to complex, species (when possible), sex and age (goffredo and meiswinkel 2004). insects were pooled according to date of collection, trap number, species and age, and pools tested for the presence of btv rna. laboratory tests serum and colostrum were tested against btv antibodies by a celisa (lelli et al. 2003) and vn test, as described in the oie terrestrial manual (oie 2018). the detection of btv rna in edta‑blood samples, organ homogenates and culicoides pools was performed by using the one step real time rt‑pcr targeting the segment 10 of btv genome, as described in the oie terrestrial manual (oie 2018). virus titration and isolation were attempted on edta‑blood samples and organ homogenates as described by savini and colleagues (savini et al. 2009). tcid 50 was calculated by using the method of reed and muench (reed and muench 1938). statistical analysis for statistical purposes, antibody titer was expressed as the reciprocal of the highest serum dilution (n) able to inhibit at least 75% of the virus cpe. data were plotted as log 2 of the mean values obtained. the results of real‑time rt‑pcr are defined by the number of the cycle threshold (ct) at which the specific amplicon was revealed. differences between the mean neutralizing antibody titers and ct per sampling day, among the different groups, were analyzed using the non‑parametric wilcoxon‑mann‑whitney test for independent groups. a probability of p < 0.05 was set as significance level. results during the entire experimental period, only 1 parous midge belonging to the obsoletus complex was collected from the blacklight trap placed between 135veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 spedicato et al. btv‑2 transplacental transmission and an interstitial myocarditis with fibrosis and degeneration were observed. lymph nodes were hyperplastic, and retropharyngeal lymph nodes were positively stained for vp7 (figure 1c). gross pathology, histology and immunohistochemistry of lambs a total of 6 lambs born from 5 ewes died (1 lamb born from a group  a ewe and 5 lambs born from group  b ewes). gross lesions were not indicative of btv infection. lambs born from ewes of group a lamb no. 32 was born dead. all tested organs were negative to btv rt‑pcr. lambs born from ewes of group b the sampled tissues collected from those lambs cutaneous haematomas, haemorrhagic mediastinic and head lymph nodes and consolidated pulmonary areas. clostridium  spp. were isolated from spleen, liver, brain, lungs and kidney, and escherichia coli and pasteurella multocida from lungs. only brain and lymph nodes tested positive for the presence of btv rna and stained positive for vp7. histologically, a severe and diffuse pneumonia figure 1. examples of tissue sections from dead ewes. all sections have been stained with haematoxylin and eosin and are shown at a magnification 40x. a. heart of a sheep of group a (infected with btv‑2 blood ) showing phlogosis and necrosis. immunohistochemistry (ihc) was negative. b. spleen of the same sheep of a), showing karyorrhexis of lymphocytes. c. retropharyngeal lymph node of a sheep of group b (infected with btv‑2 kc ), showing macrophages and lymphocytes positive for btv‑vp7 by ihc (brown spots). a b c figure 2. examples of tissue sections from dead lambs. all sections have been stained with haematoxylin and eosin and are shown at a magnification 40x. a. histological section of the lung of lamb no. 3, born from a sheep of group b (infected with btv‑2 kc ). interstitial macrophages positive for btv‑vp7 by immunohistochemistry (ihc) are shown (brown spots). b. retropharyngeal lymph node of the same lamb of a) showing macrophages and lymphocytes positive for btv‑vp7 by ihc (brown spots). a b 136 veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 btv‑2 transplacental transmission spedicato et al. collected from both animals were negative to the rt‑pcr for btv. serological and virological results in ewes the animals added to each group for monitoring btv circulation, remained negative to btv virological and serological tests throughout the entire experimental period. two animals, one of group  a and one of group  b, first showed elisa btv antibodies on day  5  pi. from day 12 pi till the end of the trial, all animals were positive to the btv c‑elisa. analogously, neutralizing antibodies were detected in all animals starting from day 12 pi till the end of the sampling period (on day 68 pi) (figure 3). the highest antibody titer was detected on day 26 pi in group a (mean 1:360) and on day 19 pi in group b (1:704) (figure 3). on day 12 pi, the mean antibody titer of group  b was significantly higher than in group  a (p  <  0.01) (figure 3). btv rna was detected in all infected ewes starting from day 3 pi in group b and day 5 pi in group a. then, viral rna concentration increased further, peaking on days 7‑10, and remaining steady throughout the sampling period. the mean ct value observed in the animals of group  a on day 5 pi was significantly higher than that observed in group  b. subsequently, the ct value trend of both groups was almost completely comparable (figure 4). all pregnant ewes had at least one‑day of infectious viraemia (from day 7 pi in group  a and from which were viraemic at birth were btv rt‑pcr positive. lamb no. 23 was viraemic from birth till death which occurred ten days later. gross lesions included pulmonary atelectasis and enteritis with ascites. e. coli was isolated from liver, ileus and ascitic fluid. except for the heart, all sampled organs were btv rt‑pcr positive. no relevant histological lesions were found. vp7 was detected in spleen, liver and retropharyngeal and meseraic lymph nodes (figure 2, a‑b). lamb no. 24 died 5 days after birth. it was viraemic from birth till death. gross lesions included pulmonary hyperaemia, splenic petechiae, enlargement of mesenteric and mediastinic lymph nodes, sub‑epicardial suffusion, hyperaemia of intestinal mucosa, renal congestion, mild ectasia of meningeal blood vessels. all sampled organs were positive to btv rt‑pcr. lamb no. 27 was weak at birth and died at the age of 2  days. gross lesions included mild enlargement of meseraic and mediastinic lymph nodes, hyperaemia of intestinal mucosa and mild ectasia of meningeal vessels. all sampled organs were negative to btv rt‑pcr and virus isolation. twin lambs no. 33 and no. 34 died at the age of 2  days. gross lesions included enteritis with ascites, sub‑epicardic petechiae, a wide superficial haemorrhage of the right ventricle. e. coli was isolated from liver, gut and ascitic fluid. organs 0 1 2 3 4 5 6 7 8 9 10 0 5 12 19 26 33 40 47 54 61 68 m ea n a n ti b o d y ti tr e (l o g 2 1 /n ) days post‑infection group a group b * * figure 3. mean neutralizing antibody titer in ewes per sampling day. group a: ewes infected with btv‑2 blood ; group b: pregnant ewes infected with btv‑2 kc . antibody titer is expressed as the reciprocal of the highest serum dilution (n) able to inhibit at least 75% of the virus cytopathic effect (cpe). data are plotted as log 2 of the mean values obtained per ewe per sampling day. group a: ewes infected with btv‑2 blood ; group b: ewes infected with btv‑2 kc . * = p < 0.01. 0 5 10 15 20 25 30 35 40 45 50 0 3 5 7 10 12 14 17 19 21 24 26 28 31 33 35 38 40 42 47 54 61 68 m ea n c t days post‑infection group a group b * * figure 4. mean cycle threshold (ct) values in ewes per sampling day. group a: ewes infected with btv‑2 blood ; group b: pregnant ewes infected with btv‑2 kc . * = p < 0.05. 137veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 spedicato et al. btv‑2 transplacental transmission severe clinical signs. in other similar studies, the clinical signs observed in sheep when infected with blood originating from a naturally btv‑infected animal, were more severe than those observed in animals infected with cell‑adapted viruses (maclachlan et  al. 2008, eschbaumer et  al. 2010, caporale et al. 2014). in this experiment, btv‑2 kc was passaged only once in culicoides cells while in all mentioned studies, the number of cell passages of the inoculum was three as a minimum, and included at least one passage on mammal cells. the exiguous number of passages to which the inoculum has been subjected or, alternatively, the type of cell lines (insect) used in this trial might therefore be at the origin of this scarce attenuation of the btv‑2. however, based on these results, it is hard to say whether it was because the strain was passaged only once on tissue culture (tc), because it was passed on culicoides derived cells only or because it was never passaged on mammal cells. on the other hands, the fact that both inocula were able to reproduce viraemia and clinical signs was fundamental in order to study the occurrence of vertical transmission of btv‑2 strains in ewes. this trial, if on the one hand demonstrated that one passage into kc cell, although not capable of reducing the virulence of btv‑2, was able to confer to the italian btv‑2 strain the capacity of crossing the placental barrier of ewes, on the other hand, confirmed that, for btv‑2 field isolates, crossing the placenta is an exceptional event. the btv‑2 strain used in this study was responsible for the most severe btv outbreak occurring in italy in 2000‑2001, causing more the 500,000 clinical cases and deaths. although many abortions were reported, btv‑2 was never detected in foetal tissues (savini et  al. 2014). in the same way, with the only exception of btv‑8 european strain, wild‑type btv strains have never been found in ruminant foetal organs (elbers et al. 2008, dal pozzo et al. 2009, méroc et  al. 2009, efsa scientific opinion on bluetongue serotype 8). transplacental infection has been, instead, frequently reported for btv strains with behind a lab story implicating several passages in an artificial culture system (maclachlan et  al. 2000, shimshony et  al. 1980, savini et  al. 2014). rasmussen and colleagues (rasmussen et al. 2013) in a recent study, injected ewes with the same strain used in this trial and proved that the italian btv‑2 strain was able to cross the placenta after being passaged once in kc cells and once in cpt‑tert cells. this study further reduced the number of cell passages of btv‑2 and proved that one passage into kc cells is still sufficient to grant the virus the competence to infect fetuses and lead to the birth of viraemic lambs. to the best of our day 5  pi in group  b). in two animals of group  a, viraemia lasted till day 14 pi, while in group  b viraemia was not longer than 12 days (3 ewes). in all infected animals btv titers never were higher than 2.3 log 10  tcid 50 /ml. except for two animals (one from group a and one from group b), btv was also detected by rt‑pcr in all the collected placentas. serological and virological results in lambs at birth, none of the lambs had antibodies against btv. subsequently, all lambs seroconverted due to passive transfer of colostral maternal antibodies. lambs born from ewes of group a all lambs born from ewes of group a were negative to btv rt‑pcr for the entire length of the trial. lambs born from ewes of group b three lambs (no. 22, no. 23 and no. 24) born from 2 ewes infected with btv‑2 kc were viraemic at birth. twin lambs no. 22 and no. 23 were born on day 22 pi and lamb no. 24 on day 23  pi. at the time of birth, the respective ewes resulted positive to btv rt‑pcr but negative to virus isolation. lamb no. 22 was btv rt‑pcr positive till 90 days of age (day 112 pi). infectious btv was isolated from edta blood samples up to 11 days of age (day 33 pi), 21 days after the latest btv isolation from its mother. the titer of neutralising antibodies detected in the colostrum was 1:160 whereas the lamb had a titer of 1:80 up to 60 days of age (last serum sampling day). lambs no. 23 and no. 24 were btv rt‑pcr positive till death (10 and 5 days of age, respectively). infectious virus was isolated from lamb no. 23 untill 6 days of age (day 28 pi; 16 days after latest btv isolation from its mother) and from lamb no. 24 untill 3 days of age (day 26 pi; 14 days after btv isolation from its mother). discussion in this study all infected ewes seroconverted and developed viraemia and rna‑aemia after btv‑2 infection. as in other experimental infections (caporale et  al. 2014), ewes infected with the btv strain grown in vitro, became viraemic earlier than those infected with the wild strain never passaged into tissue culture. both types of inoculum (btv‑2 blood or btv‑2 kc ) were also capable of causing death, and comparable 138 veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 btv‑2 transplacental transmission spedicato et al. of a single member with a defined nucleic acid sequence. rather, they are dynamic distributions of nonidentical but related members called a quasispecies. the consequence of a quasispecies is that most viral infections are initiated not by a single virion, but a population of particles. the progeny produced after this infection results from selective forces that operate inside the infected host (eigen 1993). as other rna viruses, btv also exists as a quasispecies (domingo and holland 1997, bonneau et  al. 2001). it is likely that in the btv population virions capable of crossing the placenta barrier and virions which don’t have this capacity coexist, with the latter more represented. in this study, kc cell lines have probably acted as a molecular sieve, by selecting minor viral variants with phenotypic changes which, among other characteristics, include the ability to cross the placenta. although, theoretically, phenotypic changes accumulate only over several subcultivations (kirkland and hawkes 2004, pedersen et  al. 2009, caporale et  al. 2014), in this study an unique passage into kc cells was sufficient to confer to the btv‑2 wild strain the ability to cross the placenta of ewes. this finding supports caporale and colleague hypothesis (caporale et  al. 2014) that kc cells might function as a natural source of new btv variants, because an increased variability of replication in culicoides cells allows btv to adapt faster to different selective pressures. at birth, none of the lambs had btv antibodies, and seroconversion occurred only after the ingestion of colostrum. it is known that ovine foetuses are able to mount an immune response against btv at 95‑100 days of gestation, approximately 10 days after they become infected (silverstein et  al. 1963, osburn et al. 1971, enright and osburn 1980, miyasaka and morris 1988, tizard 1996, hoffmann et al. 2013). in this trial, ewes were infected at about 120 days of gestation and viraemic lambs were born 22‑23  days post‑infection. according to theory of van der slujis and colleagues (van der slujis 2011), in this trial btv‑2 kc probably infected foetal tissues not earlier than days 12‑13  pi, so lambs did not have enough time to mount an humoral immune response detectable at birth. conclusions although, under many aspects, btv is a very well‑characterized virus, the mechanisms behind transplacental transmission are not clearly defined yet. this study proved that a single passage of btv‑2 field strain in kc cells is able to give to btv the ability to cross the placenta barrier and infect foetal tissues. it adds another piece to the intricate puzzle of knowledge, this is the first time that such a finding is reported. the effects induced on the neonatal development by btv when crossing the placental barrier, largely depend on the period of gestation and stage of maturation of the foetus. the infection may result in prenatal death with abortion or stillbirth, malformation such as hydranencephaly and blindness, abnormal behavior like tremor and ‘dummy syndrome’, or normal term neonates (oberst 1993). in this trial, ewes were infected in the last month of pregnancy, normal terms lambs were then expected. of the 5 ewes infected with btv‑2 kc , two gave birth to 3 viraemic lambs. although not unanimously accepted, the birth of viraemic offsprings can play an important epidemiological role. in northern ireland, viraemic calves from imported pcr‑negative dams were blamed as the cause of the btv introduction in the country (menzies et al. 2008). it has been hypothesized that the virus was ‘ferried’ undetected in foetal tissues (a ‘trojan horse’ mechanism). because of that, specific control measures are adopted when pregnant animals have to be moved from btv‑8 infected to btv‑8 free areas. vertical transmission has also been implicated as one of the possible strategies behind the overwintering process (takamatsu et  al. 2003, white et  al. 2005, wilson et al. 2008). it has been thought that btv can overwinter in foetal tissues to reemerge months later in viraemic offspring. in such a way, the infectious viraemic period can be extended beyond that of the dam (gibbs et al. 1979, richardson et al. 1985, de clerq et al. 2008, van der sluijs et al. 2011, savini et  al. 2012). in this trial, infectious virus was isolated in 3  lambs born from ewes infected with btv‑2 kc at 3, 6 and 11 days of age (day 33 pi), which means 14, 16 and 21 days, respectively, after the last vi positivity detected in the mother blood samples. in lamb no. 22, the only btv positive lamb which survived in this study, btv rna was instead detected until 90 days of age (day 112 pi). it is well known that an animal positive to rt‑pcr but negative to virus isolation is not infectious to vectors (maclachlan et  al. 1994, bonneau et  al. 2002), in this trial, the infectious viraemic period was then extended for a maximum of 21  days, a period probably not long enough to allow virus overwintering, even in temperate regions. of the two btv‑2 strains used in this experiment, the btv‑2 kc was the only one capable to cross the placenta of ewes. since the btv rna concentrations and the antibody response detected in the blood samples of group a and group b animals were similar, this ability could not be ascribed to different levels of viraemic titers triggered by the btv‑2 infections. in other words, this new acquired capacity of the btv‑2 kc did not depend on the host response. it is known that virus populations are not made 139veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 spedicato et al. btv‑2 transplacental transmission acknowledgments the authors wish to thank all the veterinary and technical staff of the animal experimental facilities of the izsam for their precious help in animal handling and samples collection. the authors are also grateful to the entomological unit of izsam for entomological identification and characterization; to mrs. barbara cipro and mrs. sabrina olivieri for processing biological samples; to dr. romolo salini for statistical assistance. btv transplacental transmission and improves our understanding of this phenomenon. further research, however, is needed to disclose the viral genes/proteins and the mechanisms involved in this event in order to design live vaccines not capable of crossing the placenta and infecting foetuses. backx a., heutink r., van rooij e. & van 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(http://www.oie.int/ fileadmin/home/eng/health_standards/tahm/3.01.03_ bluetongue.pdf/ accessed on 30 june 2019). wouda w., peperkamp t., roumen m., muskens j., van rijn p.a. & vellema p. 2009. epizootic congenital hydranencephaly and abortion in cattle due to bluetongue virus serotype 8 in netherlands. tijdschr diergeneeskd, 134, 422‑427. zientara s., sailleau c., viarouge c., höper d., beer m., jenckel m., hoffmann b., romey a., bakkali‑kassimi l, fablet a, vitour d & bréard e. 2014. novel bluetongue virus in goats, corsica, france, 2014. emerg infect dis, 20 (12), 2123‑2125. veterinaria italiana 2019, 55 (2), 131‑141. doi: 10.12834/vetit.1913.10140.1 spedicato et al. btv‑2 transplacental transmission 57 veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 accepted: 21.07.2021 | available on line: 16.11.2022 1istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, teramo, italy. 2asl teramo , teramo, italy. 3clinica veterinaria valdinievole, italy. *corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, teramo, italy. e‑mail: f.iannino@izs.it filomena iannino1*, guido di donato1,2, stefania salucci1, enzo ruggieri1, giacomo vincifori1, maria luisa danzetta1, paolo dalla villa1, elisabetta di giannatale1, giulia lotti3 and fabrizio de massis1 keywords campylobacter, campylobacteriosis, dog, risk factors, zoonosis, shelter, antimicrobial resistance. summary campylobacteriosis has been the most frequently reported zoonotic disease in humans in europe. the scientific literature has reported that the role of dogs may be relevant. the objectives of this work are to improve the knowledge about campylobacter spp. carriage, infection and antimicrobial resistance in household and shelter dogs in italy, and to assess risk factors at the dog/human interface. during the 2015-2016 period, rectal swabs were collected from 431 household vet-visiting dogs and 173 dogs housed in shelters. a total of 3 veterinary clinics, located in three italian regions (abruzzo, molise and tuscany) and 10  shelters, five in abruzzo and five in molise, were included in the study. relevant risk factors for the transmission of campylobacter spp. from dogs to humans were assessed by means of a questionnaire administered to owners of household dogs. for campylobacter spp. isolation, selective cultivation methods were used, followed by confirmation and species identification with the pcr method. phenotypic antibiotic resistance profiles assayed using antimicrobial susceptibility testing were combined. campylobacter spp. were isolated from 9 household dogs (2.1% ci 1.1% 3.9%) and from 13  shelter dogs (7.5 % ci 4.5% 12.4%). in household dogs c. jejuni was the most represented species (0.9%). in shelter dogs, the most common species was c. jejuni (5.2%). campylobacter spp. isolates were resistant to ciprofloxacin (22.73%), nalidixic acid (22.73%), tetracyclines (27.27%), streptomycin (9.09%) and erythromycin (4.55%). the main c. jejuni clonal complex identified in dogs were cc21, cc45, cc206, cc403, cc42 and cc658. the risk of contracting campylobacteriosis from dogs remains a concrete reality. this risk is increased in the presence of common habits, as shown by the data from the questionnaire. prevalence control of campylobacter spp. in household and shelter dogs would be important in order to reduce the transmission to humans. campylobacter and risk factors associated with dog ownership: a retrospective study in household and shelter dogs farm animals, direct contact with pets and insect as flies (adak et al. 2005, mazick et al. 2005, strother et al. 2005). the species most commonly associated with human infections are campylobacter jejuni, followed by c. coli, c. lari, and c. upsaliensis (kaakoush et  al. 2005, ibrahim et al. 2019, gahamanyi et al. 2020). in many symptomatic cases, campylobacteriosis occurs as mild and self-limiting gastroenteritis, but long-term effects such as reactive arthritis (rea), post infectious irritable bowel syndrome (ibs), guillain barré syndrome (gbs), inflammatory bowel disease (ibd) and reiter’s syndrome (rs) may be introduction in recent years, campylobacteriosis has been the most frequently reported zoonotic disease in humans in europe (efsa 2017, tam et al. 2003). the most common sources of human campylobacteriosis are the handling or consumption of contaminated/undercooked meat (especially poultry), the handling or consumption of contaminated or unpasteurized milk and dairy products, the consumption of contaminated water, person to person contact, direct contact with carrier 58 veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 campylobacteriosis and role of dogs iannino et al. each questionnaire collected data on the risk factors for zoonotic transmission at the dog/human interface, such as habits and behaviors of the hosting families of the dog and/or other pets or domestic animals. the questionnaires included 28 questions grouped in different sections (table i). the introductory questions referred to the owner’s data and the identification and description of the veterinarian and the dog. the first group of questions referred to the veterinary visit and the general conditions of the dog (purpose of the veterinary-visit, health conditions, and nutritional status). the second group of questions referred to the dog’s risk factors for carriage and infection (nutrition, travel abroad, origin, life habits, places frequented). the third group of questions referred to the human risk factors for infection (contact with humans, dog's life habits, presence of other animals, composition of the family unit). collection of samples and laboratory examinations during the 2015-2016 period, rectal swabs were collected from 431 household dogs in veterinary clinics and 173 shelter dogs. a total of 3 veterinary clinics, located in the abruzzo, molise and tuscany regions and 10 shelters, five in the abruzzo and five in the molise region, were included in the study. from each dog, 2 rectal swabs were collected. the rectal swabs were gathered from the rectum of the animals using culture swab transport system (transystem™ amies with charcoal, copan, brescia, italy). all samples were transported at 4  °c in refrigerated boxes and processed immediately upon arrival to the laboratory and not later than 72 hours after sampling (at 4 °c). the isolation of campylobacter spp. was performed according to the world organisation of animal health (woah) (oie 2008) in modified charcoal cefoperazone deoxycholate agar (mccda) (thermo scientific oxoid, milan, italy) and karmali agar (italian biolife, milan, italy). both methods involved directly plating swabs and enriching in preston broth (italian biolife, milan, italy) for 24 hours in a microaerophilic atmosphere. after enrichment, 100 microliters of the preston broth were plated in duplicate on mccda and karmali plates and all plates (directly and after enrichment) were incubated under a microaerobic atmosphere at 41.5 °c and 37 °c for 48 hours. after incubation, the plates were examined to detect the presence of suspected of campylobacter  sp. colonies. the suspect campylobacter  colonies were identified by a multiplex pcr method, as described by wang and colleagues (wang et  al. 2002) for thermotolerant campylobacter  and by sequencing of the 16s  region associated with infection (keithlin et  al. 2014, esan et al. 2017, brooks et al. 2017). the role of dogs as a source of infection could be relevant (gras et al. 2013, koene et al. 2004). owning a pet, especially a puppy, has been identified as a risk factor for campylobacter sp. infection (doorduyn et al. 2010). in many cases, dogs are asymptomatic carriers of campylobacter spp. some studies found no significant relationship between diarrhoea and campylobacter sp. infection status (acke et  al. 2009), suggesting that the organism is commensal. conversely, other studies reported an association between infection and clinical signs particularly in relatively young dogs (guest 2007, chaban 2010). animals may be more susceptible to clinical disease when stressed by concurrent disease, hospitalization, shipment, pregnancy or surgery. acute campylobacteriosis that develops in puppies and some adult dogs is characterized by mucus-laden, watery or bile-streaked diarrhoea (with or without blood and leukocytes) of five to 15 days duration, partial anorexia, and occasional vomiting. elevated temperature and leukocytosis may also be present (fox 1990, brown et al. 1999). the close relationship between humans and dogs especially family pets, can play an important role in the transmission of zoonotic agents (stull et al. 2010). the present study aimed to analyse three important aspects of campylobacteriosis: • prevalence and diversity of campylobacter species in owned and shelter dogs; • their antimicrobial resistance; • identification of possible risk factors for zoonotic transmission at the dog/human interface. materials and methods the study has been divided into three phases: • drawing up and administration of questionnaires to dog owners visiting veterinary clinics; • collection of samples and laboratory examinations; • data analysis. questionnaire design and administration a total of 431 questionnaires were administered to different dog owners and compiled in case of the owner approval. the number of completed questionnaires was  412. 59veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 iannino et al. campylobacteriosis and role of dogs table i. questionnaire completed by dog owners visiting veterinary clinics. —cont’d owner name and surname home address and telephone number owner occupation veterinarian name and surname name of veterinary clinic and address animal transponder (microchip) name breed size sex date of birth coat description reason for clinical examination  1. routine examination/vaccination  2. fever  3. diarrhoea  4. vomiting  5. trauma  6. dermatitis  7. other antibiotic administration have antibiotics been administered?  yes  no if you answered 'yes' provide details:  in the last month  in the last 3 months  in the last year trade name of the drug feeding and body condition score feeding regime wet food (canned food) 70% to 80% of moisture content  regularly (principal component of food)  weekly (one or more times per week)  monthly (occasionally)  never semi‑moist food (snacks) 15% to 40% of moisture content  regularly (principal component of food)  weekly (one or more times per week)  monthly (occasionally)  never dry food (pellets) less than 10% of moisture content  regularly (principal component of food)  weekly (one or more times per week)  monthly (occasionally)  never cooked food or food for human consumption  regularly (principal component of food)  weekly (one or more times per week)  monthly (occasionally)  never raw meat  regularly (principal component of food)  weekly (one or more times per week)  monthly (occasionally)  never body condition score  1 ribs, lumbar vertebrae, pelvic bones and all bony, prominences evident from a distance. no discernible body fat. obvious loss of muscle mass.  1.5 ribs, lumbar vertebrae and pelvic bones easily visible. no palpable fat. some evidence of other bony prominence. minimal loss of muscle mass.  2 ribs easily palpated and may be visible with no palpable fat. tops of lumbar vertebrae visible. pelvic bones becoming prominent. obvious waist.  2.5 ribs easily palpable, with minimal fat covering. waist easily observed when viewed from above. abdominal tuck evident.  3 ribs palpable without excess fat covering. waist observed behind ribs when viewed from above. abdomen tucked when viewed from the side.  3.5 ribs palpable with slight excess fat covering. waist is discernible when viewed from above but is not prominent. abdominal tuck apparent.  4 ribs palpable with difficulty; heavy fat cover. noticeable fat deposits over lumbar area and base of tail. waist absent or barely visible. abdominal tuck may be present.  4.5 ribs not palpable under very heavy fat cover, or palpable only with significant pressure. heavy fat deposits over lumbar area and base of tail. waist absent. no abdominal tuck. obvious abdominal distension may be present.  5 massive fat deposits over thorax, spine and base of tail. waist and abdominal tuck absent. fat deposits on neck and limbs. obvious abdominal distention origin of the animal breeding stray dog other family shelter other travel abroad, life habits, places frequented travel abroad in the last 3 months  yes  no how long has the dog been housed with the current family? years months where does the dog live?  only inside the household  only outside the household  inside and outside the household continued 60 veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 campylobacteriosis and role of dogs iannino et al. (ccs) were assigned by submitting the dna sequence to the campylobacter  mlst database website (http://pubmlst.org/campylobacter). data analysis for the comparison of the prevalence rates between the two groups of data (household and shelter dogs), according to the literature (berger 1985), it was decided to use a bayesian approach, using a beta distribution with the relative 95% confidence intervals (cis). results questionnaire administration table ii shows the results of the questionnaires administered to the dog owners. most owners prefer dry food, even though a not negligible percentage of owners (4.8%) more or less regularly feed their dog with raw meat. most dogs live at home (37.4%), while 32.0% live outdoor, and 30.6% in both environments. in most cases, owners have contact with their dogs during meals (51.7%), and they have been licked by from their pets on their hands and face (72.8%). most of the dogs participants in the study live in an urban environment (254 out of 412, 61.7%) and in 69.7% of cases the majority of owners do not have other pets or domestic animals. sample and data analysis campylobacter spp. were isolated from 9 out of 431 household dogs (2.1%, ci 1.1%-3.9%) and from 13 out of 173 shelter dogs tested (7.5%; ci 4.5%-12.4%) (table  iii). the 95% cis of the prevalence rates calculated by the beta distribution do not overlap, thus showing a significant difference between the two percentages. (abiprism 3500, applied biosystem) for other campylobacter spp. genomic dna was extracted using an ultraclean microbial dna kit (mo bio laboratories, solana beach, ca, usa) according to the manufacturer’s instructions and quantified using a nanoddrop spectrophotometer (nanodrop technologies, celbio srl., milan, italy). campylobacter  strain susceptibility to antibiotics was evaluated with the microbroth dilution method using sensititre® custom susceptibility plates, eucamp 2 (trek diagnostic systems, biomedical service, venice, italy). the colonies were harvested in columbia agar for 24 hours then inoculated in mueller hinton broth supplemented with blood and dispensed into eucamp microtiter plates (trek diagnostic systems, biomedical service, italy), containing known scalar concentrations of the following antibiotics: gentamicin (gm) (0.12-16 µg/ml), streptomycin (s) (0.25-16 µg/ml), ciprofloxacin (cip) (0.12-16 µg/ml), tetracyclines (te) (0.5-64 µg/ml), erythromycin (e) (1-128 µg/ml) and nalidixic acid (na) (1-64 µg/ml). after inoculation, the plates were incubated at 42 °c under a microaerophilic atmosphere for 24 hours and then screened. c.  jejuni strain nctc 11351 was used as a quality control. for the evaluation of the minimum inhibitory concentration (mic), the swin v3.3 software (trek diagnostic systems, biomedical service, italy) was used in accordance with the epidemiological cutoff values (ecoffs) defined by the european committee on antimicrobial susceptibility testing (eucast, www.eucast.org) to interpret susceptibility. multilocus sequence typing (mlst) was performed using standard protocols as previously described (dingle et al. 2001). the dna of the samples subjected to mlst was extracted from the strains using a maxwell® 16  system automatic extractor (promega, it) according to the manufacturer's indications. sequence types (oie 2008), and clonal complexes table i. questionnaire completed by dog owners visiting veterinary clinics. —cont’d do you take your dog to public area?  yes  no does the dog lick family members’ hands and face?  yes  no what do you use to collect the dog’s feces?  paper towels  plastic bags  shovel  i do not collect the feces other do you clean your hands after any food manipulation and administration?  yes  no do you wash your hands after any contact with the dog?  yes  no do you touch your dog while you consume food?  yes  no do you allow the dog to get on the sofa/bed?  yes  no living environment of the family  urban  rural does the family have other animals?  yes  no family members id member sex  f  m kinship profession date of birth 61veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 iannino et al. campylobacteriosis and role of dogs our finding showed that the principal c. jejuni clonal complex identified in dogs were cc21, cc45, cc206, cc403, cc42 and cc658 (table iv). campylobacter  sp. isolates have demonstrated resistance mainly to tetracyclines, ciprofloxacin, nalidixic acid, and streptomycin. the resistance to four out of the 9 isolates in household dogs were identified as c.  jejuni, 2 as c.  upsaliensis, 1 was identified as c. coli, 1 as c. lari and 1 as c. vulpis. in shelter dogs, nine isolates were identified as c. jejuni, 3 as c. lari and 1 as c. coli (table iii). table ii. results of the questionnaires compiled by 412 owners of dogs visiting a veterinary clinic. —cont’d description of the sample age average age: 5.60 (+/‑3.602 standard deviation) sex breed sex no. of sampled dogs % female 137 33.3% male 275 66.7% total 412 100.0% breed no. of sampled dogs % mongrel 105 25.5% purebred dogs 307 74,5% total 412 100.0% dog size body condition score size number % large sized dog (adult weight more than 25 kg) 133 32.3% medium sized dog (adult weight between 10 kg and 25 kg) 137 33.3% small‑sized dog (adult weight between 1 kg and 10 kg) 142 34.5% total 412 100.0% score number % 1 0% 2 0% 3 5 1.5% 4 22 6.4% 5 62 18.1% 6 108 31.6% 7 105 30.7% 8 37 10,8% 9 3 0.9% total 342 100.0% presence of diarrhoea in the previsit period and antibiotic administration diarrhoea antibiotic administration onset time number % in the last three months 80 64.0% in the last 6 months 21 16.8% in the last year 24 19.2% total 125 100.0% administration number % in the last month 56 77.8% in the last 3 months 8 11.1% in the last year 8 11.1% total 72 100.0% antibiotic active active ingredient frequency of use for each antibiotic % amoxicillin 28 38.9% cephalosporin 16 22.2% metronidazole 6 8.3 amoxicillin, cephalosporin 4 5.6 cephalosporin, metronidazole 4 5.6% amoxicillin, metronidazole 2 2.8% enrofloxacin 2 2.8% marbofloxacin 2 2.8% metronidazole, spiramycin 2 2.8% tylosin, metronidazole 2 2.8% amoxicillin, cephalosporin, metronidazole 1 1.4% amoxicillin, itraconazole 1 1.4% amoxicillin, metronidazole, tylosin 1 1.4% enrofloxacin, cephalosporin 1 1.4% 72 100% continued 62 veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 campylobacteriosis and role of dogs iannino et al. table ii. results of the questionnaires compiled by 412 owners of dogs visiting a veterinary clinic. —cont’d feeding regime administered monthly (occasionally) administered regularly (main component of food) administered weekly (one or more times per week) number % number % number % wet food 7 1.7% 71 17.2% 14 3.4% semi‑moist food 19 4.6% 7 1.7% 56 13.6% dry food 10 2.4% 316 76.7% 7 1.7% homemade food 46 11.2% 69 16.7% 52 12.6% raw meat 12 2.9% 5 1.2% 3 0.7% origin and living place origin where does the dog live? number % breeding 189 45.9% other family 140 34.0% shelter 40 9.7% stray dog 36 8.7% born at home 3 0.7% pet shop 1 0.2% other 3 0.7% total 412 100.0% total % only inside the household 154 37.4% only outside the household 132 32.0% inside and outside 126 30.6% total 412 100.0% habits yes no number % number % does the dog licks your hands and face? 300 72.8% 112 27.2% do you clean the hands after any food manipulation and administration? 340 82.5% 72 17.5% do you wash your hands after any contact with the dog? 186 45.1% 226 54.9% do you touch your dog while consuming your food? 213 51.7% 199 48.3% do you allow the dog to get on the sofa/bed? 184 44.7% 228 55.3% what do you use to pick up dog's feces? number % plastic bags 168 48.8% paper towels 37 10,8% shovel 106 30,8 i don't take the feces 28 8.1% other 5 1.5% total 344 100% table iii. campylobacter species in household and shelter dogs. type of dog lifestyle campylobacter species isolated no. of positive samples % household dogs campylobacter jejuni 4 0.9% campylobacter upsaliensis 2 0.5% campylobacter vulpis 1 0.2% campylobacter lari 1 0.2% campylobacter coli 1 0.2% total 9 2.1% shelter dogs campylobacter jejuni 9 5.2% campylobacter lari 3 1.7% campylobacter coli 1 0.6% total 13 7.5% table iv. c. jejuni clonal complex (cc). clonal complex (cc) sequence type (st) n°strains 21 50 2 45 538 1 2,854 1 206 3,335 1 122 1 403 403 2 177 1 42 6,532 1 658 1,044 3 63veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 iannino et al. campylobacteriosis and role of dogs encourage dog owners to carry out an appropriate collection of feces. with regard to the second point, the prevalence of campylobacter spp. recorded in shelter dogs (13 out of 173, 7.5%, ci 4.5%-12.4%) has been significantly higher than the prevalence recorded in household dogs (9 positive out of 431, 2.1%, ci 1.1%-3.9%). among dogs resulted positive for campylobacter spp., no one had diarrhoea at the time of sampling, one had diarrhoea in the previous three months, one in the previous six months and one in the last year, confirming what is reported in the literature, i.e. that infected dogs usually do not show clear symptoms. in the literature reports, campylobacter  sp. prevalence in dogs varies greatly between authors (leonard et  al. 2011, acke et  al. 2009, giacomelli et  al. 2005, holmberg et  al. 2015) depending on the sampled population, the study design, and the analysis method. however, a higher prevalence is generally reported in shelter dogs, probably due to different hygiene and life conditions, increased stress factors, cohabitation with other dogs and contact with other animals, such as mice and rats. the majority of investigated household dogs have home habits, which means a reduced risk of contracting the pathogens investigated in the present study. life conditions of shelter dogs, on the contrary, are characterized by the housing in boxes which may include a different number of dogs and the presence of open common areas, delimited by fences, allowing more frequent contacts between animal and zoonotic agent’s carriers. in both cases, however, the prevalence recorded suggests the need to adopt precise hygiene protocols in the man/ dog relationship. the prevalence of campylobacter spp. was lower than that generally found in the literature. the presence of 13.6% of household dogs treated with antibiotics in the month preceding the veterinary visit could have influenced the results of the diagnostic tests. it was not possible to obtain information on the use of antibiotics in the shelters, but it would be appropriate to investigate this circumstance with further studies. this record of campylobacter  spp. in italian dogs, and of campylobacter  jejuni in particular, further highlights the risk related to the zoonotic potential of the pathogen. its diffusion may be favored by the lifestyle in man/animal relationship and by the close contact that many companion animals have with their owners. the third aspect of this study concerns the antibiotic resistance of campylobacter spp. in veterinary practice, in the daily clinics, the antibiotics administered to pets may represent a source of molecular pressure on the microorganisms, tetracyclines was the highest, while the resistance to erythromycin was the lowest (table v). there was a high number of strains with intermediate sensitivity (10 isolates, 76.9%). discussion this study obtained information on: • the assessment of behaviors in household dogs, which can be considered risk factors for the transmission of campylobacter  and other zoonotic agents • the prevalence of campylobacter  spp. in household and shelter dogs and its characterization, including the c.  jejuni clonal complex • the antibiotic resistance of the campylobacter spp. with regard to the first point, there are few data in the literature on the living habits of households’ dogs, especially based on market studies (boya et al. 2012) or sociological studies (charles 2016). the present study evaluated some behaviors and habits as possible risk factors for the transmission of zoonotic agents. notably, among these, the “habits” section of table ii shows very high percentages associated with habits that could be considered risky in the presence of infected dogs. moreover, the habit of feeding dogs with raw meat, as reported in the “feeding regime” section of the table ii, is carried out in a non-negligible percentage of cases (4.8%), therefore representing an additional risk factor (hellgren et al. 2019). data belonging to the section “origin and living place” especially with regard to attendance in outdoor spaces, cannot be directly related to campylobacter  infection, even if they can be considered as particularly significant risk factors. again, in the section “habits”, data show that the percentage of people who declare that they do not collect feces (8.1%) is still very high, thus contributing to increase the risk of pathogen’s transmission due to environmental contamination. collection with plastic bags, which is the most valid method for a thorough removal of feces, accounts for 48%. this indicates that much still needs to be done to table v. antimicrobial resistance of the isolated campylobacter spp. no. of resistant isolates % on total isolates tetracyclines 6 27.27% ciprofloxacin 5 22.73% nalidixic acid 5 22.73% streptomycin 2 9.09% erythromycin 1 4.55% 64 veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 campylobacteriosis and role of dogs iannino et al. of particular common habits, as shown by the data from the questionnaire. the very high percentages of people who do not wash their hands after contact with their dogs, allow their dogs to lick their face and hands and allow their dogs to sleep on the bed and sofa or eat raw meat increase the risk of zoonotic disease transmission. the higher prevalence of infection recorded among shelter dogs suggests that particular commitment should be devoted to staff training from people managing these premises, especially for people who is used to direct manage dogs and for this reason are more exposed to the risk of being in contact with campylobacter spp. the risk of contracting campylobacteriosis from dogs thus remains a concrete reality. prevalence control of campylobacter  spp. in household and shelter dogs would be considered important in order to reduce the transmission to humans. funding this study is part of a project (izsam 04/13 rc) financed by the italian ministry of health, co-ordinated by the istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ (teramo, ltaly). the study design was approved by the italian ministry of health. which, if put in favorable conditions, may acquire resistance to these molecules and transmit it to their offspring. however, restriction of the use of antimicrobials due to introduction of electronic prescription (ministero della salute 2019) should further improve this condition. the prevalence of antimicrobial resistance in ciprofloxacin (22.73%), nalidixic acid (22.73%), tetracyclines (27.27%), streptomycin (9.09%) and erythromycin (4.55%) found in this study was confirmed by other studies (andrzejewska et  al. 2013). this issue is worrying as these antibiotics are also commonly used in humans. antimicrobial resistance genes can be transferred to the intestinal microbial flora, and resistant commensal bacteria can constitute a reserve of resistant genes for potential pathogens (amar et al. 2014). the principal c. jejuni ccs in dogs were cc21, cc45, cc206, cc403, cc42 and cc 658. c.  jejuni ccs cc21 and cc45 are regularly isolated from multiple animal species, hindering a human source attribution. conclusions data regarding the prevalence of campylobacter infection in household and shelter dogs confirms a real risk of transmission to humans by dogs, even though the prevalence was not very high compared to other studies. this risk is higher in the presence 65veterinaria italiana 2022, 58 (1), 57-66. doi: 10.12834/vetit.2299.15789.1 iannino et al. campylobacteriosis and role of dogs acke e., mcgill k., golden o., jones b.r., fanning s. & whyte p. 2009. prevalence of thermophilic campylobacter species in household cats and dogs in ireland. vet rec, 164, 44-47. adak g.k., meakins s.m., yip h., lopman b.a. & o'brien 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italiana 2021, 57 (4), 341-345. doi: 10.12834/vetit.2114.12867.1 accepted: 27.05.2020 | available on line: 31.12.2021 1health and biosecurity, csiro, australia. 2medical entomology lab, institut pasteur of french guiana, 23 avenue pasteur, 97300 cayenne, french guiana. 3australian animal health laboratory, csiro, private bag 24, geelong, vic 3220, australia. 4agricultural research council ‑ onderstepoort veterinary institute, private bag x05, onderstepoort 0110, south africa. 5department of veterinary and tropical diseases, university of pretoria, private bag x04, onderstepoort 0110, south africa. 6district veterinary officer, benalla, vic, australia. 7school of biological sciences, the university of queensland, st lucia, 4072, qld, australia. *corresponding author at: medical entomology lab, institut pasteur of french guiana, 23 avenue pasteur, 97300 cayenne, french guiana. e‑mail: jbduchemin@pasteur‑cayenne.fr. jean-bernard duchemin1,2*, john r white3, antonio di rubbo3, shunin shi3, gert johannes venter4,5, ian holmes6 and peter j. walker7 keywords australia, bluetongue virus, culicoides, oral susceptibility, quantitative pcr, vector competence. summary following the emerging bluetongue virus transmission in european temperate regions, we question the vector competence of the abundant culicoides austropalpalis lee and reye in south-east temperate australia. field collected culicoides midges were membrane fed with a bluetongue virus serotype 1 (btv-1). the average feeding rate was 50%. after 13 days, survival rate was 25% and virus rna presence was checked by quantitative pcr targeting viral genome segment 10. virus rna was found in 7.4% of individually tested females with relative viral rna load values lower than freshly fed females, indicating that viral replication was low or null. a second qpcr targeting viral genome segment 1 confirmed the presence of virus rna in only four out of 29 previously positive specimens. after 10 days culture on culicoides cells, none of these four confimed positive samples did show subsequent cytopathogenic effect on vero cells or btv antigen detection by elisa. as control for this virus activity detection, 12 days after microinjection of btv-1, culex annulirostris mosquitoes showed, after culture on kc cells, cytopathogenic effect on vero cells, with elisa-confirmed infection. despite its abundance in farm environment of the temperate australian regions, the results of this study make c. austropalpalis of unlikely epidemiological importance in the transmission of btv in australia. experimental bluetongue virus infection of culicoides austropalpalis, collected from a farm environment in victoria, australia became endemic across part of northern europe (carpenter et al. 2009). these events suggested that the balance of factors that have allowed australia to remain free of the disease may be relatively fragile and could be disrupted by the invasion of exotic vectors or more virulent viruses from asia, changes in the distribution of endemic viruses or vectors, and/or adaptation of viruses to vectors with a more southerly distribution. the invasion of btv and/or its vectors into southern sheep farming zones of australia would have significant impacts both on animal health and the export trade. there are critical gaps in our understanding of the genetics, biology, bluetongue (bt) is a debilitating arbovirus infection of sheep and sometimes cattle, transmitted by culicoides biting midges (diptera: ceratopogonidae) with other domestic and wild ruminants serving as asymptomatic reservoirs, mostly. multiple serotypes of bluetongue virus (btv) are enzootic in northern and eastern australia (firth et  al. 2017). however, there has been no evidence to date of naturally occurring bt in australian livestock. in 2006, btv serotype 8 (btv-8) emerged unexpectedly in northern europe. although the source of this virus is unclear, btv-8 adapted to local palaearctic culicoides species, successfully over-wintered, and short communication 342 veterinaria italiana 2021, 57 (4), 341-345. doi: 10.12834/vetit.2114.12867.1 btv-1 culicoides austropalpalis competence duchemin et al. viral molecular detection by qpcr followed the technique of veronesi and colleagues (veronesi et  al. 2013). briefly, samples were homogenized in 200  µl of minimum essential medium for insect cell culture by bead beating at 6.0 m/s for 20 s with 10  x  1.0 mm beads. an aliquot of 100 µl was stored in -  80  °c for virus isolation. the remaining 100  µl was mixed with magmax® nucleic acid extraction buffer for both dna and rna extractions. btv-1 rna presence and quantification was assessed by using a btv real-time taqman pcr assay routinely run at aahl and targets the ns3 gene expressed by virus genome segment 10 (hofmann et al. 2008). primers and probe were pre-mixed and stored in small aliquots at - 20 °c (hofmann fwd r10 189-207 5’-tggayaaagcgatgtcaaa-3’, hofmann rev r10 285-266 5’-acatcatcacgaaacgcttc-3’, hofmann probe r10 245-264 5’-6fam-argctgcattcgcat cgtacgc-tamra-3’). cycle threshold (ct) values equal or higher than 40 were considered negative. duplicates were required to yield the same results for a firm positive or negative conclusion. specimens giving a positive/negative result for each duplicate were treated as inconclusive. in each qpcr run were included a strong and a weak positive controls with standard btv rnas, giving respective ct  values around 24 and 32, and a negative control without viral rna. to exclude any major effect of midge materials presence on molecular detection, a dilution curve was composed of four 10 x serial dilutions of tissue culture super natant (tcsn) virus mixed with individual unfed midges. quality of rna extraction was internally controlled by a separate taqman assay targeting 18s ribosomal rna with specimens showing higher than 25 ct values being discarded. the mean viral rna relative index (invdiff ) was estimated with standardization after the eukayotic 18s rna by the calculation of the multiplicative inverse of the difference between btv and 18s ct values [‘invdiff’ = 1 / (btv ct 18s ct)], (figure 1). a subset of 31 positive rnas, including freshly bloodfed specimens of midges and mosquitoes, were tested for another qpcr targeting btv segment 1 (shaw et  al. 2007) with another set of primers (btvrsa 291-311for 5' -gcgttcgaagtttacatcaat3', btvrsa 387-357rev 5'-cagtcatctctctagacactctataattacg3', probe rsa-btv 341-320 5' cggatcaagttcactccacggt 3'). similar to the first qpcr, strong and weak btv rnas positive controls, as well as a negative one and 18s were used in this pcr run. the confirmed double-positive d 13 culicoides specimens, with double-positive d 0 midges and mosquitoes as positive controls, were tested for presence of viable virus. more, egg homogenates previously injected with btv-1 infected blood or pbs were included as supplementary, positive and negative controls, respectively. after ten day amplification culture on kc/fli c. variipennis cells (kindly provided by istituto ecology and distribution of btv and culicoides in australia, and the potential consequences of global warming. for a better understanding of the btv transmission potential of the culicoides species present in the southern parts of australia, we have performed btv experimental infection of culicoides caught on a farm in the southern australian state of victoria. the collection and infection procedures are detailed in venter and colleagues (venter et al. 1998). briefly, onderstepoort uv traps were set up at a farm at benalla, victoria (145.9 e, 36.6 s, 170 m above sea level) for 33 trap-nights during march 2014. three to four traps were set per night close to resting animals (sheep, horses) in paddocks. insects were collected alive and self-sorted by exiting through meshed funnel into small cardboard containers. they were provided with 10% sucrose solution on cotton pads and placed in iceboxes with cold packs before transport to the laboratory by car for 3 hours. they were acclimated for 2 to 6 days at 23 °c before experimental infections. virus experiments were conducted in a biosafety level three (bsl-3) insectary at the australian animal health laboratory (aahl). supernatant of btv serotype 1 (btv-1) strain cs 156 (st george et  al. 1980) culture was diluted in heparinised cattle blood antibody-free for btv to a final viral titre between 3  x  105 and 1  x  106 tcid 50 /ml. sucrose ad libitum was stopped 24  h before infective challenge. midges were offered an infective blood meal through one day-old chicken skin using a membrane feeding system (hemotek®) in environmental cabinets at 15  °c, 50% relative humidity and darkness and allowed to feed for 60 min. after co 2 anaesthesia and sorting on an entomological chill table, only blood-fed females were reserved in cardboard cups, with 10% sucrose and maintained at 23 °c in darkness for 13 day (d 13 ) extrinsic incubation for final testing. unfed culicoides were returned to containers for re-feeding on the following day. controls for infection experiment were defined by infection of the non-vector culex annulirostris colony mosquitoes challenged with the same btv-1 strain either orally, as negative control, or by intra-thoracic microinjection of 69 nl of the undiluted virus batch corresponding to ~ 102 tcid 50 virus load. this micro-injection bypasses the midgut barrier and makes the mosquito able of viral infection in the hemolymph and as such, considered positive control. at d 13 , live midges and mosquitoes were anesthetised with co 2 and sorted on the chill table, identified at the species level (dyce et al. 2007) and stored at - 80 °c in individual tubes. the infection dynamics was assessed by comparison of presence and relative quantification of viral rna at d 0 , in either per os or microinjection freshly challenged insects and at d 13 in midges and mosquitoes, after extrinsic incubation time. 343veterinaria italiana 2021, 57 (4), 341-345. doi: 10.12834/vetit.2114.12867.1 duchemin et al. btv-1 culicoides austropalpalis competence did not differ from per os orally challenged mosquito values (kruskal-wallis test, p = 0.22). the prevalence of positive specimens (table i) at d 13 was 7.4% (31/416) for culicoides, 0% for bloodfed mosquitoes (0/14) and 100% (9/9) for microinjected mosquitoes. of 31 ns3 qpcr positive specimens, 29 (94%) were c.  austropalpalis, one c.  marksi and one undetermined. the positive rna values for these two last species were not different from those for c.  austropalpalis. all the inconclusive samples were c.  austropalpalis. for culicoides, d 13 average viral rna relative values were 0.036 (sd  =  0.005), with positive values at 0.052, (sd  =  0.0049). both were significantly (p  <  0.0001) lower than d 0 values. for microinjected mosquitoes, the d 13 average ct values at 0.099 (sd  =  0.019) was higher than d 0 but not significantly (p  =  0.223). however, this d 13 value for micro-injected mosqutioes was significantly (p < 0.001) higher than the per-os infected mosquito at d 13 average value of 0.029 (sd = 0.002) (figure 1). of the 29 ns3 btv-1 positive d 13 -infected culicoides austropalpalis tested, only four were tested positive for rsa segment 1 qpcr, six tested inconclusive and 19 were negative. as control, two d 0 infected culicoides were positive. in total, approximately 1% of the challenged culicoides females presented viral rna from two different genes. none of these four specimens with viral rna at d 13 showed cpe on vero cells, after amplification attempt during ten days on culicoides kc cells. as control, cpe was demonstrated in 100% of d 0 -infected culicoides [n  =  4, average optical density (do) 2.178 +/0.102] and 100% of mosquitoes infected at starting stage of infection (n =  9, average od 2.229 +/0.57). we performed experimental infections on midges caught at a victorian farm of mixed cattle and sheep, and challenged more than 3,000 culicoides by membrane feeding of btv-1 infected blood meal. we obtained a feeding rate of approximately 50%, comparable to experiments conducted in europe by carpenter and colleagues (carpenter et al. 2008) on field collected midges. our 25% survival rate was lower than the 39% obtained by carpenter and colleagues (carpenter et al. 2008) but the incubation period was longer: 13 days instead of 7-10 days and a membrane-feeding instead of pad-feeding technique was used. the viral rna detection method zooprofilattico sperimentale dell’abruzzo e del molise, izsam, italy) (wechsler et  al. 1989), samples were tested for cytopathogenic effect (cpe) on vero cells, and btv antigen detection by elisa technique following stanilawek and colleagues (stanilawek et al. 1996) and hawkes and colleagues (hawkes et al. 2000), using nunc maxisorp elisa immuno-plates (thermo fisher scientifictm) and insect cell supernatants. the controls of the cell culture phase were used. statistics and graphics were obtained by using r (r core team 2017) and ggplot2 package (wickham 2016). the rate of blood-feeding for culicoides was 49.8% (n  =  3293). the rate for mosquitoes was 90.6% (n = 53). the immediate (d 0 ) mortality rate was 15% for culicoides. the percentage survival for culicoides at d 13 was 25.2% (n  =  1641), 58.3% for bloodfed mosquitoes (n  =  24), and 37.5% for microinjected mosquitoes (n = 24). among the 416 culicoides females tested by qpcr, 94.5% were culicoides austropalpalis lee and reye, 1.7% culicoides marksi lee and reye, 1.2% culicoides victoriae macfie, 0.7% culicoides bundyensis lee and reye, 0.7% culicoides ornatus grp and 1.2% were not determined. the mean ct  values for taqman pcr assay of virus and midge serial dilution (from tcid 50 101 to 104) were 26.8, 23.7, 20.1, and 16.6, respectively. the invdiff (figure  1) for freshly bloodfed culicoides (d 0 ) were 0.071 (sd = 0.007) and 0.065 for mosquitoes (sd = 0.006), and do not differ significantly (kruskal-wallis test p  =  0.27). the d 0 value for microinjected mosquitoes was 0.074 and table i. prevalence of positive, negative and inconclusive ct values for ns3 btv-1 at rt-pcr d 13 . positive (%) negative (%) inconclusive (%) culicoides blood fed (n = 416) 31 (7.4) 338 (81.3) 47 (11.3) mosquitoes blood fed (n = 14) 0 (0) 12 (85.7) 2 (14.3) mosquitoes microinjected (n = 9) 9 (100) 0 (0) 0 (0) c ul ic oi de s p er o s m o sq u it o p er o s 0.12 0.10 0.08 0.06 0.04 m o sq u it o m ic ro -i n je ct ed c ul ic oi de s p er o s m o sq u it o p er o s m o sq u it o m ic ro -i n je ct ed d 0 d 12-13 in ve rs e o f d i� (b t v c t – 18 s c t) figure 1. boxplots of normalized btv rna index standardized to the 18s ribsoomal rna [y-axis: invdiff = 1/(btv ct – 18s ct)]. on the left side, red boxplots with values at day 0 for, from the left to the right: per os infected culicoides, per os infected mosquitoes, and microinjection infected msoquitoes. on the right side, with same order, blue boxplots for day 13 values. 344 veterinaria italiana 2021, 57 (4), 341-345. doi: 10.12834/vetit.2114.12867.1 btv-1 culicoides austropalpalis competence duchemin et al. readily extrapolate our results to other serotypes and especially exotic btvs. culicoides austropalpalis is reported to be particularly abundant in eastern australia (kettle and elson 1975), in south east queensland (wild 1984) and in victoria (present work). however, its vector capacity for livestock pathogens is potentially compromised by its predominantly ornithophilic behaviour (kat et  al. 1978). similarly, african bird feeder culicoides, being found btv positive in experimental infections (paweska et  al. 2002) and isolations (nevill et  al. 1992), have been considered of low epidemiological importance. however, feeding behaviour is rarely fully restrictive and c. austropalpalis has been found to also feed on cattle at a relatively high rate of 46% (van der saag et  al. 2016), and also on marsupials (kay et al. 1978). detection of viral rna of wallal virus (an orbivirus responsible for kangaroo blindness) in c.  austropalpalis (hooper et  al. 1999) suggests it may be a vector but btv has never been isolated from pools of c.  austropalpalis (standfast et  al. 1984). therefore, despite its abundance in the farm environment in australian temperate regions, our data and its mainly ornithophilic diet indicate that c. austropalpalis is of low, if not null, epidemiological importance for btv transmission. developed by veronesi and colleagues (veronesi et  al. 2013) allowed individual testing of midges with up-scaling to more than 400  midges. the dilution curve for positive controls gave consistent ct values. seven percent of the midges tested gave positive results for the presence of viral rna 13 days after infective feeding. however, the invdiff were siginificantly lower than at d 0 , indicating that viral replication was low or null. the culex annulirostris mosquitoes used as d 0 control gave not significantly different viral rna values, despite the probable blood meal volume difference between the two insects. however, and as expected with mosquitoes and btv, results became negative with time after oral challenge. however, following microinjection and the midgut barrier bypass, increased viral rna indices, indicative of viral replication, justified the mosquito model for positive control, as with other insect models (shaw et al. 2012). differences in vector competence according to virus serotypes or strains have been described (bellis et  al. 1994). our experiments were conducted to determine for vector competence for btv-1, the most common serotype (firth et al. 2017) circulating in the adjacent more northerly state of new south wales and one of the most probable to invade southern grazing regions. however, we cannot 345veterinaria italiana 2021, 57 (4), 341-345. doi: 10.12834/vetit.2114.12867.1 duchemin et al. btv-1 culicoides austropalpalis competence bellis g.a., gibson d.s., polkinghorne i.g., johnson s.j. & flanagan m. 1994. infection of culicoides brevitarsis and c. wadai (diptera: ceratopogonidae) with four australian serotypes of bluetongue virus. j med entomol, 31 (3), 382-387. carpenter s., mcarthur c., selby r., ward r., nolan d.v., luntz a.j.m., dallas j.f., tripet f. & mellor p.s. 2008. experimental infection studies of uk culicoides species midges with bluetongue virus serotypes 8 and 9. vet rec, 163 (20), 589-592. carpenter s., wilson a. & mellor p.s. 2009. culicoides and the emergence of bluetongue virus in northern europe. trends microbiol, 17 (4), 172-178. dyce a.l., bellis g.a. & muller m.j. 2007. pictorial atlas of australasian culicoides wings (diptera: ceratopogonidae). australian biological resources study, canberra. firth c., blasdell k.r., amos-ritchie r., sendow i., agnihotri k., boyle d.b., daniels p., kirkland p.d. & walker p.j. 2017. genomic analysis of bluetongue virus episystems in australia and indonesia. vet res, 48 (1), 82. hawkes r.a., kirkland p.d., sanders d.a., zhang f., li z., davis r.j., zhang n. 2000. laboratory and field studies of an antigen capture elisa for bluetongue virus. j virol methods, 85 (1-2), 137-149. hofmann m., griot c., chaignat v., perler l. & thür b. 2008. blauzungenkrankheit erreicht die schweiz. schweizer archiv für tierheilkunde, 150 (2), 49-56. hooper p.t., lunt r.a., gould a.r., hyatt a.d., russell g.m., kattenbelt j.a., blacksell s.d., reddacliff l.a., kirkland p.d., davis r.j., durham p.j., bishop a.l. & waddington j. 1999. epidemic of blindness in kangaroos evidence of a viral aetiology. austr vet j, 77 (8), 529-536. kay b.h., boreham p.f.l., dyce a.l. & standfast h.a. 1978. blood feeding of biting midges (diptera: ceratopogonidae) ast kowanyama, cape york peninsula, north queensland. austr j entomol, 17 (2),  145-149. kettle d.s. & elson m.m. 1975. variation in larvae and adults of culicoides austropalpalis lee and reye in s.e. queensland. j natural history, 9 (3), 321-336. nevill e., erasmus b.j., venter g.j., walton t. & osburn b. 1992. a six-year survey of viruses associated with culicoides biting midges throughout south africa (diptera: ceratopogonidae) in bluetongue, african horse sickness and related orbiviruses. proc. 2nd international symposium (t.e. walton & b.i. osburn, eds). crc press, boca raton, florida, 314 319. paweska j.t., venter g.j. & mellor p.s. 2002. vector competence of south african culicoides species for bluetongue virus serotype 1 (btv-1) with special reference to the effect of temperature on the rate of virus replication in c. imicola and c. bolitinos. med vet entomol, 16 (1), 10-21. r core team. 2017. r: a language and environment references for statistical computing. r foundation for statistical computing, vienna, austria. url https:// www.r-project.org/. shaw a.e., monaghan p., alpar h.o., anthony s., darpel k.e., batten c.a., guercio a., alimena g., vitale m., bankowska k., carpenter s., jones h., oura c.a.l., king d.p., elliott h., mellor p.s. & mertens p.p.c. 2007. development and initial evaluation of a real-time rt-pcr assay to detect bluetongue virus genome segment 1. j virol methods, 145 (2), 115-126. shaw a.e., veronesi e., maurin g., ftaich n., guiguen f., rixon f., ratinier m., mertens p., carpenter s., palmarini m., terzian c. & arnaud f. 2012. drosophila melanogaster as a model organism for bluetongue virus replication and tropism. j virol, 86 (17), 9015-9024. st george t.d., cybinski d.h., della-porta a.j., mcphee d.a., wark m.c. & bainbridge m.h. 1980. the isolation of two bluetongue viruses from healthy cattle in australia. austr vet j, 56 (11), 562-563. standfast h.a., dyce a.l., st george t.d., muller m.j., doherty r.l., carley j.g. & filippich c. 1984. isolation of arboviruses from insects collected at beatrice hill, northern territory of australia, 1974-1976. austr j biol sci, 37 (5-6), 351-366. stanislawek w.l., lunt r.a,. blacksell s.d., newberry k.m., hooper p.t., white j.r. 1996. detection by elisa of bluetongue antigen directly in the blood of experimentally infected sheep. vet microbiol, 52 (1-2), 1-12. van der saag m.r., gu x., ward m.p. & kirkland p.d. 2016. development and evaluation of real-time pcr assays for bloodmeal identification in culicoides midges. med vet entomol, 30 (2), 155-165. venter g.j., paweska j.t., van dijk a.a., mellor p.s. & tabachnick w.j. 1998. vector competence of culicoides bolitinos and c. imicola for south african bluetongue virus serotypes 1, 3 and 4. med vet entomol, 12 (4), 378-385. veronesi e., antony f., gubbins s., golding n., blackwell a., mertens p.p., brownlie j., darpel k.e., mellor p.s. & carpenter s. 2013. measurement of the infection and dissemination of bluetongue virus in culicoides biting midges using a semi-quantitative rt-pcr assay and isolation of infectious virus. plos one, 8 (8), e70800. wechsler s.t., mcholland l.e. & tabachnick w.j. 1989. cell lines from culicoides variipennis (diptera: ceratopogonidae) support replication of bluetongue virus. j invertebrate pathol, 54 (3), 385-393 wickham h. 2016. ggplot2: elegant graphics for data analysis. springer-verlag, new york. https://ggplot2. tidyverse.org. wild c. 1984. the influence of meteorological factors on capture of culicoides in flight (diptera: ceratopogonidae). the university of queensland, school of biological sciences. 151 veterinaria italiana 2021, 57 (2), 151-154. doi: 10.12834/vetit.2141.12114.1 accepted: 12.05.2020 | available on line: 31.12.2021 1department of veterinary medical sciences, university of bologna, italy. 2parco nazionale dei monti sibillini, visso, macerata, italy. 3institute of veterinary pathology, vetsuisse faculty, university of zurich, zurich, switzerland. 4azienda ausl, bologna, italy. * corresponding author at: department of veterinary medical sciences, university of bologna, italy. e-mail: antoniet.difrancesco@unibo.it. antonietta di francesco1*, federico morandi2, hanna marti3, carmela santagati4 and nicole borel3 keywords chlamydiaceae, chlamydia suis, italy, pcr, tetracycline resistance genes, wild boar. summary the aim of this study was to investigate the occurrence of chlamydia suis and tetracycline resistance determinants in conjunctival swabs of italian wild boars, by pcr. extracted dna collected from 50 wild boars from northern and central italy was examined by molecular methods. one sample (2%) from the central italy was positive for c. suis. fragments of tetr(c) and tetr(c)-tet(c) resistance determinants were amplified from the same sample. further molecular investigations suggested the attribution of these tetracycline resistance determinants to c.  suis, such as the truncation of tetr(c) and absence of a intact invasion (inv)-like region. while tetracycline-resistant c.  suis is very common in domestic pigs, its occurrence has not been reported in wild boar before. wild boar might acquire tetracycline resistance determinants through direct or indirect contact with domestic pigs. chlamydia suis and tetracycline resistance genes in italian wild boar (sus scrofa) short communication domestic pigs, including c.  suis (hotzel et  al. 2004, di francesco et  al. 2013). however, the occurrence of tetracycline resistant c.  suis in wild boar has not been reported so far. the aim of this study was to investigate the occurrence of c.  suis and related tetracycline resistance determinants in two geographically different italian wild boar populations. in 2017, conjunctival swabs were collected from 50 wild boars in two italian regions: 37 free-ranging animals were sampled alive for diagnostic investigations in a rural herd in northern italy and 13 wild boars were culled during a demographic control program taking place in a regional park located in central italy. the latter wild boars lived in the same geographical region where the presence of tetracycline resistant c. suis strains has previously been assessed in domestic pigs (donati et al. 2016). dna was extracted from the conjunctival samples using a commercial kit (dneasy kit qiagen, germany). the extracted genomic dna was screened by a chlamydiaceae-specific real-time polymerase chain reaction (rt-pcr) targeting a region of the 23s rrna gene conserved among all chlamydiaceae (ehricht chlamydia suis (order chlamydiales, family chlamydiaceae, genus chlamydia) is the most common chlamydial species reported in pigs (longbottom 2004), in which it has been associated with conjunctivitis, rhinitis, penumonia, enteritis and reproductive disorders. moreover, subclinical forms are highly prevalent among domestic pigs making them more susceptible to other infections (schautteet and vanrompay 2011). a stable tetracycline resistance phenotype has been detected in porcine c.  suis strains worldwide (lenart et  al. 2001, di francesco et  al. 2008, borel et  al. 2012, schautteet et  al. 2013, donati et  al. 2016, wanninger et al. 2016). the resistance pattern has been associated with tet(c) genomic islands integrated into the chlamydial chromosome containing genes encoding a tetracycline efflux pump and a regulatory repressor (tet[c] and tetr[c], respectively), a c.  suis-specific insertion element (iscs605) and additional genes involved in plasmid replication and mobilization (dugan et al. 2004). wild boar (sus scrofa) has been suggested to represent a wildlife reservoir for the same chlamydiaceae species as those detected in 152 veterinaria italiana 2021, 57 (2), 151-154. doi: 10.12834/vetit.2141.12114.1 tetracycline resistant chlamydia suis strains in italian wild boars di francesco et al. the tetr(c)-tet(c) intergenic spacer, that was deleted in the us r19 and r27 tetracycline-resistant c.  suis strains. the sequences obtained in this study were deposited in the genbank database under accession numbers mh845047, mh893740 and mh893741. in this study, pcr examination of conjunctival swabs collected from 50 wild boars in two italian regions revealed only one (2%) c.  suis positive sample. this is much lower than that reported in a previous italian study where anti-chlamydial antibodies were detected in 110 of 173 wild boar blood samples (63.6%), with a specific reactivity to c.  suis in 44 of 173 (25%) samples (di francesco et  al. 2011). in a follow-up study (di  francesco et  al. 2013), 22 out of 44 wild boars (50%) were pcr-positive when tested for chlamydiaceae and parachlamydiaceae by pcr. sequencing of the amplicons identified c. suis and c. pecorum in twelve (27%) and five (11%) samples, respectively. both studies were undertaken in regions with high prevalence of domestic pig breeding with outdoor access suggesting possible spread of infection through direct and indirect contacts between domestic pigs and wild boar. in contrast, the low chlamydial prevalence found in this study might be attributed to limited contacts between wild boars and domestic pigs due to strict biosecurity measures or low numbers of rural pig herds. this is in line with results of a very recent study (wahdan et  al. 2020) investigating 292  wild boars with limited contacts with domestic pigs from switzerland and northern italy resulting in a low c. suis prevalence (1.4%, 4/292). antimicrobial resistance (amr) is one of the most important public health challenges of our time and its spread has been attributed to clinical or farming overuse of antimicrobials. moreover, amr has also been reported in the absence of antimicrobial treatments, such as in wildlife in which antimicrobial resistance has been increasingly investigated. tetracyclines are one of the most classes of antimicrobial agents widely used in veterinary medicine and agriculture due to their broad spectrum of activity, low cost, oral administration, and few side effects. due to their widespread and indiscriminate use, tetracycline resistance has become one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms, also in wildlife. in wild boars, the presence of tetracycline resistant organisms, such as enterococci, salmonella spp., campylobacter spp., e. coli, staphylococcus aureus, has been documented (poeta et al. 2007, zottola et al. 2013, carbonero et al. 2014, sousa et al. 2017). in this study, a conjunctival dna sample from a c.  suis positive wild boar was investigated for the presence of tetracycline resistance determinants by pcr. in theory, the most rigorous scientific et  al. 2006). samples with ct values < 40 were considered positive and re-analysed by a rt-pcr assay targeting a c.  suis-specific region 23s rrna gene (pantchev et al. 2010). dna from c. suis-positive samples was used as a template for a 1,050-bp ompa gene fragment amplification (sayada et al. 1995). two additional pcr assays were performed on the c. suis positive samples in order to assess the presence and the features of tet(c) genomic island: one pcr amplified a 608 bp fragment including the tetr(c) region and another pcr amplified a 457 bp fragment of the tetr(c)-tet(c) region. the first pcr used the primer tetr-f (5’-ttggggcaaccatttctggt-3’) (donati et  al. 2016) and the primer cs38 (5’-ccaagggatgacgacgactg-3’) (dugan et  al. 2004). the second pcr was performed using the primer tetrc-f (5’-tgcgtcgagcaacgcacgct-3’) (donati et  al. 2016) and the primer cs43 reverted (5’-caaagcggtcggacagtgct-3’) (dugan et  al. 2004). moreover, a pcr targeting a 900 bp fragment of the intact invasion (inv)-like region was applied using primers cs02 (5’-cgtttcaggaatacccacttcg-3’) and cs106 (5’-acacttcaggttttcgccgtag-3’) according to dugan et al. (2004). dna of the italian eu-21 tetracycline-resistant c.  suis (donati et  al. 2016) and the swiss 2-2 tetracycline-sensitive c. suis (wanninger et  al. 2016) field isolates were used as controls. all the amplicons were purified using a qiaquick pcr purification kit (qiagen, hilden, germany) and both dna strands were sequenced (bio-fab research, rome, italy). the sequences obtained were compared with the public sequences available in genbank using the blast server from the national center for biotechnology information (http://blast.ncbi.nlm.nih.gov/blast.cgi). all animals (n = 37) from the northern italy region were pcr-negative for chlamydiaceae. one of the 13  (7.6%) wild boars from the central italy was positive to the chlamydiaceae-specific rt-pcr. the amplicon was identified as c.  suis by the specific 23s rrna pcr. sequencing of the ompa fragment revealed a nucleotide similarity of 88% with the eu-21 italian tetracycline-resistant c. suis strain and 87% and 93% with the us r27 and r19 tetracycline [resistant c.  suis strains described by dugan and colleagues (dugan et al. 2004)], respectively. tetr(c) and tetr(c)-tet(c) fragments were both amplified from the same wild boar sample, whereas no amplification was obtained using the intact inv-like primers, similarly to the results from eu-21 italian tetracycline-resistant c. suis strain. neither tetr(c) nor tetr(c)-tet(c) were detected in the swiss tetracycline sensitive 2-2 c. suis strain, whereas a 900 bp amplicon was obtained using the intact inv-like primers. tetr(c) and tetr(c)-tet(c) sequences were identical to the corresponding sequences of eu-21 italian c.  suis strain, showing the truncation of tetr(c) and the presence of a sequence of eight nucleotides in 153veterinaria italiana 2021, 57 (2), 151-154. doi: 10.12834/vetit.2141.12114.1 di francesco et al. tetracycline resistant chlamydia suis strains in italian wild boars of the invasion (inv)-like region, which is intact and therefore amplified in tetracycline-sensitive c.  suis strains, was not amplified in the wild boar dna sample, similarly to the tetracycline resistant c.  suis strains in which the (inv)-like region is interrupted due the insertion of the tet-island. tetracycline-resistant c. suis strains are very common in domestic pigs. domestic pig husbandry with outdoor access can be a source for transmission of c.  suis and/or tetracycline resistance genes to wild boars by direct contact or fecal contamination of the environment. further studies on c.  suis isolates from wild boars are needed to gain comprehensive epidemiological data on tetracycline resistance gene transmission, in particular focussing on wild boar populations with confirmed contact to domestic pigs. approach for the evaluation of antibiotic resistance in chlamydiae consists of evaluating the behavior of bacterial isolates in cell culture in the presence and absence of the drug. here, a culture-independent approach was chosen as fresh samples were not available, a common problem when performing field studies in wildlife. due to the lack of c. suis isolation, the attribution of the tet(c) gene to c.  suis cannot be confirmed and thus, the genetic determinants of resistance might not be specific for c.  suis, but might originate from other bacteria. however, the nucleotide sequence showed that tetr(c) in the positive wild boar sample was truncated, similarly to the tetracycline-resistant c.  suis strains described by dugan and colleagues (dugan et  al. 2004) and donati and colleagues (donati et al. 2016). in addition, the 900 bp fragment 154 veterinaria italiana 2021, 57 (2), 151-154. doi: 10.12834/vetit.2141.12114.1 tetracycline resistant chlamydia suis strains in italian wild boars di francesco et al. borel n., regenscheit n., di francesco a., donati m., markov j., masserey y. & popischil a. 2012. selection for tetracycline-resistant chlamydia suis in treated pigs. vet microbiol, 156, 143-146. carbonero a., paniagua j., torralbo a., 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2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 accepted: 06.07.2020 | available on line: 27.07.2021 department of veterinary microbiology college of veterinary science tirupati-517502 andhra pradesh, india *corresponding author at: department of veterinary microbiology, college of veterinary science, tirupati-517502, andhra pradesh, india. e-mail: bollinisreedevi@rediffmail.com. prathiba yanamala, bollini sreedevi*, vinod kumar nagaram and srilatha chintamaneni keywords isolation, characterization, virulent marek’s disease virus, india. summary marek’s disease (md) is one of the most significant neoplastic diseases of poultry caused by marek’s disease virus (mdv), an oncogenic avian herpesvirus which is responsible for great economic losses to the poultry industry worldwide. md is being manifested as an acute disease with lymphomas in multiple visceral organs. in the present study, an outbreak of md was investigated in one of the poultry farms from andhra pradesh, india. the gross lesions in the affected birds included lymphomas in different visceral organs like liver, spleen, proventriculus, heart and ovaries. histopathology revealed presence of uniform lymphoblastoid cell infiltration typical of md. the isolation of the virus was carried out in duck embryo fibroblast cells. after three blind passages, the cell cultures revealed plaque formation typical of mdv. further confirmation of the virus was carried out by pcr targeting 132 bp repeats of serotype-1 mdv and the oncogenes meq and vil-8 were amplified and sequenced. the nucleotide and phylogenetic analysis of the virus confirmed the virus as virulent serotype1 mdv. the present outbreak suggests the need for change in the vaccination regimen of md vaccination with appropriate serotype1 md vaccines in indian poultry flocks as the hvt and bivalent vaccines are unable to protect the flocks against virulent mdv. isolation and molecular characterization of a virulent marek’s disease virus serotype-1 from andhra pradesh, india vaccination with herpesvirus of turkey (hvt). the disease was well controlled by wide spread use of commercially available hvt vaccines. later on, bivalent vaccines consisting of hvt and serotype-2 herpesvirus were introduced to control the marek’s disease. lately, even after vaccination with bivalent vaccines, outbreaks of md have been reported in chickens of 21-40 weeks of age with high mortality rates (sudhakar and nair 2013). the increased evidence of md in vaccinated poultry flocks could be attributed to the continuing evolution of more virulent mdv strains despite wide spread vaccination (biggs and nair 2012, prathiba et al. 2018, torres et al. 2019). there was gradual emergence of virulent pathotypes of mdv ranging from mild (m) to virulent (v), very virulent (vv) and very virulent plus (vv+) in the field in different countries of the world (witter et  al. 2005, singh et  al. 2012, torres et  al. 2019, bertzbach et  al. 2020). in the present study, an outbreak of marek’s disease in an organized poultry farm from andhra pradesh, india was investigated. isolation of marek’s disease virus and introduction marek’s disease (md) is one of the most significant neoplastic diseases of poultry caused by marek’s disease virus (mdv) responsible for great economic losses to the poultry industry worldwide. mdv is an oncogenic avian herpesvirus classified as gallid alphaherpesvirus 2, genus mardivirus, family herpesviridae, subfamily alphaherpesvirinae. mdv is  divided into three serotypes mdv-1, mdv-2 and the antigenically related meleagrid alphaherpesvirus-1 known as serotype three (herpes virus of turkey) (dunn et  al. 2014, ictv 2018). only strains of serotype 1 are capable of causing disease, while mdv-2 and mdv-3 strains are avirulent (calnek, 2001). the disease was earlier described as paralytic/classical disease which is characterized by spastic paralysis of limbs with gross enlargement of peripheral nerves. but in the recent outbreaks, md has been characterised by an acute disease with lymphomas in multiple visceral organs. initially, chickens were protected efficiently by 30 marek's disease virus in indian poultry farms prathiba et al. veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 of 132 bp repeat is 182 bp long, containing one 132 bp repeat and 50 nucleotides from primers, the length of two copies of 132 bp repeats is 314 bp, and the length of three copies of 132 bp repeats is 446 bp. meq and vil-8 genes of marek’s disease virus were amplified which are responsible for the oncogenecity and pathogenecity of marek’s disease virus. the amplicon length of meq and vil-8 were 1,081 and 887 bps, respectively. the details of the primers used are shown in table i. standardization of pcr targeting 132 bp repeat region a positive md dna sample that was obtained from directorate of poultry research, rajendranagar, hyderabad was used for standardization of pcr. the optimum conditions for pcr of 132 bp repeat region were as follows: initial denaturation at 94 °c for 4  min, 35 cycles of denaturation at 94 °c for 1 min, annealing at 56 °c for 1 min, elongation at 72 °c for 1 min, final elongation at 72 °c for 10 min and hold at 4 °c for 5 min. isolation of mdv in duck embryo fibroblast cell cultures isolation of the marek’s disease virus was attempted as per the standard method of oie (oie 2010). briefly, buffy coat cells were suspended in minimum essential medium and freeze thawed for three times in order to lyse the cell membrane. then, centrifugation was performed and the virus inoculum was made ready by filtering the supernatant with 0.45 µm syringe filter. 0.5 ml of the inoculum was inoculated on to 70% confluent def grown in 25 cm2 tissue culture flask and incubated at 37 °c for one hour. after one hour of adsorption, the cells were rinsed and 8 ml of maintenance medium were added. an uninoculated def monolayer was maintained as a control. both the inoculated and uninoculated cultures were subsequently incubated at 37 °c in an incubator with 5% (v/v) co 2 atmosphere for 3-5  days. serial passages were made on def monolayers and, at further molecular characterization of oncogenes was carried out. materials and methods collection of samples in the present study, 18 tissue samples from postmortem cases and 2 blood samples from md suspected live birds were collected from an organized poultry farm of around 1,000 birds in andhra pradesh, india. the flock consisted of 6  month old layer birds presenting high mortality rate. the history of vaccination of zero day chicks with bivalent vaccine (hvt and sb-1) at hatcheries and sudden mortality with no clinical signs but enlargement of different visceral organs was reported. heparinized blood samples were collected in sterile vacutainers from live birds in md suspected flocks and were transported to the laboratory under refrigerated conditions. different organs including liver, spleen, kidneys, ovaries, heart, sciatic nerve, proventriculus with visible lymphomatosis and enlargement were collected in sterile sample containers from dead birds during postmortem for virological and molecular examination. part of the representative organs were also stored in 10% formal saline for histopathological examination. extraction of dna from tissues, blood and cell cultures the extraction of dna was done from tissues and buffy  coat cells using alkaline lysis-phenol-chloroform method (traditional method) and qiaamp® dna blood mini kit (kit method), respectively. the extraction of dna from duck embryonated fibroblasts (def) cell cultures was done using qiaamp® dna mini kit (qiagen) method as per the manufacturer’s instructions. the absorbance of the dna at wavelengths 260 nm and 280 nm was measured using nanodrop. the viral dna extracted from mdv infected tissue showed a260/a280 ratio of 1.8 to 2. oligonucleotide primers the oligonucleotide primers which were used in the present study were procured from eurofins genomics india pvt ltd (bangalore). the primer sequences reported by tian and colleagues (tian et  al. 2011) in mdv amplification were used in the present study. as the primers of 132 bp repeat region identifies serotype-1 specific mdv’s, it was considered as diagnostic primer for the detection of md which can differentiate serotype-1 field mdv strains from vaccine strains. the length of one copy table i. primers used in pcr for detecting marek's disease virus. gene primer sequence length (nucleotides) gc content (%) md-132 – f 5’ atg cga tga aag tgc tat gga g 3’ 22 45.5 md-132 – r 5’ atc cct atg aga aag cgc ttg a 3’ 22 45.5 meq – f 5’ ggc acg gta cag gtg taa aga g 3’ 22 54.5 meq – r 5’ gca tag acg atg tgc tgc tga g 3’ 22 54.5 vil-8 – f 5’ gag acc caa taa cag gga aat c 3’ 22 45.5 vil-8 – r 5’ tag acc gta tcc ctg ctc cat c 3’ 22 54.5 31 prathiba et al. marek's disease virus in indian poultry farms veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 – xcelris (ahmedabad) (genomics.corp@ xcelrislabs. com). the sequences obtained were submitted to the genbank via sequin software. the nucleotide sequences obtained were analyzed with genbank sequences by national center for biotechnology information – basic local alignment search tool (ncbi – blast) (www.ncbi.nlm.nih.gov/blastn) to check for homology. the nucleotide sequence of meq gene of ap-01 mdv was compared with the sequences of 26 reference and other mdv strains (table ii) using mega 10.0 software. the nucleotide sequence of vil-8 gene of ap-01 mdv was compared with the sequences of 14 reference and other mdv strains (table iii). phylogenetic analysis of the mdv isolate a phylogenetic tree of meq and vil-8 genes was constructed with the aligned sequences of reference mdv strains and ap-01 mdv isolate using mega 10.0 software by neighbour joining method using tajima nei model. results clinical signs and gross lesions birds with signs of paleness of combs and wattles, feather loss at neck region with enlargement of feather follicles were suspected for md. from these md suspected birds, blood samples were collected and buffy coat was separated. grossly, in majority of the md cases, the liver, spleen, proventriculus and kidneys were most commonly affected. sciatic nerve, ovaries, heart, mesentery and lungs were, less commonly, every passage, infected defs were checked for cytopathic effect and were preserved at 80 °c for further passages. standardization of pcr for oncogenes (meq and vil8) the pcr for amplification of oncogenes i.e. meq and vil-8 was standardized as per the method described by tian and colleagues (tian et  al. 2011). the optimum conditions for pcr of meq and vil8 genes were as follows: initial denaturation at 94  °c for 4 min followed by 35 cycles of denaturation at 94 °c for 1 min, annealing at 59.4 °c for 1 min, elongation at 72 °c for 1 min and final elongation at 72 °c for 10 min and hold at 4 °c for 5 min. sequence analysis of oncogenes pcr products of 132 bp repeat region, meq and vil-8 genes were sent for sequencing to genomics corp table ii. marek’s disease virus (mdv) reference strains of meq gene published in genbank. s. no. mdv strains virulence geographic origin accession number 1 cu-2 mild usa dq534538 2 cvi988 commercial vaccine netherland dq534538 3 bc-1 virulent usa ay362707 4 jm virulent usa ay243331 5 567 virulent usa ay362709 6 571 virulent usa ay362710 7 573 virulent usa ay362711 8 617a virulent usa ay362712 9 ga virulent usa m89471 10 md-5 very virulent usa af243438 11 rb-1b very virulent usa ay243332 12 549 very virulent usa ay362714 13 643p very virulent usa ay362716 14 595 very virulent usa ay362715 15 l very virulent plus usa ay362717 16 n very virulent plus usa ay362718 17 new very virulent plus usa ay362719 18 w very virulent plus usa ay362723 19 x very virulent plus usa ay362724 20 648a very virulent plus china af493558 21 584a very virulent plus usa dq534532 22 tn-n1 very virulent tamil nadu hm749324 23 tn-n2 very virulent tamil nadu hm749325 24 ind/ka12/02 isolate tamil nadu kp342383 25 ind/tn11/01 isolate tamil nadu kp342384 26 ind/tn12/03 isolate tamil nadu kp342385 table iii. marek’s disease virus (mdv) reference strains of vil-8 gene published in genbank. s. no mdv strains virulence geographic origin accession number 1 cu-2 mild usa eu499381 2 cvi988 commercial vaccine netherland dq534538 3 ls virulent china hq638183 4 lms virulent china hq658622 5 571 virulent usa dq534531 6 ind/tn11/01 virulent tamil nadu kp644421 7 ind/tn12/03 virulent tamil nadu kp644420 8 ind/ka12/02 virulent tamil nadu kp644422 9 ga virulent usa af147806 10 md5 very virulent usa af489275 11 rb1b very virulent usa ef523390 12 595 very virulent usa dq534533 13 648a very virulent plus usa dq534534 14 584a very virulent plus usa dq534532 32 marek's disease virus in indian poultry farms prathiba et al. veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 cells in between hepatic cords resulting in loss of hepatic architecture and replacement of hepatic parenchyma by infiltration of pleomorphic cells (figure 7). massive proliferation of lymphoblast cells and infiltration of pleomorphic cells were noticed in the spleen (figure 11). this led to lack of differentiation between red and white pulp and also lack of differentiation between splenic corpuscles and germinal centers. in the proventriculus, infiltration of pleomorphic cells into interstitial connective tissue with cystic dilatation and atrophy of proventricular glands was observed (figure 10). in the kidney, diffuse proliferation and infiltration of lymphocytes, plasma cells and macrophages in the interstitial connective tissue and partial to complete loss of renal architecture i.e. renal tubules and glomerulus were noticed (figure 9). ovary revealed affected. all these organs showed enlargement with discrete greyish white nodules of various sizes. in all observed cases, liver was enlarged several imes beyond its normal size with multifocal greysh white nodules of 2.5 in diameter (figure  1). multifocal nodules were observed in lungs (figure  2). enlargement of the organ upto the size of a small egg and numerous nodules/ lymphomas of the size of a pipoint to 1 mm distributed throughout the surface of the spleen were noticed in the majority of the samples (figure 3). in proventriculus, the wall was thickened and in few birds, ulceration and congestion of proventricular mucosa were noticed (figure 5). white multi focal nodules of 1 mm in size were observed in the heart (figure 6). in sciatic nerve, edematous lesion with loss of cross striations was observed in few birds (figure 4). histopathological changes the histopathological examination of liver showed extensive proliferation of pleomorphic lymphoid figure 1. enlarged liver with multifocal whitish nodules of a marek's disease virus infected chick. figure 2. multifocal nodules in lungs of a marek's disease virus infected chick. figure 3. spleen showing greyish white multifocal nodules of a marek's disease virus infected chick. figure 4. sciatic nerve showing edema and loss of cross striations of a marek's disease virus infected chick. 33 prathiba et al. marek's disease virus in indian poultry farms veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 in def, mdv positive samples showed cytopathic effect. the cpe included appearance of round refractile cells and after incubation for three days extensive infiltration of stromal tissue around graffian follicle and corpus luteum obliterating normal parenchyma of ovary (figure 8). pcr targeting 132 bp repeat region of serotype-1 mdv dna isolated from different tissue and blood samples was subjected to pcr targeting 132 bp repeat region as mentioned earlier. out of 18 tissue and 2 blood samples tested, all were positive for serotype-1 specific md yielding a 314 bp product in pcr amplification which indicates presence of two copies of 132 bp tandem repeats (264 bases) along with 50 bases of primers (figure 12). isolation of marek’s disease virus in duck embryo fibroblast cell cultures buffy coat lysate was inoculated into def monolayer and serial passages were made. after five passages figure 5. thickened pro-ventricular wall of a marek's disease virus infected chick with ulceration of mucosa. figure 6. multifocal greyish white nodules over the epicardial surface of heart of a marek's disease virus infected chick. figure 7. liver of a marek's disease virus infected chick showing proliferation of pleomorphic lymphoid cells with loss of hepatic architecture h & e x280. figure 8. ovary of a marek's disease virus infected chick with pleomorphic cell infiltration with obliteration of ovarian parenchyma h & e x280. figure 9. kidney of a marek's disease virus infected chick showing infiltration of lymphoblasts with obliteration of renal tubules h & e x280. 34 marek's disease virus in indian poultry farms prathiba et al. veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 characteristic cpe specific to mdv. dna extraction was performed using qiaamp® dna mini kit (qiagen). pcr was performed using primers targeting 132 bp repeat region of mdv. the sample showed a 314 bp pcr product confirming the presence of md serotype 1 virus in the cell culture fluids. appropriate negative controls were incorporated in the test. the isolate was designated as ap-01 mdv. post infection, each plaque was surrounded by the additional refractile cells at periphery with formation of syncytia. further incubation showed development of clear areas in the centre of the plaques six days post infection (figures 13 to 15). confirmation of mdv serotype-1 from cell cultures the cells were then freeze thawed three times and the cell culture fluids were harvested from cells showing figure 10. proventriculus of a marek's disease virus infected chick with infiltration of lymphoblasts in between proventricular glands h & e x280. figure 11. spleen of a marek's disease virus infected chick showing pleomorphic lymphoid cell infiltration h & e x280. figure 12. screening of marek's disease suspected field samples using pcr for 132 bp repeat region. lane m = molecular weight marker (100 bp); lane 1 = positive control; lane 2 = negative control; lane 3 to 6 = field samples positive for marek's disease virus serotype-1. figure 13. duck embryo fibroblast cell monolayer – normal. figure 14. duck embryo fibroblast cell monolayer infected with marek's disease virus (mdv) serotype-1 showing syncytia formation on day 3-pi. figure 15. duck embryo fibroblast cell monolayer infected with marek's disease virus (mdv) serotype-1 showing plaque formation with zone of clearance on day 7-pi. 35 prathiba et al. marek's disease virus in indian poultry farms veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 comparison of ap-01 mdv nucleotide sequence of vil-8 gene with genbank sequences comparison of the nucleotide sequence of vil-8 gene of ap-01 mdv showed from 99.44 to 99.87% homology with other sequences of mdv. the sequence of ap-01 had the highest homology (99.87%) with ls and lms virulent strains and had the lowest homology (99.44%) with cvi988 strain. when ap-01 mdv nucleotide sequence of vil-8  gene was compared with the reference strain lms, one variation at position 106 (tc) was shown. nucleotide sequences of vil-8 gene of ap-01 mdv showed 100% similarity with sequences of field isolates of tamil nadu (ind/tn11/01 and ind/ tn12/03) and karnataka (ind/ka12/02). comparison of ap-01 mdv amino acid sequences of oncogenes with genbank sequences the total amino acid length of meq protein was 339 amino acids and the total length of vil-8 protein was 134 amino acids. comparison of meq and vil-8 gene amino acid sequences of ap-01 mdv was done with those of reference strains and field isolates of tamil nadu and karnataka. the amino acid sequence of meq gene of ap01 was neither mild type nor very virulent plus mdv. there was a mutation at position 71 (sa) in the meq gene which is associated with higher virulence in the mdv strains. compared to closely related strains rb-1b (vv) and ga (v), ap01 showed variations in 77 (k to e), 80 (d to y), 139 (t to a), 209 (l to p) amino acid positions. compared to closely related strains 571 and 573 (v), ap01 showed variations in 115 (a to v), 139 (t to a), 176 (h to p), 209 (l to p) amino acid pcr and sequence analysis of oncogenes (meq and vil-8) from ap-01 mdv the dna obtained from ap-01 isolate was further subjected to pcr for amplification of oncogenes (meq and vil-8). the dna samples were found to be positive for the presence of oncogenes (meq and vil-8) and yielded amplicons of 1,081 bp and 887 bp, respectively (figures 16 and 17). the purified pcr products of meq and vil-8 were sent for sequencing to genomics corp – xcelris, ahmedabad. a total of 989 nucleotides of meq and 821 nucleotides of vil-8 were read. the obtained nucleotide sequences of meq and vil-8 genes were verified by ncbi-blast for homology analysis. the nucleotide sequences showed homology with meq and vil-8 genes of different strains of serotype-1 specific mdv strains. the nucleotide sequences of meq and vil-8 genes were submitted to genbank database. the accession number for meq gene is kt246100 and vil-8 is kt272874. comparison of ap-01 mdv nucleotide sequence of meq gene with genbank sequences comparison of the nucleotide sequence of meq gene of ap-01 mdv showed from 84.5 to 99.6% homology with other sequences of mdv. the sequences of ap01 had the highest homology with rb1b (99.6%) and ga  (99.5%) and had the lowest homology with cvi988 strain (84.5%). ap-01 showed nucleotide mutations at positions 229 (ag), 238 (gt ), 415 (ag), 626 (tc) of 1,020 meq gene in comparison with rb1b (very virulent) and ga (virulent) strains. ap01 nucleotide sequence showed variations at three positions 262 (ag), 278 (ga) and 626 (tc) with closely related tamil nadu mdv isolates. figure 16. screening of marek's disease suspected field samples using pcr for 132 bp repeat region. lane m = molecular weight marker (100 bp); lane 1 = positive control; lane 2 = negative control; lane 3 to 6 = field samples positive for marek's disease virus serotype-1. figure 17. screening of marek's disease suspected field samples using pcr for 132 bp repeat region. lane m = molecular weight marker (100 bp); lane 1 = positive control; lane 2 = negative control; lane 3 to 6 = field samples positive for marek's disease virus serotype-1. 36 marek's disease virus in indian poultry farms prathiba et al. veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 mdv nucleotide sequence with different reference and other strains in the genbank (n = 26) formed a cluster with 571, 573 virulent and other mdv strains from tamil nadu and karnataka as shown in the figure 18. the phylogenetic analysis of vil8 gene of the ap-01 mdv nucleotide sequence with different reference and other strains in the genbank (n = 14) formed a cluster with ls and lms virulent mdv strains and those of tamil nadu and karnataka as shown in the figure 19. discussion md almost devastated the poultry industry in the 1960s but the disease was brought under control after introduction of live vaccine containing hvt. subsequently, severe cases caused by more virulent strains of mdv which could not be controlled by using hvt vaccination occurred. hence, bivalent vaccines consisting of hvt and attenuated serotype-2 were developed and used in the field. the widespread use of vaccines against marek's disease has been suggested to have led to the evolution positions. the deduced amino acid sequences of meq protein of ap01 mdv showed three variations in the amino acid sequence at positions 88 (ta), 93 (rq) and 209 (lp) compared with field isolates of tamil nadu (ind/tn11/01 and ind/tn12/03) and karnataka (ind/ka12/02) (table iv). alignment analysis of the deduced amino acid sequence of vil-8 gene of ap01mdv and 14 published mdvs was performed. comparison of amino acid sequence of vil-8 protein of ap01 with that of reference strains like cu-2, ga, rb1b, 648a, 584a, md5, 595, 571 and cvi988 showed that mutations exists at position 4 (ls) and 31 (dg). the amino acid mutations at position 4 and 31 were similar to the virulent reference strains lms and ls. the amino acid sequence was 100% identical with field isolates of tamil nadu (ind/tn11/01 and ind/ tn12/03) and karnataka (ind/ka12/02) (table v). phylogenetic analysis of meq and vil-8 genes of mdv the phylogenetic analysis of meq gene of the ap-01 table iv. aminoacid substitutions in the meq protein of marek's disease virus serotype-1 (mdv-1) field strains with reference strains. s. no virulence & field samples strain name amino acid substitutions in the meq protein of mdv-1 field strains and reference strains 71 77 80 88 93 115 119 139 153 176 180 209/ 268a 217/ 276a 277/ 336a 283/ 342a 320/ 379a 326/ 385a 1 vv strain rb1b a k d a q v c t p p t l p l a i t 2 v strain ga . . . . . . . . . . . . . . . . . 3 v strain 567 . e y . . . r . . . . . a . . . . 4 v strain 617a . e y . . . r . . . . . a . . . . 5, 6 v strain 571, 573 . e y . . a . . . h . . . . . . . 7 field sample ap01 meq . e y . . . . a . . . p . . . . . 8 tn isolate ind/tn11/01 . e y t r . . a . . . . . . . . . 9 tn isolate ind/tn12/03 . e y t r . . a . . . . . . . . . 10 ka isolate ind/ka12/02 . e y t r . . a . . . . . . . . . 11 vv isolate tn-n1 . . . . . . . . . . . p . . . . . 12 vv isolate tn-n2 . . . . . . . . . . . p . . . . . 13 v strain bc-1a s a . . . a . . . . . . . . . . . 14 v strain jma s a . . r a . . . . . . . . . . . 15 m strain cu-2a s e . . . . . . . . . . . . . . . 16 vaccine strain cvi988a s e . . . . . . . . . . . . . . i 17 vv strain md5 . . . . . . . . . . . . a . v t . 18 vv+ strain w . . . . . . . . . . . . a . v t . 19 vv strain 643p . . . . . . r . q a a . a f . . . 20, 21 vv+ strain new, 584a . . . . . . r . q a . . a . v t . 22, 23 vv strain 549, 595 . . . . . . r . q a a . a . . . . 24, 25 vv+ strain l,x . . . . . . r . q a a . a . . . . 26 vv+ strain 648a s e . . . a . . . . . . . . . . . 27 vv+ strain n . . . . . . r . q a a . a p . . . a denote strains containing 59 amino acid proline rich repeat amplification 37 prathiba et al. marek's disease virus in indian poultry farms veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 nerve, ovaries, heart, mesentry and lungs were also found involved in some of the birds. pcr targeting 132 bp repeat region was standardized and all the samples yielded a 314 bp product in pcr indicating the presence of two copies of 132 bp tandem repeats (doosti and golshan 2011, tian et al. 2011). it has been reported that no comparable 132 bp repeat region exists in either serotype-2 or hvt (silva et  al. 2004). the results of sequencing confirmed the presence of pathogenic serotype-1 of field viruses with greater virulence. a number of pathotypes classified as vmdv, vvmdv, and vv+mdv have in fact been isolated (witter 2001, witter et al. 2005). these more virulent strains could overwhelm the protection conferred by currently available vaccines (witter 1997). these virulent strains of mdv are resulting in production of lymphomas in different visceral organs. in the present study, 18 tissue samples from postmortem cases and 2 blood samples from md suspected live birds were collected from an organized poultry farm, tirupati, andhra pradesh. some of the birds were found dead without showing clinical signs. some birds were found depressed with paleness of combs and wattles, feather loss at neck region with feather follicle enlargement. lately, clinical picture of md is slowly changing from the classical form of marek’s disease characterised by birds showing nervous symptoms with unilateral paralysis with typical posture of one leg stretched forward and other leg backward. in the current outbreak, the birds were apparently normal without any clinical manifestation as the disease progression is more towards the formation of lymphomas rather than the classical nervous manifestation. similar observations were also reported by other researchers where the involvement of oncogenic more virulent serotype-1 mdv strains was reported (okwor and eze 2011, sudhakar and nair 2013, puro et al. 2018). the postmortem examination of the md suspected birds in the present investigation revealed the involvement of liver, spleen, proventriculus and kidneys in majority of the cases. organs like sciatic table v. amino acid substitutions in the vil-8 protein of marek's disease virus serotype-1 (mdv-1) field and reference strains. s.no virulence and field samples strain name 4 31 67 81 1 v strain lms s g m e 2 v strain ls . . i g 3 field sample ap01 vil-8 . . i . 4 vv ind/ka12/02 . . i . 5 v ind/tn11/01 . . i . 6 v ind/tn12/02 . . i . 7 v strain 571 l d i . 8 v strain ga l d i . 9 vv strain rb1b l d i . 10 vv strain md5 l d i . 11 vv strain 595 l d i . 12 vv + strain 584a l d i . 13 vv + strain 648a l d i . 14 m strain cu-2 l d i . 15 vaccine strain cvi988 l d i . figure 18. phylogenetic analysis of meq gene nucleotide sequence of ap-01 with other reference sequences. the phylogenetic tree was constructed using the mega version 10.0 by the neighbor joining method with 1,000 bootstrap replicates using tamura nei model. figure 19. phylogenetic analysis of vil8 gene nucleotide sequence of ap-01 with other reference sequences. the phylogenetic tree was constructed using the mega version 10.0 by the neighbor joining method with 1000 bootstrap replicates using tamura nei model. 38 marek's disease virus in indian poultry farms prathiba et al. veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 china ( zhang et  al. 2013). the deduced amino acid sequences of meq and vil8 genes of ap01are hence comparable to virulent/very virulent mdvs. in the very virulent plus mdvs, there was an amino acid mutation at position 119 (cr). higher virulence (vv and vv+) mdvs had point mutations at the positions 153 (pq), 176 (pa) and 217 (pa) which were not present in the amino acid sequences of ap-01in the present study. the alignment analysis of the deduced amino acid sequences of vil-8 gene of ap-01 was similar to the lms and ls (virulent) reference strains and other virulent mdvs reported from india. the present study showed that meq is the most important oncogene which shows variable mutations in mdvs of different virulence. the meq gene is present only among mdv oncogenic strains. the strongest association with the observed increased virulence is the polymorphisms identified in the major oncoprotein and transcription factor meq. despite the rather low evolutionary rate of double-stranded dna viruses (firth et  al. 2010, trimpert et  al. 2019), it has been reported that the meq gene is evolving at a much faster rate than most genes in double-stranded dna viruses (padhi and parcells 2019). the vaccine strains i.e. hvt and mdv serotype-2 lack meq gene, it is therefore likely that the changes in meq gene might have occurred as a result of functional selection for their effects on mdv and/or cellular gene expression. hence, it can be concluded that mutations in the putative oncogene meq appear to be correlated with increased mdv virulence. similar results were recorded by kumar and colleagues (kumar et  al. 2012) and suresh (suresh 2013). in the present study, the samples were collected from md outbreaks of chicken which received hvt and sb-1 bivalent md vaccines. based on the nucleotide, amino acid, phylogenetic analysis and the vaccination status of the affected flocks, the mdv strain obtained in the present outbreak of andhra pradesh could be designated as virulent/ very virulent mdv. the control strategies for mdv in poultry flocks includes the use of live vaccines and maintenance of good hygiene and management in the poultry farms. initially, the hvt live vaccine was developed and administered for several years in india. as the protection conferred by this vaccine was not optimum, a bivalent vaccine including hvt and an avirulent serotype 2 mdv was developed. as mdv strains evolved becoming more virulent, bivalent vaccines were not anymore capable of protecting poultry flocks. in most of the european countries, usa and china, more efficacious serotype-1 md vaccine with cvi988/rispens strain is being successfully used (kumar et  al. 2012, dunn and gimeno 2013). the increasing virulence of mdv may pose a threat to the standard md prevention mdv strains in the field samples tested in the present study. hence, pcr targeting 132 bp repeat region can be used for specific detection of serotype-1 mdv strains and can differentiate the pathogenic mdv strains from that of vaccine strains. attempts for the isolation of the mdv was carried out in def cell cultures. after three subsequent blind passages, a characteristic cpe of serotype-1 md virus was observed in the infected cell cultures. after three day of inoculation, the monolayers showed the appearance of round refractile cells with formation of syncytia. after further 3 days of incubation clear areas developed in the centre of plaques. the cpe was more pronounced and clear in further passages (5th to 7th). similar cpe was reported in def monolayers by gopal and colleagues (gopal et  al. 2012) and gong and colleagues (gong et  al. 2013). the presence of serotype-1 mdv in the cell culture fluid was confirmed by extraction of dna and pcr for 132 bp repeat region. the genome of serotype-1 mdvs contains several oncogenes which are important in viral pathogenesis and in the clinical manifestation of marek’s disease viruses in the field outbreaks. in the present outbreak, affected birds showed the presence of lymphomas in different visceral organs. hence, further studies were carried out for characterization of viral oncogenes. the nucleotide sequence of meq gene of ap01 had the highest homology with rb1b (99.6%) and ga (99.5%) strains and had the lowest homology with cvi988 strain (84.5%). the nucleotide sequence of vil8 gene of ap01 showed the highest homology (99.87%) with the ls and lms virulent strains and the lowest homology (99.44%) with cvi988 strain. it was 100% identical with the sequences of ind/tn11/01, ind/ka12/02 and ind/tn12/03. the deduced amino acid sequences of meq and vil-8 genes of ap-01 were compared with those of the reference and other previously reported mdv strains. the amino acid variations are mostly located at nine positions i.e. 71, 77, 80, 115, 119, 139, 153, 176 and 217 (tian et  al. 2011, gong et  al. 2013, sathish et al. 2014). low virulence mdv strains including cu-2, bc-1, jm and cvi988 showed 59 amino acid insertions with proline-rich repeats in the deduced amino acid sequences of meq gene. the amino acid sequences of ap-01 mdv were of 339 amino acids without the 59 amino acid proline rich insertion indicating that these strains belong to more virulent mdv strains. in  ap-01, the amino acid positions 71 and 77 showed the presence of alanine and glutamate, respectively. the amino acid mutations at positions 80 (aspartatetyrosine), 139 (threoninealanine) were also observed. the mutation in positions 80, 115, 139, 176 of meq protein could be used as virulent genetic characteristic of the circulating mdvs in 39 prathiba et al. marek's disease virus in indian poultry farms veterinaria italiana 2021, 57 (1), 29-39. doi: 10.12834/vetit.2197.13279.1 in india, we are still practicing single or bivalent vaccines and serotype-1 md viruses are not yet used for vaccination in the field. in the context of emergence of more virulent md viruses in the southern parts of india and failure of present md vaccines, it is appropriate to recommend usage of more efficient serotype-1 mdv vaccine strains in the poultry flocks to reduce the marek’s disease incidence in the country. strategy, progressively reducing the success of vaccine protection, especially for programs based on hvt strain vaccines. research and continuing surveys may provide answers regarding the epidemiology of md, the evolving virulence of circulating mdv strains, and might enable determining the best fit vaccination protocols and strategy (bertzbach et al. 2020). biggs p.m. & nair v. 2012. the long view: 40 years of marek’s disease research and avian pathology. avian pathol, 41, 3-9. doosti a. & golshan m. 2011. molecular study for detection of marek´s disease virus (mdv) in southwest of iran. sci res essays, 6 (12), 2560-2563. dunn j.r. & gimeno i.m. 2013. current status of marek’s disease in the united states and worldwide based on a questionnaire survey. avian dis, 57, 483-490. gong z., zhang l., wang j., chen l., shan h., wang z. & ma h. 2013. isolation and analysis of a very virulent marek’s disease virus strain in china. virol j, 10, 115. gopal s., manoharan p., kathaperumal k., chidambaram b. & divya k.c. 2012. differential detection of avian oncogenic viruses in poultry layer farms and turkeys by use of multiplex pcr. j clin microbiol, 50, 2668-2673. kumar p., dong h., lenihan d., gaddamanugu s., katneni u., shaikh s., hotz p.t., reddy s.m., peters w. & parcells m.s. 2012. selection of a recombinant marek’s disease virus in vivo through expression of the marek’s ecori-q (meq) – encoded oncoprotein: characterization of an rmd5-based mutant expressing the meq of strain rb-1b. avian dis, 56, 328-340. okwor e.c. & eze d.c. 2011. outbreak and persistence of marek’s disease in batches of birds reared in a poultry farm located in nsukka, south east nigeria. int j poult sci, 10 (8), 617-620. prathiba y., sreedevi b., vinod kumar n. & srilatha ch. 2018. molecular characterization and phylogenetic analysis of oncogenes from virulent serotype i marek’s disease virus in india. acta virol, 62, 277-286. sathish g., divya k.c., parthiban m. & aruni a.w. 2014. in silico analysis show incidence of very virulent marek’s references disease virus (mdv). int j comput bioinfo in silico model, 3 (3), 374-380. silva r.f., reddy s.m. & lupiani b. 2004. expansion of a unique region in the marek's disease virus genome occurs concomitantly with attenuation but is not sufficient to cause attenuation. j virol, 78 (2), 733-740. singh s.d., barathidasan r., kumar a., deb r., verma k. & dhama k. 2012. recent trends in diagnosis and control of marek’s disease (md) in poultry. pak j biol sci, 15, 964-970. sudhakar s. & nair a.j. 2013. marek’s disease: the never ending challenge – a review. int j pharm bio sci, 4 (2),  6-11. suresh p., dorairajan n., balachandran c. & manohar b.m. 2013. incidence of marek's disease in namakkal, tamil nadu. cheiron, 19 (3), 143-144. tian m., zhao y., lin y., zou n., liu c., liu p., cao s., wen x. & huang y. 2011. comparative analysis of oncogenic genes revealed unique evolutionary features of field marek’s disease virus prevalent in recent years in china. j virol, 8, 121-131. witter r.l., calnek b.w., buscaglia g., gimeno i.m. & schat k.a. 2005. classification of marek’s disease viruses according to pathotype: phylosophy and methodology. avian pathol, 34 (2), 75-90. world organisation for animal health (oie). 2010. manual of diagnostic tests and vaccines for terrestrial animals. paris, oie. zhang d., dai y., zhao r., hu x., hou h., pan x. & zhou x. 2013. molecular cloning and sequence analysis of meq gene of marek’s disease virus. int j food agricul environ, 11 (3-4), 1005-1008. 1 a r t i c l e a h e a d o f p r i n t veterinaria italiana 2022, xx (x), xxx‑xxx. doi: 10.12834/vetit.2442.15397.1 accepted: 02.09.2021 | available on line: xx.xx.2022 1national veterinary research institute, nigeria. 2faculty of veterinary medicine, ahmadu bello university zaria, nigeria. 3faculty of veterinary medicine, university of ilorin, nigeria. 4 national veterinary research institute, vom, nigeria. *corresponding author at: national veterinary research institute, nigeria. e‑mail: yinkadeji@yahoo.com. adeyinka adedeji1*, paul abdu2, olatunde akanbi3 and pam luka4 keywords histopathology, nigeria, marek’s disease, pcr, vaccinated. summary marek’s disease (md) is a devastating neoplastic disease of poultry caused by md virus (mdv). md is one of the several diseases limiting the thriving nigerian poultry industry. md is mostly diagnosed in nigeria based on history and gross lesions without laboratory investigations leading to underreporting of the disease. this study investigated md outbreaks in poultry farms using polymerase chain reaction (pcr) and histopathology. tumourous visceral organs were collected from dead chickens presented to veterinary clinics from 110 farms in plateau state, north central nigeria from april 2013 to august 2014. clinical signs observed in affected chickens were paralysis, stunting and uneven growth. whilst the gross lesions observed were hepatomegaly, splenomegaly with lymphoma, prominent peripheral nerves and cachexia. the meq gene of mdv‑1 was detected by pcr in 55.0% (n = 11/20) of broilers and 71.1% (n  =  64/90) of vaccinated layer chicken samples collected. microscopy revealed severe diffuse lymphocytic infiltrations in the heart, spleen and liver of chickens with tumourous gross lesions. based on history, gross lesions, detection of meq gene of mdv‑1 by pcr and histopathology results, md was confirmed in the affected farms. despite vaccination, outbreaks of md still occurs in poultry farms in nigeria. this study represents the first confirmatory diagnosis of md in vaccinated poultry in nigeria please refer to the forthcoming article as: adedeji a. et al. 2022. molecular and pathological investigations of marek’s disease outbreaks in vaccinated poultry farms in plateau state, north central‑nigeria. vet ital. doi: 10.12834/vetit.2442.15397.1. molecular and pathological investigations of marek’s disease outbreaks in vaccinated poultry farms in plateau state, north central‑nigeria belong to serotype 1 (nair et al. 2020). furthermore, pathogenic mdv serotype 1 or mdv‑1 are classified into four pathotypes i.e. mild mdv (mmdv), virulent mdv (vmdv), very virulent mdv (vvmdv) and very virulent plus mdv (+vv mdv) (payne 2004, gimeno and pandiri 2013). chickens are known to be the most important natural and susceptible host to md, although the disease has been reported in quails, turkeys, pheasants, game fowls, ducks, sparrows, partridges, pigeons, and red crown cranes (murata et  al. 2012, schat and nair 2013, schock et  al. 2016, lian et al. 2018, adedeji et al. 2019). md is distributed worldwide with annual economic losses estimated to be $1‑2 billion dollars (morrow and fehler 2004, introduction marek’s disease (md) is a highly contagious and lymphoproliferative neoplastic disease of poultry associated with severe economic impact caused by the ubiquitous md virus (mdv) (biggs and nair 2012, bertzbach et  al. 2020). mdv belongs to the order herpesvirales, family herpesviridae, subfamily alphaherpesvirinae and genus mardivirus (davison 2010, gimeno and schat 2018). there are three members of the genus mardivirus; gallid herpesvirus 2 or mdv serotype 1 (mdv‑1), gallid herpesvirus 3 or mdv serotype 2 (mdv‑2) and herpesvirus of turkeys (hvt) or mdv serotype 3 (nair 2005, gimeno and schat 2018). all virulent or oncogenic strains of mdv 2ar ti cl e ah ea d of p ri n t veterinaria italiana 2022, xx (x), xxx‑xxx. doi: 10.12834/vetit.2442.15397.1 are layer stock for egg production (fao 2019). most of the hatcheries in nigeria are concentrated in the south‑western part of the country, where 80% of the day old chicks (doc) are hatched and transported to the rest of country (akinwumi et  al. 2010, jwander et  al. 2015). the hatcheries routinely vaccinate against md using either hvt or a combination of mdv rispens‑cvi988 and hvt. since the first report of md in nigeria in 1962, several outbreaks of md have reported with increasing prevalence of the disease in poultry farms (hill and davis 1962, owoade et al. 2008, okwor and eze 2011, jwander et al. 2015, sani et al. 2017). inasmuch md is not a reportable disease, veterinary clinics in nigeria regularly records cases of the disease in farms. hence the impact of md on poultry production in nigeria is unknown and coupled with the fact that diagnosis on the field is not supported by laboratory confirmation. there is a paucity of data on the md outbreaks in nigeria and description of the epidemiological features are not documented, particularly in relations to the possible role of poultry husbandry systems as drivers of the disease. hence, md poses a constant threat to poultry farms in nigeria. this study presents the molecular and pathological investigations of md outbreaks in poultry farms in plateau state, nigeria. materials and methods study area plateau state is located in north central nigeria with 17 local government areas (lga) and population of 3,500,000 people. the study area covers five local government areas (lga) of plateau state; jos‑north, jos‑south, jos‑east, bassa and bakin ladi (figure  1). most of the poultry farms in plateau state are located in the above mentioned lgas. plateau state is situated approximately on latitude 9.60 n, 10. 20 n and longitude 8.50 e, 9.10 e with a more temperate climate than the rest of nigeria. the state is best suited for poultry production with average monthly temperatures of 21 °c‑25 °c that sometimes drops as low as 11 °c. livestock production particularly poultry farming is a major economic activity in plateau state with over 3,000 poultry farms. the poultry production system in plateau state are small‑holder backyard and commercial farms with population of 50‑50,000 chickens per farm (maduka et al. 2015). a few poultry hatcheries are also located in plateau state which supplies day old chicks (doc) mainly to the northern parts of the country (akinwuni et al. 2010). majority of the poultry farmers in plateau state keep layers or are egg producers, while it is a common practice also for farmers to keep multi‑type and species of poultry like broilers, layers and turkeys within the same premises or poultry pen. nair 2018). marek’s disease virus is transmitted horizontally by direct or indirect contact between chickens mainly through the airborne route (davidson & borenshtain 2002, schat and nair 2013). mdv‑1 and mdv‑2 are contagious, but hvt does not spread easily between infected chickens early in life (islam et al. 2007). natural infection of mdv is through inhalation of viral infected epithelial cells of the keratinized layer of feather follicles (baigent and davison 2004). feathers/dander with mdv are infectious in contaminated poultry dust/house for several months (denesvre et  al. 2013). clinical presentation of md may vary in chickens with lymphoma or paralysis syndromes, but few lesions are specific to md (gimeno and pandiri 2013). in affected flocks, birds appear unthrifty with ruffled feathers and stunted growth, other non‑specific signs include emaciation, paleness, anorexia, and diarrhea especially in birds with chronic disease (schat and nair 2013). mdv can induce immunosuppression resulting in affected chickens succumbing to secondary infections or other pathogens which may mask the virus (haq et  al. 2013, gimeno and schat 2018). the gross lesions commonly observed in the classical and acute cases of md are enlargement of the peripheral nerves, formation of lymphoma in various organs and tissues (payne 1985, schat and nair 2013). in the field, diagnosis can be complicated because of similarities of md or co‑infection with other viral neoplastic diseases of poultry such as avian leukosis (al) and reticuloendotheliosis (re) (nair et al. 2020). hence, confirmatory diagnosis is a multi‑step process, involving a combination of flock history, clinical signs, gross, histopathologic findings and nucleic acid detection (zelnik 2004, gimeno and pandiri 2013). for molecular assay such as polymerase chain reaction (pcr), detection of mdv meq gene is important, it is the gene responsible for oncogenicity of mdv which is present in less and very virulence strains of the virus (davidson 2020). however, in resource limited countries like nigeria, confirmatory diagnosis of md is usually a challenge because of poor access to laboratory facilities by poultry veterinarians. control and prevention of md is based on a combination of biosecurity and vaccination. all md vaccines are live vaccines which can be classified as 1st generation vaccines i.e hvt a mdv‑3, 2nd generation vaccines, a combination of hvt and sb‑1 a mdv‑2, and finally 3rd generation vaccines an mdv‑1 isolate called rispen‑cvi998 which is the gold standard md vaccine (atkins et al. 2013, ralapanawe et al. 2016, schat 2016). in recent years combination of mdv‑1 rispen‑cvi998 and hvt stored in liquid nitrogen is popular and effective (diaz 2014, schat 2016). with 180 million chickens reared on extensive or free‑range (46%), semi‑intensive (33%) and intensive/commercial (21%) husbandry systems, majority of the commercial poultry farms xxxxxxxxxxxxxxxxxxxxxxx adedeji et al. 3 ar ti cl e ah ea d of p ri n t veterinaria italiana 2022, xx (x), xxx‑xxx. doi: 10.12834/vetit.2442.15397.1 adedeji et al. nick title investigation of marek’s disease outbreaks on poultry farms visit to selected poultry farms with clinically diagnosed cases of md was carried out based on consent and availability of the farmers. the farms visited were located in jos‑north, jos‑south, jos‑east, bassa lga (figure 1b). during farm visit, history was recorded including age, type of bird, source of birds, population, date of onset, vaccination history, mortality rate and egg production history. necropsy was carried out on dead chickens on the farms and samples collected. the geographic co‑ordinates of veterinary clinics/ hospitals and some of the affected farms visited were recorded. histopathology necropsy was performed, and histological processing was done as previously described (schat and nair 2013, akanbi et  al. 2020). briefly, tumourous visceral organs of clinically diagnosed cases of md namely heart, lung, liver, spleen, were collected at necropsy. the organs were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm. they are thereafter mounted on clean glass slides and stained with hematoxylin and eosin case history, gross pathologic findings, and sample collection samples were collected from chicken carcasses submitted to four veterinary clinics located in jos north (1) and jos south (3) lgas (figure 1) of plateau state nigeria from april 2013 to august 2014. the farms that submitted sick dead chickens for necropsy were from jos‑north, jos‑south, jos‑east, bassa and bakin ladi. the case definition was clinical diagnosis of md based on history, onset of disease before ≤ 14 weeks of age with neoplastic/tumourous lesions in visceral organs and gross lesions on peripheral nerves. the age, clinical signs, vaccination history and gross lesions observed were recorded. tumours of the liver, spleen, lungs, ovaries, heart, intestine, skin and kidney observed during necropsy were collected. samples from different dead chickens but from the same flock/farm were pooled together and gross lesions of chickens from the same farm were also recorded together. samples were collected in two parts, one part in sample bottles for conventional pcr. the second part of the samples were placed in 10% buffered formalin and stored at appropriate conditions at viral research division, national veterinary research institute, vom, nigeria until used. figure 1. a. map of nigeria showing plateau state. b. map of plateau state showing the study area, veterinary clinics and farms were samples were collected. nick title first author et al. 4 veterinaria italiana 2022, xx (x), xxx-xxx. doi: 10.12834/vetit.xxxxxar ti cl e ah ea d of p ri n t (h&e) stains for microscopic examination using low and high powered field of carl zeiss binocular microscope. polymerase chain reaction tumourous tissue samples were homogenized and dna extracted using qiaamp dna mini kit (qiagen, hilden germany) according to manufacturer’s protocol. conventional pcr protocol targeting the meq gene which is the oncogene of mdv‑1 using the following primer sequences forward 5’‑ttccctcttctgccctcc‑3’, reverse 5’ tcctgttcgggatcctcg‑3’ with the expected amplified pcr product of 200 bp (woz´niakowski et al. 2013). briefly, the pcr was carried out in a 25 µl reaction mixture containing 20 pmol of each primer, 12.5  µl (thermo scientific dream taq®) green pcr master mix 2x) and dna template 2 µl. the thermo cycling conditions include initial denaturation at 94  °c for 3 minutes followed by 35 cycles each consisting of 90 s at 94 °c, 1 min at 60 °c and 2 min at 72 °c, and final extension of 72 °c for 7 min. the positive controls used was (virulent mdv serotype 1) provided by dr. aly fadly of the avian diseases and oncology laboratory, east lansing, mi, usa). results clinical signs and gross pathologic findings one hundred and thirty six chicken (136) carcasses were examined from 110 farms consisting of 90 layer farms and 20 broiler farms during necropsy. the farms were located in jos north (12), jos east (4) jos south (88), bassa (5), bakin ladi (1). farm records revealed onset of the disease was from 3‑14 weeks of age in layer farms and 6‑11 weeks in broilers farms (table i). also 5.6% (n  =  5) of layer farms with clinically diagnosed cases of md had 2‑3 flocks of different ages affected by the disease on the same farm (table i). records showed that 16.6% (n  =  15) of layer farms revaccinated their chickens using hvt fc‑126 strain vaccine once. while 11.1% (n = 10) revaccinated with hvt fc‑126 strain vaccine twice between 3‑21 days of age, irrespective of the vaccination history from the hatchery. another 2.2% (n  =  2) of layer farms revaccinated with combined mdv‑1 rispens‑cvi988 and hvt fc‑126 strain vaccine (table i). there were no history of vaccination against md in all the broiler farms. common clinical signs observed were stunted and uneven growth within the flocks, undeveloped combs, paralysis of the legs (figure 2a, table i), anorexia and diarrhea. the common gross lesions observed during necropsy were cachexia, (figure  2c) hepatomegaly table i. summary of history and gross pathologic findings of chickens clinically diagnosed as cases of marek’s disease in plateau state, nigeria. history and clinical signs layer (n=90) broiler (n=20) age at onset of disease 3-14 weeks 6-11 weeks history of marek’ s disease 7 (6.4%) repeat md vaccination at farm hvt once 15 (16.6%) hvt twice 10 (11.1%) rispens +hvt 2 (2.2%) gross pathologic findings layer (n=90) broiler (n=20) total (n=110) hepatomegaly with lymphoma 50 (55.5%) 9 (36%) 59 (53.6%) splenomegaly with lymphoma 54 (60.0%) 8 (32%) 62 (56.4%) heart with lymphoma 9 (10.0%) 5 (20%) 14 (12.7%) lungs tumours 4 (4.4%) 2 (8%) 6 (5.5%) skin tumours 3 (15%) 3 (2.7%) prominent/enlarged sciatic nerve 15 (16.7%) 2 (8%) 17 (15.5%) enlarged provetriculus 16 (17.7%) 16 (14.5%) weight loss/emaciation with tumour in visceral organ(s) 37 (41.1%) 37 (33.6% intestinal tumours 2 (2.2%) 2 (1.8%) tumours of the muscles 2 (2.2%) 2 (1.8%) lymphoma of the ovaries 4 (4.4%) 4 (3.6%) underdeveloped ovaries @18 weeks > 8 (8.8%) 8 (7.2%) atrophy of the spleen 10 (11.11%) 10 (9.1%) lymphoma of the bursa with lymphoma (53.6%, n = 59) which were several times the normal size, diffuse, nodular or both, with grey or white discoloration (table i, figure 2d). splenomegaly with lymphoma (56.4%, n  =  62) (figure  2e), heart with multiple nodular tumours (12.7%, n = 14) (figure 2f ), and tumour of the lungs (5.5% n = 6). the proventriculus was thickened and firm on palpation with prominent glands (1.7%, n  =  16), prominent sciatic nerves (15.5% n  =  17), skin tumours (2.7%, n  =  3) and weight loss/severe emaciation with tumour on visceral organ (s) (33.6%, n  =  37) (table i, figure 2f ). other gross lesions observed were tumours of the intestine (1.8%, n = 2), muscles (1.8%, n = 2) and ovaries (3.6%, n = 4). also observed was atrophy of the spleen (9.1%, n = 10). outcome of field investigation on twenty poultry farms clinically diagnosed with marek’s disease farm visit to twenty (20) poultry farms revealed (figure 1), one of the farms had two flocks and two other farms had three flocks all affected by md. all first author et al. nick title 5veterinaria italiana 2022, xx (x), xxx-xxx. doi: 10.12834/vetit.xxxxx ar ti cl e ah ea d of p ri n t figure 3. a. heart, severe, diffuse, lymphohistioplasmacytic myocarditis. h&e x 100. b. spleen, diffuse infiltration of lymphohistioplasmacytic cells, occasionally forming nodules. h&e x 100. c. liver, mid‑zonal hepatocellular necrosis with bridging lymphohistioplasmacytic cellular infiltration. h&e x 100. d. liver, multifocal to diffuse hepatocellular necrosis and destruction of hepatic cords with lymphohistioplasmacytic cellular infiltration in inter‑hepatic cord and sinusoidal spaces. h&e x 400. e. intestine, severe, diffuse, lymphohistioplasmacytic infiltration, h&e x 100. f. lung, bronchiopneumonia, with lymphohistoplasmacytic accumulation in large pulmonary airway, h&e x 100. farms visited had birds on deep litter management system. the populations of chickens on the farms visited were 800‑10,000 chickens. based on farm records 60% (n  =  12) had onset of the md at 5‑8 weeks of age of the chickens. the chickens were stunted with uneven growth within flocks of the same age (figure 2a). the average mortality rate was 20%‑60% which was over a period of several weeks due to the prolong nature of md. all the farms visited were layer farms and egg production performance by flocks affected by md ranged between 36% and 80%, with 20% (n  =  4) of the farms at 80% egg production performance and for the rest it was 36%‑75%. in addition, 20% (n = 4) of the farms visited had previous history of md, while the rest did not indicate any previous history of md. flock history also showed that 80% (n  =  16) of the farms visited repeated vaccination against md at least once before 21 days of age using hvt vaccine, however some of the farmers vaccinated twice using hvt vaccine. ninety percent (90% n  =  18) of the farms sourced replacement stock from southwest nigeria and the birds are transported over 12‑14 hours by road to plateau state. other husbandry practices observed include keeping of multi‑age flock in the same premises and lack of all‑in, all‑out management system or fallowing of farms. md was confirmed on the farms based on history, clinical figure 2. a. twenty five (25) week old layers farm diagnosed with marek’s disease, note stunted and uneven growth, only few birds had developed comb. b. fifteen (15) week old pullet with classical leg paralysis or ‘hurdle jumper position’ as result of marek’s disease. c. carcass of a layer chicken with prominent kneel bone as a result of severe emaciation. d. the liver of broiler chicken with hepatomegaly and diffuse lymphoma. e. severe splenomegaly in layer chicken. f. heart of a 9 week old broiler chicken with multifocal lymphoma nodules. signs, histopathology and results of conventional pcr. there was clustering of farms visited in bassa, jos south and jos east lgas (figure b). histopathology results microscopically, lesions observed in tumours or lymphoma of visceral organs were mainly proliferating lymphocytes, lymphoblasts and macrophages admixed with some inflammatory polymorphonuclear cells. in the heart (figure 3a), severe diffuse lymphocytic (lymphocytes and lymphoblast) infiltration with myocarditis and vasculitis. microscopic lesions in the spleen were periarteriolar lymphoid cellular accumulation, vasculitis with lymphoblast and; macrophages infiltration (figure 3b). while in the liver, there was bridging lymphohistioplasmacytic cellular infiltration with multifocal to diffuse hepatocellular necrosis and destruction of hepatic cords (figure 3c‑d). nick title first author et al. 6 veterinaria italiana 2022, xx (x), xxx-xxx. doi: 10.12834/vetit.xxxxxar ti cl e ah ea d of p ri n t report were enlarged liver with lymphoma and enlarged spleen with lymphoma in dead chickens at necropsy. lymphoma of the visceral organs in md is usually several times the normal size similar to the findings in the study (nair 2018). lesions on the peripheral nerves are considered pathognomonic for md, were observed in 15.5% of dead chickens examined in this study. visceral tumours or lymphoma can occur without corresponding gross peripheral nerve lesions in md cases as previously reported and observed in this study (table i) (schat and nair 2013). thus the absence of lesions on nerves cannot be used as an exclusion criteria for md clinical diagnosis (nair et  al. 2020). another finding was severe emaciation with visceral organ tumours in 33.6% carcasses examined at necropsy. weight loss, emaciation and chronic wasting are observed in prolong course of md due to starvation or dehydration (schat and nair 2013, mcpherson and delany 2016). histopathologic results revealed infiltration of lymphocytes, lymphoblasts and macrophages in visceral organs samples collected. these are characteristic microscopic lesions of md as previously described and confirmed the md in the samples analyzed (adedeji et al. 2019, nair et al. 2020). the virulent mdv‑1 was detected in 68.2% of the tumourous samples collected from 110 poultry farms. detection of the virulent mdv‑1 in samples alongside gross lesions and histopathologic lesions confirmed md in poultry farms in plateau state, nigeria. md was confirmed in both broilers and vaccinated layer farms in this study. md outbreaks were recorded in layer flocks despite vaccination against md at the hatchery and revaccination by poultry farmers. whilst, broiler type chickens are not routinely vaccinated against md in nigeria even at the hatchery, because broilers are not kept beyond 10 weeks to reduce of production cost. broilers are commonly kept alongside layers in nigeria which exposes them to virulent mdv‑1 (jwander et al. 2015, maduka et  al. 2016). despite the successes of md vaccines, it has been suggested that the advent of md vaccine drives mdv field viruses towards greater virulence, nonetheless little evidence supports this theory since vv mdv strains were first described (schat et  al. 2016, reddy et  al. 2017). suggested polymerase chain reaction results the samples analyzed were from broilers (18.2% n = 20), and layers (81.8%, n = 90). of the tumorous samples analyzed, meq gene of mdv‑1 was detected in 68.2% (75/110) comprising 55.0% (n  =  11/20) of broiler samples and 71.1% (n  =  64/90) of layer samples. discussion it is estimated that over 70 percent of nigerians directly or indirectly depend on the poultry industry for their livelihood, it is the most commercialized sector of the agricultural industry in nigeria with a net worth of usd 1.7 billion per year (fao 2019). however, growth of the poultry sector is seriously hampered by several poultry diseases including md. previous reports have suggested that nigeria is one of the poultry producing countries in the world with increasing prevalence of md, despite routine vaccination poultry with md vaccines (gimeno 2004, dunn and gimeno 2013). but, there are no reports that investigated md in vaccinated poultry flocks in nigeria. in this study, md outbreaks were confirmed in vaccinated and unvaccinated poultry farms in plateau state, north‑central nigeria based on history, clinical signs, gross lesions, histopathology and detection of meq gene of mdv‑1 by pcr. all chicken carcasses sampled in this study had tumour or lymphoma lesions in at least one visceral organs i.e. liver, spleen, heart, kidneys and lungs (figure 3, table i). cases of visceral tumours or lymphoma in chickens < 14 weeks was used as criteria for sample collection. due to the similarities of md and other neoplastic diseases of chicken such as al and re, age is an important criteria for differentiation and diagnosis of these diseases in the field (schat and nair 2013). mdv‑1 is considered the cause of tumours of visceral organs in chickens <  14 weeks of age, whereas al and re cause tumour/lymphoma in chickens older than 14 weeks of age (gimeno and pandiri 2013). the gross lesions in this study were consistent with what has been observed with md in other reports (schat and nair 2013). the most common gross lesions observed in this figure 4. agar gel electrophoresis of pcr product of virulent mdv‑1 strain. lanes 116 are the visceral and feathers samples collected from clinically diagnosed cases of marek’s disease. the positive samples were amplified at 200bp. l = a 100 bp dna marker (qiagen®). +ve = positive control. -ve = negative control. first author et al. nick title 7veterinaria italiana 2022, xx (x), xxx-xxx. doi: 10.12834/vetit.xxxxx ar ti cl e ah ea d of p ri n t pathogens due to the immunosuppressive nature of md. even so, these mortalities were directly or indirectly attributed to md and they occur over a period of several weeks due to the prolong course of md in some flocks or farms. in addition, there was also clustering of farms confirmed with md in this study (figure 1). in order to control md, biosecurity practices must be enhanced particularly a massive change in husbandry practices. in as much as vaccination against md is important, it can only be effective when augmented with good biosecurity measures. further studies should conducted on the economic impact of md in nigeria and mdv strains circulating in poultry flocks should be characterized. conclusions this study reports a concise molecular, pathologic and epidemiologic features of md in vaccinated and unvaccinated in poultry farms in nigeria. md was confirmed in vaccinated layer and unvaccinated broilers farms where samples were collected. in order to mitigate economic losses associated with md, prompt diagnosis, implementation of biosecurity measures and proper vaccination are recommended. acknowledgement the authors acknowledges assistance of dr. t.m. joannis, dr. i. shittu, dr. dauda mari, dr. enoch ishaku, dyek yohanna dyek and adrian maguda. reasons for failure of vaccines to protect against md, particularly in some african countries includes; poorly administered vaccines, the wrong type of vaccine, poor biosecurity practices, early chick exposure to pathogenic mdv and contaminated md vaccines (morrow and fehler 2004, gimeno 2008, shittu et al. 2019). poor biosecurity and management practices such as keeping of multiage flocks and not fallowing farms leads to high contamination which were observed in this report. these husbandry practices in poultry farms in nigeria exposes chicks early to virulent mdv‑1 and may negate the merit of vaccination. in addition, these unwholesome practices are also drivers of not only md but other economically important poultry diseases in nigeria. albeit, revaccination of chickens with md vaccines have been reported to be beneficial and it is a widely practiced protocol (wu et  al. 2009). but, it appears not to be beneficial based on findings from this study, because 11.1%‑ 16.6% of farms vaccinated or revaccinated with hvt still experienced outbreaks of md. likewise, sourcing of doc from long distances which are transported by road, thereafter vaccinated after a few days may contribute to failure of the vaccines to protect against md. the major economic burden of md is attributed to both direct losses from chicken morbidities, mortalities and egg production losses, whilst indirect losses result from the use of expensive md vaccines (rozins et  al. 2019). the twenty farms visited experienced 20%‑60% mortality rates in their poultry flocks, although this may have exacerbated by secondary nick title 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52‑58. 49 veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 accepted: 31.01.2020 | available on line: 27.07.2021 1virology department, ankara university, veterinary medicine, ankara, turkey 2virology departmen, hatay mustafa kemal university, veterinary medicine, hatay, turkey. 3virology department, van yuzuncuyıl university, veterinary medicine, van turkey. *corresponding author at: ankara university, veterinary medicine, ankara, turkey. e-mail: sevalbilge@hotmail.com. seval bilge dağalp1*, firat dogan2, ali riza babaoglu3, touraj aligholipour farzani1 and feray alkan1 keywords bovine herpesvirus type 4, glycoprotein b (gb), pcr, sequencing, thymidine kinase (tk), virus isolation. summary bovine herpesvirus type 4 (bohv-4) is a common virus in the world that is detected in clinically ill or in apparently healthy cattle. this study provides a molecular characterization of bohv-4 strains from 24 cattle some showing respiratory and/or reproductive problems and some without any apparent clinical sign. this study also reported the growth properties of five bohv-4 field isolates. the 24 sampled cattle came from 13 different herds in 10  provinces collected between 2007 and 2018. phylogenetic analysis using partially amplified nucleotide sequences of orf8 genes coding glycoprotein b (n = 24) and orf3 genes coding thymidine kinase (n =  9), demonstrated genetic variability among the bohv-4 strains analysed. the partial gb gene sequences clustered in three different genotypes (genotype i, ii and iii) were located within the genotype i cluster, such as movar strain. the analysis of the five bohv-4 strains isolated from vaginal swabs (n = 2), nasal swab (n = 1), and brain samples (n = 2) revealed no significant differences in their growth properties in mdbk cell culture. genetic variability of bovine herpesvirus type 4 (bohv-4) field strains from turkish cattle herds of the virion, essential for viral infectivity (little et  al. 1981, lomonte et  al. 1997). it is one of the most studied genes because its region is one of the most highly conserved among herpesviridae family members (goltz et  al. 1994, wellenberg et  al. 2001). tissue tropism instead is most likely associated with variations in virion surface glycoproteins (frazier et al. 2002, wellenberg et al. 2001). another frequently studied gene is orf3, which codes for thymidine kinase (tk). it helps in understanding the precise regulation of specific genes, their promoter regions, and transactivation mechanisms (kit et  al. 1986, lomonte et al. 1992). in turkey, previous studies have shown that bohv-4 infection is prevalent in dairy cattle herds (bilge dağalp et  al. 2007, bilge dağalp et  al. 2010, bilge dağalp et  al. 2012, gur and dogan 2010, kale et  al. 2011), with seropositivity levels reaching 56.8% and 44.9% in sampled herds with reproductive disorders and healthy appearance, respectively (bilge dağalp et  al. 2007). while several reports have detected bohv-4 from cattle with or without clinical symptoms, the genetic characterization of local viruses is not yet well documented. the two introduction bovine herpesvirus type 4 (bohv-4), a member of the herpesviridae family, subfamily gammaherpesvirinae in the rhadinovirus genus, is found worldwide among cattle populations (roizman et  al. 1992, zimmerman et  al. 2001). the bohv-4 genome is a double-stranded dna of 144 ± 6kb and a long unique coding region (lur) or light dna (l-dna) with a size of approximately 108 kb. based on restriction endonuclease patterns, almost all american strains belong to the dn-599 group whereas european strains belong to the movar 33/63 group (bublot et  al. 1990). however, the restriction patterns of some strains do not completely match with those belonging to the dn-599 or movar 33/63 groups. these are the lvr 140 strain, known as the belgian reference strain (thiry et  al. 1992, wellemans et  al. 1986), and the taiwan strain isolated from bovine vascular endothelial cells (lin et al. 1997). several bohv-4 genes have been examined in detail by various researchers (egyed et al. 1996, goltz et al. 1994). bohv-4 open reading frame 8 (orf8) codes for glycoprotein b (gb), which is a major component 50 genetic variability of bohv-4 bilge dağalp et al. veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 bilge dağalp et al. 2012). considering that the arrival dates of these samples are old, the specimens did not propagate in cell cultures and were discarded. as a result of these elimination only isolates and bohv-4 suspected samples that were delivered to our laboratory in recent years were amplified and sequenced. they were selected as representative of different herds, clinical disorders, and sampling years. they included diagnostic samples originated from nasal and vaginal swabs, milk, and necropsy samples collected between 2007 and 2018 from cattle with severe reproductive disorders, abortions, and/or respiratory system disorders from 13 different dairy cattle herds of 10 provinces (table i). pcr dna extraction was carried out according to sambrook and colleagues (sambrook et  al. 1989). the detection of bohv-4 dna was performed using oligonucleotide primers specific for bohv-4 orf8 gene purposes of this study are therefore i) analysis of the gene regions coding gb and tk in local bohv-4 strains from cattle suffering reproductive and respiratory disorders; ii) investigation of their growth characteristics in cell cultures. materials and methods samples for this study, 24 cattle samples in which bohv-4 was identified were selected previously (bilge dağalp et al. 2010, bilge dağalp et  al. 2012). those samples that were still positive when re-tested in this study were used for analysis of the genes coding gb. the field isolates (n = 5) and the samples that were delivered to our laboratory in the last years were also used. the sequences used in this study were obtained from the samples that were targeted for the amplification of the gb gene region or were found positive against gb in previous two projects (bilge dagalp et al. 2010, table i. details of the bovine herpesvirus type 4 positive samples . herd no/ location virus code clinical signs year samples sequences of gb genes sequences of tk genes2 genbank acc.no genotype genbank acc.no. i bursa k339 metritis 2007 vaginal swab1 eu055543 ii jx644990 ii aksaray kcvs12 metritis, upper respiratory disease 2007 vaginal swab1 gq246864 jx644991 kcorg abortus 2011 organ mk543547 nd kcb12 neonatal death 2009 brain1 jx644988 jx644992 kcvs2 repeat breeder, upper respiratory disease 2009 vaginal swab mk543548 nd iii kars kbsb9 abortus 2007 brain gq246865 nd iv kirklareli tg1 metritis 2007 leukocyte gq246866 nd tgvs550 repeat breeder 2011 vaginal swab mk543551 nd tgvs549 repeat breeder 2011 vaginal swab mk543550 nd tgvs29 abortus 2011 vaginal swab mk543549 nd tg4 metritis 2007 vaginal swab gq246863 nd v balikesir t6 repeat breeder 2007 leukocyte gq246867 nd vi yozgat yl abortus 2007 leukocyte gq375280 nd vii sivas u-ns66 upper respiratory disease 2009 nasal swab1 jx644989 jx644993 viii i̇zmir iz1lk136 abortus 2010 leukocyte mk543546 nd ix ankara ank.ns repeat breeder, upper respiratory disease 2014 nasal swab kx192364 nd ank.br. neonatal death 2015 brain1 kx192363 kx352279 x ankara ank.teat. mammary lesions, mastitis 2014 teat lesion kx192365 kx352280 xi kirklareli b1ac4 upper respiratory disease, pneumonia 2017 lung mh318579 mh614620 b2ac4 upper respiratory disease, pneumonia 2017 lung mh318580 mh614621 xii kastamonu kas21 repeat breeder 2017 leucocyte mg181944 i negative kas24 repeat breeder 2017 leucocyte mg181945 negative kas25 repeat breeder 2017 leucocyte mg181946 mg242137 xiii ankara 9870milk mastitis 2018 milk mh605274 iii negative 1 bohv-4 field strains isolated from these samples; 2 all clustered in genotype i; nd = not done 51 bilge dağalp et al. genetic variability of bohv-4 veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 tested for bohv-4 by direct immunofluorescence (ifa) technique, using a commercial conjugate (bio028, biox, belqium) as described by nettleton and colleagues (nettleton et al. 1982) and/or pcr as described in the material and method section in this study. samples showed cytopathic effects in mdbk cell cultures, 5-7 days after 3rd passage. after isolation and identification procedure, the supernatants from third passages of isolates (n = 5) were used for titration. the virus titer was calculated using the method of reed and muench (reed and muench 1938). to investigate the replication kinetics, five bohv-4 strains isolated in this study were used. these bohv-4 isolates (n = 5) were inoculated into mdbk cells grown in 24-well plates (greiner bio-one, frickenhausen, germany) at a multiplicity of infection (moi) of 0.1. cell cultures were incubated at 37 °c with 5% co 2 atmosphere and observed daily for the presence of cpe. the supernatants were harvested at 24, 48, 72, 96, and 120 h post-infection (hpi) and frozen at -  80  °c for further viral quantitation, as described by verna and colleagues (verna et al. 2016). the percentage of cpe was recorded at the time of supernatant collection. five replicates were performed to establish the kinetics of viral replication for each experiment (virus strain and harvest time points). negative controls were included in each experiment. to determine the replication kinetics of these local isolates (n = 5), the infectivity titers were estimated using viral suspensions collected at 24, 48, 72, 96, and 120 hpi from every mdbk cell culture (reed and muench 1938). results pcr, sequencing, and phylogenetic analysis after pcr, gb gene region of 24 samples and tk region of 9 of 24 samples were also amplified. in pcr, the local viruses produced the expected size of products (615 bp) for the gene coding gb and produced the expected size of products (567 bp) for the gene coding tk (table i). all amplicons from the local strains along with the reference strain (dn-599) were commercially sequenced and their genome sequences were deposited in genbank under the accession numbers in table i. phylogenetic analysis, performed using the gb coding gene sequences of the local viruses (n = 24) and bohv-4 strains with different genotypes in genbank, revealed that genotype ii was prevalent in the local isolates (20/24, 83.3%) and sampled herds (11/13, 84.6%). the other two genotypes (genotype  i and iii ) were identified in other two codifying gb (5’-cccttctttaccaccacctaca-3’ and 5’-tgccatagcagagaaacaatga-3’) (goltz et  al. 1994), and for bohv-4 orf3 gene codifying tk (5’-gttgggcgtcctgtatggtagc-3’ and 5 ’ atg tatg ccc a a a ac t tata atatg acc ag 3 ’ ) (egyed et al. 1996) pcr was conducted as described by wellenberg and colleagues (wellenberg et  al. 2001) with some minor modifications. briefly, 3  μl dna was subjected to thermocycling in a 30  μl reaction mixture containing 2.5  u taq dna polymerase (fermentas, lithuania), 3.5 mm dntp mix (fermentas, lithuania), 10 pmol of each primer, 1.5 mm mgcl 2 , 1  x  pcr buffer, and 6% dmso. thermal cycling conditions were 6 min at 96 °c, followed by 40 cycles of 56 °c for 45 sec, 72 °c for 2 min, and 95 °c for 1 min. the reaction tubes were kept for a further 10 min at 72 °c for final extensions. pcr products were visualized in a transilluminator after electrophoresis in 1% agarose gel containing ethidium bromide. the north american (dn-599) reference strain of bohv-4 was used as positive control. the reference virus strain was kindly provided by dr. g.j. wellenberg (lelystad, netherlands). sequencing and phylogenetic analysis pcr amplicons with the expected sizes (615 bp and 567 bp for genes coding gb and tk, respectively) were subjected to sequence analysis. the amplicons were firstly gel purified using a commercial kit (genemark, taiwan) and sequenced by a commercial enterprise. the gb and tk gene sequence assembly and editing were performed using bioedit (version 7.0.5.3) and clustral w (hall 1999). the sequences were then compared with those of the genbank and sequenced database for similarities using the basic length alignment search tool (blast) software of the national center for biotechnology information (ncbi) (althsul et al. 1997). the obtained analysis data were compared with different bohv-4 strains by using neighbor joining method (1,000 replication) and they were phylogenetically analyzed in mega 6.0 software program under kimura 2  parameter (tamura et al. 2011). virus isolation and determination of the replication kinetics of isolates in cell culture all samples (n = 24) were inoculated (100 µl) onto mdbk cell cultures for virus isolation. the cells were washed 3 times with pbs-m before adding medium. the monolayers were incubated at 37 °c in a 5% co 2 atmosphere and checked daily for the appearance of cytopathic effects (cpe). the monolayers were passaged at weekly intervals for a total of three passages. the supernatants of all cell cultures were 52 genetic variability of bohv-4 bilge dağalp et al. veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 98% and 96.5%, respectively (figure 5 and table iii). considering the sequences for the two genes at aa and na level, the differences in orf8 sequences were greater than the differences for the orf3 gene sequences between our viruses and bohv-4 from other countries, and also between our local viruses. figure 6 shows the amino acid sequence alignments of orf8 genes coding gb of our bohv-4 samples and other strains, including the reference viruses (movar and dn -599). virus isolation and replication kinetics out of 24 samples previously confirmed as positive for bohv-4 by pcr on orf8 gene amplification, five showed cytopathic effects in mdbk cell cultures, 5-7 days after the 3rd passages. these bohv-4 strains were then successfully isolated and identified by immunofluorescence and/or pcr. the origin of these samples was as follows: two vaginal swabs (k339 and kc-vs12), one nasal swab (u-ns66-tr2009), and two brain samples (kc-b12 and ank.br.tr2015) from animals in herds  i, ii, vii, and ix (table i). figure  7 shows the infectivity titers of 5 isolates which grew in cell culture (n = 5). discussion bohv-4 appears capable of significant genome drift, as demonstrated by the variability in the different herds (figure 1 and table i). the grouping in the phylogenetic tree is described by verna and colleagues (verna et al. 2012) and areda and colleagues (areda et al. 2018). the phylogenetic analysis of bohv-4 based on sequences of the orf3 gene coding tk revealed three distinct genotypes: genotype i, ii, and iii (figure 2). as shown in figure  2, phylogenetic comparisons with the previously described bohv-4 tk gene sequences indicated that all of our bohv-4 field viruses belonged to genotype  1, which includes the european (movar-like) reference strains, along with the italian, japanese, and argentinian bohv-4 strains. we also compared the nucleic acid (na) sequences for the genes coding gb and tk of the local bohv-4 samples both among themselves (figures 3 and 4) and with the sequences deposited in genbank from other countries (tables ii and iii). figure 5 shows the amino acid (aa) similarity rates for genes coding tk. for genotype i, the similarity rates for genes coding tk were 100% among the bohv-4 strains detected in this study and 98.5-100% with the other viruses. the similarities with local bohv-4 identified as genotype ii and genotype iii were mg181944kas21-lok-tr2017 mg181945kas24-lok-tr2017 mg181946kas25-lok-tr2017 mg264405 isolate d1611430-2.5 gb mg264400 d1611430-2.8 gb mg264401 d1611430-2.15 gb mg264402 d1611430-2.27 gb kp209018 isolate 08 209 gb mg264396 isolate d1300426-1.1 gb af318573 gb mh318580 (b2 ac4-tr2017) mh318579 (b1 ac4-tr2017) gq246867 t-6 kp209029 isolate dn 599 gq246865 sb-9 ay847334 movar33/63 z15044 bohv-4 gb kx192364 ank-ns-tr2014 eu055543 k339 kx192365. ank-teat-tr2014 gq246864 strain kc-12 mk543547 tr2011-kcorg mk543546 tr2010-iz1lk136 jx644989 u-ns66-tr2009 kx192363 ank-br-tr2015 jx644988 kc-b12-tr2009 mk543548 tr2009-kc-vs2 mk543549 tr2011-tgvs29 mk543550 tr2011-tgvs549 mk543551 tr2011-tgvs550 gq375280 yl gq246863 tg-4 gq246866 strain tg-1 mg264399 isolate d1611430-1.88 gb mh605274 (9870milk-tr2018) kp209016 isolate 07 435 gb kp209024 isolate 09 227 gb mg264397 isolate d1609492-1.6 gb mg264404 isolate d1611430-2.48 gb 46 54 99 63 60 83 58 64 82 65 ge no ty pe i ge no ty pe ii ge no ty pe ii i 2 figure 1. phylogenetic tree based on nucleotide sequences of the gb gene of turkish bohv‑4 strains shown in round () shape and bohv‑4 strains from various countries. ab035516 japon 1999 movar 33/63 ab035517 japon 1999 ab035515 japon 1999 mh614621 (b2ac4tk-tr2017) mh614620 (b1ac4tk-tr2017) kx352280 ank-teat-tr2014 tk kx352279 ank-br-tr2015 tk mg242137 kas25tk-lok-tr2017 jq838059 argentina 2012 jx644990 k339-tr2009 jx644992 kc-b12-tr2009 tk jx644991 kc-12-tr2007 tk jx644993.1 u-ns65-tr2009 tk kt166423 italy 2015 to-1 tk eu244699 brazil 2007 eu244698 brazil 2007 eu244697 brazil 2007 eu244700 brazil 2007 kp209015 argentina 2014 ku180422 argentina 2015 ku180419 argentina 2015 kp209014 argentina 2014 jq838047 argentina 2012 jq838057 argentina 2012 jq838062 argentina 2012 dn599 jq838052 argentina 2012 jq838053 argentina 2012 jq838046 argentina 2012 jq838056 argentina 20120.005 ge no ty pe i ge no ty pe ii ge no ty pe ii i figure 2. phylogenetic tree based on the nucleotide sequences of the tk gene of turkish bohv‑4 strains showed in squared () shape and bohv‑4 strains from various countries. 53 bilge dağalp et al. genetic variability of bohv-4 veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 aborted foetus tissue samples, and dead calves (mk543547, jx644988, gq246865, kx192363), and tissue and leucocyte samples from cattle with respiratory and/or fertility system disorders (mh31879-mh31880, kx192365, mk543546, gq375280, gq246866-gq246867) (table i). our field viruses that clustered in genotype i (mg181944, mg181945, mg181946) came from the leucocyte samples of repeat breeder cows in one specific herd (herd no:  xi), which aligned with argentinian and american bohv-4 strains. for genotype iii, only one field virus (mh605274) was detected in the milk sample from a cow with mastitis, which aligned with several north american bohv-4 strains in the phylogenetic tree (figure 1). thus, the phylogenetic analysis shows that our local viruses sequences of several gene regions coding gb and tk in the bohv-4 field isolates from turkish cattle with different clinical signs (frazier et al. 2002, areda et al. 2018, verna et al. 2016, gagnon et al. 2017). our molecular analysis of the partial gb gene sequences obtained from 24 local bohv-4 strains revealed that turkish bohv-4 isolates clustered into three genotypes, with the separation of several viruses in different branches in genotype ii (figure 1). the majority of the local viruses were classified in genotype ii, together with several european and north american bohv-4 strains. turkish bohv-4 strains in genotype ii (n = 20) were from various clinical samples, specifically nasal swabs (kx192364, jx644989), vaginal swabs (gq246863, gq246864, mk543548mk543551, eu055543), figure 3. nucleotid identity (%) of gb gene sequences of some turkish bohv‑4. figure 4. nucleotide identity (%) of partial tk gene sequences of some turkish bohv‑4 strains. 54 genetic variability of bohv-4 bilge dağalp et al. veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 revealed three main clusters, which included european (movar-like, genotype i) and american strains isolated from cattle, the african strain isolated from buffalo (dn599-like, genotype ii), and argentinean and brazilian strains (genotype iii) from cattle. the majority of samples (20/24) and herds (11/13) in this study were identified as genotype ii. these herds were either small family herds of 1-10 cattle (iii, vi, viii, xii, xiii) or state farms (i, ii, iv, v, vii, ix-xi) of about 1,000 cattle, which had been restocking from internal animal sources, transfering animals between herds, or occasionally introducing imported have high genetic similarities with bohv-4 reported from different geographical regions worldwide, specifically europe, and south and north america. most of the local bohv-4 strains with different clinical disorders were located in genotype ii while bohv-4 identified as genotype i and iii were only detected in cows with repeat breeder and a cow with mastitis, respectively (table i). despite these findings, we cannot yet claim a definite association between the genotypes and clinical cases due to the differences in the numbers of bohv-4 detected with different genotypes and in different clinical findings. the phylogenetic tree for the gene coding gb table ii. nucleic acid identity (%) of gb gene sequences of turkish bohv‑4 strains and bohv‑4 strains selected as representative for different genotypes from genbank database. genotype virus code acc number genotypes i ii iii mg264402 mg264401 kp209029 dn_599 ay847334 movar33/63 mg264396 mg264404 mg264399 i kas25 mg181946 100 100 99.7 99.3 99.7 92.7 92.3 kas21 mg181944 100 100 99.7 99.3 99.7 92.7 92.3 kas24 mg181945 100 100 99.7 99.3 99.7 92.7 92.3 mg264402 100 99.7 99.3 99.7 92.7 92.3 mg264401 100 99.7 99.3 99.7 92.7 92.3 ii dn_599 kp209029 99.7 99.1 99.7 100 93.0 92.7 movar33/63 ay847334 99.3 99.3 99.7 99.7 92.7 92.3 mg264396 99.7 99.7 100 99.7 93.0 92.7 u-ns66 jx644989 97.0 97.0 97.3 97.0 97.3 90.3 90.0 yl gq375280 97.7 97.7 98.0 97.7 98.0 91.0 91.3 ank-br kx192363 99.0 99.0 99.3 99.0 99.3 92.3 92.7 kc-b12 jx644988 99.0 99.0 99.3 99.0 99.3 92.3 92.7 tg-4 gq246863 98.7 98.7 99.0 98.7 99.0 92.0 92.3 tg-1 gq246866 98.7 98.7 99.0 98.7 99.0 92.0 92.3 kc-12 gq246864 99.3 99.3 99.7 99.3 99.7 92.7 92.3 ank-teat kx192365 99.3 99.3 99.7 99.3 99.7 92.7 92.3 k339 eu055543 99.3 99.3 99.7 99.3 99.7 92.7 92.3 ank-ns kx192364 99.3 99.3 99.7 99.3 99.7 92.7 92.3 sb-9 gq246865 99.3 99.3 99.7 99.3 99.7 92.7 92.3 t-6 gq246867 99.7 99.7 100 99.7 100 93.0 92.7 b1 ac4 mh318579 99.7 99.7 100 99.7 100 93.0 92.7 b2 ac4 mh318580 99.7 99.7 100 99.7 100 93.0 92.7 iz1lk136 mk543546 99.3 99.3 99.7 99.3 99.7 92.7 92.3 kcorg mk543547 99.3 99.3 99.7 99.3 99.7 92.7 92.3 kcvs2 mk543548 99.0 99.0 99.3 99.0 99.3 92.3 92.7 tgvs29 mk543549 98.7 98.7 99.0 98.7 99.0 92.0 92.3 tgvs549 mk543550 98.7 98.7 99.0 98.7 99.0 92.0 92.3 tgvs550 (mk543551) 98.7 98.7 99.0 98.7 99.0 92.0 92.3 iii 9870milk mh605274 91.7 91.7 92.0 91.7 92.0 99.0 98.7 mg264404 92.7 92.7 93.0 92.7 93.0 99.7 mg264399 92.3 92.3 92.7 92.3 99.7 55 bilge dağalp et al. genetic variability of bohv-4 veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 table iii. nucleic acid identity (%) of tk gene sequences of turkish bohv‑4 strains and bohv‑4 strains selected as representative for different genotypes from genbank database. virus code acc number genotypes i ii iii ab035516 movar33/66 ab035517 jq838059 jq838062 dn-599 jq838047 jq838057 jq838046 jq838056 b1ac4 mh614621 99.5 100 100 98.0 98.0 98.0 96.5 96.5 b2ac4 mh614620 99.5 100 100 98.0 98.0 98.0 96.5 96.5 ank-teat kx352280 99.5 100 100 98.0 98.0 98.0 96.5 96.5 ank-br kx352279 99.5 100 100 98.0 98.0 98.0 96.5 96.5 u-ns66 jx644993 99.5 100 100 98.0 98.0 98.0 96.5 96.5 kc-12 jx644991 99.5 100 100 98.0 98.0 98.0 96.5 96.5 kc-b12 jx644992 99.5 100 100 98.0 98.0 98.0 96.5 96.5 k339 jx644990 99.5 100 100 98.0 98.0 98.0 96.5 96.5 kas25 mg242137 99.5 100 100 98.0 98.0 98.0 96.5 96.5 figure 5. amino acid identity of tk gene sequences of turkish bohv‑4 and bohv‑4 strains selected as representative for different genotypes from genbank database. figure 6. amino acid differences of gb gene sequences of turkish bohv‑4 and bohv‑4 strains selected as representative for different genotypes from genbank database. 56 genetic variability of bohv-4 bilge dağalp et al. veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 then clinical cases (figure 1 and table i). we believe that further epidemiological studies of bohv-4 will reveal the relationship between the genotypes and geographical regions, and the association between molecular characterization and pathogenesis of infection. this study was able to characterize nine bohv-4 field strains for the sequences coding tk. based on phylogenetic analysis of the tk gene sequences, these isolates (n = 9) were identified as genotype i, which includes the european (movar-like) reference strains, together with the italian, japanese and argentinian bohv-4 strains (figure 2). these field strains were detected in vaginal swabs (jx644990, jx644990), nasal swab (jx644993), teat lesion samples (kx352280), lungs (mh614620, mh614621), leucocytes from cows with respiratory and/or reproductive system disorders (mg242137), and brain tissue (kx352279 and jx644992) from calves that died after birth (table i). regarding the classification of the tk gene, the local strains were identical to each other (100%) and highly similar to other virus strains in genotype i (99.5-100%), genotype ii (98%), and genotype iii (96.5%) (tables i and iii, figure  4). consequently, these genotypes cannot imply a possible association with clinical signs. however, some researchers (verna et al. 2016, gagnon et al. 2017) have argued that the existence of different genotypes suggests some association between genetic variations and specific pathogenic potential. in our study, all bohv-4 strains in genotype ii and iii, based on sequences for the orf3 gene, came from argentina or brazil (figure 2). this origin suggests an association between genotypes and geographic regions similar to that suggested for the gb gene sequences. this may be important for pursuing the traces of domestic or international trade. we found nine bohv-4 strains with both the animals. there is clear animal trade between either small family herds and/or industrial herds. we can speculate that animal movements between different geographical regions in turkey and also from other countries, such as argentina or uruguay, especially since 2010, may have influenced the distribution of the different genotype bohv-4 strains and their genetic resemblances. it is noteworthy that all turkish bohv-4 samples classified in genotype  i and iii, which were detected in diagnostic materials sampled in 2017 and 2018, showed close similarities with argentinian and american bohv-4 strains. in contrast, genotype ii has been detected in cattle since at least 2007 (table i). given these results, the introduction into turkey of live animals from other countries may be an important risk factor, as previously reported for bohv-4 and several other infections (fichtelova and kovarcik 2010, houe 1995). it should also be remembered that the gene region coding for gb is quite highly conserved among bohv-4 strains (campos et  al. 2014, bellino et  al. 2015). it is therefore not useful for providing clues about the strains’ geographical origins. thus, the gb gene sequences (gq246866, mk543551, mk543550, mk543549, gq246863) of bohv-4 strains from cattle with reproductive disorders, sampled at two different time points (2007 and 2011) in herd iv were placed in the same branch in the genotype  ii cluster (figure 1), with the nucleotide similarities of  100%. this also supports claims that the gene coding gb is quite highly conserved, with similarities rates of (91.7-100%) among all the bohv-4 strains analyzed in this study, despite being classified in three different genotypes (figure 3). additionally, the location on the different branches and/or clusters of the sequences of the samples in the phylogenetic tree from cattle with respiratory system and reproductive system disorders from different herds and years also supports claims that these genetic differences are more related to the herds sampled 24h ank-br-tr2015 6.0 5.5 5.25 5.25 4.75 5.75 4.25 3.75 4.75 4.25 3.75 4.75 4.5 4.0 3.25 4.75 3.5 4.0 3.0 kc-b12-tr2009 kc-vs12 k339/bursa-tr2009 u-ns66-tr2009 7 6 5 4 3 2 1 0 48h 72h 96h 120h 5.5 4.0 figure 7. replication kinetics of cythopathic bovine herpesvirus type 4 isolates in mdbk cell culture. 57 bilge dağalp et al. genetic variability of bohv-4 veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 to respiratory and reproductive system disorders, and two brain tissue samples from calves (kc-b12 and ank.br.tr2015) were representative of the 24 different herds (herd no: i, ii, vii, ix) and different sampling years (table i). the replication kinetics of the bohv-4 isolates showed some differences across the five sampling points. of bohv-4 isolates, four (kc-vs12, ank-br-tr2015, kc-b12 and u-ns66-tr2009) had the highest titers at 96 hpi whereas isolate k339’s titer increased constantly till 120 hpi. the highest virus titers were obtained with k339. verna and colleagues (verna et al. 2016) reported clear evidence that in  vitro characterization (biological and molecular) of viral replication in cell culture correlates with in  vivo virulence. perez and colleagues (perez et al. 2011) concluded that the viral strain is only weakly correlated with clinical manifestations. we therefore believe that the significant genetic variability in gb gene region sequences and the results on the in vitro biological behavior of our bohv-4 field isolates provides an important resource for further studies into the relationship between pathogenesis and the molecular characterization of bohv-4 from animals with different clinical signs or from apparently healthy cattle. unfortunately, all the isolates that we used to investigate biological behavior in mdbk cells were genotype i for gb gene sequences, although we also identified bohv-4 in genotypes ii and iii. conclusions in conclusion, we provided a precise biological and molecular characterization of the orf8 and orf3 gene regions coding gb and tk of bohv-4 field strains from turkish cattle with different health status. we hope that these findings may help future researchers to understand more clearly the molecular epidemiology of bohv-4 infection in cattle and the possible association between the virus and pathogenesis of infection. acknowledgements the authors would like to thank g. wellenberg for kindly providing the north american reference strain. funding this study was supported by a grant from the scientific research projects (project no: 2004 0810 068 and 10b3338005), ankara university. sequences for genes coding gb and tk. of these, eight were identified as genotype ii and one as genotype i for gb sequences while all were genotype i for tk genes (table i). we hope that further molecular characterization studies using larger number of samples and targetting some other genes along with tk and gb genes will be able to evaluate the role of these gene(s) in the pathogenesis of bohv-4 infection. in our study, the nucleotide and amino acid differences were more pronounced across the gb gene than the tk gene (figures 3-6). all the local bohv-4 samples located in genotype i had a change in the amino acid (aa) sequences of the gb gene at positions 44 (kq), similar to other genotype  i bohv-4 strains. the sequences (mh605274) of one field bohv-4 sample from milk, which was the only strain clustered in genotype iii, had changes at position 53.aa. (sd). turkish bohv-4 in genotype ii had one aa change at 57.aa (at). it was detected in seven strains (jx644989, mk543546, mk543547, gq246864, kx192365, eu055543, kx192364). these formed a branch within genotype ii in the phylogenetic tree. this is important given the variety of specimen types and their origin from seven different herds (figure  6, table i). the greatest aa change was detected in the gb sequences of bohv-4 strains from a nasal swab (jx644989) and a leucocyte sample (gq375280) from cattle in two different herds (figure 6). these results regarding aa change, especially in the seven genotype ii bohv-4 samples, reveal traces of domestic and/or international trade, although the latter change (a57t) did not match the sequences of any bohv-4 deposited in genbank. the main conclusion from our results is that there are significant differences between the bohv-4 strains circulating in turkey, although it is not yet clear how clinical cases are related to their molecular genetic characteristics. verna and colleagues (verna et al. 2012) and altamiranda and colleagues (altamiranda et al. 2015) reported similar high variability among bohv-4 strains in argentina. of the 24 samples used in this study, bohv-4 was isolated in five of them. these were then included in a virus isolation study to investigate their biological behavior using mdbk cell cultures. several factors may explain our failure to isolate the virus from 19 pcr-positive samples, including the poor quality of the samples or the inherent difficulty of isolating bohv-4 from samples (lin et  al. 1997, wellenberg et  al. 2002). we were lucky that our five positive samples, including one nasal swab (u-ns66-tr2009) and two vaginal swab samples (k339 and kc-vs12) from cattle with different clinical disorders attributed 58 genetic variability of bohv-4 bilge dağalp et al. veterinaria italiana 2021, 57 (1), 49-59. doi: 10.12834/vetit.2095.11150.1 altamiranda e.g., manrique j.m., pérez s.e., ríos g.l., 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319 veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 accepted: 17.01.2020 | available on line: 31.12.2021 1university of life sciences, faculty of veterinary medicine, department of preclinical veterinary sciences, sub‑department of veterinary microbiology, akademicka 12, 20‑033 lublin, poland. 2state veterinary laboratory, droga męczenników majdanka 50, 20‑325 lublin, poland. *corresponding author at: university of life sciences, faculty of veterinary medicine, department of preclinical veterinary sciences, sub‑department of veterinary microbiology, akademicka 12, 20‑033 lublin, poland. tel.: 0048 81 445 60 93, e‑mail: sebastian.gnat@up.lublin.pl. dominik łagowski1, sebastian gnat1*, aneta nowakiewicz1, marcelina osińska1 and przemysław zięba2 keywords alpaca, diagnostic procedure, dermatophytosis, infection source, trichophyton benhamiae. summary alpacas (vicugna pacos) are growing in popularity and are increasingly being presented for veterinary care. literature reports indicate that dermatophytosis occurring in alpacas accounted for about 3% of dermatological diagnoses. however, there are no reports regarding species of dermatophytes associated with alpacas and reservoirs of infection. in this study, we investigate the diagnosis and epidemiological origin procedure and the virulence enzymes activities of trichophyton benhamiae isolates obtained from alpacas from a breeding farm. identification was carried out traditionally by correlating clinical manifestations with micro and macroscopic examination, and molecular differentiation methods based on internal transcribed spacer (its) sequences. epidemiological analysis was carried out on the basis of melting point pcr (mp-pcr) and amplified fragment lenght polymorphism (aflp) genotyping. the production of virulence factors was evaluated phenotypically using specific test media. the results obtained from diagnostic tests indicated that the etiological factor of dermatophytosis is t. benhamiae. the same species was also isolated from cowsheds and insects. the mp-pcr and aflp analyses indicated high invariability of the genomes of the strains isolated from the animals, cowsheds, and insects. in conclusion, animal husbandry outside the natural ecological niche may increase predisposition to dermatophytosis. the treatment of animals alone is insufficient, one should be aware that only elimination of all fungal sources is a long-term success and the key point of therapy. diagnostic and epidemiological analysis of trichophyton benhamiae infection on an alpaca (vicugna pacos) farm in poland alpacas together with llamas (lama glama) are domesticated species of south american camelids (sacs), whereas guanaco (lama guanicoe) and vicuna (vicugna vicugna) are wild species of sacs (halsby et al. 2017). camelids are regarded as rather exotic animals in europe (twomey et al. 2014, halsby et  al. 2017, foster et  al. 2007). as a rule, they live in zoological gardens, although in some countries, e.g. in great britain or poland, alpacas are already farmed and raised due to the quality of their wool. some are kept as pets, for trekking, guarding livestock, and in open farms (halsby et  al. 2017). in england and wales, there is also a small but developing market for alpaca meat products (twomey et al. 2014). literature reports indicate occurrence of dermatophytosis in alpacas (foster et  al. 2007, introduction alpacas (vicugna pacos, formerly lama pacos) are growing in popularity and are increasingly being presented for veterinary care (scott et  al. 2011, halsby et al. 2017). these animals often present skin disorders that constitute diagnostic, therapeutic, and epidemiology challenges for practicing veterinarians (foster et  al. 2007). additionally, alpacas may have regular and close human contact (halsby et  al. 2017). understanding infectious diseases associated with these animals and the possible risks to human health is important for alpaca keepers and breeders, veterinary professionals, and others involved in recreational and therapeutically activities (halsby et al. 2017, scott et al. 2011). 320 veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 trichophyton benhamiae infection on an alpaca farm łagowski et al. difficult (sabou et al. 2018). in the present study, we identified and analysed t.  benhamiae infection in alpacas from a breeding farm. the aim of this study was to investigate the epidemiological origin of t.  benhamiae isolates using morphological traits in combination with molecular analysis and to assess their capability to produce different enzymes. materials and methods strains and sampling methods strains were isolated in the department of veterinary microbiology, university of life sciences in lublin, poland from clinical samples sent to our diagnostic laboratory for identification by an alpaca breeder and veterinarian in sterile petri dishes. forty-one samples were obtained from alpacas, including 11 with symptomatic dermatophytosis (each isolate from another animal), 10 collected from the cowshed (from mulch), and twenty from insects that were identified as the housefly (musca domestica; insects collected from catching tapes suspended in the cowsheds). the farm was located in south-eastern poland and comprised 41 animals aged 1-5 years kept in an outdoor breeding system (standard maintenance and nutrition conditions). the alpacas were imported 6  years earlier from chile and no new animals were introduced into the farm ever since. no case of dermatophytosis was diagnosed on the farm earlier. in eleven alpacas, in september 2018, round alopecia sites or ca. 3 cm excoriations covered with thickened scaling epidermis were noticed at the border of the head and neck with a distinct tendency to hair loss (from three to five in each of the suspected animals). the clinical changes were diagnosed as dermatophytosis. hair from the margins of the clinical changes was collected for mycological diagnostics. at the same time, a routine veterinary assessment for superficial fungal infections of the other alpacas was made on the farm, and material from the neck and trunk taken with the brush technique was sent to the laboratory. before the cowshed was disinfected, material from the bedding was collected for epidemiological purposes. during the cleaning works, rodent droppings were noticed in the cowshed, but no rats or mice were found. mycological studies were also made in material taken from a dog and two cats living on the farm as well as from the nail of the breeder and veterinarian. at a later stage of the studies, a flytrap that was suspended in the cowshed with 20 insects stuck to it was subjected to mycological analysis. d’alterio et  al. 2006, halsby et  al. 2017). this fungal infection accounts for about 3% of dermatological diagnoses (d’alterio et al. 2006); nevertheless, in the animal health and veterinary laboratories agency, only five cases of dermatophytosis in alpacas were reported between 2000 and 2015 (halsby et  al. 2017). normally, symptoms of ringworm in alpacas are similar to these in other farm animals and include mild skin alopecia, with skin rarely covered by crust or hyperkeratotic areas (scott et  al. 2011). however, there are no reports regarding species of dermatophytes associated with alpacas and reservoirs of infection. the dermatophyte trichophyton benhamiae is an important zoonotic pathogen, and the infection rates have been increasing worldwide over the last 15 years (sabou et al. 2018, drouot et al. 2009). a study performed in germany between march 2010 and march 2013 showed that t. benhamiae had already become the most frequent zoophilic dermatophyte responsible of human infections, with a prevalence of 2.9% (jochen brasch et  al. 2015). the histories of many zoonotic infections suggest that guinea pigs are the main source of transmission. however, other small rodents (jochen brasch et  al. 2015), rabbits (nakamura et  al. 2002), dogs (sieklucki et  al. 2014), or even porcupines (takahashi et  al. 2008) have also been associated with t.  benhamiae infection, but no comparative investigations of different small animals have been published yet. so far, no t. benhamiae infections in south american camelids and other farm animals have been described. t.  benhamiae was formerly known as trichophyton species of arthroderma benhamiae; this species was considered part of the t. mentagrophytes species complex (gnat et al. 2019c). in the past, t. benhamiae was often misdiagnosed as microsporum canis (mayser et  al. 2013, nenoff et  al. 2014) or t.  mentagrophytes var. porcellae (sabou et  al. 2018) because of a similar colony color or as t. interdigitale and microscopic similarities (nenoff et al. 2014). two phenotypes have been described for t. benhamiae: yellow and white (brasch et  al. 2015, nenoff et  al. 2014, sabou et al. 2018). the first is characterized by strains with downy, pleated mycelium with a slow growth rate and poor sporulation on sabouraud medium with rare microconidia or absence of macroconidia (hiruma et  al. 2015, sabou et  al. 2018). the white phenotype strains are powdery to floccose with a rapid growth rate (sabou et  al. 2018). numerous spherical to clavate microconidia and sparse, thin-walled, cigar shaped macroconidia are present in microscopic preparations (hiruma et al. 2015). due to its more than three different names, two different phenotypes, and several different possible hosts, the identification of t.  benhamiae is 321veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 łagowski et al. trichophyton benhamiae infection on an alpaca farm mixture with primer powaagct (25 pmol, 5’-ctcactctcaccaacgtcgacagctt-3’; genomed, warsaw, poland). the aflp analysis were performed with few modification as previously described by graser and colleagues (gräser et  al. 2000). briefly, one restriction enzyme ecori (thermo fisher, usa) and one primer with three selective nucleotides ecori-atg (5’-gac tgc gta cca att cat g; genomed, warsaw, poland) were used. the pcrs were carried out according to the source description in a tgradient thermal cycler (analytik jena ag, jena, germany). electrophoresis of all pcr products was carried out in 3% agarose gels. all analyses were made at least in triplicate. evaluation of production of virulence factors the production of virulence factors was evaluated using specific test media. firstly, the dermatophytes were cultured onto sabouraud glucose agar (becton dickinson, franklin lakes, nj, usa) and incubated at 37 °c for seven days. next, an inoculum with a 5 mm diameter from the edge of each culture was transferred onto plates containing the test medium (second passage). the following tests were performed: production of keratinase, phospholipase, lipase, elastase, protease, and gelatinase and detection of haemolytic activity as previously described (gnat et  al. 2018c). each test was performed in triplicate and each strain was tested in duplicate in each experiment. the enzymatic activities were expressed as a difference between the total diameter of the colony plus the zone of precipitation and the colony diameters. in the case of detection of haemolytic activity, a transparent clearance zone around the colony indicated complete haemolysis. the enzymatic activity of dermatophyte isolates was statistically analysed with the r program (free software foundation, boston, usa). statistical inference was performed based on the results of the student t test. results in this study, 77 samples were investigated: 41  samples were collected from alpacas with and without clinical symptoms of trichophytosis, 10 from the cowshed where the alpacas were kept, 20 from a fly-stick hanging in the cowshed, one from a dog, two from cats, two from the breeders, and one from the veterinarian. sixteen of these samples (21%) were t.  benhamiae positive, as demonstrated by morphology and its sequencing (table i). the first group of strains included isolates from animals with dermatophytosis. on average, 27% of laboratory diagnostic procedure after the material was delivered to the laboratory, the first stage of the diagnostics consisted in direct examination of unstained slides in a 10% koh solution, which was then viewed at a magnification of 40x10 (nikon coolpix ys100). simultaneously, cultures were inoculated with the bilayer tile method onto sabouraud glucose agar (becton dickinson, new jersey, usa), incubated at 37 °c for 14-21 days, and examined twice a week. dermatophytes were identified based on colony texture and production of typical spores (hoog et al. 2000). confirmation of the initial identification was performed retrospectively by sequencing the internal transcribed spacer (its) region (including parts of 18s and 28s rdna, as well as the whole of its1, 5.8s rdna, and its2) of the ribosomal dna as previously described (gnat et  al. 2018a). briefly, dna was isolated from the dermatophytes with the phenol-chloroform method (gnat et al. 2017). the its amplification reaction was carried out in a t personal thermal cycler (biometra gmbh, goettingen, germany) using qiagen taq pcr master mix (qiagen, hilden, germany), 10 pmol of each primer: its1 (5’-tccgtaggtgaacctgcgg-3’) and its4 (5’-tcctccgcttattgatatgc-3’) (white et  al. 1990) (genomed s.a, warsaw, poland), and 50  ng of dna template. electrophoretic separation of pcr products was carried out in 1% agarose gels. the gels were documented and analysed in geldoc 2000 (bio-rad, hercules, california, usa). the its sequencing reaction was carried out using a bigdye terminator cycle sequencing kit (life technologies, carlsbad, california, usa) and primers its1 and its4. two separate reactions were carried out for each primer. pcr was performed in a t-personal thermal cycler (biometra gmbh). the amplicon was purified using an exterminator kit (a&a biotechnology, gdynia, poland) and then the dna sequence was read in the 3500 genetic analyzer (life technologies, carlsbad, california, usa). the identification was made using the blast (basic local alignment search tool) in the genbank database. epidemiological analysis epidemiological analysis was carried out on the basis of mp-pcr (melting-point-pcr) and aflp (amplified fragment length polymorphism) genotyping of whole genomes of the clinical isolates. the mp-pcr procedure was optimized for dermatophyte differentiation by leibner-ciszak and colleagues (leibner-ciszak et al. 2010). in this study, approximately 100 ng of dna, endonuclease hindiii (thermo fisher, usa) and a mixture of two oligonucleotides helper and ligated (15 pmol; helper: agctgtcgacgttgg, ligated: ctcactctcaccaacaacgtcgac) were used. pcr was carried out in a 25-µl reaction 322 veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 trichophyton benhamiae infection on an alpaca farm łagowski et al. the third group were strains isolated from flies collected on the adhesive tape of the flytrap. three strains were isolated from the 20 collected flies (15%). they were two strains with a beige obverse and one with a white phenotype. the analyses of the strains showed distinct transition from the dominant hyphal form on day 4 to the sporous form dominating from day 14, especially in the case of the white-phenotype strain. a comparative analysis of the its sequences of the isolated and reference strains available in the ncbi (nucleotide centre of biotechnology information) database was employed for a correct identification of the dermatophyte species. the its sequences obtained for all 16 isolates (accession number mk922472-mk922478, mk922512-mk922519, mk940328) using the blast (basic local alignment search tool) software available in the ncbi (national center for biotechnology information) database exhibited approximately 99% similarity to the t. benhamiae cbs623.66 sequences. the mp-pcr and aflp methods were used to determine the genomic differentiation of the t.  benhamiae isolates. the agarose gel electrophoregrams indicated high invariability of their genomes (figures 3 and 4). although the dermatophytes were isolated from the different animals, samples taken from cowsheds, and insects, no different genomic profiles were obtained. the virulence activities of the dermatophyte isolates the alpacas on the farm were affected with typical clinical symptoms. furthermore, no dermatophytes were isolated from the asymptomatic animals (alpacas, dog, cats) and from three farm employees. direct analysis of the material sampled from the clinical lesions in the symptomatic alpacas revealed the presence of arthrospores (figure 1). the positive result of the examination of the culture confirmed the initial diagnosis. the macromorphological image of the isolated t.  benhamiae showed two types of colonies: four isolates with a fluffy texture, a beige obverse, and a yellow reverse and the other type with a friable, powdery, white obverse and a brown reverse (figure 2). the size of the colony was in the range from 5 to 8 mm. the edges of the colony were softly corrugated with prominent protrusions extending from the centre. the micromorphological image on the microscope slides revealed visible circular to clavate microconidia, which were much less numerous in the colonies with the beige phenotype (figure 2). there were no macroconidia or spiral hyphae. the second group of strains comprised isolates from the cowshed litter. t.  benhamiae was isolated in 2 of the 10 collected samples (20%). both strains represented the beige phenotype. the sporulation rate in these isolates was definitely lower than that observed in the samples collected from the animals. the emerging microconidia were not sufficiently abundant to dominate the preparation and the hyphal form was profusely visible. table i. isolates of dermatophytes obtained from symptomatic animals, cowshed and insects with description of isolation source, location of changes and accession numbers of its sequences. isolates host isolation source location of changes phenotypic type accession numbers of its sequences identification consistent with the ncbi database tba1 alpaca clinical lession neck beige mk922475 trichophyton benhamiae cbs623.66 (accession number ab088677) tba2 alpaca clinical lession head beige mk922476 tba3 alpaca clinical lession head, neck beige mk922477 tba4 alpaca clinical lession head, neck beige mk922478 tba5 alpaca clinical lession head, neck white mk922514 tba6 alpaca clinical lession head, neck white mk922515 tba7 alpaca clinical lession head white mk922516 tba8 alpaca clinical lession head, neck white mk922517 tba9 alpaca clinical lession neck white mk922518 tba10 alpaca clinical lession head, neck white mk922519 tba11 alpaca clinical lession head beige mk940328 tbb1 cowshed litter ‑ white mk922512 tbb2 cowshed litter ‑ beige mk922472 tbc1 insect outer shell ‑ beige mk922473 tbc2 insect outer shell ‑ beige mk922474 tbc3 insect outer shell ‑ white mk922513 ncbi = national center for biotechnology information. 323veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 łagowski et al. trichophyton benhamiae infection on an alpaca farm discussion dermatophytes are generally cosmopolite, but in recent years, there has been a notable tendency towards limitation of the geographical range of some species and the strict connection with a sensitive animal host (sabou et  al. 2018, gnat et  al. 2019a). nonetheless, population migration, including the import of animals that do not naturally occur in a given climate, improvement of the hygiene of animal breeding, changes in the human lifestyle, and the increase in physical activity in the company of animals have an impact on the geographical distribution and reservoirs of dermatophytes (hiruma et al. 2015, sabou et al. 2018). were screened in a phenotypic assay, i.e. production of enzymes and determination of the haemolytic activity of the strains (table ii). all the clinical isolates of t.  benhamiae showed keratinase, phospholipase, lipase, gelatinase, and protease activity. there were no statistically significant differences in the keratinase, gelatinase, and lipase activities between all the isolates. no elastase activity was detected in any of the isolates. the statistically highest phospholipase and protease activities, i.e. with a diameter of 9.1 mm and 9.9 mm, respectively, were noted for the t. benhamiae isolates derived from the animals. in addition, regardless of the source of the isolate, all strains caused type β haemolysis. small differences between the distribution zones around the colonies were noted for the haemolytic activity of isolates obtained from the alpacas and the cowshed. definitely weaker haemolytic properties were demonstrated by the t.  benhamiae isolates derived from the insects. figure 1. microscope slides from direct analysis of material sampled from clinical lesions in the alpaca (pictures taken with light microscopy at 400x, nikon coolpix ys100). figure 2. micro‑ and macroscopic morphology of dermatophytes isolated after 14 days of incubation, a ‑ the beige phenotype, b ‑ the white phenotype (pictures taken with light microscopy at 400x, nikon coolpix ys100). a, b. macromorphology (obverse and reverse), a’, b’. micromorphology (stained with lactophenol blue). figure 3. electrophoretic profile obtained with the mp‑pcr fingerprinting method in 3% agarose gel. m = molecular weight marker generulertm (1000bp; thermo fisher, usa); tba1‑tba11 = strains isolated from alpacas; tbb1‑tbb2 = strains obtained from litter; tbc1‑tbc3 = strains isolated from insects. figure 4. electrophoretic profile obtained with the aflp method in 3% agarose gel. m = molecular weight dna marker ‑ marker 3 (3000bp; a&a biotechnology, gdynia, poland); tba1‑tba11 = strains isolated from alpacas; tbb1‑tbb2 = strains obtained from litter; tbc1‑tbc3 = strains isolated from insects. 324 veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 trichophyton benhamiae infection on an alpaca farm łagowski et al. 2010, brillowska-dabrowska et  al. 2007, gnat et  al. 2018a, shehata et al. 2008). our study also revealed that the pcr-based band profiles obtained by mp-pcr and aflp were identical in all the clinical isolates studied. the homology of the genomes in the t.  benhamiae isolates obtained in this analysis indicates a high probability of the same origin of the etiologic agent of the dermatophytosis in the alpacas and its source location in the cowshed or originating from insects. interestingly, this pathogen was not detected in the two cats and the dog kept on the same farm. this may be related to the greater mobility of these animals and the possibility of chasing away flies trying to sit on their fur, or the impossibility to enter the cowshed. bartosch and colleagues (bartosch et  al. 2019) showed that the high infection rate of 58% confirms that guinea pigs are a main reservoir for t.  benhamiae, which was in agreement with previous studies (nenoff et  al. 2014). furthermore, it is suggested that although rats, mice, and even rabbits are less susceptible to t. benhamiae infection than guinea pigs, the spread of these pathogens is much easier due to the lack of biosecurity in this animal stock (bartosch et al. 2019, nenoff et al. 2014). because the animals are rodents or are kept only as feed animals, most often no skin infection therapy is applied. moreover, brasch and colleagues (brasch et  al. 2016) noted that the spread of t.  benhamiae might be difficult to control, as infected guinea pigs are often free of clinical signs of dermatophytosis. although there are no similar data for rodents, it is important in this case that they are usually unnoticed by breeders on the farm. in our study, t. benhamiae was isolated from the litter in the cowshed where the alpacas were kept. although no rodents were noticed on the farm, the presence of probably rats’ droppings was noted during the cleaning work in the cowshed. therefore, guinea pigs and rodents, at least in breeding stocks and pet shops, should be screened regularly for dermatophytes and adequate biosecurity measures should be implemented to prevent this ignored zoonosis (brasch et al. 2016). the housefly has the potential for dissemination of microorganisms associated with animal faeces, skin, fomites, and natural environment (soil, water, seeds, and nuts) (korniłłowicz-kowalska et  al. 2013, zarrin an important factor influencing the epidemiology of dermatophytosis is the knowledge of its possible transmission source. it is thought that transmission can take place after direct contact with an infected individual, even if asymptomatic, or indirectly via fomites, scales, and animal hairs (moriello et al. 2017). particularly dangerous in terms of epidemiology is the fact that infectivity may persist as long as two years (contet-audonneau et  al. 2010). to date, only few reports on other potential sources of indirect infection, i.e. soil, water, plants, rodents, insects, etc., are available (korniłłowicz-kowalska et al. 2013). this contributes to the spread of infection, since they are neither identified as a potential source of infection for the animal milieu nor screened or removed from animal surroundings. furthermore, trichophytia infections in animals are often misdiagnosed at the initial presentation and therefore wrongly treated, for example with steroids, modifying the clinical findings and resulting in difficulty in further diagnosis (brillowska-dabrowska et  al. 2010). improperly diagnosed treated or recurrent superficial mycoses are a potential source of infection via direct and indirect transmission to other animals and humans (gnat et al. 2018b). for this reason, epidemiological studies on new potential sources of dermatophyte infections are an important element of knowledge for veterinarians. based on the increasing role of t.  benhamiae in the development of dermatophytosis in various breeding and companion animals as well as humans, we believe that it is important to carry out epidemiological analysis of this pathogen from an outbreak on an alpaca farm. morphological analysis failed to discover the source of dermatophyte infection; nevertheless, it resulted in highly probable species identification for all 16 t. benhamiae isolates investigated in this study and confirmed the species through its sequencing. therefore, we used the mp-pcr and aflp genotyping methods. currently, pcr-fingerprinting techniques are believed to have sufficient suitability for epidemiological investigation of the origin of dermatophyte infection. many investigators show that these methods can be a useful tool for analysis of the diversification of dermatophyte genomes in full agreement with identification based on both cultureand its-techniques (leibner-ciszak et  al. table ii. enzymatic activity in vitro of isolates obtained from alpacas, cowshed, and insects expressed by the mean (in mm) of the diameter of the clear zone around the colonies after 21 days of incubation. origin of isolates isolates keratinase phospholipase lipase elastase gelatinase protease haemolysis alpacas tba 8.4 (0.6) 9.1 (1.1)+ 8.8 (0.9) 0 1.1 (0.3) 9.9 (0.5)+ 11.2 (1.9) cowshed tbb 7.5 (1.0) 7.4 (0.8) 7.9 (0.9) 0 1.0 (0.5) 8.4 (1.2) 11.8 (0.8) insects tbc 5.9 (0.8) 3.1 (0.5) 4.0 (1.1) 0 0.7 (0.5) 5.2 (0.9) 6.7 (1.0) standard deviation in brackets; +statistically significance in column. 325veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 łagowski et al. trichophyton benhamiae infection on an alpaca farm different hosts (gnat et  al. 2018c, figueredo et  al. 2011). therefore, the pathogenic potential of a dermatophyte depends on its ability to produce various enzymes (sharma et  al. 2007). in turn, variations in the enzymatic profile and its potential might be responsible for the differences in the pathogenic effects in different hosts (dalis et  al. 2014). in this context, interesting data are provided by studies of the enzymatic activity of strains isolated from another source. some investigators suggest that the diversified enzymatic activity of dermatophytes is an adaptive trait and can be partially explained by the effect of substrate availability (figueredo et  al. 2011). however, in our study, statistically significantly higher phospholipase and protease activity was obtained in the case of the alpaca infection in comparison to strains isolated from the cowshed and insects. in turn, the keratinase activity was much lower in the dermatophyte from the insects. probably, there is a relationship between the enzymatic activity and the affinity of dermatophytes for species-specific keratin, and every change in the life niche weakens the fungal virulence. in this case, there is a time to adapt between the transfer of infectious dermatophyte structures from the vector to the sensitive individual and the occurrence of the infection symptoms (gnat et al. 2019a). appropriate hygienic activities provided to the animal at this time can prevent the development of an infection. this issue requires more extensive research and deeper discussion. zoophilic dermatophytes tend to be contagious; therefore, collaboration between a dermatologist and a veterinary is advisable as soon as the source of infection has been identified. this report implies that rodents and insects can be a reservoir or vector for zoophilic dermatophyte infection, also in the case of breeding animals. the treatment of the patients alone is insufficient, and cleaning of contaminated items and areas needs to be performed simultaneously in every case of dermatophytosis. therefore, one should be aware that only elimination of all fungal sources ensures a long-term success and is the key point of therapy. et al. 2007). these insects have been shown to feed on secretions and other human and animal wastes, thus becoming ideal carriers for transmitting various pathogenic microorganisms. not only has the association of insects and bacterial diseases been documented but also transmission of fungi has been confirmed by several reports (zarrin et al. 2007, gilliam et  al. 1974, da costa et  al. 1998). da costa and oliveira (da costa et  al. 1998) isolated various species of penicillium from mosquito vectors of tropical diseases. zarrin and colleagues (zarrin et al. 2007) verified the predominance of fungi from the genera aspergillus and penicillium in adult insects from the family muscidae captured in an abattoir. interestingly, two species of dermatophytes, i.e. trichophyton mentagrophytes and microsporum gypseum, were identified among filamentous fungi in their study. our study has demonstrated that the housefly can be a carrier of trichophyton benhamiae fungal spores. the epidemiological danger of this situation is associated with the fact that these flies were caught on the alpaca farm, where there was an outbreak of dermatophytosis caused by this etiological factor. these results are in contradiction to the findings reported by ysquierdo  and  colleagues (ysquierdo et al. 2017) suggesting that dermatophyte arthroconidia are not acquired and disseminated by houseflies. while adherence of arthroconidia to human skin is strongly time dependent, it is unclear whether this holds true for cuticular surfaces (ysquierdo et al. 2017). it is worth noting that the host is most contagious prior to hair loss, while denuded lesions are definitely less contagious. the housefly collection in the study conducted by ysquierdo and colleagues (ysquierdo et al. 2017) was sampled from cattle with hair loss, which was less than optimal in terms of contagion. given these circumstances, the formulated conclusions do not have to be definitive. analyses of various superficial mycosis cases based on the severity of infection have shown that dermatophyte strains may vary in their ability to cause infection in animals and humans (sharma et  al. 2007, gnat et  al. 2019b). this phenomenon can be associated with the synthesis of enzymes that enhance dermatophyte survival in tissues of 326 veterinaria italiana 2021, 57 (4), 319-327. doi: 10.12834/vetit.1967.10530.2 trichophyton benhamiae infection on an alpaca farm łagowski et al. bartosch t., agnes f., günther c., silke uhrlaß, heydel t., nenoff p., baums c. & schrödl w. 2019. trichophyton benhamiae and t. mentagrophytes target guinea pigs in a mixed small animal stock. med mycol case rep, 23, 37-42. brasch j., beck-jendroschek v., voss k., uhrlass s. & nenoff p. 2016. arthroderma benhamiae strains in germany: morphological and physiological characteristics of the anamorphs. hautarzt, 67, 700-705. brasch j. & wodarg s. 2015. morphological and physiological features of arthroderma benhamiae anamorphs isolated in northern germany. mycoses, 58, 93-98. brillowska-dabrowska a., 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m., denis j., boulanger n., forouzanfar f., glatz i., lipsker d., poirier p., candolfi e. & letscher-bru v. 2018. molecular identification of trichophyton benhamiae in strasbourg, france: a 9-year retrospective study. med mycol, 56, 723-734. scott d.w., vogel j.w., fleis r.i., miller w.h. jr. & smith m.c. 2011. skin diseases in the alpaca (vicugna pacos): a literature review and retrospective analysis of 68 cases (cornell university 1997-2006). veterinary dermatology, 22, 2-16. sharma r., hoog s.c., presber w. & gräser y. 2007. a virulent genotype of microsporum canis is responsible for the majority of human infections. j medical microbiol, 56, 1377-1385. 5 review veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 accepted: 02.02.2022 | available on line: 29.06.2022 1national and woah reference centre for brucellosis, istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. 2direzione generale della sanità animale e dei farmaci veterinari, ministero della salute, viale g. ribotta 5, 00153 rome, italy. * corresponding author at: national and woah reference centre for brucellosis, istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. e‑mail: f.demassis@izs.it. fabrizio de massis1*, flavio sacchini1, antonio petrini1, fabio bellucci1,2, margherita perilli1, giuliano garofolo1, giovanni savini1 and manuela tittarelli1 keywords brucellosis, brucella canis, dog, zoonosis. canine brucellosis due to brucella canis: description of the disease and control measures summary brucellosis is a contagious disease caused by bacteria of the genus brucella, which can affect different animal species. dogs may occasionally be infected with b. abortus, b. melitensis or b. suis, or by the endemic form of the disease, caused by b. canis. among the brucellosis‑affecting domestic animals, that of the dog is certainly the least frequent, but also the least studied. canine brucellosis due to b. canis represents the dog‑specific brucellosis, both because it is the main susceptible animal species, and because it constitutes its fundamental reservoir of infection. the disease can also affect humans, although its course does not assume the characteristics of severity typical of the infection determined by the ‘classical’ species of the genus brucella. in italy, there are frequent imports of dogs from countries where the disease is present, often with non‑controlled movements and without sanitary controls. considering that the zoonotic potential of the disease can be favored by the close cohabitation between man and dog, which occurs especially in urban environments, canine brucellosis has to be regarded as a public health problem susceptible to introduction and spread in the italian territory. dog‑specific brucellosis. dogs in fact are the main susceptible animal species, and the fundamental reservoir of infection. the disease can also affect humans, although its course does not assume the characteristics of severity typical of the infection due to the ‘classical’ species of the genus brucella; however, in countries where the disease is present (particularly in the american continent) it is considered an authentic zoonosis. in italy, there are frequent imports of dogs from countries where the disease is present, often with non‑controlled movements and without sanitary control. considering that the zoonotic potential of the disease can be favored by the close cohabitation between man and dog, which occurs especially in urban environments, canine brucellosis has to be regarded as a public health problem susceptible to introduction and spread across the italian territory. introduction brucellosis is a contagious disease caused by bacteria of the genus brucella, which can affect different animal species. dogs may occasionally be infected with b.  abortus, b.  melitensis or b.  suis, or be affected by the endemic form of the disease, caused by b.  canis. among the brucellosis‑affecting domestic animals, that of the dog is certainly the least frequent, but also the least studied. canine brucellosis due to b.  abortus or b.  melitensis is uncommon in dogs living in close contact with infected ruminants. in these cases, it only represents an epiphenomenon of the infection circulating in the affected farm, and the dog does not play the role of disease reservoir. the same applies for dog brucellosis due to b. suis, which is rarely detected in dogs and, in any case, always in connection with a coexisting infection in pig farms. canine brucellosis due to b.  canis represents the 6 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. investigation on several outbreaks of abortion and infertility in dogs occurring in different areas of the united states of america (carmichael 1966). subsequently, b.  canis was isolated in different countries of the world, while in others its presence has been suspected based on serological positivity. recently, hensel and colleagues (hensel et  al. 2018) have collected the information available from the international literature regarding serological investigations carried out for canine brucellosis due to b. canis worldwide (figure 1). these studies show that there is a wide level of variability in serum positivity (from 1% to 28%), depending on the country studied and the sample taken (hensel et al. 2018). seroprevalence is strongly influenced by the type of serological test used and the interpretation of its results, due to the fact that b.  canis shares antigen determinants with all r‑phase (rough) brucellae and with microorganisms of other genera not related to the genus brucella (carmichael 1990). furthermore, apart from the true disease prevalence in the area under study, the wide spread of high level of seroprevalence recorded could also be attributed to the sampling design and the algorithm used for the interpretation of test results (hensel et al. 2018). in italy, several surveys reported the presence of seropositivity at various levels and in different geographical areas (tolari and pizzirani 1978, colella et  al. 1980, ciuchini et  al. 1982, vesco et  al. 1987, valente et al. 1991, buonavoglia et al. 1992, prosperi et al. 1994, casalinuovo et al. 1996). the only clinical case described in scientific literature dates to 2008, when corrente and colleagues (corrente et al. 2010) detected b. canis by pcr in a half‑breed dog suffering from chronic prostatitis and discospondylitis. in 2020, b.  canis has been detected and isolated for the first time in a commercial breeding kennel (de massis et al. 2021). decription of the disease etiology brucellae are gram‑negative coccobacilli or short rods measuring from 0.6 to 1.5 µm long and from 0.5 to 0.7 µm wide. they are usually arranged singly, and less frequently in pairs or in small groups. the morphology of brucella is fairly constant, except in old cultures where pleomorphic forms may be evident. bacteria of the genus brucella are normally non‑motile. they do not form spores, or pili; true capsules are not produced. they are aerobes and asporigenic. with regards to b.  canis, it has no biovar, does not require carbon dioxide to grow in first isolation, and grows in presence of thionin but not with basic fuchsin. in first isolation, colonies of b.  canis always occur in phase r (rough) or m (mucoid), while their existence in phase s (smooth) has never been reported. b. canis does not agglutinate in the presence of brucella a‑ and m‑ monospecific antisera, while it agglutinates with specific antisera towards the r antigen of b. ovis (reo 198 strain). furthermore, cross‑reactions with surface antigens of other species of the genus brucella in a non‑smooth phase are also possible. b. canis does not produce h 2 s, does not oxidize the substrate based on l‑asparagine or d‑xylose, does not reduce nitrates to nitrites, and does not show lysis in the reaction with different phages, except with the phage r/c (corbel and brinkley‑morgan 1989). epidemiology geographical distribution b.  canis was first reported in 1966 during an figure 1. location and outcome of serological investigations for the detection of antibodies against b. canis recorded in the international scientific literature. each point represents a published study; the color of the point represents the identified source (from hensel et al. 2018) seroprevalence (%) 0-6 6.1-20 20.1-≈30 7veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis of infected males is high; the elimination of the microorganism in low concentrations for a period exceeding 60 weeks was also observed (george et al. 1979). under experimental conditions, the isolation of b.  canis from prostate and epididymis was possible up to more than two months after the end of bacteremia, and the venereal transmission of the disease continued for at least two years in clinically normal appearing dogs. even after castration, males may remain a source of infection because the bacterium can persist in the prostate and lymphatic tissues (carmichael 2012). urinary elimination begins a few weeks after the onset of bacteremia and continues for at least three months. in the urine of infected males, concentrations of microorganisms from 103 to 106 colony forming units (cfu)/ml have been found, while for females this number is lower (serikawa and muraguchi 1979). the bacteria have also been isolated from saliva, nasal and ocular secretions of dogs (carmichael and joubert 1988, moore 1969). unlike what occurs in the male, it seems that in females urine is not important for the transmission of the disease, due to the low number of microorganisms present (carmichael 1990). the fact that the male eliminates the microorganism with urine in greater quantities is probably related to the specific localization of b.  canis in the prostate and epididymitis or contamination with seminal fluid (carmichael and joubert 1988). despite this evidence, according to henesel and colleagues (hensel et  al. 2018), the role of urine as a means of transmission of canine brucellosis from b. canis has not yet been fully clarified (hensel et al. 2018). pathogenesis and pathological lesions although the pathogenesis of b. canis infection has not yet been fully elucidated, it is likely it follows the general pattern of brucella infection common to other animal species. the route of entry of the pathogen are the conjunctival, oral, and genital mucous membranes (carmichael 1990). in the dog, the minimum oral infectious dose is around 2 × 106 cfu, while the minimum infectious dose via venereal route has not been determined yet, although it is assumed to be slightly lower (carmichael and joubert 1988). the infectious dose via conjunctival route is between 104 and 105 cfu (serikawa and muraguchi 1979). after oral infection, the incubation period is variable, however a bacteremia associated with leukocytes can normally be detected one or two weeks after infection (carmichael and joubert 1988). a characteristic feature of the infection, also significant from a diagnostic point of view, is the prolonged bacteremia. commonly, it is possible to susceptible species unlike s‑phase (smooth) brucellae, which are able to infect different animal species, b.  canis has a limited range of possible hosts. dogs and wild canids are thought to be the only significant hosts for b. canis. cattle, sheep and swine were found to be highly resistant to the infection. cats appear to be moderately sensitive and, following oral exposure to b. canis, can develop bacteremia with low antibody titers. guinea pigs, mice, rats, and non‑human primates are also susce ptible to experimental infection. (carmichael 1990). the rabbit is more sensitive than other laboratory species, developing orchitis and peritoneal abscesses following high doses inoculated through intraperitoneal route (carmichael and bruner 1968). humans can get b.  canis infection through direct contact with infected dogs, with their reproductive secretions, or with their blood (lawaczeck et  al. 2011, lucero et  al. 2010). clinical disease has been described. transmission canine brucellosis due to b.  canis is especially common among stray dogs, in shelter kennels, in commercial breeding kennels, or in places where they live in large groups (carmichael and joubert 1988). when b.  canis is introduced into a kennel, the spread is rapid (carmichael and bruner 1968). b.  canis can be transmitted from infected to healthy adult males after a few weeks or months of cohabitation. conversely, it is not transmitted between non‑sexually mature males or females (carmichael and joubert 1988). a number of natural pathways of transmission of canine brucellosis have been observed, but the most common is the contact with placenta, fetal tissues and vaginal discharges resulting from abortion due to b. canis infection. moreover, transmission is also possible during estrus or mating (carmichael 1990). vaginal discharge can contain more than 1010 microorganisms per ml, and elimination by this route can continue for several weeks after abortion (carmichael and joubert 1988). other body secretions contain lower concentrations of b.  canis and are less important for the spread of infection. most puppies become infected in the uterus. infected mothers or their milk also represent a potential source of infection for those which survive infection. seminal fluid and urine of dogs harboring b.  canis in the prostate and epididymis have been demonstrated as an important source for disease spreading. during the first 6‑8 weeks of infection, the concentration of b.  canis isolated from semen 8 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. is a unilateral or bilateral lymph node enlargement at sites of bacterial entry (usually retropharyngeal lymph nodes for the oral route and superficial inguinal lymph nodes for the vaginal route). the lymphadenopathy is the only detectable symptom in the non‑pregnant adult female, while prostatitis and epididymitis can be observed in the adult male. in pregnant females, the main symptom is late abortion, which generally occurs between the 45th and 55th day of gestation (carmichael 2012). the abortion is followed by a yellow to brown odorless vaginal discharge, that persists from one to six weeks (carmichael 1968). cases of embryonic resorption or abortion from the 10th to the 20th day of mating have also been described. they often go unnoticed in practice, as they are normally considered as an infertile mating (carmichael 1990). in exceptional cases, pregnancy can be brought to the end, with simultaneous birth of both live and dead puppies (feldman and nelson 1996). most live‑born puppies die within a few hours or days; those that survive normally show a generalized increase in lymph node volume, which is the main symptom until they reach sexual maturity (carmichael 1990). these puppies may be bacteriemic but apparently healthy (carmichael 2012). in addition, as a systemic manifestation of b. canis infection, surviving puppies usually show persistent hyperglobulinemia, and some of them may have transient fever, leukocytosis, or convulsions (carmichael 2012). it is possible that apparently healthy puppies born from infected mothers spread b.  canis to other dogs and humans (dentinger et al. 2012). as with brucellosis affecting other animal species, b. canis infection in dogs does not interfere with the normal oestrus cycle. actually, it has been found that more than 85% of females which have had abortions because of b.  canis infection can have normal gestations with regular births, while the remaining females may still experience reproductive problems, which can also occur intermittently. more rarely, infected females can have abortions more than four consecutive times, or have more than three unsuccessful mating (carmichael 1990). these animals represent a reservoir of infection for the still healthy dogs inside the kennel (carmichael 1968, carmichael 2012). in infected adult males, epididymitis, unilateral or bilateral testicular atrophy and scrotal dermatitis are normally found. palpation of the scrotum or testicle generally does not cause acute pain. generally, there is a decrease in the volume of ejaculate without loss of libido by the subject; however, it is possible to notice some suffering of the subject at the time of ejaculation. the seminal liquid of infected males find in the blood the presence of a number of bacteria even more than 103 per ml, starting 2‑3 weeks after infection. bacteremia commonly persists for at least one or two years. in some cases, it has also been observed for a period of five years (carmichael et al. 1984). the bacteremia, constant in the acute phase of the disease, becomes intermittent in the chronic phase (alton et al. 1988). b. canis can be isolated for several months from spleen, lymph nodes, bone marrow, prostate, and epididymis even when the bacteria cannot be found in the blood any more (carmichael 1990). no specific lesions due to b.  canis infection have been described; lymph node hypertrophy and splenomegaly can be observed in infected adults and  surviving puppies. acute or chronic inflammatory lesions (carmichael 1990) can also be found in the genital system. microscopically, a generalised lymphoreticular hyperplasia affecting all lymphoid organs is costantly observed despite the bacteremia (serikawa and muraguchi 1979). in chronic bacteremia, lymph node and splenic sinuses can show infiltration of plasma cells and macrophages containing phagocytised bacteria. in all organs of the urogenital system, a widespread lymphocytic infiltration can be observed at the submucosa level, which mainly affects the prostate, epididymis, renal pelvis and uterus (carmichael 2012). subacute or chronic endometritis, granulomatous prostatitis, and testicular atrophy  and fibrosis are common in chronically infected dogs. other lesions may involve the kidney, with ialine thickening of the glomerulus basal membrane, with poor cell infiltration. other described alterations include focal hepatic necrosis, myocarditis, and meningoencephalitis (carmichael 1990). alterations at the eye level are represented by granulomatous iridociclitis and exudative retinitis, characterized by widespread infiltration of lymphocytes, plasma cells and neutrophils (saegusa et al. 1978). aborted fetuses show subcutaneous edema, congestion, and hemorrhages in the subcutaneous areas of the abdominal region (carmichael 2012). placental annexes might contain foci of coagulative necrosis in the chorionic villi, and a large number of bacteria within epithelial trophoblastic cells (feldman and nelson 1996). symptomatology although b. canis infection is systemic, infected adult dogs rarely show obvious symptoms (carmichael 1990). apart from rare cases, the disease evolves in the absence of fever (carmichael and bruner 1968). there are no pathognomonic symptoms (hensel et al. 2018). a common symptom for both males and females 9veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis affecting the fractions β and  γ can be indicative of b. canis chronic bacteriemia. urine biochemical tests are generally normal, despite the possible presence of bacteriuria. in cases of meningoencephalitis, analysis of cerebrospinal fluid reveals the presence of neutrophils and an increase in protein concentration, changes that are not evident if only discospondylitis is present. radiological findings of intervertebral disc infection should be followed by serological testing for b.  canis and, where possible, bacteriological confirmation (carmichael 2012). indeed, the examination of the seminal fluid may be of greater help in the presumptive diagnosis of the disease. sperm abnormalities which may affect more than 90% of spermatozoa, start being present 5 weeks after infection and become evident from the 8th week onwards even after 20 weeks (george et al. 1979). in any case, the sperm disorders induced by b. canis infection are not specific, so the detection of an abnormal spermogram is not indicative of brucellosis (berthelot and garin‑bastuji 1993). laboratory diagnosis direct methods isolation the isolation of the bacterium represents the only proof of b. canis infection. it is not difficult to isolate b.  canis since the microorganism grows in the culture media normally used for all other species of the genus brucella (nicoletti and chase 1987b). isolation of b.  canis is possible from blood, fresh samples (vaginal discharged material, placental and fetal tissues, urine, semen, milk) and samples taken from necropsy (lymph nodes, spleen, prostate, epididymis, uterus, bone marrow, eye, intervertebral discs) (feldman and nelson 1996). blood culture is the method of choice for confirming b.  canis infection, provided that they have not been treated with antibiotics (carmichael 2012). in dogs experimentally infected per os, bacteremia begins 7 days after exposure, while blood culture begins to provide positive results from 2‑3 weeks after exposure. if in the meantime the subject has not been treated with antibiotics, the positivity to blood culture usually persists for at least 6 months, and may last up to 2 years (carmichael 1990). in this regard, the international scientific literature also reports cases of experimentally infected dogs that remained positive to the blood culture for a period of 5.5 years (carmichael et al. 1984). has a considerable number of sperm abnormalities and inflammatory cells, especially during the first three months of infection. chronic infection can lead to bilateral testicular atrophy with consequent aspermia (carmichael 2012). in addition to symptoms affecting the reproductive sphere, although less frequently, b.  canis infection can cause disorders in other organs as well. in some subjects, for example, generalized lymphadenopathy may be accompanied by splenomegaly (carmichael 2012). a well‑known and described clinical manifestation of b. canis infection is discospondylitis (henderson et al. 1974). this can arise in apparently healthy dogs or those which have had history of reproductive disorders and have been treated with antibiotics (kervin et  al. 1992, hurov et  al. 1978). dogs initially experience pain in the spinal cord, then, if compression of the spinal cord increases, they present paresis and ataxia (kornegay 1983). the incidence of discospondylitis is higher in males than in females, probably due to the localization of b. canis in the prostate, which can cause intermittent bacteremia even in castrated males (carmichael and joubert 1988, kerwin et al. 1992, hurov et al. 1978). cases of osteomyelitis affecting the appendicular skeleton and resulting in lameness on the affected limb have also been described (smeak et  al. 1987). although the onset of meningoencephalitis has only been observed as a consequence of an experimental infection, some authors have detected the presence of behavioral changes, anisocoria, ataxia, hyperesthesia and circle movements in dogs with b.  canis infection, in which such neurological signs started three weeks after mating (carmichael 2012). in some dogs with chronic b.  canis infection, the onset of recurrent anterior uveitis with corneal edema has also been described (saegusa et al. 1978, reike and rhodes 1975). other manifestations of b. canis infection may be represented by polyarthritis (carmichael 2012). diagnosis clinical diagnosis the clinical signs may only allow a suspicion of b.  canis infection. since b.  canis infection is one of the most common causes of reproductive disorders in dogs, it should be ruled out before investigating other causes of infertility or abortion (carmichael 2012). however, if reproductive failure is not observed, canine brucellosis can be difficult to diagnose. the hematological and biochemical values remain generally unchanged or in any case they don't show characteristic changes. a hyperglobulinemia 10 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. given the potential impact that a positive or negative result can have on individual dogs, on the dog population of origin, on a single customer or on a kennel operator, quality control and assurance of the b.  canis diagnostic tests are fundamental, in particular for pcr (cosford 2018). assuming that the accuracy of the results can be improved, the advantages of pcr include: the possibility of detecting the species and (sometimes) the brucella biovar involved in the infection, the high sensitivity and specificity, the minimum requirements needed in terms of biological containment, the relatively short time to obtain the results, and the possibility of performing a genetic fingerprinting which is useful for epidemiological investigations aimed at controlling the disease (yu and nielsen 2010). most of the pcr assays reported in the literature are based on the identification of the brucella genus. in the past, multiplex pcrs have also been set up to distinguish between some brucella species, to avoid the excessive development of contaminants, blood samples should be sowed on selective media (carmichael 2012). blood samples should be collected in a sodium citrate or sterile lithium heparin blood collection tube, stored at refrigeration temperature (not frozen) and submitted within 24 hours at the laboratory, where they can be cultured on a farrell's medium or on a modified thayer‑martin medium (carmichael and greene 2006, cfsph 2010, gda 2020). at necropsy lymph nodes and spleen are the recommended organs for culturing the bacterium. it is also possible to collect samples from the eye in case of uveitis or the intervertebral disc in case of discospondylitis (feldman and nelson 1996). in all these cases, the negativity of the culture does not exclude the presence of infection (carmichael 1990). unfortunately, the chances of growing this microorganism in culture depend on several factors, including b.  canis concentration in the collected sample, the intermittent germ elimination from infected subjects, due to the quality of samples, the sample handling, the forms of the microorganism, or how culture media have beeen used (carmichael and greene 2006). therefore, a negative culture cannot exclude the presence of infection, since the low sensitivity of the test can lead to relatively high number of false negatives. although culture is to be considered inappropriate as a screening test, it still represents the ideal test for confirming infection. the test can give positive results as early as 2‑4 weeks after infection and the dog can remain positive for several years (hollett 2006). isolating the bacterium from samples is the best method for the diagnosis of early infection in dogs that have not received antibiotic treatment. expected results of the culture according to the time of sampling and the material examined are summarized in table i. polymerase chain reaction (pcr) several pcr primers have been used to detect b. canis dna in whole blood, vaginal secretions and semen. due to its high sensitivity and specificity, pcr can be used as a rapid screening test or as confirmation test for serum positive dogs (keid et al. 2007, kauffman et  al. 2014, kang et  al. 2014). there is a good correlation between pcr test results and those obtained with the 2me‑rsat test (mol et  al. 2020). the matrix of choice is the whole blood taken with anticoagulant sodium citrate (light blue cap tubes). pcr testing on serum has little diagnostic value as it provides unsatisfactory results in terms of sensitivity, much lower than those of blood culture, whole blood pcr, rsat and 2me‑rsat (keid et al. 2010). table i. possible results of the culture of b. canis in relation to the time at which it is performed and the material under examination (adapted from feldman & nelson 1996). culture material optimal moment of cultivation expected results post-abortion discharges when present + + + placenta when present + + fetus when present possible negativity seminal fluid 3-11 weeks pi + + + 12-60 weeks pi reduction in number > 60 weeks pi blood 5-30 weeks pi 100% + after 30 weeks pi intermittently 6-12 months pi >80% + 28-48 months pi 50-80% + 48-58 months pi 25-50% + > 58 months pi < 25% + epididymis 35-60 weeks pi 50-100% + > 100 weeks pi urine 8-30 + weeks pi emission of more microorganisms in males than in females prostate up to 64 weeks pi usually + lymph nodes, bone marrow and spleen during bacteremia usually + during the non-bacteriemic phase phase +/eye in case of uveitis + + + intervertebral disks in case of discospondylitis +/pi = post-infection; + = positivity; = negativity. 11veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis serological tests aimed at detecting antibodies against b. canis should be based on a wall antigen of b.  canis or b.  ovis (complete cross‑reaction), or on a cytoplasmic antigen of b. abortus or b. canis. whichever test or antigen is used, b. canis antibodies in the blood are usually detectable not earlier than 5‑8  weeks after infection (carmichael 1990). the results then may be negative in the first 3‑4  weeks of infection, although the dog may be bacteremic as early as two weeks after infection (carmichael and greene 2006). when the bacteremia ceases, the antibody titer starts descreasing. however, level of detectable antibodies can be found for further 4‑6 months. after that period, serological tests have negative or doubtful results (carmichael 1990). the antibody titer may fluctuate even in the presence of persistent bacteremia and its value does not reflect the disease progress. furthermore, a possible decrease of this value does not necessarily indicate the efficacy of the therapy (feldman and nelson 1996). the characteristics of the main serological tests for b.  canis antibodies have been summarized by carmichael and shin (carmichael and shin 1996, table iii), and by cosford (cosford 2018, table iv). rapid slide agglutination test (rsat and 2me‑rsat ) rsat in the original technique elaborated by george and carmichael (george and carmichael 1978) uses a heat‑inactivated b.  ovis or b.  canis, colored with rose bengal. ebani and colleagues (ebani et al. 2003) performed the test with an antigen from heat‑inactivated and rose bengal‑stained b.  ovis strain 63/290 prepared according to alton and colleagues (alton et  al. 1988). currently, the strain recommended by the oie for the production of including b. canis (lópez‑goñi et al. 2011). in recent years, specific pcrs for b.  canis (kaden et  al. 2014, kauffman et  al. 2014, kang et  al. 2014, boeri et  al. 2018) have also been developed. the performance of these assays however needs to be completed in order to assess sensitivity and specificity. until then, data from pcr trials should only be used in conjunction with clinical and serological information (cosford 2018). cosford (cosford 2018) has recently reviewed the state‑of‑the art of the different researches available to date on pcr for b. canis (table ii). indirect methods several serological methods are available for detecting antibodies against b.  canis. the rapid slide agglutination test (rsat), the 2‑mercaptoethanol‑rapid slide agglutination test (2me‑rsat), and the tube agglutination tests (tat, and 2me‑tat) represent, in countries where the disease is present, the tests most widely used in the field. the agar gel immuno‑diffusion test (agid), that uses either cell‑wall antigens (agidcwa), or cytoplasmic antigens (agidcpa), is used as confirmatory test. other available tests include indirect fluorescent antibody assay (ifa), complement fixation test (cft) and immunoenzymatic tests (enzyme‑linked immunosorbent assay, elisa). they, however, have only been used in experimental studies (carmichael and shin 1996, ebani et  al. 2003, carmichael and greene 2006, hollett 2006, keid et al. 2009). it is important to note that b.  canis does not have smooth‑phase cell wall antigens such as b.  abortus, b.  melitensis or b.  suis, but only rough antigens (carmichael and bruner 1968), like b.  ovis. therefore, table ii. pcr assays for the detection of brucella canis in dogs from cosford, edited (cosford 2018). 16s-23s rdna interpace region (44) brucella genus whole blood 100% 100% 16s-23s rdna interpace region (45)a brucella genus vaginal swabs not available not available 16s-23s rdna interpace region (46)a brucella genus semen not available not available 16s-23s rrna sequence (47) brucella genus whole blood 100% 100% 16s-23s rrna interpace region (48) brucella genus inguinal lynph nodes 100% 100% 16s-23s rrna interpace region (49) brucella genus whole blood serum whole blood: 97.14% serum: 25.71% intergenic spacer is711 823)b brucella genus males: proputial swabs, semen or urine female: vaginals swabs not available not available gene fragment on chromosome 1 (23)b brucella canis specie-specific as intermediately above not available not available b. canis outer membrane protein 25 dna quantitative pcr (50) brucella canis specie-specific vaginal swabs whole blood vaginal swabs: 92.31% whole blood: 16.67% bcan_b0548-0549 region in chromosome 2 of brucella canis (51)c brucella canis specie-specific whole blood buffy coats not available not available apcr on vaginal swabs and semen in these studies correlates with blood pcr and blood coltures assessed by a kappa-coefficient and the mc nemar test. bboth brucella canis genus-based and brucella canis specific pcrs used in swedish outbreak investigation. cbrucella canis inoculated samples; pcr on buffy coat separated from whole blood was approximately 100 times more sensitive that from whole blood. 12 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. table iv. comparison of traditional serological tests for the diagnosis of canine brucellosis. from cosford, edited (cosford 2018). test antigen sensitivity specificity how to use the test rsat b. ovis (m-) strain b. canis moderate to high low to moderate screening testolder studies suggest high older studies suggest 40%-50% newer studies suggest 70.58% newer studies suggest 83.34% 2-me-rsat (m-) strain of b. canis lower than rsat (31.76% vs. 70.58%) higher than rsat (100% vs. 83.34%) confirmatory test tat b. canis high low screening test ifa anti-canine immunoglobulin (ig)g directed against antibodies to b. canis unknown unknown screening test agid cwa lipopolysaccharide antigen from the cell wall of b. canis high lower than agid cpa screening test agid cpa lps-free soluble, internal cytoplasmic proteins extracted from b. canis or b. abortus low high confirmatory test52.94 sensitivity 100% 47.06 false negatives cross‑reactions between the antigen used and specific antibodies, possibly present in the tested serum. possible cross‑reactions have been observed against bacteria, such as pseudomonas  spp., bordetella  spp., streptococcus  spp. and, more generally, some enterobacteriaceae (carmichael 2012). in order to reduce the incidence of false positivity, the rsat test was then modified to include the addition of 2‑mercaptoethanol (2me‑rsat) to the test serum before mixing with the antigen. 2‑mercaptoethanol reduces the incidence of false positivity substantially because it inactivates the less specific igm antibodies (badakhsh et  al. 1982). the b.  ovis antigen is b.  ovis reo 198 (oie 2018). rsat is a quick, easy to perform and read test, commercially available (e.g. d‑tec cb, synbiotics, san diego ca, usa). positive reactions are detectable as early as 3‑4 weeks after the onset of infection (hollett 2006). the test has a sensitivity (defined as the probability of the test not to give rise to false negative reactions) of 99% (carmichael 1990). on the contrary, specificity (defined as the probability of the test not to give rise to false positive reactions) is rather limited, counting false positive rates that commonly range from 20% up to even 50% (carmichael and shin 1996). false positive results would apparently be produced by table iii. pcr assays for the detection of brucella canis in dogs (cosford 2018). test nature of the antigen positivity limitsa comments 2me-rsat cell wall from 5-8 sept. pi up to 3 months after the cessationof the bacteremia (followed by variable results) high sensitivity (99%); low specificity (50%-80%); quick and easy performing. tat cell wall similar to 2me-rsat. false positivity as in 2me-rsat; semi-quantitative; titles above 1:200 are indicative of infection in progress. 2me-tat cell wall similar to 2me-rsat. slightly higher specificity than tat;longer laboratory process. agid cpa cell wall similar to 2me-rsat,possibility of detection 1-2 weeks before. higher sensitivity than rsat; permanence of frequent non-specific reactions; complexity of execution; difficulties of interpretation agid cwa cytoplasm from 8-12 weeks to at least 12 monthsafter the end of the bacteremia, up to 36 months greater specificity (97%); but less sensitivity; reveals chronic cases negative to other tests; reveals infections from other brucellae. elisa cell wall cytoplasm unknown, believed to be similar to tat technique in experimental phase; high specificity when using wall antigens of b. canis in phase m or cytoplasmic antigens. ifa cell wall unknown unpublished data;appears to have less sensitivity than 2me-tat. atimes are approximate; rsat = rapid slide agglutination test; me = test with 2-mercaptoethanol; tat = tube agglutination test; agid = agar gel immunodiffusion test; elisa = immunoenzymatic test; ifa = immunofluorescence assay; pi = post-infection; + = positive; = negative. 13veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis other species of the genus brucella (agidcpa), b.  abortus in particular (carmichael and shin 1996). the technique has been described by zoha and carmichael (zoha and carmichael 1982). the use of cytoplasmic‑derived antigen further increases the specificity of the test, lowering the percentage of false positivity to 3% (zoha and carmichael 1982). conversely, the same test performed using the cell wall antigen, suffers from the same false positivity problems as the agglutination tests previously described (carmichael 2012). to produce the antigen, ebani and colleagues (ebani et al. 2003) used a hot saline extract (hse) of b.  canis strain rm6/66. the lipopolysaccharide wall antigen is less specific than the cytoplasmic antigen. therefore, agidcwa has a high sensitivity but it still has the probability to give false positive results. positive results appear 8‑12 weeks after infection and can remain for 3‑4 years (hollett 2006). the assay which uses cpa antigen is more specific but less sensitive since it can react with antibodies against other brucella species (e.g. b. canis, b. abortus, b. suis) (hollett 2006). the disadvantage when using cytoplasmic‑derived antigen is the long period of time necessary for the test to become positive, which is around 8‑12 weeks after the exposure. this makes the test not indicated for revealing the early stages of the infection. however, the assay remains positive for a longer period, about 12 months after the cessation of bacteremia. so this test can be more useful in detecting chronic infections (carmichael 2012). in the literature, positive results to this test have been reported to persist more than 5 years after infection (hollett 2016). agidcpa test is the most effective technique in kennels infected with b.  canis. provided that the definitive diagnosis of b. canis infection always requires confirmation by blood culture, agidcpa test it can be used as a confirmatory test for those sera resulting positive to the agglutination tests (carmichael and shin 1996, cosford et al. 2018). a limited number of veterinary diagnostic laboratories are capable of carrying out agid (hollett 2006) due to the difficulties of antigen preparation and purification, as well as the requirement of specialized personnel (feldman and nelson 1996, hollett 2006). indirect immunofluorescence assay (ifa) this test can be considered as alternative when rsat and tat are not avalaible (weber and hussein 1976). however, results from the cornell university's diagnostic laboratory indicate a high rate of false positive reactions with the ifa test (wanke 2004). and, since the sensitivity of the ifa is not yet fully known, there is also a probability that infected dogs rsat test has been further modified by replacing the b. ovis antigen with an antigen derived from b. canis in phase m (mucoid) (carmichael and joubert 1988). this resulted in a reduction of the rate of false positivity to about 10%. therefore, since false negativity to this test is rare, it can be used as screening test to identify and separate negative subjects (wanke 2004). tube agglutination test (tat ) the technique has been described by carmichael and kenney (carmichael and kenney 1968) and alton and colleagues (alton et  al. 1975). the tat is able to detect antibodies to b.  canis in dogs tested positive for rsat or 2me‑rsat. the test begins to provide positive results already 2‑4 weeks after exposure (hollett 2006). tat consists in the addition of a fixed dose of heat inactivated b. canis antigen to different test serum dilutions. it is able to determine the antibody titer (feldman and nelson 1996). the test is sensitive but not very specific, allowing false positive results (hollett 2006). as for rsat, the addition of 2‑mercaptoethanol (2me‑tat) reduces false positive reactions (carmichael 1990). although more data on the reliability of this test are needed, the serum samples are considered negative when the agglutinating titer is less than 1:50 and doubtful when agglutinating titers are between 1:50 and 1:200. titers above 1:200 are considered as positive. however, blood culture is always required to confirm the infection (fredrickson and barton 1974, rhoades and mesfin 1980, flores‑castro and carmichael 1977, henderson et  al. 1974, carmichael and shin 1996). there is a good correlation between tat titer ≥  1:200 and the isolation of the microorganism by blood culture (hollett 2006). in the united states and in countries where the disease is present, 2me‑tat is no longer used in laboratories. they prefer to use 2me‑rsat which has the same diagnostic accuracy, is easier to perform, standardizable and capable of giving comparable results between laboratories (carmichael and shin 1996). being however a semi‑quantitative test, 2me‑tat is still used in kennels where brucellosis, due to b.  canis infection, has been diagnosed. it allows to indirectly evaluate the response to antibiotic therapy, through the decrease of the agglutination antibody titer (carmichael and shin 1996), although this correlation has not yet been sufficiently demonstrated (nicoletti and chase 1987a). agar gel immunodiffusion test (agid) agid can employ two different types of antigens: a b.  canis cell wall antigen (agidcwa) or antigenic proteins extracted from cytoplasm of b.  canis or 14 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. table v. comparison of elisa tests for the diagnosis of canine brucellosis. from cosford, edited (cosford 2018). antigens sensitivity specificity lipopolysaccharides-free cytoplasmic proteins of b. abortus 92% 96.7% hot-saline extract of b. canis containing outer membrane antigens 92% 94.3% luminaze synthase of brucella spp. 81% 96.7% 18kda cytoplasmic protein of b. canis 87%a 98%b bacterial whole cell extract from wild isolate of b. canis used as solid phase antigen 95% 91% heat soluble bacterial extract from wild isolate b. canis antigenc 91.18% 100% m-strain b. canis antigen 100% 98.8% b. ovis strain #11 antigen 100% 98.8% b. abortus rb51 strain antigen 100% 98.8% areal sensitivity not reported as percentage was calculated considering 26/30 known cases that tested positivie with this elisa. bspecificity not reported as percentage was calculated from the data set as 2/103 animal tested falsely positive with this elisa in the healthy population. cheat soluble extract were more useful than ultrasonic homogenates of bacteria isolates to generate candidate capture antigens, a sonicated antigens were associated with more cross reactivity, and, therefore, false positives in both elisa and western blotting. 1975, weber and krauss 1977, ebani et al. 2003). the antigen used by ebani and colleagues (ebani et  al. 2003) was a hse of b. ovis strain 63/290. immunoblotting (ib) ebani and colleagues (ebani et  al. 2003) explored the performances of sodium dodecyl sulfate‑polyacrilamide gel electrophoresis (sds‑page) using b. ovis and b. canis hse. the results of the study suggest that immunoblotting may be the most specific serodiagnostic method for detecting antibodies to b. canis. the authors had no false positive reactions with any of the evaluated sera. serum samples positive to agid or cft were all negative to immunoblotting test. immunogenic bands were evidenced with both b. canis and b. ovis hse antigens only when positive control sera were tested. on the basis of the results obtained, the authors recommended to use the immunoblotting test as a confirmatory test. however, because of the intensive labor and time required for running the test, ib is not performed in laboratories on a routine basis (ebani et al. 2003). interpretation of diagnostic tests the different serological tests available have different levels of sensitivity and specificity depending on the stage of the disease and the type of method and antigen used. medical history and clinical data, where available, should always be used in conjunction with the laboratory results to achieve a definitive diagnosis (wanke 2004). false negative results may occur following sampling carried out prior to seroconversion or due to low titers of circulating antibodies in some chronically infected subjects (carmichael and greene 2006). false positive results, on the other hand, are the major problem when using these serological tests. these drawbacks can depend on specific and non‑specific cross reactions with surface antigens of other microorganisms, such as pseudomonas aeruginosa, bordetella bronchiseptica, actinobacillus equuli, streptococcus spp., staphylococcus spp., moraxella‑type microorganisms and gram‑negative bacteria (carmichael and greene 2006, hollett 2006, cfsph 2020, yu and nielsen 2010). these tests can be used as screening tests but positive results should be confirmed by a high‑specific confirmatory test such as 2me‑rsat or agidcpa (carmichael and greene 2006, hollett 2006, keid et al. 2009, cosford 2018). it has been reported that agglutinating antibodies may not protect the dog from infection (pollock 1979) or from bacteriemia (serikawa and muraguchi 1979, cdc 1977). may not be detected when tested (carmichael and greene 2006). immunoenzymatic test (elisa) data on the performance of the elisa for the detection of b.  canis antibodies have recently been summarized by cosford (cosford 2018) (table v). these tests have been developed by using either cell wall of b. canis (m− and rm 6/66), or cytoplasm of b.  abortus, as antigens (baldi et  al. 1997). the cytoplasmic antigens, common to all strains of the genus brucella, have the advantage of not showing cross‑reactivity with bacteria belonging to genera other than brucella spp. however, it cross reacts with all bacteria of the genus brucella. elisas which use cell wall antigens of brucella strains in phase m− are highly specific but not highly sensitive (serikawa et  al. 1989, mateau de antonio et  al. 1993). high false positive rates were instead observed when the elisas which use the rm 6/66 strain as antigen, were employed (mateau de antonio et  al. 1993, ebani et al. 2003). complement fixation test (cft ) the complement fixation test was described by alton and colleagues (alton et  al. 1975), and weber and krauss (weber and krauss 1977). altough showing a good correlation with tat (weber and krauss 1977), cft is not used on a routine basis since dog serum often is anticomplementary (alton et al. 15veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis seem to be desirable, since it would interfere with the serological tests carried out to identify infected subjects (carmichael 1990). direct prophylaxis management of kennels free from b. canis and prevention of spreading between kennels since a valid immunological product is not available, the prevention of canine brucellosis due to b. canis, in territories where the disease is present, should be based on the classic sanitary measures of direct prophylaxis, based on regular serological testing of the animals hosted in the kennel. strict biosecurity measures should be implemented, together with adequate kennel management and strict environmental controls (cleaning, disinfection, ensuring adequate temperature and humidity). it is necessary to take special care to properly clean and disinfect on a daily basis the delivering sites and the spaces where the newborn puppies are housed (usda 2015). quarantine kennels free from b.  canis should keep any newly introduced dog in quarantine, subjecting them to at least two specific serological tests carried out at four weeks of distance, admitting them to be in contact with the others only if both tests are negative (carmichael 1996). the best way to keep brucellosis out of the kennel is then to isolate and test all incoming dogs, and testing them negative before placing into the kennel. this is best achieved by isolating newly purchased dogs in a separate building or facility, away from the rest of the population, for a minimum of eight weeks. all incoming dogs should be tested for b. canis on arrival and again after eight weeks. only after having obtained two negative screening tests on all dogs of the isolation facility, they can be safely transferred in contact with the rest of the kennel population. if during this eight‑week isolation period a newly introduced and isolated dog results positive to b.  canis serology, it should be immediately removed from the facility. the eight‑week isolation period will then restart for the remaining dogs in isolation. the quarantine isolation approach combined with laboratory tests has been shown to be the safest way to introduce new dogs into an established reproductive population without the fear of introducing brucellosis or other infectious agents (usda 2015). control of breeders b.  canis infection is of major relevance for breeders therapy treatment of canine brucellosis due to b.  canis is possible, although the results are often disappointing, because of the intracellular localization of the bacterium for long periods, and its ability to generate episodic bacteremia (carmichael 1990). for this reason, although in vitro b.  canis is sensitive to different antibiotics, often the therapy is not effective and relapses of infection are common. among the various proposed therapeutic protocols, the scheme which combines tetracycline (25 mg/kg t.i.d., po) for four weeks, and dihydrostreptomycin (10 mg/kg b.i.d., im) in the first and last week of therapy gave the best results (carmichael 1996). this experimental therapeutic scheme led to serological negativization of 94% of treated animals within two months after treatment (nicoletti 1991). the therapy is less successful in males than in females, probably due to the greater difficulty encountered in eliminating foci of infection from the male genital tract, especially from the prostate (carmichael 1990). despite the treatment, male subjects can still develop irreversible sterility and this, along with the difficulty of eliminating the infection from the prostate, suggests that these subjects should be excluded from reproduction anyway (carmichael 2012). the variability of the results obtained by the various authors in the application of experimental therapeutic protocols (flores‑castro and carmichael 1981, zoha and walsh 1982, nicoletti and chase 1987a, nicoletti, 1991) also depends on the criterion adopted to define recovery (i.e. negativization vs bacteriological or serological examinations, respectively) (carmichael and shin 1996). in any case, in light of the fact that the infection can reoccur even in weeks or months after the end of therapy, it is recommended to carry out serological monitoring of the dogs for at least three months after completing the therapeutic cycle, repeating it in case serological positivity is found (carmichael 1996). in addition, sterilization of the subject is recommended (carmichael 1990). the clinician should inform the owner about the problems associated to the therapy, its costs, length, and possible failures (feldman and nelson 1996). prophylaxis indirect prophylaxis as far as indirect prophylaxis is concerned, all attempts to produce an efficacious vacine have not provided encouraging results. on the other hand, the existence of a vaccine for some aspects does not 16 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. isolation and disinfection at delivery it would also be appropriate to isolate females at the time of delivery, as well as the regular disinfection of kennel premises, particularly the delivering rooms (berthelot and garin‑bastuji 1993). management of infected kennels in case the infection is identified in a kennel, the entire existing population should be confined (prohibition of entry/exit of animals) and subjected to serological tests. all the dogs present should be considered suspected cases. protection of operators in order to reduce the likelihood of exposure to b.  canis, personnel who work in infected kennels should wear disposable protective gloves when assisting delivery, including handling newborn puppies, placenta, fetal membranes or possible contact with urine or vaginal secretions. extreme care should be taken when handling miscarriage, including dead or partially developed puppies, their fetal membranes and placentas. protective gloves should also be used during assistance for insemination, both natural and artificial. to prevent b.  canis infection during cleaning and disinfection of premises or handling of animals in quarantine and isolation situations, appropriate use of personal protective equipment (i.e. respiratory and ocular protection) would be advisable in addition to the simple use of gloves. it is also recommended that veterinarians, staff, owners, and laboratory personnel be careful when collecting and handling blood, serum, fluids or tissues for laboratory testing (usda 2015). census of animals present before starting the checks, a thorough survey of the correspondence of the kennel register and the animals really hosted should be carried out. subsequently, a register of controls should be set up where animals are distinguished by sex, race, and age, taking care to specify for each one of them the date of birth, so that it would be possible to easily identify the subjects to be monitored. animal tracing it is necessary to identify the source of entry of the infection and its possible origin. similarly, all animals having left the infected premise should be tracked. the animals and the sheltering kennel traced in this way should be subject to the same provisions as those adopted in the premise of origin. as it is normally a problem that forces to end their reproductive career (carmichael 1990). in breeding kennels located in areas where the disease is endemic, annual serological testing of breeding dogs should be carried out, combined with a further check to be performed at least three weeks before each mating (feldman and nelson 1996). ideally, breeding dogs should never leave the breeding facilities except to be visited by a veterinarian for the necessary care (e.g. caesarean section, serious injuries or illnesses). it is advisable to keep the breeding dog population sheltered in the kennel, and to avoid sending females out for mating. these subjects could pose a risk of introduction of the disease; therefore, any dog that leaves the structure for mating or for any reason other than a caesarean section should be tested 90 days after its return to confirm its negative status. it is best to isolate these subjects from the rest of the kennel population upon their return, although doing so may not be practical. it may in fact require isolation during gestation and delivery, which may be problematic in some facilities (usda 2015). an alternative approach to genetic improvement of the breeding kennel population could be the use of artificial insemination (ai) on breeding females, using semen obtained from external breeding dogs proven negative for b.  canis (at least eight weeks prior to semen collection). if male breeding dogs are subjected to outdoor mating, the safest approach would be to offer this service only through the use of ai, using semen collected in the kennel and then shipped to the requesting kennel without the female being introduced into the premises or the male leaving them (usda 2015). control of other introductions stray dogs should be prevented from entering breeding kennels. likewise, contact with animals or groups of animals of unknown or doubtful health status should be avoided, particularly in the case of competitions or mating (carmichael 1996). visitors (including the customers themselves) should not have visited any other breeding kennel on the same day; they should wear clean clothing, disinfect their shoes, wear disposable protective shoe covers and wash their hands properly. ideally, visitors should not touch or handle dogs or equipment. one solution could be to provide direct contactless access using a video display of the mother and father, as well as the litter during the period of parental care. this would eliminate most of the direct risk of transmission of the disease until the puppy is moved to its new home (usda 2015). 17veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis of all dogs present, for whatever reason, until the kennel reaches the b. canis‑free status. depending on the epidemiological situation of the kennel and on the problems that may be present in relation to the welfare of the hosted dogs, official veterinarians should verify whether a suspicious or infected kennel should be subject to a strict quarantine (which means a ban on the sale or transfer of any subject until the achievement of the b. canis‑free status) or to a partial quarantine, in which it is forbidden to sell or transfer only positive animals and puppies born to positive mothers. the private veterinarians responsible for the kennel should closely link up with the respective official veterinarians to agree on how to manage quarantine in relation to the suitability of the facilities, periodic serological checks, the removal and isolation of positive animals and compliance with the rules on animal welfare. destruction of fetal membranes and aborted fetuses females should deliver in separate rooms that are properly washable and disinfectable. the placenta and fetal membranes should be removed and destroyed. similarly, aborted fetuses or puppies that die before weaning should be removed and destroyed. cleaning and disinfection b.  canis does not survive for long periods in the environment, and it is normally sensitive to common disinfectants, such as ammonium quaternary salts and iodophores (carmichael 1990) or to direct sunlight (usda 2015). brucella is also sensitive to 1%, sodium hypochlorite, to 70% ethanol, alcohol/ iodine solutions, glutaraldehyde and formaldehyde (hollett 2006). in the presence of organic debris, b. canis remains stable in the environment for up to two months (proper cleaning and disinfection are therefore essential). b.  canis is resistant to drying in the presence of organic debris. it can withstand freezing and can survive in water, dust and soil. the combination of organic debris, high humidity, low temperatures and little or no sunlight promotes the organism’s survival (this corresponds in most kennels to winter conditions). an important and often neglected part of kennels management is the correct cleaning and disinfection of environments. when dealing with brucellosis or other diseases, cleaning and disinfection serve to limit the spread of infections and are fundamental components for their prevention. proper cleaning and disinfection needs time and should be carried controls on animals once brucellosis is diagnosed, the only way to regain the freedom from the disease is to remove all positive dogs from the kennel. all dogs over six weeks of age should be serologically tested with a first screening test (e.g. rsat, 2me‑rsat or tat). dogs to be tested must not have received any antibiotic treatment in the three months prior to sampling (hollett et al. 2006). dogs tested negative to the first screening test should undergo a second screening test to be carried out at least 4 weeks later. dogs that tested negative to this second test can be considered as not infected with b. canis. dogs that test positive to the first serological test should be classified as suspected of b. canis infection (usda 2015). they should be isolated and subjected to a confirmation test (e.g. agidcwa) to be carried out not earlier than 4 weeks. the dogs should remain isolated until the second response is obtained. if the second test is also positive, these dogs should be considered suspected of infection. they should be removed from the colony. to confirm or definitively rule out brucellosis, blood culture (feldman and nelson 1996), or a third diagnostic test (e.g. agidcpa) could be performed eight weeks after the second test (usda 2015). puppies born to positive mothers or in any case younger than 6 weeks at the time of control should undergo three blood cultures carried out every 24  hours. puppies with positive blood culture should be removed from the plant. puppies over six weeks at the time of control must follow the same diagnostic protocol as adults (hollett 2006). in order to reach the status of kennel free from b.  canis, the tests should be repeated with the remaining dogs every four weeks, until there are two consecutive negative tests on the entire population of the kennel (usda 2015, carmichael 1996). isolation of positive animals – ban on mating and sale dogs infected or suspected of infection should be kept physically isolated from negative dogs. however, this may not be feasible or sufficient to prevent the spread of the disease, even if strict hygiene measures are maintained (carmichael and joubert 1988). for this reason, it is recommended to remove from the plant all animals infected (wanke 2004). in kennels infected or suspected to be infected with b. canis, it is recommended to put in place isolation procedures for all positive dogs, and to stop any trade or exchange of positive subjects or puppies born to positive mothers. in addition to this, it is strongly recommended to stop all sales or transfer 18 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. because of the zoonotic potential of brucella, dogs that have been confirmed positive for brucellosis should not be given for adoption. the decision regarding the possible adoption of b.  canis positive dogs can only be taken with the authorization of the competent health authorities both in the areas of origin and destination (usda 2015). if a decision to authorize the adoption of b.  canis positive dogs is taken, they should be subject to ovarian‑hysterectomy or castration, as well as to appropriate long‑term antibiotic therapy, with appropriate supervision by an official veterinarian. this should include periodic lifetime laboratory tests for b.  canis. canine brucellosis is considered a potentially long‑life infection; even after undergoing surgical spaying and long‑term use of antibiotics, both male and female dogs can continue to eliminate the bacterium intermittently. new owners should be made aware of the potential risk these dogs may pose over the course of their lives regarding infection of humans, other dogs and other susceptible animal species they could come in contact (usda 2015). training and information for operators official or private veterinarians managing dog breeding kennels infected from b.  canis should discuss in depth with the staff and the owners of the facility the potential for legal liability (beyond damaging the reputation of the kennel) that would inevitably accompany cases of zoonosis from b. canis as a result from the sale of infected puppies or adult dogs. these puppies or adult dogs commonly come in contact with children, the elderly or other who may be immunocompromised (usda 2015). a recent example was recorded in new york city in 2012 involving a 3‑year‑old girl. it was the first documented case of transmission of b.  canis from a puppy to a child in the united states (dentinger et al. 2015). public health aspects b.  canis can cause disease in humans, which can acquire infection through direct contact with infected dogs, their reproductive secretions, or their blood (lawaczeck et al. 2011, lucero et al. 2010). sources of infection a potential source of spread of b. canis are breeding kennels, both for the nature of the disease, and for the fact that the animals are housed in close contact with each other, and for the constant movement of dogs for reproduction or for sale (brower et al. 2007). recent outbreaks in kennels in the usa, hungary, out correctly in order for a kennel to be considered truly disinfected. it is important to remember that a clean kennel is not always a disinfected kennel. the kennel consultant veterinarian should be sure that operators thoroughly understand the entire cleaning and disinfection process, including the correct dilution and storage of detergents and disinfectants, as well as the fact that compliance with the expected contact times and proper rinsing are absolutely necessary. the frequency required for cleaning and disinfection (daily or weekly) should be thoroughly discussed with the operators. the structure should always be cleaned and disinfected following an order of susceptibility of animals to the disease, starting first of all from the areas of the kennel that host the most sensitive animals (puppies and lactating females), followed by the areas that house healthy adults and finally the areas that house animals in poor health or in isolation (usda 2015). treatment and relocation of positive animals in countries where the disease is present, although the infection does not expose the dog to a lethal risk, in the view that antibiotic therapy does not guarantee the bacteriological recovery of infected dogs and that they may represent a source of infection both for other dogs and for humans, euthanasia is recommended. possibly an attempt of therapy may be reserved exclusively for high‑value breeding animals (carmichael 1996). however, although cases of partially successful treatments have been reported, no treatment has been shown 100% effective, and puppies born to mothers with chronic brucellosis, if they survive, are often infected. for this reason, it is essential that dogs positives to diagnostic tests are not maintained as breeders, even if they have a high genetic value (wanke 2004). if the owner does not intend to choose euthanasia, an attempt of therapy could be recommended by providing from the beginning the necessary information on the zoonotic potential of the disease, especially underlining that even a sterilized dog subjected to specific therapeutic treatment could still be a source of contagion for humans (feldman and nelson 1996). it is important to emphasize that canine brucellosis due to b. canis is currently not considered a curable disease in dogs. attempts at therapeutic treatment led to very disappointing results, with relapses commonly occurring. the attempt at treatment can also mask diagnostic test results, and has been shown to be an important contributing factor to the spread of the disease. the impact of this evidence for dog breeding kennel owners is that animals infected with b.  canis must be removed from the breeding population (usda 2015). 19veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 de massis et al. canine brucellosis due to brucella canis people would have a higher risk of contracting the disease (dentinger et  al. 2012, marzetti et  al. 2013, tosi and nelson 1982, lucero et al. 2010). three cases have been reported in children under 4 years of age (dentinger et  al. 2012, marzetti et  al. 2013, tosi and nelson 1982). in one of the reports, dentinger and colleagues (dentinger et  al. 2012) described the transmission of b.  canis to a child by an infected puppy that had been purchased from a pet store and had been believed healthy during the preliminary veterinary visit. the child showed fever and b. canis infection was diagnosed with blood culture. the isolated strains from the child and the puppy were sent to the centers for disease control and prevention (cdc, usa) where the two strains showed a close genetic similarity, suggesting that the puppy had been the source of infection. however, clinical signs did not develop in four adults belonging to the same family, all exposed to the puppy. several recent reports of b.  canis infection in patients with hiv infection also highlight this population as at risk (lawaczeck et  al. 2011, lucero et  al. 2010, moreno et  al. 1998). these cases of b.  canis infection have been linked to the owning of non‑spayed dogs that had a history of reproductive failure and a following diagnosis of b. canis infection by serology and blood culture (lawaczeck et al. 2011, moreno et al. 1998). symptomatology symptoms of b.  canis infection in humans are generally similar to those of brucellosis caused by other brucella species (e.g. b. abortus or b. melitensis) (usda 2015). symptoms are often non‑specific and may include one or more of the following: fever (often periodic and nocturnal), fatigue, headache, weakness, general malaise, nausea, chills, sweating, loss of weight, hepatomegaly, splenomegaly and lymphadenopathy (swenson et  al. 1972, usda 2015). endocarditis, meningitis, arthritis and visceral abscesses can represent further complications, however rare (carmichael 2012). although there are reports in the literature that the course of the disease would still be less severe when compared to the infection caused by the ‘classical’ species of the genus brucella (swenson et  al. 1972, polt et  al. 1982), however, severe manifestations have been also described. these include septic arthritis, aortic valve vegetation, osteomyelitis, epidural abscess, pleural effusion, oral lesions, lower limb aneurysms and culture‑negative endocarditis (nasphv 2012). diagnosis the diagnosis of infection in humans, as well as in dogs, is based on serological examination followed by blood culture. however, in humans, diagnosis sweden and colombia highlight the link between outbreaks and the inter‑regional or international movement of breeding dogs (kaden et  al. 2014, castrillón‑salazar et  al. 2013, gyuranecz et  al. 2011, brower et  al. 2007). uncontrolled handling of puppies or non‑spayed dogs is a known risk factor for the spread of infectious diseases, and this has led to human infection with b. canis (dentinger et al. 2015, brower et al. 2007). compared to owned dogs, stray dogs are more likely to be not spayed, and may have a higher level of seropositivity to b.  canis (flores‑castro and segura 1976, brown et al. 1976). a high incidence of canine brucellosis in stray dog populations may cause a spillover in the human population, especially in areas with a large number of non‑spayed stray dogs, as these dogs are brought into shelter kennels or placed in other types of premises awaiting adoption. in the united states, 30% of pet dogs are adopted from animal shelters, and testing for b.  canis is not a standard procedure before adoption (brower et al. 2007). however, there is no evidence of a direct link between the number of fertile stray dogs in an area and the potential for exposure to humans. another potential source of b.  canis infection may be represented by laboratory accidents. brucella spp. is considered a high‑risk pathogen and it requires manipulation in a specialized laboratory at biosecurity level 3 (bsl 3), which if not used may result in laboratory acquired exposure (yagupsky and baron 2005). dentinger and colleagues (dentinger et  al. 2012) described an incident in which 31 laboratory technicians were exposed to b.  canis after handling an unknown gram‑negative bacterium on the bench. no one has fallen ill with clinical disease, even those classified as having had high‑risk exposures (according to cdc guidelines) or who have declined post‑exposure prophylaxis (5 out of 21 of those at high risk) (dentinger et  al. 2012). a case of laboratory‑acquired exposure has been documented in a technician who used oral pipetting to re‑suspend an m‑phase strain of b.  canis; the technician showed symptoms despite this particular strain being considered non‑virulent in dogs (wallach et al. 2004). categories at risk laboratory staff, veterinarians and animal keepers are the categories at greater risk of exposure to b.  canis (lucero et  al. 2010, marzetti et  al. 2013, krueger et al. 2014). in addition to these, several reports in the literature highlight pet dog owners as possible categories at risk (swenson et al. 1972, munford et al. 1975, lucero et  al. 2010, dentinger et  al. 2012, tosi and nelson 1982). in particular, children and immunosuppressed 20 veterinaria italiana 2022, 58 (1), 5‑23. doi: 10.12834/vetit.2561.16874.1 canine brucellosis due to brucella canis de massis et al. therapy unlike canine brucellosis, the disease in humans can be quickly and effectively treated with tetracycline therapy administered for two to three weeks (carmichael 1990). prophylaxis in countries where the disease is present, it is recommended that veterinarians always inform the owners of infected animals about the potential zoonotic risk of cohabitation with their pets and to use the outmost caution and hygiene when visiting dogs suspected of infection, especially the female dogs who have aborted in the recent past (carmichael 2012). similarly, caution is recommended in the laboratory when handling samples to be subjected to diagnostic tests for b. canis (carmichael 2012). acknowledgements the authors would like to thank giulia colacicco for the english text revision. is often complicated due to non‑specific signs and symptoms and it is therefore associated with a low rate of disease suspicion by many physicians. if the disease is placed in a differential diagnosis, blood culture is the only test available to confirm b.  canis infection in humans. however, confirmation is not straightforward, due to intermittent and low‑level bacteremia (rumley and chapman 1986). regarding serology, human antibodies to b. canis react with the same antigens used in dog serological tests, while they do not react with the b. abortus‑derived antigens (brucella abortus strain 99, weybridge or b.  abortus strain 1119‑3, usda), which are used in routine tests for the diagnosis of human brucellosis caused by smooth strains (carmichael 1990). therefore, even if the physician may suspect b.  canis infection on the basis of clinical findings, the diagnosis may not be supported by serological tests available on the market, as these are aimed at detecting antibodies produced against brucella in a smooth phase and do not detect antibodies against b.  canis (lucero et  al. 2005). serological tests for the detection of b.  canis infection developed on dogs 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viruses in cattle and camels in southern egypt. vet ital. 10.12834/vetit.2361.14459.2. 1division of infectious diseases, animal medicine department, faculty of veterinary medicine, south valley university, qena 83523, egypt. *corresponding author at: division of infectious diseases, animal medicine department, faculty of veterinary medicine, south valley university, qena 83523, egypt. e‑mail: mhassan@vet.svu.edu.eg. hassan y.a.h. mahmoud*, alsagher o. ali (fourichon et al. 2005) in farm animals, the bvd virus is rna-virus which belongs to the pestivirus genus of the flaviviridae family. bvdv can be transmitted from animals to another animal iatrogenically and through nasopharyngeal secretions (lindberg and houe 2005). the acute infection typically remains inapparent or causes only moderate disease and establishes a strong immunity. the significance of bvdv is due to the occurrence of persistently infected animals, resulting from infections of pregnant cows throughout gestation before the development of introduction bovine viral diarrhea virus (bvdv) and bovine alphaherpesvirus 1 (bohv-1) are important pathogen of cattle that cause a large economic loss due to reproductive disasters, several calf mortalities, enteric and respiratory disease. bovine viral diarrhea and infectious bovine rhinotracheitis are globally distributed and tend to be endemic in most animal populations (houe 1999, lindberg 2003). bovine viral diarrhea is responsible for huge economic losses keywords bvd, camel, cattle, ibr and southern egypt. veterinaria italiana 2022, 58 (4), 399-404. doi: 10.12834/vetit.2361.14459.2 accepted: 15.01.2021 | available on line: 31.12.2022 summary in this study, the elisa procedure was used to detect antibodies against bovine viral diarrhea and infectious bovine rhinotracheitis (ibrv ) viruses. the bvdv serological survey in aswan province in southern egypt was carried out on 184 unvaccinated cattle and camels. the overall seroprevalence was 18.48% (34/184), in cattle and 2.18% (2/92) in camels. the serological survey on infectious bovine rhinotracheitis virus ibrv antibodies for was conducted on 460 unvaccinated cattle from three different provinces (qena, luxor, and aswan). the overall seroprevalence was 60.00% (276/460). the infection rate in aswan was a higher (83.70%) than luxor and qena, 54.65.% and 53.63%, respectively. epidemiological status was established to clarify the influence of location and management systems for the increased rate of infection in animals. this study aims to investigate the seroprevalence rate of bovine alphaherpesvirus 1 and bovine viral diarrhea virus in different animals and localities in southern egypt. epidemiological investigation on bovine alphaherpesvirus 1 and bovine viral diarrhea virus in cattle and camels in southern egypt epidemiological investigation on ibr and bvd in southern egypt mahmoud et al. 400 veterinaria italiana 2022, 58 (4), 399-404 doi: 10.12834/vetit.2361.14459.2 the immune system of the fetus, the virus is not recognized and the animal after birth will spread the virus long-lasting. persistently infected cattle may also enhance fatal mucosal disease (bachofen et al. 2008). the isolation of bvdv and the prevalence of persistently infected animals have been documented in a range of free-ranging and captive species (frölich et al. 2002, vilcek and nettleton 2006). bovine alphaherpesvirus 1 (bohv-1) is an important pathogen of cattle, affecting the respiratory and genital tracts. it can cause abortion, infertility, encephalomyelitis conjunctivitis, mastitis, enteritis and dermatitis (straub 2001, muylkens et al. 2007). bohv-1 and different viral infections can play a direct or indirect role in the etiology of bovine mastitis (wellenberg et al. 2002, barkema et al. 2009), thereby affecting milk parameters (halasa et al. 2007). it has been observed that milk parameters of subclinical mastitis had a significantly greater somatic cell count and lower fat content compared with those of healthy cows (tomazi et al. 2015). bohv-1 can stay latent throughout the lifetime of the host in the trigeminal ganglion, pharyngeal tonsils and the sacral ganglia following a main infection of the conjunctiva, nasal cavities, and genitalia, (ackermann and wyler 1984, winkler et al. 2000) or can be reactivated via factors that cause stress or alter the immune system of the animal such as parturition, transportation, mixing or movement of animal (jones and chowdhury 2010), inclement weather, overcrowding (sylvia van drunnen, 2006) or following therapy with corticosteroids (winkler et al. 2000). bvdv and bohv-1 long-time eradication programs have been executed in some european countries, which is based on barring the use of vaccines, recognizing and removing infected animals, collectively with accelerated herd biosecurity. the national bvd programs in the scandinavian countries, as well as the regional programs in a few other nations in europe, have had success with control of bvdv and are pointing toward eradication (synge et al. 1999, bitsch et al. 2000). material and methods the hypothesis of the study southern part of egypt (upper egypt) is a naive area for different kinds of research. investigating the seroprevalence of bohv-1 and bvd in different animals and localities would clear the picture of infectious diseases burden and give a chance for strong preventive and control measures. animals and geographic locations a total of 644 serum samples were collected from apparently and clinically healthy animals including cattle and camels of different locations, breeding systems and sex. serum samples were randomly collected between may/ 2017 to june/ 2019 from animals of different villages in aswan, qena and luxor governorates in southern egypt (figure 1). the animals were not subjected to vaccination program agains amongst individual owners and smallholder farms located in similar environmental and husbandry conditions which were characterized by hot and dry weather. data collection data including breed, age, sex, body condition, temperature, respiratory rate, and mucous membranes were recorded. blood sampling blood samples were collected through vein puncture from each animal in glass tubes without anticoagulant and serum was separated by centrifugation and stored at −20c until use. elisa procedure two commercial elisas (monoscreen ab elisa bvdv, ns3, bio-x diagnostics, belgium; id screen ibr gb competition, france) were used to examine the collected sera. the protocols described by the kit manufacturer were followed and also the results were expressed according to the instructions of the manufacturers. data management and analysis the collected data were analyzed using microsoft 2016 excel. results all animals appear generally healthy and all general parameters including temperature, mucous membrane examination and pulse rate were in normal ranges. for bvd, serum samples were collected from 184 animals including cattle and camels from aswan in southern egypt (figure 1). the bvd overall seroprevalence was 18.48% (34/184), 34.78% (32/92) and 2.18% (2/92) in camel (table i). mahmoud et al. epidemiological investigation on ibr and bvd in southern egypt veterinaria italiana 2022, 58 (4), 399-404. doi: 10.12834/vetit.2361.14459.2 401 for infectious bovine rhinotraheitis (ibr), serum samples were collected from 460 cattle from three governorates qena, luxor, and aswan (figure1 and table ii) was 60.00% (276/460). regarding possible factors which may act as a predisposing factor to increase the rate of infection in animals (table iii), it appeared that location and breeding system might play an important role. aswan had a higher infection rate (83.70%) than luxor (54.65.%) and qena (53.63%), while intensive breeding system had a higher infection rate than the individual breeding system. table i. percentage of bovine viral diarrhea virus infection of cattle and camels in aswan. table iii. infectious bovine rhinotracheitis infection of cattle in regard to location, age, sex, and breeding system. animals sex location age breeding system positive no. (%) negative no. (%) total no. cattle female aswan 3-5 years individual breeding system 32 (34.78%) 60 (25.22%) 92 camel male aswan 3-5 years individual breeding system 2 (2.18%) 90 (98.82%) 92 total 34(18.48%) 150(81.52%) 184 location sex age breeding system positive no. (%) negative no. (%) total no. qena female 3-5 years intensive breeding system 133 (52.36 %) 121 (47.64%) 254 male 3-5 years intensive breeding system 15 (68.18%) 7 (31.82%) 22 luxor female 3-5 years individual breeding system 35 (42.17%) 48 (57.83%) 83 male 3-5 years individual breeding system 7 (77.78%) 2 (22.22%) 9 aswan female 3-5 years intensive breeding system 74 (84.10%) 14 (15.90%) 88 male 3-5 years intensive breeding system 3 (75.00%) 1 (25.00%) 4 total 267(60.00%) 193 (40.00%) 460 table ii. percentage of infectious bovine rhinotracheitis in cattle in southern egypt. location positive no. (%) negative no. (%) location qena 148 (53.63 %) 128 (46.37%) 276 luxor 42 (54.65%) 50 (54.35%) 92 aswan 77 (83.70%) 15 (16.30%) 92 total 267 (60.00%) 193 (40.00%) 460 figure i. map of egypt showing the location of qena, luxor and aswan governorates where the samples have been collected. epidemiological investigation on ibr and bvd in southern egypt mahmoud et al. 402 veterinaria italiana 2022, 58 (4), 399-404 doi: 10.12834/vetit.2361.14459.2 as well as increasing the susceptibility of the animals to other respiratory and enteric pathogens (mehmet et al., 2016). various factors contribute to the calving interval variation. some of them depend primarily on the genetic material and others only on the environment or the relationship between genetic and environmental variables (abdalla et al. 2017). the prevalence of infectious agents in cattle herds can be attributed to many variables: health care of livestock and herds, method of diagnosis, nature of the samples to be tested and the processing system (walz et al. 2015). the target animals and vaccination systems for vaccines against bohv-1 and bvd are very alike. polyvalent vaccines can be used with different vaccinations schemes including the booster (álvarez et al. 2007, saravanajayam et al. 2015, gethmann et al. 2015, santman-berends et al. 2018). no definitive research has so far been performed on the efficacy of bohv-1 / bvdv vaccination in reducing reproductive losses caused by these diseases in cattle. also, there is an apparent risk that bvdv modified live vaccines cause fetal deaths, so it is an important dilemma for practicing veterinarians working in the field to determine whether or not vaccination can be carried out (schumaher et al. 2019). conclusions this study provides valuable data on the high prevalence of bvdv and ibr in cattle and camels in southern egypt; that will assist in the development of prevention and control strategies for the disease. the high level of bvdv and ibrv infection in cattle may be the principal factor to limit the cattle industry in egypt . more researches and efforts are needed from governmental and non-governmental partners to minimize the economic losses caused by viral infection. authors contributions both authors contributed equally in sampling collection, experiment design, the data analysis, interpretation of results, and writing the manuscript. acknowledgments we appreciate the finical support by south valley university, higher education, and scientific research sector. discussion infection with bvdv is mutually recognized throughout the globe as one of the most important causes of reproductive disorders (bolin and ridpath, 1996). the number of samples tested in the present study was not large, because sampling was restricted to animals for which data on age, sex, breed, and vaccination history were survey overall seroprevalence was 18,48% (34/184), in 34.78% (32/92) in cattle and 2.18% (2/92) in camels. this study provides useful information on the current bvdv antibody prevalence in southern egypt as no data are currently available for cattle and camels. the bvd infection is commonly transmitted between animals by inhalation or ingestion of nasal and ocular secretions, saliva, urine, and feces. the infection is additionally transmitted by semen from an infected bull, or by transfer of contaminated embryos. the fetus infection can cause abortions, congenital malformations, persistently infected animals, mummifications, and embryonic absorption (houe 1995). latent infections are possible, making it difficult to regulate the exposure of recent animals by relying on clinical examination and quarantine. in our result, the ibr overall seroprevalence was 60.00% (276/460) in this study aswan had a higher infection rate (83.70%) than luxor (54.65 %) and qena (53.63%). this result for farm animals of the southern part of egypt lead us to indicate that there is no risk factor neither from the breeding system or geographic location in southern egypt, this may be due to there is no high difference in temperature and climate in this area. bohv-1 could be a generally spread pathogen showing critical differences in regional incidence and prevalence concerning the geographical area and breeding management (ackermann and engels 2006). the best method for the control of ibr infection in cattle herds has been vaccination of the animals. the profitability of cattle herds relies upon cost control and the creation of saleable items. disease with bohv-1 or bvdv can diminish reproductive efficiency (inui et al. 2000). bohv-1 which harms the production output of infected cattle herds contributes to substantial economic losses for cows (renault et al. 2018). bvdv is the pathogen that affects the reproductive system most in cattle, contributing to low rates of pregnancy, abortions, and congenital abnormalities, mahmoud et al. epidemiological investigation on ibr and bvd in southern egypt veterinaria italiana 2022, 58 (4), 399-404. doi: 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822-831. wellenberg g.j., van der poel w.h. & van oirschot j.t. 2002 viral infections and bovine mastitis: a review. vet microbiol, 88, 27-45. winkler m.t., doster a. & jones c. 2000. persistence and reactivation of bovine herpesvirus 1 in the tonsils of latently infected calves. j virol, 74, 53375346. & dos santos mv.2015. bovine subclinical intramammary infection caused by coagulasenegative staphylococci increases somatic cell count but has no effect on milk yield or composition. j dairy sci, 98, 3071-3078. van drunen littel-van den hurk s. 2006. rationale and perspectives on the success of vaccination against bovine herpesvirus-1. vet microbiol, 113, 275-282. vilcek s. & nettleton p.f. 2006. pestiviruses in wild animals. vet microbiol, 116, 1–12. walz p.h., edmondson m.a., riddell k.p., braden 123 introduction mycoses in the hyalohyphomycosis group are heterogeneous, defined by the presence of hyaline hyphae in tissues. the number of organisms causing hyalohyphomycosis is increasing and the most clinically important genera are fusarium spp., scedosporium spp., acremonium  spp., scopulariopsis  spp., purpureocillium and paecilomyces spp. (tortorano et  al. 2014). fusarium (order hypocreales) is a large genus with at least 200 species divided into 20 complexes, and many members are commonly found in the environment, where they are isolated from soil, plants and water systems, distributed worldwide and encompassing saprotrophic, biotrophic‑pathogenic or endophytic fungi. agents of any type of fusariosis are mainly found in three species complexes: f. solani complex, f. oxysporum complex and f. fujikuroi complex. fusarium  spp. are notorious as pathogen to plants, animals and humans and as a producer of secondary metabolites causing toxicoses (fumonisin mycotoxins, toxic and/or carcinogenic to human and domesticated animals) or invasive disease (brown and proctor 2013, nuñez otaño et  al. 2014, wellehan and divers 2019). human isolates mostly cause a broad spectrum of opportunistic superficial infections in immunocompetent individuals (i.e. onychomycosis or keratitis), but in recent years they have been increasingly associated with invasive and disseminated infections, predominantly in severely immunocompromised patients, diabetics, cancer patients taking cytotoxic drugs, and burn patients, becoming the leading mycosis affecting immunocompromised patients, and the second most common cause of filamentous fungi infections after aspergillosis, with high morbidity and mortality rates (montali et  al. 1981, brown and proctor 2013, al‑hatmi et  al. 2018, leu et  al. 1995, alastruey‑izquierdo et al. 2008). fusarium  spp. are frequently isolated from marine mammals: fusarium  solani in captive california sea lions (zalophus californianus) and grey seals (halichoerus grypus) (montali et al. 1981), white‑sided dolphin (lagenarhynchus acutus), pigmy sperm whale (kogia breviceps) and harbour deals (phoca 1department of veterinary medicine, university of bari ‘aldo moro’, bari, italy. 2centro recupero tartarughe marine ‘luigi cantoro’, torre guaceto, brindisi, italy. 3dipartimento di medicina animale, produzioni e salute, university of padova, padova, italy. *corresponding author at: department of veterinary medicine, university of bari ‘aldo moro’, bari, italy. e‑mail: antonella.tinelli@uniba.it . keywords fusarium solani, hyalohyphomycosis, caretta caretta, multidrug resistance, zoonosis. summary fusarium spp. are pathogens plants, animals and humans, isolated from soil, plants and water systems. they are distributed worldwide and include saprotrophic, biotrophic‑pathogenic or endophytic fungi, or producers of mycotoxins (fumonisins). human isolates are becoming the leading mycosis affecting immunocompromised patients and frequently involved in mycoses of aquatic mammals and reptiles, included sea turtles or their eggs. here reported are three severe cases of unusual localizations of fusarium in loggerhead sea turtle (caretta caretta) and their diagnostic, therapeutic and clinical output. in the clinical practice, correct genus‑level identification of fusarium species is critically important to enable correct treatment as in vitro antifungal susceptibility testing is mandatory for each fusarium‑like isolate. for this reason, susceptibility testing can significantly help the practitioner in choosing the most appropriate therapeutic protocol. olimpia lai1, antonella tinelli1*, simona soloperto2, giacomo marzano2, massimiliano tosches1, rosa leone1, donatella gelli3, chiara belloli1 and giuseppe crescenzo1 fusarium solani hyalohyphomycosis in loggerhead sea turtles (caretta caretta): a diagnostic and therapeutical challenge veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 accepted: 18.05.2020 | available on line: 08.10.2020 case report 124 veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 fusarium in caretta caretta lai et al. with geographic region (wang et  al. 2011) and different species have different drug susceptibility patterns (nucci and anaissie 2007), accurate species assignment is important for epidemiological studies and to guide clinical management. f. solani and f. verticillioides are usually resistant to azoles and exhibit higher amphotericin b minimal inhibitory concentration (mics) than other fusarium  spp. (nucci and anaissie 2007, wang et  al. 2011). in contrast, f.  oxysporum may be susceptible to voriconazole and posaconazole. (nucci and anaissie 2007, tanaka et al. 2012). under this respect, in  vitro antifungal susceptibility testing is mandatory for each fusarium‑like isolate causing a confirmed systemic infection (lass‑flörl et  al. 2010, summerbell 2002, johnson 2008, perkhofer 2010). itraconazole and other azole drugs may need to be replaced, supplemented with amphotericin b, or discontinued as useless against the pathogen. empirically, a combination of terbinafine with either posaconazole or voriconazole may be used while identification, speciation, and sensitivity results are pending, but intrinsic resistance interferes with antifungal therapy, clearly challenging treatment owing to the limited therapeutic options, and finally resulting in high mortality rates in patient (brown and proctor 2013, sharma et al. 2017, al‑hatmi et al. 2018, mader 2019). whenever feasible, infected tissue should be excised surgically (wellehan and divers 2019). case history, clinical signs, pathological findings, and diagnosis case 1 a juvenile cold stunned loggerhead sea turtle (caretta caretta) stranded in february on the coast of adriatic sea near to san foca (lecce, italy), with average sea temperature of 9  °c. the animal was referred to ‘torre guaceto’ marine turtle rescue centre (carovigno, italy), and immediately moved to the department of veterinary medicine of bari (italy) for diagnostics and treatment because of severe signs ascribable to cold stunning (hypothermia). at admission the turtle weighted 5 kg, and the straight carapace length resulted 33 cm. the animal was lethargic and severely emaciated. blood samples were collected in heparinized tubes from cervical sinus for complete haematological and plasma chemistry analyses. afterward, x‑ray examination was performed in order to check for foreign objects and/or pneumonia because of abnormal buoyancy. both resulted negative. after determination of glycemia (resulted within the ranges), fluids were intracoelomically administered vitulina) (frasca et al. 1996) or other marine organisms: american horseshoe crabs limulus polyphemus (tuxbury et  al. 2014), wild narrow‑clawed crayfish astacus leptodactylus (salighehzadeh et  al. 2019), black gill disease in cultured kuruma prawn penaeus japonicas (bian and egusa 1981) and giant tiger prawn p. monodon (khoa et  al. 2004), captive lined seahorses hippocampus erectus (salter et  al. 2012). fusarial infections in freshwater and marine fish include deep mycoses, ocular and skin lesions, and fatal ulceration and necrohemorrhagic dermatitis (yanong 2003, noga 2010). fusarium  spp. has been identified as causative agent of cutaneous hyalohyphomycosis in captive loggerhead sea turtles, where the lesions were described as superficial, white and scaly or ulcerative (austwick et al. 1981, cabañes et al. 1997), and in a stranded kemp’s ridley sea turtle with pulmonary hyalohyphomycosis (orós et  al. 2004), where fungal pneumonia was associated with cold stunning (knotek and divers 2019). fusarium  solani has also been found in sea turtle eggs, associated with mass mortalities (up to 83.3%) in natural and relocated nests of the sea turtle caretta caretta (sarmiento‑ramìrez et al. 2014a). colonization of eggs with fusarium is considered among the main causes of globally declining turtle populations (chelonia mydas, caretta caretta, eretmochelys imbricata, lepidochelys olivacea, dermochelys coriacea, and natator depressus) (sarmiento‑ramìrez et al. 2014b). treatment of fusariomycosis is very challenging. fusarium  spp. shows a remarkably high degree of intrinsic multidrug resistance to a wide spectrum of commonly used antifungals azoles, echinocandins and polyenes. the triazole group of antifungals works by inhibiting the fungal lanosterol 14α‑demethylase in the ergosterol biosynthesis pathway. the polyenes include amphotericin b, which binds to ergosterol thereby forming pores and damaging the cell membrane. finally, the echinocandins, namely caspofungin, micafungin and anidulafungin, affect cell wall synthesis by inhibiting 1,3‑β‑d‑glucan synthase. fusarium species share their high degree of intrinsic resistance to most currently used antifungal agents with acremonium and trichoderma, which belong to the same order hypocreales, and with microascus, scopulariopsis, scedosporium and lomentospora in the adjacent order microascales. intrinsic multiresistance of these fungi is unique among the ascomycota and suggests that this property was acquired in a common ancestor of the two orders (al‑hatmi et al. 2018). for practical purposes in the clinical practice, correct genus‑level identification of fusarium  species is a critically important action to enable correct treatment and infection control. since the distribution of fusarium  species varies 125veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 lai et al. fusarium in caretta caretta incubated at 25  °c in air for 14 days. whitish gray cottony colonies suggestive of fusarium  spp. were isolated from all samples. successive subcultures performed on potato dextrose agar in the dark showed sickle‑shaped multiseptated macroconidia, and one‑ to two‑celled microconidia formed from unbranched phialides, conidiophores, and chlamydospores typical of fusarium  solani (de  hoog 1995) (figure 2). this evidence, its reported in  vitro and in  vivo resistance to most of the available antifungal drugs, together with the lack of clinical outcome of the current therapy, suggested to perform susceptibility test on the isolate. voriconazole, posaconazole, itraconazole, ketoconazole, fluconazole and amphotericin b were tested. isolates of f. solani resulted resistant to all the antimycotic drugs tested, so itraconazole was discontinued, and only topical iodopovidone ointment was administered. tissue samples submitted to the unit of pathology for histology were fixed in 10% neutral buffered formalin for routinely process, embedded in paraffin, sectioned at 5 microns, stained with hematoxylin and eosin (he) and examined by light microscopy. histological analysis revealed necrosis and the presence of inflammatory cells (heterophils, monocytes, and lymphocytes) at the level of the basement membrane and in the dermis, together (1 part dextrose 5%, 1 part saline, 1 part electrolytic rehydrating solution, 1 part sterile water) at 1% b.w., then the body temperature was increased 3 °c/day until it reached 24  °c. antimicrobial and antifungal therapy was initiated when the turtle’s temperature reached 19 °c to 20 °c (norton and walsh 2012). the turtle presented several head, shell and plastron traumatic injuries, probably resulting from repeated dashes against rocks. the shell showed several disseminated superficial and ulcerous lesions, involving keratinized scales and the underlying bone plates, and one large lesion in the column region that continued on the right and left side of carapace, with necrosis of column bones, carapacial bone plates and ribs too (figure 1a). also, the head and plastron showed deep lesions with loss of tissue and exposition of bone superficies. lesions were sampled using a sterile lancet collecting material from the advancing edges of the lesions, after disinfection of the surface with iodopovidone and local anaesthesia induced by injection of a solution of 2% lidocaine hydrochloride; the bioptic samples were used for microbial and mycological culturing, and for histologic examination. standard prophylactic antimicrobial and antimycotic treatment was started (mader 2019, manire et  al. 2003, paré and jacobson 2007, mcarthur et  al. 2004), with marbofloxacin (2 mg/kg, im q 24 h; lai et  al. 2009) and itraconazole (5 mg/kg, po, q 24  h administered with assisted feeding; mader 2019). topical iodopovidone ointment was applied on shell and plastron lesions. the samples for bacteriological testing were cultured on tryptic soy blood agar and mac conkey agar, incubated for 24 h at 37 °c. on the basis of biochemical reaction and morphological characteristics, two different isolates were noticed, then identified with the biochemical system api 20 ne. vibrio fluvialis and aeromonas hydrofila were isolated from lesions and tested for susceptibility to several antibiotic drugs (ampicillin, ceftazidime, cephalothin, cefuroxime, doxycycline, norfloxacin, ciprofloxacin, enrofloxacin, marbofloxacin). the isolates resulted susceptible to ceftazidime, norfloxacin, ciprofloxacin, enrofloxacin, marbofloxacin and resistant to ampicillin, cephalothin, cefuroxime, doxycycline. on the basis of that result, marbofloxacin was continued for 60  days, and suspended after bacteriological cultures resulted negative. the samples for mycological examination were stained with calcofluor white and examined with fluorescent microscope. few single septate nonbranching hyphae bright greenish stained were noticed, so the presence of a fungal infections was supposed. thus, samples were cultured onto sabouraud agar chloramphenicol 0.05% and figure 1. a. carapacial lesions of the loggerhead sea turtle at admission; b. at release (case 1). 126 veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 fusarium in caretta caretta lai et al. animal was lethargic and severely emaciated. the standard procedure for the treatment of cold stunning was started (see above). samples of the lesions were obtained for microbial and mycological culturing, and histologic examination (see above), together with blood samples for haematological and biochemical exams (tables i and ii). standard prophylactic antimicrobial and antimycotic treatment was started (see above). once recovered from hypothermia, the young turtles started feeding voluntarily, and never stopped. prevotella rettseri, serratia marcescens, pseudomonas aeruginosa, proteus spp. were isolated from lesions and tested for susceptibility (see above); all of them resulted susceptible to fluoroquinolones. on the basis of that result, marbofloxacin was continued for 50 days, then suspended after bacteriological cultures resulted negative. in about a month the lesion extended as area and depth until it involved the underlying bone planes, with multifocal necrosis and severe haemorrhages, with presence of moderately basophilic hyphae; consequently an elective stain with stain periodic acid‑schiff (pas) was decided. pas staining revealed hyphae morphologically similar in all samples, with thin, generally parallel walls and variably spaced septa, frequent acute and right angle branching septate hyaline hyphae (figure 3), with optional sporulation, and occasional invasion of blood vessels during the period of hospitalization, the conditions of the turtle worsened. after two months of autonomous feeding with an average daily assumption of 200 g anchovies, the turtle became progressively anorectic due to the progressive avulsion of upper and lower ramphoteca, so a permanent oesophagostomy tube was applied under general anaesthesia (figure 4) (di bello et  al. 2011). prophylactic marbofloxacin (2 mg/kg po, q 24 h, marìn et al. 2009) was administered for as long as the tube was maintained, together with the food (2‑3% of b.w. of homogenized fish supplemented with vitamins). the permanent oesophagostomy tube was well tolerated by the turtle, which began voluntary feeding in one month, after the complete avulsion of ramphoteca, which leaved the maxillary and mandibular bones exposed and partially eroded on the right side (figure 5a). the tube was left in place for further 7 days, then it was removed after a light sedation with medetomidine (0.05 mg/ kg, im) reversed with atipamezole (0.25 mg/kg, im) in order to apply stitches to close oesophagostomy. the recovery was uneventful. fungal testing, performed every month, resulted negative after 4 months since the admission, then all the lesions progressively healed, including the beak, which started to show signs of new growth of the corneal part. complete beak repair took further 4 months (figure 5b), and the carapace lesions repaired with scar tissue to cover the bone plates (figure 1b). at that time, the turtle was back to ‘torre guaceto’ marine turtle rescue centre for summer release. case 2 a juvenile cold stunned loggerhead sea turtle (caretta caretta) stranded in august on the coast of adriatic sea near to jesolo (venice, italy), with an unusually low average sea temperature of 15 °c. the animal was referred to ‘torre guaceto’ marine turtle rescue centre, and immediately after moved to the department of veterinary medicine of bari for diagnostics and treatment because of extensive shell lesion (probably boat impact) limited to corneal scales. at admission the turtle weighted 1.2 kg, and the straight carapace length resulted 25 cm. the figure 2. culture on potato dextrose agar showing sickle-shaped multiseptated macroconidia and one/two celled microconidia formed from unbranched phialides, conidiophore and chlamydospores. figure 3. micrograph of necrotic tissue with fusarium solani hyphae invasion (arrows). periodic acid schiff stain, 40x (case 1). 127veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 lai et al. fusarium in caretta caretta which remained uncovered after the corneal scales detached (figure 6). mycological culturing isolated fusarium  solani from all samples. also, in this case susceptibility test on the isolate was performed, and f. solani resulted resistant to all the antimycotic drugs tested, so itraconazole was discontinued, and only topical iodopovidone ointment was administered. histological analysis revealed necrosis and a general situation similar to case 1, as in both subjects the colonization interested corneal structures. fungal testing, performed every month, resulted negative after 5 months since the admission, and the lesion gradually repaired with scar tissue. at that time, the turtle was back to ‘torre guaceto’ marine turtle rescue centre for summer release. case 3 a juvenile cold stunned loggerhead sea turtle (caretta caretta) stranded in march on the coast of adriatic sea near to lecce (italy), with average sea figure 4. permanent oesophagostomy tube in place (case 1). figure 5. a. eroded maxillary and mandibulary bones after complete avulsion of upper and lower ramphoteca; b. complete repair of beak 4 months after ramphoteca avulsion (case 1). table i. haematological results. turtle 1 turtle 2 turtle 3 reference values* median min max hct (%):17 18.00 24.00 23.00 28 17 45 rbc rbc (x106/µl):17 2 2.66 2.54 1.87 0.3 6 wbc wbc (x10³/µl):17 3.20 4.30 5.80 5.9 2 18.9 heterophils (%):18 64 73 79 75.8 51.61 88.6 lymphocytes (%):18 30 22 16 18.41 4.4 30.92 monocytes (%):18 0 1 1 1.2 0 5.3 eosinophils (%):18 6 4 4 4.5 0 29.4 basophils (%):18 0 0 0 0 0 0 plt estimated adequate adequate adequate table ii. plasma chemistry results. turtle 1 turtle 2 turtle 3 reference values* median min max ast (iu/l):17 460 653 389 194 < 10 844 alt (iu/l):17 14 35 18 24 < 10 258 alp (iu/l):17 45 71 64 67 51 562 cpk(iu/l):20 188 538 874 534 3 1,899 total bilirubin (mg/dl):17 0.21 0.32 0.01 0.2 0.2 0.5 glucose (mg/dl):17 79 98 77 129 19.8 291.9 total cholesterol (mg/dl):17 63 73 52 139 50.2 397.7 uric acid (mg/dl):20 0.8 0.9 1.9 2.4 0.7 4.2 creatinin (mg/dl):17 0.32 0.45 0.22 0.4 0.3 0.8 total protein (g/dl):17 2.60 4.3 1.9 2.4 2 11 albumin (g/dl):17 1.2 1.3 0.8 1.1 1 1.4 calcium (mg/dl):17 6.8 7.2 8.1 8 2.8 12.4 phosforum (mg/dl):17 4.6 4.5 5.1 6.4 4.1 7.9 ionized na (meq/l):20 146 151 134 156 135 175 ionized k (meq/l):20 3.7 5.8 3.2 5.1 3.3 13.9 ionized cl (meq/l):20 112 125 109 130 107 158 128 veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 fusarium in caretta caretta lai et al. samples were sent to the pathology unit of department of veterinary medicine of bari for histologic examination. aeromonas hydrofila was isolated from lesions and tested for susceptibility (see above), resulted susceptible to fluoroquinolones. on the basis of that result, marbofloxacin po was continued for 40 days, and then suspended after bacteriological cultures resulted negative. mycological culturing isolated fusarium solani from all samples. in this case too susceptibility test on the isolate was performed, and f. solani resulted resistant to all the antimycotic drugs tested, so itraconazole was discontinued, and only topical iodopovidone ointment was administered. in case 3 the lesions were very advanced, with extensive destruction of soft and bone tissues, replaced by repair tissue colonized in depth by the fungal hyphae and with extensive microvasculitis caused by the invasion of the microcirculation. the extensive injuries did not recover, so the proposal of live food (crustaceans and fish) was attempted before considering euthanasia. the subject responded positively, by actively preying on living preys, so the tube was removed, and the turtle was back to ‘torre guaceto’ marine turtle rescue centre for summer release. discussion dermatomycoses occur regularly in reptiles and are largely underdiagnosed, as lesions are indistinguishable from those caused by bacterial infections at a gross examination, so they are often misdiagnosed as such. mixed fungal and bacterial infections are also common, and it may be difficult to establish which of the two is the primary offender, so both microbiological and mycological diagnostics have always to be recommended (paré and jacobson 2007). temperature of 11  °c. the animal was referred to ‘torre guaceto’ marine turtle rescue centre, and immediately moved to the department of veterinary medicine of bari for diagnostics and treatment because of severe signs ascribable to cold stunning. at admission the turtle weighted 8.7 kg, and the straight carapace length resulted 45 cm. the animal was lethargic and emaciated. the standard procedure for the treatment of cold stunning and prophylactic antimicrobial and antimycotic treatment were started (see above). the severe emaciation was probably due to the extensive injury to the rostrum and the nasal cavities that the animal presented to admission, with complete absence of soft and cartilaginous tissues of the nose and of the rearward conchae (figure 7). in addition to the usual hematologic, bacteriological and mycological procedures, x‑ray and computed tomographic examination of the head were performed. x‑ray exam revealed extensive loss of bone tissues in the nasal cavity with absence of the nasal conchae. for the ct exam, images were acquired with the turtle placed in ventral recumbency under general anaesthesia (propofol 2 mg/kg iv; mader 2019). contiguous transverse slices were obtained from the basisphenoid to the external nares, showing severe osteolysis of the prefrontal bone, destruction of the cartilage of nasal septum, erosive changes of the dorsal and ventral conchae, and soft tissue opacification of the right dorsal nasal concha sinus and left ventral concha sinus. the turtle rejected various proposed foods (anchovies, squid, cuttlefish, mussels) for several days, probably because of complete loss of the olfactory capacity, so the esophagogastric tube was applied. prophylactic marbofloxacin (2 mg/kg po, q 24 h; marìn et al. 2009) was administrated. samples of the lesions were obtained for microbial and mycological culturing. some of the bioptic figure 7. extensive injury to the rostrum and the nasal cavities (case 3). figure 6. shell damages after corneal scales detachment (case 2). 129veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 lai et al. fusarium in caretta caretta in the reported cases, no particular immunosuppressed status has been noticed in hematologic exams (tables 1 and 2). in fact, all the parameters were within the normal ranges, if compared with bibliographic data on juvenile loggerhead sea turtles (casal et al. 2007, casal et al. 2009, gelli et  al. 2009) and comparable with data reported for stranded loggerheads (deem et  al. 2009). no particularly compromised scenery was depicted, so the severity of the fusarium  solani infection has to be ascribed to other potential factors that may facilitate fungal invasion: concomitant viral or bacterial infections, bacteria‑infected traumatic lesions, large areas of devitalized skin, nutritional nitrogen imbalance and stressors associated with stranding (frasca et al. 1996, lass‑flörl 2010). unlike most fungi species, in which conidiation is stimulated by emergence of the fungus through the water‑air interface, the conidiation of fusarium spp. typically occurs both in submersion and upon exposure to air. this explains the rapid seeding of these infections to numerous capillary beds, especially in the skin, a process that gives rise to the widespread ecthyma gangrenosum‑like lesions that characterize disseminated fusarial infections (summerbell 2002). angioinvasion, with vascular thrombosis and tissue infarction, explains the evolution of lesions to necrotic and bone‑involving levels. therefore, fusarium  spp. infection has not to be undervalued as it can generate extensive and ravaging lesions (salter et al. 2012). due to the critical role of immune response in the outcome of fusariosis, the optimal treatment strategy for patients with severe fusarium  spp. infection remains unclear. reversal of immunosuppression is recommended whenever possible. early therapy of localized lesions (including surgical debridement) is important to prevent progression to a more aggressive or disseminated infection (tortorano et al. 2014). the definitive diagnosis requires isolation of fusarium spp. from infected sites (culture, histology, molecular probes). the repeatedly positive cultures of biopsies from the lesions for fusarium  solani, along with evidence of tissue invasion with mycelial elements having the configuration of fusarium spp., indicate that this fungus was acting as a pathogen in the three turtles. in invasive infection fusarium  solani can also be isolated from blood cultures in up to 40‑60% of human cases (tortorano et al. 2014), but in the present cases blood was not used to attempt isolation. culture identification is important because of the histopathological similarities between fusarium spp. and other hyalohyphomycetes. although the genus fusarium  can be identified by culture by the mycoses seem particularly prevalent in sea turtles (mader 2019, manire et al. 2003, paré and jacobson 2007, cabañes et  al. 1997, duguy et  al. 1998), most commonly related to immunosuppressed status, traumatic lesions, captivity, and cold‑stunning. in particular, some authors (cafarchia et  al. 2019) have postulated that from an epidemiological point of view the origin of this opportunistic infection has to be considered related to the presence of f. solani in rescue center tanks and to immunosuppression due to the traumatic lesions suffered, surgical treatment applied, and other stressful conditions associated with transportation or rehabilitation of these marine turtles. the finding that animals admitted to the center for more than 20 days were more frequently colonized also suggested an association between fusarium‑like organisms and the skin lesions that occurred, given the presence of f.  solani in the tank at the rescue centers, a recognized risk factor of infection in receptive adult turtles. this finding suggested that the environmental conditions and management at the rescue center might favor fusarium  spp. growth and might be the source of colonization. this latter hypothesis was supported by the fact that sand from the filter of the two cited centers was positive for fusarium spp. on the contrary, in the present cases the lesions were already present and invaded by the fusarium  spp. before admission to the center. in particular, analysis of the sands of the center's filtering plant (equipped with a skimmer and uv passage for the sterilization of the filtered water before returning to the tanks) did not reveal any contaminating pathogenic organisms, so the hypothesis posed by the cited authors cannot be accepted and should not be generalized. otherwise, sudden drops in seawater temperature often result in hypothermia in juveniles or sub‑adult sea turtles. these cold‑stunned turtles typically become weak, float, and strand, with different possible types of wounds and lesions, and their rehabilitation can become long and difficult because of multiple metabolic disorders and opportunistic infections due to immunosuppression. severe mycotic infections can be common sequelae in these animals, and antifungal drugs are part of the standard pharmacological treatment of this syndrome (mader 2019, manire et al. 2003, paré and jacobson 2007). fungi as fusarium spp. in particular are often implicated, but other species of scarce or little‑known pathogenicity, such as colletotrichum acutatum, have caused disseminated mycoses in cold‑stunned turtles (manire et  al. 2003), indicating how severely immunocompromised these animals may become. nonetheless, fungal infections have also been reported in wild stranded sea turtles in florida unrelated to cold stunning events (paré and jacobson 2007). 130 veterinaria italiana 2020, 56 (2), 123‑132. doi: 10.12834/vetit.2035.13422.1 fusarium in caretta caretta lai et al. finally, the receptivity of man to fusariosis must not be underestimated, as fusarium  spp. can act as zoonotic agent for the operators or the visitors of sea turtle rescue centres. opportunistic fusarium  species cause a broad spectrum of infections predominantly in immunocompromised individuals via direct inoculation and airborne uptake as the most common routes of infections. the clinical signs of fusariosis depend largely on the immune status of the host and the portal of entry, which include paranasal sinuses, lungs and skin, with neutropenia as one of the most important risk factors for acquiring disseminated disease (tortorano et al. 2014); therefore the operators of the centres must be informed of the risk and equipped accordingly for the management of the infected subjects, while the visitors must not be admitted to the tanks of subjects with active fusariosis. production of hyaline, crescent or banana‑shaped multicellular macroconidia, species identification is difficult and may require molecular methods, although they should be used only to supplement conventional laboratory tests (tortorano et al. 2014). since the distribution of fusarium  species varies with geographic region 51 and different species have different drug susceptibility patterns (nucci and anaissie 2007) accurate species assignment is important for epidemiological studies and to guide clinical management. even though there is no correlation between f.  solani species complex and antifungal susceptibility, f. solani and f. verticillioides are usually resistant to azoles and exhibit higher amphotericin b minimal inhibitory 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endangered sea turtles. plos one, 9 (1), e85853. sarmiento‑ramìrez j.m., van der voort m., raaijmakers j.m. & diéguez‑uribeondo j. 2014b. unravelling the microbiome of eggs of the endangered sea turtle eretmochelys imbricate identifies bacteria with activity against the emerging pathogen fusarium falciforme. plos one, 9 (4), e95206. sharma c. & anuradha c. 2017. molecular bases of antifungal resistance in filamentous fungi. int j antimicrob agents, 50 (5), 607‑616. 155 veterinaria italiana 2021, 57 (2), 155-159. doi: 10.12834/vetit.2341.14461.1 accepted: 02.02.2021 | available on line: 31.12.2021 1department of microbiology, infectious and parasitic diseases, faculty of veterinary medicine, trakia university, stara zagora, bulgaria. 2department of infectious diseases, military medical academy, sofia, bulgaria. 3department of general and clinical pathology, faculty of veterinary medicine, trakia university, stara zagora, bulgaria. 4department of infectious pathology, hygiene, technology and control of foods from animal origin, faculty of veterinary medicine, university of forestry, sofia, bulgaria. 5department of occupational medicine, faculty of public health, medical university, sofia, bulgaria. 6unit of medical statistics and molecular epidemiology, universita campus bio-medico di roma, rome, italy. 7queens park, london, united kingdom. *corresponding author at: department of infectious diseases, military medical academy, sofia, bulgaria. e-mail: dr.baymakova@gmail.com. ilia tsachev1, magdalena baymakova2*, kiril k. dimitrov3, krasimira gospodinova1, plamen marutsov1, roman pepovich4, todor kundurzhiev5, massimo ciccozzi6 and harry r. dalton7 keywords hepatitis e virus, industrial farms, northern bulgaria, pigs. summary the purpose of the present study was to investigate pigs in northern bulgaria for serological evidence of hepatitis e virus (hev). sera from 225 individuals from three industrial farms were tested for anti-hev igg antibodies. the overall hev seroprevalence was 36% (81/225); weaners 6.8% (5/74); fattening pigs 38.7% (29/75) and in sows 61.8% (47/76). compared to weaners, hev positivity was higher in fattening pigs and sows: or = 8.70 (95% ci: 3.14-24.12) and or = 22.37 (95% ci: 8.07-61.96), respectively. these data confirm that hev is endemic in pigs throughout bulgaria, and can be a public health problem due to the transmission of hеv to humans through the consumption of pork meat and pork products. serological evidence of hepatitis e virus infection in pigs from northern bulgaria (ecdc 2017). in total, 28 deaths associated with hev infection were reported from five countries between 2005 and 2015 (ecdc 2017). these were almost all locally acquired and were thought to be porcine enzoonotic infections: the main route of infection is considered to be consumption of infected pig meat. recent estimates suggest that there may be at least two million human infections with hev in europe every year, most of which are asymptomatic (easl 2018). in bulgaria, the first evidence of human hev infection was reported in 4 patients in 1995 (teoharov et  al. 1995). afterwards many other cases of human hev infection were recorded in bulgarian citizens (baymakova et al. 2016, bruni et al. 2018, cella et  al. 2019). baymakova and colleagues reported serological evidence of anti-hev igm and anti-hev igg antibodies in 20 patients out of 806 patients showing acute viral hepatitis (baymakova hepatitis e virus (hev) is a single-stranded positive-sense rna virus (ictv 2018, purdy et  al. 2017). since the first report of its genomic sequence (tam et al. 1991), strains have been widely detected, not only in humans, but also in a great number of animal species. hev is classified into hepeviridae family which has recently been divided in two genera: ortohepevirus and piscihepevirus (ictv 2018). ortohepevirus includes four species: orthohepevirus a (genotypes: hev-1 – hev-8), orthohepevirus b (avian hev genotype 2), orthohepevirus c (germany rat hev, vietnam rat hev and ferret hev) and orthohepevirus d (germany bat hev) (ictv 2018, pepovich et  al. 2019). genus piscihepevirus has only one species – piscihepevirus  a, and one genotype – cutthroat trout hev (ictv 2018). the number of laboratory-confirmed cases of hepatitis e in europe has dramatically increased in recent years, from 514 in 2005 to 5,617 cases in 2015 short communication 156 veterinaria italiana 2021, 57 (2), 155-159. doi: 10.12834/vetit.2341.14461.1 hepatitis e virus in pigs from northern bulgaria tsachev et al. humans through the consumption of pork meat and derivative products. two hundred and twenty-five pigs (n = 225) from three commercial farrow to finish pig farms of northern bulgaria (goliyamo vranovo, nikolovo and pleven, 38,000, 15,000 and 700 heads of pigs, respectively) were enrolled (figure 1). pigs included in the study were divided into three age groups: weaners (age: 30-100 days), fattening pigs (101-160 days) and sows (> 365 days). pigs showed no clinical signs at sampling time point. the sex (weaners and fattening pigs) was not recorded. the enrolled pigs were randomly selected for sampling. the collection of the samples was planned in context of farm capacity. swine blood samples (up to 5 ml per individual) were taken by puncture of the sinus ophthalmicus. blood collection tubes without anticoagulant were kept at room temperature (20 °c) until clot retractionwas visible. then they were centrifuged at 1,500 g for ten minutes, and the serum was separated and stored at - 20 °c until testing. the serum samples were tested for hev antibodies in the laboratory of infectious diseases, faculty of veterinary medicine, trakia university, stara et  al. 2016). bruni and colleagues found 103 hev igm positive serum samples collected from hospitalized patients with acute hepatitis from all over bulgaria (bruni et  al. 2018). the same authors performed phylogenetic analysis (n = 64 patients) and genotyped hev-1 in 2% of the cases (1/64), hev-3e subtype in 62% (39/63), hev-3f subtype in 24% (15/63), hev-3c subtype in 13% (8/63), and hev-3hi subtype in 2% (1/63). a  recent analysis of 2,257 cases of human hepatitis in bulgaria (1995 to 2018) showed that 13.1% were caused by hev, predominantly genotype 3 (baymakova et al. 2019). the first preliminary data for swine hev infection in bulgaria were published in 2018 (pishmisheva et  al. 2018), showing an overall seroprevalence of anti-hev antibodies of 40% (34/85). in 2019, a detailed seroprevalence study of hev infection in pigs from southern bulgaria (tsachev et  al. 2019), documented an overall hev seroprevalence of 60.3% (217/360). the aim of our study was to investigate pigs in northern bulgaria for serological evidence of hev, in order to obtain a complete nationwide picture of viral exposure in this important primary host. this could indeed amplify the transmission of hеv to romania serbia north macedonia greece turkey 50 km 30 mi vidin goliyamo vranovo 31.1% pleven 0% pishtigovo 50% kostinbrod 88.8% kalchevo 58.8% lovech so�a province pernik kyustendil blagoevgrad smolyan plovdiv kardzhali haskovo stara zagora burgas gabrovo varna shumen silistra dobrichrazgrad targovishtevellko tarnovo sliven nontana vratsa yambol 43.4% nikolovo 58.9% figure 1. geographic distribution of hev infection in pigs from bulgaria. the data from the current paper are shown in grey. the investigated farms were located in the northern bulgarian plains (approximately 43°40’n and 43°84’n latitude, 24°62’e and 25°95’e longitude); the climate is continental (mean annual temperature 10-12 ºc, precipitation approximately 630 mm/m2). data from our previous study in southern bulgaria are shown in black, for comparison (tsachev et al. 2019). 157veterinaria italiana 2021, 57 (2), 155-159. doi: 10.12834/vetit.2341.14461.1 tsachev et al. hepatitis e virus in pigs from northern bulgaria was found in goliyamo vranovo (16.7%; 5/30). the highest hev seropositivity in fattening pigs and sows was documented in nikolovo – 96.7% (29/30) and 80% (24/30), respectively. the overall prevalence of anti-hev antibodies in each farm was: goliyamo vranovo 31.1% (28/90); nikolovo 58.9% (53/90); and pleven – 0% (0/45) (figure 1). there were significant differences in hev seropositivity between pig age and farms (table i). to estimate the risk for hev seropositivity, the odds ratio (or) in different age groups was performed by binary logistic regression. the or of anti-hev antibodies occurrence in fattening pigs and sows was determined comparing to group weaners (table  ii). we found that the odds of hev infection was nearly 8-times higher in fattening pigs and 22-times higher in sows than in weaners. the prevalence of hev-antibodies in both pigs and humans is quite variable between and within countries. this variability may, at least in part, be influenced by the study design, diagnostics methods and tested population. various laboratory tests also influenced the final results (krumbholz et  al. 2013). nevertheless, it seems that many pigs worldwide are infected with hev and represent zoonotic risk for transmission to humans. the seroprevalence in our study (36%; 81/225) is similar to data found in several other countries, including taiwan (37.1%; 102/275) (hsieh et al. 1999); serbia (34.6%; 109/315) (lupulovic et  al. 2010); usa (34.5%; 29/84) (withers et  al. 2002); croatia (32.9%; 469/1,424) (jemersic et al. 2017); south korea (40.7%; 57/140) (meng et al. 1999); france (31%; 2,035/6,565) (rose et  al. 2011); thailand (30.7%; 23/75) (meng et al. 1999); romania (42.7%; 65/145) (savuta et al. 2007) and india (42.9%; 122/284) (arankalle et al. 2002). sows had the highest seroprevalence (61.8%) in our study. martinelli and colleagues found 70.6% hev-prevalence in sows, also the highest seroprevalence in their study (martinelli et al. 2011), whereas danish investigation presented 73.2% positive sows for hev-igg (breum et  al. 2010). the results of our study showed an age-dependent seroprevalence (or = 8.70, p < 0.001; or = 22.37, zagora, bulgaria. a commercial enzyme-linked immunosorbent assay (elisa, priocheck hev ab porcine, mikrogen gmbh, neuried, germany) was used, according to the manufacturer’s instructions. the priocheck hev ab porcine is a diagnostic test for detection of hev-specific antibodies in porcine serum and meat juice samples. a microtiter plate is coated with recombinant hev antigen of the open reading frame 2 (orf2) and orf3 of genotypes hev-1 and hev-3. the test has 91.0% sensitivity and 94.1% specificity. the cut-off, as well as positive, negative and borderlie results were calculated as described by the manufacturer. borderline results were repeated and those remaining in the borderline range were considered negative. hev positive results among different swine age groups and farms were compared by using the chi-square test. binary logistic regression was used to evaluate the risk of positive results according to age group. statistical analysis was performed by spss statistics 19.0 (ibm corp., armonk, new york, usa). a p-value < 0.05 was considered statistically significant. the study was approved by the ethics committee in animal experimentation and animal welfare at trakia university, stara zagora (bulgaria) and was conducted according to the ethical principles of animal experimentation, adopted by the bulgarian ministry of agriculture, food and forestry. anti-hev iggs were detected in 81 (36%) of the 225 tested sera (table i). the overall seropositivity in weaners, fattening pigs and sows was 6.8% (5/74), 38.7% (29/75) and 61.8% (47/76), respectively (table ii). the highest hev seropositivity in weaners table i. seroprevalence of hev infection according to category groups in pigs from northern bulgaria. age groups age, days investigated pigs, n hev positive, n (%) chi-square df p-value goliyamo vranovo weaners 30-100 30 5 (16.7) 45.52 2 < 0.001 fattening pigs 101-160 30 0 (0.0) sows > 365 30 23 (76.7) nikolovo weaners 30-100 30 0 (0.0) 66.18 2 < 0.001 fattening pigs 101-160 30 29 (96.7) sows > 365 30 24 (80.0) pleven weaners 30-100 14 0 (0.0) na na na fattening pigs 101-160 15 0 (0.0) sows > 365 16 0 (0.0) hev = hepatitis e virus; df = degrees of freedom; na = not applicable. table ii. logistic regression showing the relationship between hev positive pigs and age. age groups investigated pigs, n hev positive, n (%) pe se p-value or 95% ci weaners 74 5 (6.8) na na na 1.00 na fattening pigs 75 29 (38.7) 2.16 0.52 < 0.001 8.70 3.14-24.12 sows 76 47 (61.8) 3.11 0.52 < 0.001 22.37 8.07-61.96 hev = hepatitis e virus; pe = parameter estimate; se = standard error; or = odds ratio; ci = confidence interval; na = not applicable. 158 veterinaria italiana 2021, 57 (2), 155-159. doi: 10.12834/vetit.2341.14461.1 hepatitis e virus in pigs from northern bulgaria tsachev et al. our study shows that many pigs in northern bulgaria have been exposed to hev, but the frequency of exposure is somewhat lower than that documented in southern bulgaria. these data confirm that hev is endemic in pigs throughout bulgaria, and so it is a nationwide public health concern. in addition, compared to other european countries, our results are similar to those of some southern european countries such as france (31%) (rose et  al. 2011), croatia (32.9%) (jemersic et  al. 2017), serbia (34.6%) (lupulovic et al. 2010) and romania (42.7%) (savuta et  al. 2007), and differed greatly from the results of continental and northern europe such as switzerland (58.1%) (burri et  al. 2014), germany (68.6%) (wacheck et  al. 2012), finland (86.3%) (kantala 2017) and norway (90%) (lange et  al. 2017). in conclusion, we think that the present study complemented the knowledge about hev infection both nationally and regionally. acknowledgments this study was funded by the trakia university, 6000 stara zagora, bulgaria (grant n. 12/2018). p < 0.001). similar observations have been reported in other studies (breum et  al. 2010). hev infection occurs in all age groups of pigs and it seems most likely that infection occurs in nursery and fattening periods (pavio et  al. 2010). most pigs become infected at 6-8 weeks of age, while virus in feces peaks at 12-14 weeks and declines at 20-22 weeks (pavio et  al. 2010). hev-iggs appear at 8-9 weeks, increase in frequency, and approximately all infected pigs at 22-24 weeks have hev-igg (pavio et al. 2010). the established hev seroprevalence in pigs from northern bulgaria is lower (mean 36%, and 38.7% for fattening) than that reported from southern bulgaria (mean 60.3%, and 75.8% for fattening) (tsachev et al. 2019). there is a number of possible explanations for this observation, relating to geographical differences in pig farm density, husbandry conditions, model of pig farming, animal contact with the environment, sewage systems and water facilities. in the present study, the involved farms were typical industrial farms with low and moderate density of animals, longer farming for fattening and small herds. these factors may have contributed to the lower hev seroprevalence in pigs from northern bulgaria, compared to pigs from southern bulgaria. 159veterinaria italiana 2021, 57 (2), 155-159. doi: 10.12834/vetit.2341.14461.1 tsachev et al. hepatitis e virus in pigs from northern bulgaria arankalle v.a., chobe l.p., joshi m.v., chadha m.s., kundu b. & walimbe a.m. 2002. human and swine hepatitis e viruses from western india belong to different genotypes. j hepatol, 36, 417-425. baymakova m., popov g.t., pepovich r. & tsachev i. 2019. hepatitis e virus infection in bulgaria: a brief analysis of the situation in the country. open access maced j med sci, 7, 458-460. baymakova m., sakem b., plochev k., popov g.t., mihaylova-garnizova r., kovaleva v. & kundurdjiev t. 2016. epidemiological characteristics and clinical manifestations of hepatitis e virus infection in bulgaria: a report on 20 patients. srp arh celok lek, 144, 63-68. breum s.o., hjulsager c.k., de deus n., segales j. & larsen l.e. 2010. hepatitis e virus is highly prevalent in the danish pig population. vet microbiol, 146, 144-149. bruni r., villano u., equestre m., chionne p., madonna e., trandeva-bankova d., peleva-pishmisheva m., tenev t., cella e., ciccozzi m., pisani g., golkocheva-markova e. & ciccaglione a.r. 2018. hepatitis e virus genotypes and subgenotypes causing acute hepatitis, bulgaria, 2013-2015. plos one, 13, e0198045. cella e., golkocheva-markova e., sagnelli c., scolamacchia v., bruni r., villano u., ciccaglione a.r., equestre m., sagnelli e., angeletti s. & ciccozzi m. 2019. human hepatitis e virus circulation in bulgaria: deep bayesian phylogenetic analysis for viral spread control in the country. j med virol, 91, 132-138. european association for the study of the liver (easl). 2018. easl clinical practice guidelines on hepatitis e virus infection. j hepatol, 68, 1256-1271. european centre for disease prevention and control (ecdc). 2017. hepatitis e in the eu/eea, 2005-2015. stockholm (ecdc). garcia-hernandez m.e., cruz-rivera m., sanchez-betancourt j.i., rico-chavez o., vergara-castaneda a., trujillo m.e. & sarmiento-silva r.e. 2017. seroprevalence of anti-hepatitis e virus antibodies in domestic pigs in mexico. bmc vet res, 13, 289. hsieh s.y., meng x.j., wu y.h., liu s.t., tam a.w., lin d.y. & liaw y.f. 1999. identity of a novel swine hepatitis e virus in taiwan forming a monophyletic group with taiwan isolates of human hepatitis e virus. j clin microbiol, 37, 3828-3834. international committee on taxonomy of viruses (ictv). 2018. herpeviridae (virus taxonomy: 2018b release). https://talk.ictvonline.org (accessed on july 20, 2020). jemersic l., keros t., maltar l., barbic l., vilibic-cavlek t., jelicic p., rode o.d. & prpic j. 2017. differences in hepatitis e virus (hev) presence in naturally infected seropositive domestic pigs and wild boars an indication of wild boars having an important role 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1991. hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome. virology, 185, 120-131. teoharov p., tiholova m., draganov p., lilyanova v., ivanova r., varleva t. & dimitrova t. 1995. first cases of hepatitis e virus infection in bulgaria. infectology (sofia), 32, 17-18. tsachev i., baymakova m., ciccozzi m., pepovich r., kundurzhiev t., marutsov p., dimitrov k.k., gospodinova k., pishmisheva m. & pekova l. 2019. seroprevalence of hepatitis e virus infection in pigs from southern bulgaria. vector borne zoonotic dis, 19, 767-772. withers m.r., correa m.t., morrow m., stebbins m.e., seriwatana j., webster w.d., boak m.b. & vaughn d.w. 2002. antibody levels to hepatitis e virus in north carolina swine workers, non-swine workers, swine, and murids. am j trop med hyg, 66, 384-388. 353 veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 accepted: 03.08.2021 | available on line: 31.12.2022 1department of veterinary medical sciences, alma mater studiorum, university of bologna, ozzano dell’emilia (bo), italy. 2synlabs, veterinary pathology group, temple point, bullerthorpe ln, colton, leeds, united kingdom. *corresponding author at: deparment of veterinary medical sciences, university of bologna, via tolara di sopra 50, ozzano dell’emilia 40060 (bo), italy. tel.: +39 051 2097506, fax: +39 051 2086178, e‑mail: armando.foglia2@unibo.it. sara del magno1, marta gruarin2, armando foglia1*, veronica cola1, chiara agnoli1, roberta galuppi1, francesco dondi1 and luciano pisoni1 keywords aspergillosis, aspergillus terreus, osteomyelitis, pyelonephritis, systemic mycosis, ureteral obstruction. summary a female, 1.5 years old, mixed-breed dog, was presented for left hind limb lameness. radiographs revealed an irregular periosteal proliferation on the left iliac wing. the clinical condition worsened with generalised enlargement of the lymph nodes, azotaemia, and pyelonephritis. the magnetic resonance imaging of the pelvis and a surgical biopsy diagnosed a mycotic myositis and osteomyelitis of the iliac wing and gluteal muscles. aspergillus terreus was isolated from culture of urine and lymph nodes aspirates. the antifungal susceptibility test showed moderate sensitivity to itraconazole. after one month of therapy with itraconazole, the dog presented discospondylitis of l1-l2 and partial ureteral obstruction due to mycotic bezoar that was resolved with medical treatment and itraconazole dose elevation. after twelve months, itraconazole was suspended; a severe osteomyelitis of the left femur developed, and the dog was euthanised. the necropsy confirmed the presence of mycotic osteomyelitis of the iliac wing and femur, discospondylitis, lymphadenitis and severe granulomatous pyelonephritis. systemic aspergillosis has rarely been reported in the literature, especially in italy. the pelvic bone involvement is rare both in dogs and humans. although itraconazole treatment allowed remission of the clinical signs for one year, it was not able to cure the dog. disseminated aspergillosis in a german shepherd mixed breed dog with unusual initial localization to the iliac wing in dogs, the disseminated form has a haematogenous spread and clinical signs are initially mild and nonspecific as fever, anorexia, weight loss, vomiting, and lethargy. specific signs are also reported according to the localisation of the infection. common sites of embolic dissemination of fungal hyphae are the intervertebral discs, kidneys, and uveal tracts. central nervous system, lungs and long bones represent other common localisations of the infection. orthopaedic symptoms may be present as a consequence of osteomyelitis and discospondylitis, while signs of renal involvement can be related to pyelonephritis (schultz et al. 2008). due to the variety of nonspecific clinical signs, the diagnosis of systemic aspergillosis is usually belated. therefore, most of the dogs are severely ill at the time of diagnosis. the prognosis is usually poor introduction disseminated aspergillosis is a rare disease in dogs, more frequently reported in usa (corrigan et al. 2016, starkey and mcloughlin 1996, van wie et  al. 2013, schultz et  al. 2008), rather than in other countries (bruchim et al. 2006). systemic aspergillosis has been reported recently in italy as a disease in three dogs (morabito et al. 2020). aspergillus spp. are saprophytic and ubiquitous fungi, which usually colonise immunocompromised humans and animals (bieganska et al. 2014). german shepherd dogs seem to be predisposed to the localised sino-nasal (seyedmousavi et  al. 2015) and generalised forms, possibly because of heritable abnormalities of the humoral and cellular immune responses (schultz et al. 2008). case report 354 veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 disseminated aspergillosis in a mixed breed dog del magno et al. toulouse, france) and omeprazole (0.7 mg/kg q24, mepral, bracco, milano, italy) for 10 days, without improvement. during treatment, the dog presented hyperthermia (39.2  °c), anorexia and peripheral lymph nodes enlargement. full blood count (fbc), serum chemistry profile, fine needle aspirates of the prescapular and popliteal lymph nodes and magnetic resonance imaging (mri) of the pelvis were performed. a mild leucocytosis consisting of mature neutrophilia was observed on the fbc. the serum chemistry profile revealed mild azotaemia, increased concentration of total protein and c-reactive protein (table i, t 0 ). cytological examination of the lymph nodes showed a granulomatous lymphadenitis associated with the presence of fungal hyphae (figure 2). the mri showed a wide lesion of the deep and middle gluteal, longissimus lombi, piriformis, and partially of the iliopsoas muscles. the iliac even when a specific therapy is instituted (schultz et  al. 2008, morabito et  al. 2020). the antifungal therapies available for animals are limited because of the costs of last generation antimycotic drugs, like voriconazole and posaconazole (koehler et al. 2013, corrigan et al. 2016). in humans and dogs, obstructive uropathy related to aspergillosis is a very rare condition and, only few cases are reported in the literature (yoon et al. 2010, najafi et  al. 2013, starkley and mcloughlin 1996, schultz et al. 2008). this report describes a case of disseminated aspergillosis in a young female german shepherd mixed breed dog in italy. case description a spayed female, 1.5 years old, german shepherd mixed breed dog was referred for an orthopaedic consultation for lameness on the left hind limb of 2 week duration. the physical and orthopaedic examination revealed a painful swelling on the left iliac wing region. the radiographs of the pelvis showed an irregular margin of the crest of the left iliac wing (figure 1) and through the ultrasonographic examination an enthesitis of the middle gluteal muscle was suspected. treatment initially consisted of firocoxib (5 mg/kg q24h, previcox, merial, figure 1. radiograph, ventro-dorsal projection of the pelvis of a dog with lameness, slight swelling of the gluteal region and pain on palpation. blue arrow indicates the periosteal reaction of the left iliac wing. figure 3. magnetic resonance imaging (mri) of the pelvic region, post-contrast t-1 weighted sequence. the blue arrow indicates the increased contrast uptake of the deep and middle gluteal, longissimus lombi, piriformis and partially of the iliopsoas muscles muscles. note the irregular appearance of the iliac bone with periosteal reaction and bone lysis. the asterisk indicates severe lymphadenomegaly of the left sacral lymph node. figure 2. fine needle aspirate, prescapular lymph node. evidence of a fungal septate hypha of aspergillus terreus embedded in necrotic material. may-grünwald giemsa stain. x100 objective. 355veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 del magno et al. disseminated aspergillosis in a mixed breed dog became progressively depressed and presented vomit, and polyuria and polydipsia. abdominal ultrasonography and urinalysis were performed. a bilateral dilatation of the renal pelvis (right renal pelvis diameter 26 mm, left renal pelvis diameter 12  mm) and the ureters (diameter 6 mm) with scattered hyperechoic content associated with enlargement of the mesenteric lymph nodes were noticed. urinalysis revealed isosthenuria and microscopic examination of a wet-mount of urine sediment revealed pyuria wing presented periosteal proliferation and bone lysis, and an enlargement of the sacral lymph node was observed (figure 3). a surgical biopsy of the left middle and deep gluteal muscles and of the iliac wing was performed and a severe chronic granulomatous, necrotising myositis, and severe chronic panosteitis and bone lysis were diagnosed. the presence of fungal hyphae in the biopsy samples was documented on histopathology. after the mri, the clinical condition of the dog worsened. the dog table i. clinicopathological findings of the described aspergillosis case evaluated upon admission and during the follow up period. variable units t0 t1 t2 t3 reference interval haematology haematocrit value % 49.6 47.7 36.6 52 37.0-55.0 rbc ×1012/l 7.5 7.2 5.7 7.6 5.5-8.5 wbc ×109/l 18.5 17.5 20.6 13 6.0-17.0 neutrophils ×109/l 13.1 12.8 14.4 5.9 3.0-12.0 lymphocytes ×109/l 3.1 3.4 4.9 4.8 1.0-4.8 monocytes ×109/l 1.3 0.8 1.1 0.5 0.1-1.4 eosinophils ×109/l 0.6 0.4 0.3 1.5 0-0.7 plt ×109/l 34.6 25.5 21.7 26.4 16.0-50.0 chemistry ck u/l 200 50-290 alt u/l 20 25 67 38 20-55 ast u/l 35 91 46 31 20-42 alp u/l 66 52 123 42-180 ggt u/l 1.8 1.4 2.0 0–5.8 glucose mg/dl 86 142 81 98 65-115 total bilirubin u/l 0.21 0.2 0.32 0.07-0.33 creatinine mg/dl 2.08 1.29 4.3 1.64 0.65-1.35 urea mg/dl 67 32.23 86.48 38.91 18-55 total proteins g/dl 8.21 8.16 6.88 5.60-7.90 albumin g/dl 2.91 2.82 3.13 2.80-3.70 a:g 0.55 0.53 0.83 0.60-1.30 crp mg/dl 2.98 0.01 0-0.8 calcium mg/dl 10.5 9.9 9.8 9.0-11.8 phosphate mg/dl 4.6 6.3 4.3 2.6-4.90 sodium meq/l 141 146 146 144 143-154 chloride meq/l 104 111 113 108.0-118.0 potassium meq/l 3.9 4.2 4.3 4.7 3.9-5.3 urinalysis usg 1,012 1,018 1,016 1,020 > 1,030 ph 5.5 5.5 6 5.5 protein (dipstick) mg/dl 0 0 0 0 absent urine sediment mycotic hyphae, leucocytes erythrocytes and rare leucocytes mycotic hyphae, erythrocytes and leucocytes mycotic hyphae upc 0.3 0.1 0.0-0.5 culture urine aspergillus terreus lymph nodes aspergillus terreus a:g = albumin-to-globulin ratio; alp = alkaline phosphatase; alt = alanine aminotransferase; ast = aspartate aminotransferase; at, antithrombin; ck, creatinine kinase; crp = c-reactive protein; ggt = gamma-glutamyltransferase; plt = platelets; upc = urinary protein to creatinine ratio; rbc = red blood cells; t 0 = presentation; t 1  = 7 days later, preoperative; t 2 = 7 days postoperative; t 3 = 12 days postoperative; usg = urine specific gravity; wbc = white blood cells. 356 veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 disseminated aspergillosis in a mixed breed dog del magno et al. and the presence of fungal hyphae (figure 4) (table i, t 0 ). the final diagnosis was systemic mycosis with osteomyelitis localised to the left iliac wing and fungal pyonephrosis/pyelonephritis. the dog was hospitalised and antifungal therapy with itraconazole (10 mg/kg q24h os, itraconazolo teva, milano, italy) was started along with intravenous  (iv) fluid therapy. the culture from the urine and the lymph nodes resulted positive for moulds belonging to aspergillus terreus group, based on phenotypic characteristics, according to raper and fennell (raper and fennell 1965) (figure 5). the antifungal susceptibility test showed moderate sensitivity to itraconazole, resistance to fluconazole and a good sensitivity to voriconazole. the dog progressively improved clinically, as the dilation of the renal pelvis diminished (right renal pelvis diameter 14 mm, left renal pelvis diameter 3.9 mm) and azotaemia resolved (table i, t 1 ). the patient was discharged with antimycotic therapy (itraconazole 10 mg/kg q24h po). figure 5. a. terreus from urine: a. culture on sabouraud dextrose agar; b. compact, biseriate conidial heads; c. aleurioconidia produced directly on the submerged hyphae lactophenol cotton blue stain. figure 4. wet-mount urine sediment preparation. two septate hyphae of aspergillus terreus surrounded by scattered leucocytes and red blood cells. x40 objective. figure 6. descending urography, ventro-dorsal projection of the abdomen. the blue arrow indicates the dilated left renal pelvis and proximal portion of the left ureter due to obstruction by suspected fungal bezoars (red arrow). yellow arrow underlines very reduced contrast uptake of the right kidney. green arrow indicates the severe progression of bone lysis of the left iliac wing in comparison to the first x-ray. after one month, the dog was presented for acute vomiting. moreover, the owner noted a reduced urine production in the previous 24 hours. physical examination detected pain at palpation of the abdomen, renal area, and lumbar region. a discitis at the l1-l2 intervertebral disc was evident on the radiographs. acute kidney injury was suspected based on increased serum creatinine concentration, on the persistence of hyphae in the urine sediment (table i, t 2 ) and on the results of abdominal ultrasonography. in particular, the latter revealed worsening of the bilateral pyelonephritis/pyonephrosis (right renal pelvis diameter 24 mm, left renal pelvis diameter 9.3 mm) and proximal hydroureters (proximal right ureter diameter 14 mm, proximal left ureter diameter 10 mm), floating hyperecoic intraluminal debris and retroperitoneal effusion. a descending intravenous urography was performed and evidenced a partial obstruction of the left ureter associated with dilatation of the ipsilateral pelvis. based on the excretory urography the right kidney appeared very poorly perfused. mycotic bezoars were suspected to be the cause of the ureteral obstruction (figure 6). a urine culture and susceptibility test were repeated and the sensitivity to itraconazole appeared decreased. voriconazole was proposed as a more efficient therapy, but the owner refused due to financial restraints. the dosage of itraconzole was then increased at 7 mg/kg q12h and conservative treatment for ureteral obstruction was started with 357veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 del magno et al. disseminated aspergillosis in a mixed breed dog approximately one year. the physical examination was unremarkable, except for the palpation of an irregular margin on the left iliac wing. the dog was staged, based on the iris staging of chronic kidney disease, as stage 2, non-proteinuric (table i), normotensive. a mild bilateral dilatation of the renal pelvis and of the proximal ureters associated with hyperechoic content and enlargement of the mesenteric lymph nodes persisted at every abdominal ultrasound re-examination. after 12 months from the beginning of treatment, itraconazole was temporarily discontinued because of severe vomiting. the clinical condition of the dog progressively deteriorated and, after two weeks, the dog was presented for acute and non-weight bearing left hind limb lameness. radiographic examination revealed a lytic and proliferative lesion of the shaft of the left femur, along with a severe periosteal reaction. a mycotic osteomyelitis was strongly suspected (figure 7). the dog was humanely euthanised and the necropsy identified a severe mycotic bilateral pyelonephritis with granulomatous lesions in the right renal parenchyma (figure 8), renal, lombo-aortic and sacral lymphadenitis, severe osteomyelitis to the left iliac wing and left femur (figure 9) and discospondylitis of l1-l2. from all these tissues, a. terreus was isolated. iv fluids (ringer lactate 3 ml/kg/h braun, melsungen, germany) and analgesia (buprenorphine 15 mcg/kg every 6 h, temgesic, indivior italia srl, milano, italy). the dog developed post-obstructive polyuria 24 h after the beginning of the conservative treatment and the bilateral dilatation of the renal pelvis and ureters partially resolved (right renal pelvis diameter 7 mm, left renal pelvis diameter 3.5 mm). renal function improved (table i, t 3 ) and the dog was discharged after one week of hospitalisation. the dog was re-examined periodically for figure 7. radiograph, ventro-dorsal projection of the pelvis and femurs. the blue arrow indicates the severe focal periosteal reaction and mild bone lysis in the proximal third of the left femoral diaphysis due to mycotic osteomyelitis. the arrow-head underlines the bone lysis and periosteal reaction of the left iliac wing due to mycotic osteomyelitis. note the decreased muscle mass of the left tight in comparison to the right one. figure 8. post-mortem sagittal section of the kidneys. note a large mass invading the right renal pelvis consistent with mycotic granuloma (asterisks). note in both pelvis the presence of yellow material compatible with mycotic bezoars (blue arrows). figure 9. post-mortem longitudinal section of the left femur with mycotic osteomyelitis of the proximal third of the shaft of the femur. 358 veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 disseminated aspergillosis in a mixed breed dog del magno et al. urinalysis and cytological examination of the fine needle aspirates of peripheral lymph nodes were helpful for the final diagnosis in the present case. indeed, since urinary excretion of mycotic hyphae can occur in renal involvement, their observation at sediment examination is possible, as already reported by morabito and colleagues (morabito et  al. 2020). moreover, lymph node involvement is frequently reported (morabito et  al. 2020) and the cytological observation of fungal hyphae in their aspirates can be a useful and quick way to obtain the diagnosis of systemic mycosis. the sterile sampling of urine and lymph nodes could represent easily collectable specimens to perform fungal culture and even susceptibility test that can guide the treatment, as in our case. the sensibility test is an important step for the treatment because of the intrinsic amphotericin b resistance of a. terreus and the report of isolates with reduced azole-susceptibility (zoran et al 2018). a bony prominence, the iliac crest, appeared to be the first affected site in the present case. no previous trauma or surgery were reported by the owners. consequently, a possible direct inoculation of a. terreus after an undetected penetrating puncture or lesion can be suspected. other entry routes appeared less probable because of the absence of respiratory and enteric signs of the disease. very likely, a secondary haematogenous dissemination of the infection took place and involved the kidneys and lymph nodes; while discospondylitis developed after the beginning of the antifungal therapy with itraconazole, indicating a lack of control of the infection. long-term remission was partially achieved after antifungal therapy and drug dosage review, which carried no side effects for several months. femoral osteomyelitis developed after 12  months from the initial presentation, during a period of withdrawal of itraconazole. this last localisation again confirmed the inability of itraconazole to control the dissemination of aspergillosis, which within a few days from discontinuation of the antimycotic drug gained another haematogenous spread to the bone. the trend of initial improvement or apparent remission and subsequent relapse has already been reported for different antimycotic drugs like itraconazole, amphotericin b, ketoconazole and posaconazole (schutz et al. 2008, corrigan et al. 2016). this unsatisfactory response to antifungal therapy is not completely understood; however, an inadequate concentration of these drugs in the urine and affected tissues, along with a lack of response of the immune system of affected dogs can explain the poor outcome with medical therapy (corrigan et  al. 2016). moreover, the difficulties encountered in the diagnosis of the subtle and nonspecific signs, can prolong the time of diagnosis and treatment, although this do not seem to be the case for the discussion disseminated aspergillosis is considered an uncommon disease in dogs, and it usually carries a poor prognosis. the fungal species involved in this disease is more frequently a. terreus, unlike the more frequent sino-nasal aspergillosis that is primarily caused by a. fumigatus (schultz et al. 2008). conversely, a. terreus species complex appears to be a rare cause of invasive aspergillosis in human beings (where the most frequent agent is a.  fumigatus), and often the isolates display a polyene resistance (risslegger et  al. 2017). its incidence as cause of invasive aspergillosis in humans is higher in innsbruck (austria) and houston (texas-usa) hospitals than worldwide. studies performed in tyrol concluded that in this region patients develop a. terreus infections more often, because this fungus is more prevalent in the environment (lackner et al. 2016). the dog of the present report was born in emilia-romagna region and lived in an apartment in bologna. in italy, canine generalised aspergillosis appears to be a very rare disease, with three reported cases with a short follow-up before euthanasia, to the best of the authors’ knowledge (morabito et al. 2020). however, it cannot be excluded that the real prevalence of the disease is currently underestimated. female german shepherd dogs seem to be overrepresented in the literature about systemic aspergillosis (schultz et  al. 2008, van wie et  al. 2012, perry et  al. 2012, morabito et  al. 2020). the dog of the current case report was a young female mix-breed dog, linked to german shepherd dog. she did not have any other evident predisposing factors to fungal infection, like evident causes of immunosuppression or previous surgery (bieganska et  al. 2014). these data are in accordance with the previous veterinary literature, where most of the reported cases had no historical evidence of immunosuppression (schultz et  al. 2008). canine transplacental aspergillosis transmission from an infected mother appears possible (elad et  al. 2008), however no information about the littermates was available for this dog. at the time of first presentation, the dog was in good general condition and the initial clinical signs were just related to the iliac bone involvement causing lameness; only after generalisation of the disease, the dog showed nonspecific and systemic signs of generalised illness. the abnormalities detected in the initial blood work were only neutrophilia and hyperglobulinaemia, two common but also nonspecific findings in dogs with systemic aspergillosis (schultz et  al. 2008). raised creatinine and urea concentrations reflected kidney involvement, another common event in dogs with the generalised infection (schultz et al. 2008). 359veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 del magno et al. disseminated aspergillosis in a mixed breed dog fungal hyphae were observed at urine sediment examination. the main hypothesis remains the haematological spread of the infection, however an ascending form cannot be excluded. the ureteral obstruction observed in the dog of the present report was successfully managed by conservative treatment; however in case of no response, surgical treatment is advised. although the use of a nephrostomy tube to temporarily decompress the renal parenchyma can be necessary, secondary bacterial infections have been reported (starkley et  al. 1996). in humans, the endoscopic removal of the ureteral obstruction is typically performed; however, to the best of the authors’ knowledge, this treatment has never been reported in dogs (najafi et  al. 2013). the placement of a ureteral stent can also be considered, even if it could maintain the infection and it could be obstructed by the hyphal casts as already reported in humans (kuntz et  al. 2015, najafi et al. 2013). in conclusion, this report describes an uncommon presentation of disseminated aspergillosis sustained by a. terreus in a dog with an initial localisation of the mycotic lesions to the wing of the ilium. a further systemic involvement was recognised with severe renal lesions and ureteral obstruction. despite a prompt diagnosis and treatment with high doses of itraconazole, that gave a long-term clinical remission, the recurrence appeared after one year, soon after temporary discontinuation of antifungal drug. disseminated aspergillosis remains a very difficult disease to treat with often a negative prognosis in the long term. an aggressive approach, when possible, including both medical and surgical management could lead to a better control of the infection. dog here reported. the costs for last generation azoles limit their use in veterinary medicine, since prohibitive for many owners, as in this case. in humans, even in cases of disseminated infection, surgical debridement is suggested as an important part of the multimodal treatment strategy together with systemic antimycotic drug administration (koehler et  al. 2013). in our case, a wide biopsy was performed in the iliac wing; unfortunately, dissemination already occurred, and a surgical debridement was not performed. probably a more aggressive surgical treatment could have improved the penetration of itraconazole in the primary bone affected site and potentially decreased the haematogenous spread. to the best of the authors’ knowledge, the iliac crest has never been reported as site of first localisation of disseminated aspergillosis in dogs and pelvic bone involvement is rarely described in cases of mycotic infection both in dogs and humans (koehler et  al. 2013, schultz et  al. 2008). on the contrary, discospondylitis seems to be the most frequent skeletal lesion reported in dogs, followed by osteomyelitis of the long bones like humerus, tibia, femur and scapula (schultz et  al. 2008). in humans, the vertebrae are also the most affected site, but they are followed by the cranium, long bones, and joints (reischies and hoenigl 2014, koehler et al. 2013). aspergillosis of the urinary tract may occur in 3 patterns: haematogenous spread with renal parenchymal involvement, aspergillus casts of the renal pelvis leading to the formation of fungal bezoar, and ascending infection (najafi et  al. 2013). in the case here described, both a parenchymal involvement of the kidney and involvement of the renal pelvis and ureter were documented. moreover, 360 veterinaria italiana 2022, 58 (3), 353-360. doi: 10.12834/vetit.2260.15764.2 disseminated aspergillosis in a mixed breed dog del magno et al. biegańska m., dardzńiska w. & dworecka-kaszak b. 2014. fungal colonization – an additional risk factor for diseased dogs and cats? ann parasitol, 60, 139-146. bruchim y., elad d. & klainbart s. 2006. disseminated aspergillosis in two dogs in israel. mycoses, 49, 130-133. corrigan v.k., legendre a.m., wheat l.j., mullis r., johnson b. & bemis d.a. 2016. treatment of disseminated aspergillosis with posaconazole in 10 dogs. j vet intern med, 30, 167-173. elad d., lahav d. & blum s. 2008. transuterine transmission of aspergillus terreus in a case of disseminated canine aspergillosis. med mycol, 46, 175-178. koehler p., tacke d. & cornely o.a. 2013. aspergillosis of bones and joints – a review from 2002 until today. mycoses, 57, 323-335. kuntz j.a., berent a.c., weisse c.w. & bagley d.h. 2015 double pigtail ureteral stenting and renal pelvic lavage for renal-sparing treatment of obstructive pyonephrosis in dogs: 13 cases (2008-2012). j am vet med assoc, 246, 216-225. lackner m., coassin s., haun m., binder u., kronenberg f., haas h. d., jank m., maurer e., meis j.f. & lass-flörl c. 2016. geographically predominant genotypes of aspergillus terreus species complex in austria: s microsatellite typing study. clin microbiol infect, 22, 270-276. morabito s., specchi s., auriemma e., ferro s., kuhnert p. & zini e. 2020. computed tomographic and ultrasonographic findings of abdominal arterial pseudoaneurysms caused by systemic mycosis in dogs. j small anim pract, 61, 300-307. najafi n., shokohi t., basiri a., parvin m., yadegarinia d., taghavi f., hedayati m.t. & abdi r. 2013. aspergillus terreus-related ureteral obstruction in a diabetic patient. iran j kidney dis, 7, 151-155. perry j.j., rech r.r., camus m.s. & howerth e.w. 2010. pathology in practice. j am vet med assoc, 236, 173-175. raper k.b. & fennell d.i. the genus aspergillus. the williams & wilkins company, baltimore, 1965. reischies f. & hoenigl m. 2014. the role of surgical debridement in different clinical manifestations of invasive aspergillosis. mycoses, 57, 1-14. risslegger b., zoran t., lackner m., aigner m., sánchez-reus f., rezusta a., chowdhary a., taj-aldeen s.j., arendrup m.c., olivieri s., kontoyaiannis d.p., alastruey-izquierdo a., lagrou k., lo cascio g., meis j.f., buzina w., farina c., drogari-apiranthitou m., grancini a., tortorano references a.m., willinger b., hamprecht a., johnson e., klingspor l., arsic-arsenijevic v., cornely o.a., meletiadis j., prammer w., tullio v., vehreschild j.j., trovato l., lewis r.e., segal e., rath p.m., hamal p., rodriguez-iglesias m., roilides e., arikan-akdagli s., chakrabarti a., colombo a.l., fernández m.s., martin-gomez m.t., badali h., petrikkos g., klimko n., heimann s.m., houbraken j., uzun o., edlinger m., fuente s. & lass-flörl c. p. 2017. a prospective international aspergillus terreus survey: an efisg, isham and ecmm joint study. clin microbiol infect, 23, 776-e1-776-e5. seyedmousavi s., guillot j., arné p., de hoog g.s., mouton j.w., melchers w.j.g. & verweij p.e. 2015. aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease. med mycol, 53, 765-797. schultz r.m., johnson e.g., wisner e.r., brown n.a., byrne b.a. & sykes j.e. 2008. clinicopathologic and diagnostic imaging characteristics of systemic aspergillosis in 30 dogs. j vet intern med, 22, 851-859. starkley r.j. & mcloughlin m.a. 1996. treatment of renal aspergillosis in a dog using nephrostomy tubes. j vet intern med, 10, 336-338. van wie e., chen a.v., thomovsky s.a. & tucker r.l. 2013. successful long-term use of itraconazole for the treatment of aspergillus diskospondylitis in a dog. case reports vet med, 907276. http://dx.doi. org/10.1155/2013/907276. yoon y.k., kang e.h., in k.h. & kim m.j. 2010. unilateral ureteral obstruction caused by aspergillus, subgenus nidulantes in a patient on steroid therapy: a case report and review of the literature. med mycol, 48, 647-652. zoran t., sartori b., sappl l., aigner m., sánchez-reus f., rezusta a., chowdhary a., taj-aldeen s.j., arendrup m.c., olivieri s., kontoyannis d.p., alastruey-izquierdo a., lagrou k., lo cascio g., meis j.f., buzina w., farina c., drogari-apiranthithou m., grancini a., tortorano a.m., willinger b., hamprecht a., johnson e., klingspor l., arsic-arsenijevic v., cornely o.a., meletiadis j., prammer w., tullio v., vehreschild j.j., trovato l., lewis r.e., segal e., rath p.m., hamal p., rodriguez-iglesias m., roilides e., arikan-akdagli s., chakrabarti a., colombo a.l., fernández m.s., martin-gomez m.t., badali h., petrikkos g., klimko n., heimann s.m., uzun o., roudbary m., de la fuente s., houbraken j., risslegger b., sabino r., lass-flörl c. & lackner m. 2019. corrigendum: azole-resistance in aspergillus terreus and related species: an emerging problem or a rare phenomenon? front microbiol, 28, 516. 287 veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 accepted: 23.08.2021 | available on line: 31.12.2022 1faculty of veterinary medicine, ahmadu bello university p.m.b 1045 zaria, kaduna state, nigeria. 2faculty of veterinary medicine, university of ilorin, p.m.b 1515 ilorin, kwara state, nigeria. 3faculty of veterinary medicine, university of abuja, p.m.b 117 gwagwalada. abuja, nigeria. 4 woah reference laboratory for contagious bovine pleuropneumonia, istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. *corresponding author at: : faculty of veterinary medicine, ahmadu bello university p.m.b 1045 zaria, kaduna state, nigeria. e-mail: markfrancis4u@gmail.com. markus isa francis1, mashood abiola raji2, clara nna kwanashie1, jibril adamu1, lushaikyaa allam1, james agbo ameh3, godwin onyemaechi egwu2, katiuscia zilli4, flavio sacchini4 and massimo scacchia4 keywords cattle, contagious bovine pleuropneumonia, mycoplasma mycoides subspecies mycoides, pcr-rflp, nigeria. summary this study aimed to perform molecular typing of mycoplasma mycoides subsp. mycoides from slaughtered cattle in adamawa and taraba states, north-eastern nigeria. a total of four hundred and eighty (480) samples of lung tissues, nasal swabs, ear swabs and pleural fluids were collected from cattle at slaughter and processed according to standard laboratory protocols. identification and confirmation were achieved with specific pcr and pcr-rflp. an overall m.  mycoides subsp. mycoides isolation rate of 6.87% (33/480) was obtained. in adamawa state, 12 (10.91%) isolates of m. mycoides subsp. mycoides came from both, lung tissues and pleural fluids. while in taraba state, 5 (7.14%) and 4 (5.71%) isolates of m. mycoides subsp.  mycoides came from lung tissues and pleural fluids, respectively. the samples from nasal and ear swabs from the study states were negative for m. mycoides subsp. mycoides. thirty-three out of the 37 culture positive isolates were confirmed to be mycoplasma mycoides subspecies mycoides with the production of a band equivalent to 574-bp. molecular typing with restriction endonuclease vsp1 results in the two bands of 180-bp and 380-bp. in conclusion, the study has established an isolation rate of 6.87% for m. mycoides subsp.  mycoides. measures to strengthen movement control in order to minimise the spread of this dreaded disease of cattle were recommended. identification of mycoplasma mycoides subspecies mycoides from slaughtered cattle in two transboundary states of north‑eastern nigeria mycoplasma mycoides subsp.  mycoides infects lung tissues and can be transmitted through inhalation of infectious aerosols of the organism by a susceptible animal (oie 2018). severe inflammatory, exudative lesions at lung and pleural membranes characterize the disease. in calves, however, infection of m.  mycoides subsp.  mycoides results mainly in arthritis and associated lameness, but rarely in pulmonary lesions (oie 2015). other factors such as movement of trade cattle, seasonal migration, transhumance and normadism also enhance the spread of the infection (aliyu et al. 2000). the woah recommended complement fixation test (cft), competitive enzyme-linked immunosorbent assay (c-elisa) and polymerase chain reaction for the diagnosis of cbpp (oie 2018). isolation introduction mycoplasma mycoides subspecies  mycoides (m.  mycoides subsp.  mycoides) is an important bacterium pathogen of cattle causing contagious bovine pleuropneumonia (cbpp), which is one of the most severe endemic infectious disease in sub-saharan africa. the disease is causing major social and economical impact, due to the animal losses and international trade restrictions (mariner et  al. 2006, dupuy et  al. 2012, fischer et  al. 2015, parker et al. 2018). m. mycoides subsp. mycoides was the first mycoplasma to be described, as extracellular bacterium that lives in close association with their host cells (dupuy et  al. 2012). the bacterium lacks of cell wall, and has the capacity of self-replication (westberg et al. 2004, di teodoro et al. 2020). 288 veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 m. mycoides subsp. mycoides in nigeria francis et al. border. adamawa and taraba states have a land area of about 91,390 km2 with tropical wet and dry climate. the two states are among the lead producers of livestock in nigeria with an estimated cattle population of 3.5 million (francis et al. 2018b). sample collection and storage the samples used for this study were obtained from cattle slaughtered at abattoirs within the areas under study between 2016 and 2017. the samples were collected for a period of thirteen (13) months. a total of four hundred and eighty (480) samples of pneumonic suspected lung tissues (180), nasal swabs (180), ear swabs (60) and pleural fluid (60) were collected from 190 cattle heads at slaughter, when they were showing pathological lesions suspected for cbpp. two hundred and eighty four (284) and one hundred and ninety six (196) samples from cattle were collected from the abattoirs in adamawa and taraba states, respectively (table  i). all the collected samples from abattoirs in yola and jalingo were transported to the national veterinary research institute zonal offices in yola and jalingo, respectively. samples were processed in 2 ml sterile nalgene® cryo vials containing 1.5 ml of pleuropneumonia-like organism (pplo) broth (difco) supplemented (horse serum, sodium pyruvate and fresh yeast extract) and were later transported in refrigerated coleman box to the mycoplasma laboratory, national veterinary research institute (nvri) vom, plateau state and then stored at - 20 °c prior to further processing. and identification of mycoplasmas is still the gold standard even though it requires a well-equipped laboratory with expertise for these very fastidious and slow-growing organisms (mcauliffe et  al. 2005, egwu et  al. 2012). the use of pcr for the rapid and specific identification of m. mycoides subsp. mycoides has its limitations in developing countries, but detection of these species by pcr proved to be highly efficient with better results than culturing techniques (egwu et al. 2012, wade et al. 2015). the disease threatens livestock production, limits international trade and is therefore of huge economic concern in affected countries (fadiga et al. 2013). in nigeria, estimated cbpp morbidity and mortality rate of up to 50% and 25%, respectively, have been documented with annual economic losses of more than 2.2 billion nigerian naira (fadiga et al. 2013). outbreaks of the disease still occur in the northern region which harbours three-quarter of the country’s 19.5 million cattle population (tambuwal et al. 2011). several epidemiological studies have been conducted in nigeria to assess the situation of cbpp in the north-eastern region (ameh et al. 1998, aliyu et  al. 2000, francis et  al. 2018a) but there was no report on the molecular typing of the causative agent. recent information about the disease is scanty and no extensive work been done in the study area. the present study was aimed to perform molecular identification and typing of m.  mycoides subsp. mycoides from slaughtered cattle in adamawa and taraba states, north-eastern nigeria, with the aim to help in clarifying the characteristics of the strains circulating in the study area. materials and methods study area adamawa and taraba states are located in the south-eastern part of nigeria (figure 1). they lie between latitude 7°0’0” and 11°0’0”n and between longitude 9°0’0” and 13°0’0”e. the yola modern abattoir in adamawa state is located on the latitude 9°13’33.3”n and longitude 12°27’10.1”e. an average of 70-80 cattle is slaughtered daily in the abattoir, while numbers are higher during festivity period. jalingo abattoir in taraba state is located on the latitude 8°54’12.1”n and longitude 11°21’14.3”e with an average of 40-50 cattle slaughtered daily. adamawa state shares boundaries with taraba, borno, gombe states while taraba state is bounded by gombe, bauchi, plateau, nassarawa and benue states. they both share an international boundary with the cameroon republic along the southeastern figure 1. map of adamawa and taraba state, north‑eastern nigeria showing the study areas. 289veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 francis et al. m. mycoides subsp. mycoides in nigeria agarose gel, stained with ethidium bromide (van kuppeveld et  al. 1994). this pcr was able to detect mycoplasma spp. in general and it is routinely used to confirm the presence of mycoplasmas in samples. isolates that yielded positive to mycoplasma species underwent additional testing using a mycoplasma specific pcr. identification of mycoplasma mycoides subspecies using pcr this protocol is a pcr-based test for the specific identification of m.  mycoides subspecies according to bashiruddin and colleagues (bashiruddin et  al. 1994). for molecular characterization, the nucleic acid of the samples were further amplified using a set of primers: mm450 (forward) 5’-gtattttcctttctaatttg-3’ and mm451 (reverse): 5’-aaatcaaattaagtttg-3’ targeting the cap-21 genomic region of 16s rrna. an initial denaturation at 94 °c for 5 minutes was followed by 40 cycles at 94 °c for 30 seconds, 50 °c for 30 seconds, and 72 °c for 30 seconds, and then a final extension at 72 °c for 5 minutes. mycoplasma mycoides sub-clusters were used as positive controls and the expected positive pcr products of 574 bp were visualized on a 1% agarose (musa et al. 2016). the pcr products were electrophoresed on 1% agarose gel and the samples run for 30 minutes. the resultant bands were stained with ethidium bromide and visualised under ultraviolet light (bashiruddin et al. 1994). differentiation of mycoplasma mycoides subspecies mycoides using pcr‑rflp the typing method was adapted from the protocol used in woah reference laboratory for cbpp and that of bashiruddin and colleagues (bashiruddin et al. 2005), but varied with respect to vsp1 (8-12 u/µl concentration) which replaced asn1 endonuclease in this study. the pcr-rflp was performed according to the method described by musa and colleagues (musa et al. 2016). results out of the 480 samples tested, 39 (8.13%) were positive for mycoplasma species by culture. in adamawa state, 25 (8.80%) isolations were made from 12 (10.91%) lung tissues and 13 (11.82%) pleural fluid. in taraba state, 8 (11.43%) lung tissues, 5 (7.14%) pleural fluid and 1 (3.57%) ear swab yielded a characteristic ‘fried egg’ colonies, typical of mycoplasma, with an isolation rate of 7.14% (n = 14). the thirty nine (39) pure isolates of mycoplasma isolation of mycoplasmas the samples were processed according to the method described by thiaucourt and colleagues (thiaucourt et  al. 1992). the pplo broth (difco) supplemented, containing various samples were filtered by passing through 0.45 µm millipore filters (millipore merck, burlington, ma, usa) which is permeable to organisms smaller than 0.45 µm in size. the filtrate was then incubated at 37 °c in screw-capped bottles containing supplemented pplo broth (difco) for three (3) days under 5% co 2 and later cultured onto pplo agar (difco) supplemented. these were incubated at 37 °c in a 5% co 2 atmosphere with maximum humidity, and examined daily for a period of 1-2 weeks for evidence of growth, using a stereomicroscope. isolated colonies having the classical ‘fried eggs’ appearance with dense raised centres (nipples) were triple-cloned into 10 ml of pplo broth (difco), and incubated for one week. both, broth and agar media were considered negative if no comet or mycoplasma-like colonies were observed after 14 days of incubation. cloned isolates were stored at + 4 °c. these isolates were later transported refrigerated at 2-8 °c by express courier to the woah reference laboratory for cbpp at the istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ (izsam), teramo, italy. upon receipt, the isolates were stored at +  4  °c, verified and then registered under the exotic disease unit before being processed. identification and confirmation of mycoplasma mycoides subsp. mycoides isolates dna extraction deoxyribonucleic acid was extracted from 500  µl of pplo broth presenting comet colonies using maxwell® 16 tissue/cell dna purification kits (promega, wincosin, usa) with maxwell® 16  instrument (promega, wincosin, usa) according to the manufacturer’s instructions and the extracted dna was kept at - 20 °c until analyzed. identification of mycoplasma species using specific pcr the nucleic acid from each sample was tested using a pcr specific for mycoplasma species as previously described (van kuppeveld et al. 1994). the mycoplasma group-specific primer set which amplifies a 280-bp fragment, consists of the forward primer 5’-gggagcaaacaggattagataccct-3’ and the reverse primer 5’-tgcaccatctgtcactctgttaacctc-3’. the aliquots were analyzed by electrophoresis on 2% 290 veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 m. mycoides subsp. mycoides in nigeria francis et al. area were negative for m. mycoides subsp. mycoides (table i). thirty-three (33) out of the thirty-seven (37) mycoplasma isolates were positive for pcr, showing a band of 574-bp (figure 3). all the pcr positive samples were characterized as m. mycoides subsp. mycoides by pcr-rflp method (figure 4). discussion the isolation rate of 6.87% of m.  mycoides subsp.  mycoides found in this study was slightly higher than the previous investigations carried out in northern and central nigeria (ankeli et al. 2016, musa et al. 2016, tambuwal and egwu 2017). the increased rate observed in the same agro-ecological zone may be connected to the social security situation in the north-eastern nigeria, where there are fragmented veterinary services due to insurgency (aliyu species were shipped to the above mentioned woah reference laboratory for cbpp, where 37  isolates were confirmed on both pplo media and pcr specific for mycoplasma species. thus, 19 (10.56%) lung tissues, 17 (9.44%) pleural fluid and 1 (1.67%) ear swab yielded positive results, leading to an overall mycoplasma species recovery rate of 7.70% (37/480), out of which adamawa state had 25 (8.80%) and taraba states had 12 (6.12%). more isolates came from adamawa state compared to taraba state. the isolation and recovery rates of mycoplasma species are shown on table i. mycoplasma mycoides subsp. mycoides (figure 2) was the dominant species 33 (6.87%). in adamawa state, 12 (10.91%) isolates of m. mycoides subsp. mycoides came from both, lung tissues and pleural fluids. the study revealed that in taraba state 5  (7.14%) and 4  (5.71%) isolates of m.  mycoides subsp.  mycoides came from lung tissues and pleural fluid, respectively. the samples from nasal and ear swab from the study table i. isolation rates of mycoplasma species and identification of m. mycoides subsp. mycoides (mmm) from cattle in adamawa and taraba states, nigeria. source of sample sample type no. of samples collected no. of mycoplasma cultured (%) no. of mycoplasma confirmed (%) no. of mmm identified (%) adamawa state lungs 110 12 (10.91) 12 (10.91) 12 (10.91) pleural fluid 110 13 (11.82) 13 (11.82) 12 (10.91) nasal swab 32 0 (0.00) 0 (0.00) 0 (0.00) ear swab 32 0 (0.00) 0 (0.00) 0 (0.00) sub-total 284 25 (8.80) 25 (8.80) 24 (8.45) taraba state lungs 70 8 (11.43) 7 (10.00) 5 (7.14) pleural fluid 70 5 (7.14) 4 (5.71) 4 (5.71) nasal swab 28 0 (0.00) 0 (0.00) 0 (0.00) ear swab 28 1 (3.57) 1 (3.57) 0 (0.00) sub-total 196 14 (7.14) 12 (6.12) 9 (4.59) total 480 39 (8.13) 37 (7.71) 33 (6.87) figure 2. colonies of m. mycoides subspecies mycoides from lung sample on pplo agar showing characteristic ‘fried egg shape’ colonies, dense raised central area ‘nipple’ (n) and transparent periphery (t) growth on pplo agar isolated at nvri vom after 72 hours (a) and recovered from isolates shipped to izsam teramo, italy after 5 days (b) of incubation viewed under stereomicrospe (x35). a b fried egg colony with dark centre n t 291veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 francis et al. m. mycoides subsp. mycoides in nigeria fragments delineated m.  mycoides subsp.  mycoides from m.  mycoides subspecies capri. this finding corroborates the earlier reports (bashiruddin et  al. 1999, musa et  al. 2016) and it further revealed strains of m. mycoides subsp. mycoides circulating in adamawa and taraba states, nigeria. in conclusion, the present study has established the occurrence of m.  mycoides subsp.  mycoides north-eastern part of nigeria. also, a high number of m. mycoides subsp. mycoides field strains have been isolated which is to the best of our knowledge the first figure of this kind in nigeria. it is recommended to strenghten animal movements control at nigerian national and international borders, in order to minimise the spread of this dreaded and internationally recognised disease of cattle. statement of animal rights as at the time of kick-starting this study, ahmadu bello university zaria, nigeria did not have any committee mandated to authorise research works. though, the research procedure employed in this study was presented to the postgraduate board of faculty of veterinary medicine, which considered ethical and welfare standards before approval was given. acknowledgements the authors appreciate the staff of mycoplasma laboratory, national veterinary research institute vom, plateau state nigeria and the management of woah reference laboratory for cbpp at istituto zooprofilattico sperimentale dell’abruzzo e molise ‘g caporale’, campo boario teramo, italy for free bench space and technical support for this work. et  al. 2000, francis et  al. 2018a). to the best of our knowledge, this is the highest reported isolation rate of m.  mycoides subsp.  mycoides from field samples in nigeria. the high isolation rate of m.  mycoides subsp. mycoides obtained in this study may indicate that the disease is very active and endemic in the study area. the increased and unrestricted cattle movements, as well as the porous nature of borders with neighbouring countries, might have among other factors contribute to the high rate observed in this study (aliyu et al. 2000). mycoplasma mycoides subspecies  mycoides was not isolated from the nasal and ear swab samples collected, even though woah recommended these sites for sampling in live animals (karahan et  al. 2010, oie 2015). this may be due to contamination of such sites with debris, ear mites, other bacterial and mycoplasma flora, thus suppressing m. mycoides subsp.  mycoides from thriving in such anatomical sites (santos et al. 2009). compared to nasal and ear swabs, the broncho-alveolar lavage (bal) method of sampling is the most preferred method for isolation of mycoplasma in live animals, to avoid sample contamination. however, the bal sampling method was much more difficult to perform under field conditions, and animal owners are usually reluctant to allow this procedure (karahan et  al. 2010, akan et al. 2014). the results obtained in the present study are in contrast with the previous report of ankeli and colleagues (ankeli et  al. 2016), who reported isolation of m. mycoides subsp. mycoides from the ear canal of apparently healthy cattle in plateau state. molecular typing of thirty-three isolates out of the original thirty-seven was quite significant and showed that cbpp caused by m.  mycoides subsp. mycoides was active, endemic and widespread in the study area. this finding is in agreement with previous reports (bashiruddin et  al. 2005, muuka et  al. 2013, wade et  al. 2015, musa et  al. 2016). the digested restriction endonuclease 380-bp figure 3. pcr amplification of m. mycoides subspecies cap‑21 genomic region (574‑bp) on 1% agarose gel for identification using primers mm450 and mm451. lane m = molecular size marker 100-bp; lanes 1-4 = samples showing mm subspecies; pc = positive control; nc = negative control. figure 4. pcr‑rflp amplicons on 3% agarose gel for the identification of m. mycoides subspecies mycoides following digestion of pcr product with restriction endonuclease vsp1. lane m = molecular size marker 100-bp; lanes 1-5 = positive samples showing m. mycoides subsp. mycoides profile; lane pc1 = m. mycoides subsp. mycoides positive control; lane pc2 = mm subsp. capri positive control. m 1 2 3 4 pc nc m 600 500 400 574 bp m 1 2 3 4 pc1 pc2 m5 400 300 150 380 bp 230 bp 180 bp 150 bp 292 veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 m. mycoides subsp. mycoides in nigeria francis et al. aliyu m.m., obi t.u. & egwu g.o. 2000. prevalence of contagious bovine pleuropneumonia (cbpp) in northern nigeria. prev vet med, 47, 263-269. akan m., babacan o., torun e., mustak h.k. & oncel t. 2014. diagnosis of mycoplasma bovis infection in cattle by elisa and pcr. kafkas university vet j, 20, 249-252. ameh j.a., nawathe d.r. & emmans t.a. 1998. the role of agar gel diffusion test (agpt) in diagnosis and eradication of contagious bovine pleuropneumonia. bull anim hlth prod afri, 43, 187-189. ankeli p.i., fagbamila i.o., ikpa l.t., muraina i.a., luka p.d., raji m.a., kazeem h.m., odugbo m.o., umaru n.j., nwankiti o.o. & nwankpa n.d. 2016. isolation and identification of mycoplasma mycoides subspecies mycoides from the ear canal of cattle in plateau state, north-central nigeria. vet sci res rev, 2, 52-59. bashiruddin j.b., de santis p., vacciana a. & santini f.g. 1999. detection of mycoplasma mycoides subsp. mycoides sc in clinical material by rapid colorimetric pcr. mol cell prob, 13, 23-28. bashiruddin j.b., taylor t.k. & gould a.r. 1994. a pcr-based test for the specific identification of mycoplasma mycoides subspecies mycoides sc. j vet diagn investn, 6, 428-434. bashiruddin j.b., de santis p., persson a., ball h. & regalla j. 2005. detection of mycoplasma mycoides subspecies mycoides sc in bovine lung and lymph node tissues by culture, sandwich elisa and polymerase chain reaction systems. res vet sci, 78, 199-205. di teodoro g., marruchella g., di provvido a., d’angelo a.r., orsini g., di giuseppe p., sacchini f. & scacchia m. 2020. contagious bovine pleuropneumonia: a comprehensive overview. vet pathol, 57 (4), 476-489. dupuy v., manso-silvan l., barbe v., thebault p., dordet-frisoni e., citi c., poumarat f., blanchard a., breton m., sirand-pugnet p. & francois t. 2012. evolutionary history of contagious bovine pleuropneumonia using next generation sequencing of mycoplasma mycoides subsp. mycoides small colony. plos one, 7, e46821. egwu g.o., adamu m., mshelia g.d. & bukar-kolo y.m. 2012. isolates of mycoplasma mycoides subspecies mycoides  (sc) in small ruminants in sahel zone of nigeria and its implications on disease control. afri j biotech, 11, 6396-6401. fadiga m., jost c. & ihedioha j. 2013. financial costs of disease burden, morbidity and mortality from priority livestock diseases in nigeria: disease burden and cost-benefit analysis of targeted interventions. nigeria integrated animal and human health management project final report. ilri reasearch report 33, nairobi, kenya: ilri. fischer a., santana-cruz i., hegerman j., gourle h., schieck e., lambert m., nadendla s., wesonga h., miller r.a., vashee s., weber j., meens j., frey j. & jores j. 2015. high quality draft genomes of the mycoplasma mycoides subsp. mycoides challenge strains afade and b237. standards genom sci, 10, 89-98. references francis m.i., oragwa a.o., ankeli p.i., liba j.w., ejeh e.f., raji m.a., ameh j.a. & egwu g.o. 2018a. prevalence of contagious bovine pleuropneumonia based on gross lesions in cattle at slaughter in adamawa state, nigeria. sokoto j vet sci, 16, 31-37. francis m.i., kalang j.j., raji m.a. & egwu g.o. 2018b. vaccination coverage of contagious bovine pleuropneumonia in adamawa state, northeastern nigeria. nig vet j, 39, 161-167. karahan m., kalin r., atil e. & cetinkaya b. 2010. detection of mycoplasma bovis in cattle 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zambia: a case study. j vet sci med diagn, 2, 4. office international des epizooties (oie). 2018. manual for diagnostic tests and vaccines for terrestial animals, chapter 3.4.8., 1097-1118. office international des epizooties (oie). 2015. world animal health information database (wahid) [database online]. contagious bovine pleuropneumonia. paris, oie, 2015. http://www.oie.int/wahis2/ public/wahid. php/ diseaseinformation/ statuslist. accessed on 16 november 2015. parker a.m., sheehy p.a., hazelton m.s., bosward k.l. & house & j.k. 2018. a review of mycoplasma diagnostics in cattle. j vet int med, 1, 1-12. santos s.b., nascimento e.r., faccini j.l.h., barreto m.l. & pereira v.l. 2009. potentially pathogenic mycoplasmas in the external ear canal of clinically normal cattle in southeast brazil: first report. braz j microbiol, 40, 455-457. tambuwal f.m., egwu g.o., shittu a., sharubutu g,h., umaru m.a., umar h.u., mshelia p.c. & garba s. 2011. vaccination coverage and prevalence of contagious bovine pleuropnuemonia (1999-2008) in two transboundary states of north-western nigeria. nig vet j, 32, 169-173. tambuwal f.m. & egwu g.o. 2017. occurrence of mycoplasma mycoides subspecies mycoides small 293veterinaria italiana 2022, 58 (3), 287-293. doi: 10.12834/vetit.2331.16051.3 francis et al. m. mycoides subsp. mycoides in nigeria wade a., yaya a., el-yuguda a.d., unger h., nafarnda w.d., ikechukwu e.s. & egwu g.o. 2015. the prevalence of contagious bovine pleuropneumonia in cameroun: a case study garoua central abattoir, cameroun. j vet med res, 2, 1029-1034. westberg j., persson a.a., holmberg a., goesmann a., lundeberg j., johasson k.e., petterson b. & uhlem m. 2004. the genome of mycoplasma mycoides subsp. mycoides sc type strain pg1, the causative agent of contagious bovine pleuropneumonia (cbpp). genom res, 14, 221-227. colony, mycoplasma bovis from respiratory tracts of cattle presented for slaughter in north-western nigeria. direct res j vet med anim sci, 2, 115-121. thiaucourt e., guerin c., mady v. & lefevre p.c. 1992. diagnosis of caprine contagious pleuropneumonia: recent improvements. rev sci techoff intl epiz, 11, 859-865. van kuppeveld f.j.m., johannson k.e., galama j.m.d., kissing j., bolske g., van der logt j.t.m. & melchers w.j.g. 1994. detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific pcr. appl environ microbiol, 60, 149-152. 335 veterinaria italiana 2021, 57 (4), 335-340. doi: 10.12834/vetit.2172.13765.1 accepted: 23.09.2020 | available on line: 31.12.2021 §these authors contributed equally to this work. 1university of pisa, department of veterinary sciences, pisa, italy. 2department of surgery, anesthesiology and radiology, faculty of veterinary medicine, mansoura university, 35516 mansoura, egypt. 3department of surgery, anesthesiology and radiology, faculty of veterinary medicine, kafrelsheikh university, kafrelsheikh, el geish street, 33516, egypt. 4animal reproduction research institute, agriculture research centre, 12556 haram, giza, egypt. *corresponding author at: university of pisa, department of veterinary sciences, pisa, italy. e-mail: francesca.bonelli@unipi.it. francesca bonelli1*§, awad rizk2§, irene nocera1, luca turini1, alaa ghazy3, ahmed elmesiry4 and ayman atiba3 keywords forelimb, claw length, claw trimming, sole, heel bulb, buffalo cow. summary the aim of this study was to compare forelimb claw dimensions in buffalo cows, and to establish a reference values for functional claw trimming. medial and lateral healthy front feet claws were evaluated from 20 young (yb) and 20 mature (mb) egyptian buffalo cows (bubalus bubalis). the dorsal wall length (tl), dorsal wall angle (ta), heel length (hbl), height (hbh) and width (hbw), sole (sl) and claw length (cl) and sole width (sw) were measured. differences between yb and mb were evaluated using anova test, while those between the lateral and medial claws with a paired t test. before and after trimming, the hbl, hbw, hbh, sl and cl were significantly higher for both claws in mb, while the ta was significantly lower. before trimming the lateral hbw and hbh were significantly higher than the medial, while the sl and cl of the medial claw were significantly higher than the lateral claw. after trimming, the lateral claw hbl was significantly higher in mb. the sw of the lateral claw was significantly higher than the medial claw in both yb and mb, and it did not change after trimming. these results can be considered as a guidance during front feet trimming in buffaloes. standard measurement of the normal forelimb claw dimensions in egyptian buffalo cows (bubalus bubalis) before and after functional trimming: a post-mortem study under intensive management conditions (khan and coskun 2018). intensive farming of buffaloes can increase their production, but it can also predispose to those herd problems typical of dairy cows under intensive management (cramer et al. 2008, guccione et al. 2014, guccione et al. 2016). fertility problems, mastitis and lameness due to foot disorders represent the most important health related cause of economic loss in dairy industry. financial losses are mostly due to decreasing in milk yield, reducing of reproductive performance, and increasing of culling rate (guccione et  al. 2016, kshandakar et  al. 2017). beside the economic aspects, lameness is usually associated with long duration pain and discomfort, thus it represents also introduction the domestic buffalo (bubalus bubalis) represents an important source of meat, skin, traction power and milk, especially in tropical and subtropical countries. domestic buffalo are represented by river buffalo, mostly used for milking production in india, pakistan, egypt, and europe, and swamp buffalo, especially found in eastern asia and used as a draft animal and for meat purposes (de rosa et al. 2009). in many european countries, especially in italy, buffaloes’ milk and milk products are particularly well-known and appreciated by the stakeholders (borghese and moioli 2016). dairy cows and buffaloes are usually bred and managed with similar farming system. this similarity led to an increasing of the buffalo population bred 336 veterinaria italiana 2021, 57 (4), 335-340. doi: 10.12834/vetit.2172.13765.1 claw dimensions in egyptian buffalo cows related to trimming bonelli et al. only the feet without any claw affections were included in this study. ready after collection, the feet were divided based on the previous mentioned aging categories (40 feet obtained from yb and 40 feet from mb), and they were thoroughly cleaned and the hair around the coronet was clipped (figure 1, a-d). the measuring techniques were performed according to previous studies on cattle (nuss and paulus 2006, nuss et al. 2011). all the measurements were carried out by a single expert operator (ag) using a goniometer and calipers (future electronics, egypt) with an accuracy of 0.05 mm. the mean value of three consecutive measurements for each variable was taken. in order to standardize the sole thickness between all claws, two drilling of 10 mm of diameters were performed through the sole at the level of the corium (figure 2, a-b). the thickness of the sole was measured before trimming. the first step of the applied trimming method was to reduce the claw length. then, the thickest claw was trimmed and a symmetry between the medial and lateral claws was established. at the level of the axial wall, the trimmer made a dish in order to minimize the local pressure at the solar region. trimming of the solar aspect was made by proceeding from the heel to the toe, and the cut was made in a sweeping action slightly across the sole in an axial-to-abaxial direction (toussaint raven 2002). ready after trimming, the sole was thinned using a grinder until it reached a 5 mm thickness at the apical end of the hole near the tip and 8 mm thickness at the palmar end of the hole near the heel to create a flat sole surface. the toe was measured from the border between the skin and the coronet to the distal end of the dorsal wall a major concern for dairy animals’ welfare (fjeldaas et al. 2007, de rosa et al. 2009). claw disorders are considered the main important cause of lameness in dairy animals (perez-cabal and charfeddine 2016). thus, corrective foot trimming is important as a preventive and therapeutic procedures and needs to be carried out regularly (greenough et  al. 1997, guccione et  al. 2016). in order to achieve a good trimming procedure and to maintain a good health of the whole buffaloes’ herd, hoof measurements need to be established, as it has been suggested for cows (greenough et  al. 1997, nuss and paulus 2006, nuss et al. 2011). the aim of the present study was to compare measurements of the forelimb claw dimensions in young and mature buffalo cows, and to establish a reference values for functional claw trimming. materials and methods all the procedures in this study were conducted at the department of surgery, anaesthesiology and radiology, faculty of veterinary medicine, kafrelsheikh university, egypt. this study was performed on a total of 80 front claws obtained at the slaughterhouse from 40 clinically healthy egyptian water buffaloes. twenty out of 40 were young buffalo cows (yb), while 20 out of 40 were mature animals (mb). the median age of the yb was 27 months, ranging between 24 and 36 months, while the median age of mb was 90 months, ranging between 60 and 120 months. the median weights for the buffaloes’ carcass were 400 kg, ranging between 350 and 450 kg for yb, and 530 kg, ranging between 450 and 600 kg for mb. figure 1. all the feet collected from the slaughterhouse from 20 young buffaloes’ cows and 20 mature buffaloes’ cows were thoroughly cleaned (a, b) and the hair around the coronet was clipped (c, d). a b c d 337veterinaria italiana 2021, 57 (4), 335-340. doi: 10.12834/vetit.2172.13765.1 bonelli et al. claw dimensions in egyptian buffalo cows related to trimming and parallel to the digital axis (figure 3a) in order to evaluate the dorsal wall length, or the toe length (tl). before the evaluation of the toe angle wall (ta), a dotted line was drawn 1 cm from the interdigital space on the dorsal wall of the horn capsule, then the ta was measured between this line and the sole (figure  3, b-c). the heel bulb length (hbl) was measured between the caudal ends of the sole to the highest area of the heel bulb. heel bulb height (hbh) was measured along a line perpendicular to an imaginary caudal extension of the sole to the highest point of the heel bulb. heel bulb width (hbw) was measured from the axial to the abaxial borders between the horn and the haired skin (figure 4,a-b). the diagonal sole length (sl) was measured from the tip of the claw to the palmar-most point of the sole (figure 5a). sole width (sw) was measured following a line that intersected sole length perpendicularly and ran from the axial to the abaxial border of the claw, 25% of the sole length away from the tip of the sole (figure 5a). claw length (cl) was measured along the line of the diagonal sole length from the tip of the claw to the heel bulb (figure 5, b-c). statistical analysis the software used for statistical calculation was spss version 20 (spss for windows, version 20.0; spss inc., chicago, il, usa). the explorative data analysis and normal distribution with the kolmogorov-smirnov test were used for the evaluation of the distribution of data. differences between yb and mb were evaluated using analysis of variance (anova) and bonferroni-corrected pair-wise comparisons. the differences between the lateral and medial claws were evaluated using a paired t test. differences were considered statistically significant at p < 0.05. figure 2. the sole was drilled at the level of the corium with 10 mm of diameters holes in order to standardize the sole thickness between all claws (a, b). ba figure 3. measurement of the toe length was evaluated starting from the border between the skin and the coronet to the distal end of the dorsal wall and parallel to the digital axis (a). measurement of the toe angle wall was evaluated between the dotted line on the dorsal wall of the horn capsule and the sole (b, c). the dotted line was marked with a dot 1 cm from the interdigital space. ba c figure 4. measurement of the heel bulb length was evaluated between the caudal ends of the sole to the highest area of the heel bulb (a). heel bulb height (hbh) was measured along a line perpendicular to an imaginary caudal extension of the sole to the highest point of the heel bulb (a). heel bulb width was measured from the axial to the abaxial borders between the horn and the haired skin (b). ba figure 5. measurement of the diagonal sole length was made from the tip of the claw to the palmar-most point of the sole (a). measurement of the sole width was done following a line that intersected the sole length perpendicularly and ran from the axial to the abaxial border of the claw, 25% of the sole length away from the tip of the sole (a). claw length (cl) was measured along the line of the diagonal sole length from the tip of the claw to the heel bulb (b, c). b a c 338 veterinaria italiana 2021, 57 (4), 335-340. doi: 10.12834/vetit.2172.13765.1 claw dimensions in egyptian buffalo cows related to trimming bonelli et al. before trimming, the lateral ta were significantly higher than the medial one in both yb and mb, while after trimming, they did not differ. the ta of both the medial and lateral claw was significantly increased after trimming in both yb and mb. the medial and lateral ta were significantly lower in mb compared to yb before and after trimming. the lateral hbl were not significantly higher than the medial heel bulbs in both yb and mb before and after trimming, while it was significantly higher after trimming in mb. the hbl was significantly higher in mb compared to yb before and after trimming. before trimming, the lateral hbw was significantly wider than the medial claw in both yb and mb. the hbw of both medial and lateral claw was not significantly changed after trimming in yb and mb. before trimming, the hbh of the lateral claw was higher than the hbh of the medial claw in mb. the medial and lateral hbw and hbh were significantly higher in mb compared to yb before and after trimming. before trimming, the sl and cl of the medial claw were significantly higher than the sl and cl of the lateral claw in both yb and mb. the sl and cl of both medial and lateral claw were significantly decreased after trimming. the sl and the cl of both medial and lateral claw were higher in mb compared to yb before and after trimming. before and after trimming, the sw of the lateral claw was significantly higher than the medial claw in both yb and mb. the sw did not significantly change after trimming. discussion buffaloes and dairy cows seem to be predisposed to similar herd problems, probably because the breeding systems and most of breeding technologies results all the feet were examined before collection for healthiness; thus, no claws were excluded from the study and measurements took place in a total of 80  forelimb claws. all the measurements of the claws were easy to perform. data showed a normal distribution; thus, the results were expressed as mean and standard deviation. data concerning the tl and the ta, the heal measurements and the sole measurement of the forelimb claw of yb and mb before and after trimming were reported in table i, ii and iii, respectively. before trimming, the mean tl of the medial claws were significantly longer than the tl of the lateral claw in both yb and mb, while after trimming the medial and lateral tl did not differ. both the medial and lateral tl were significantly reduced after trimming compared to the measures before trimming. also, the medial and lateral tl were significantly higher in mb compared to yb before and after trimming. table i. toe measurements of the forelimb claw of young buffalo cow (yb) and old buffalo cow (mb) before and after trimming. the sole thickness has been standardized. parameter before trimming after trimming bonferr test medial lateral medial lateral toe length yb 113.7 ± 5.6c 101.2 ± 4d 89.95 ± 1.4fg 88.7 ± 1.3g 5.1 mb 144.9 ± 8.7a 124.8 ± 8.7b 95.7 ± 1e 93.8 ± 1.2ef toe angle yb 38.9 ± 1.2d 40.7 ± 1.3c 43.1 ± 0.9ab 44.2 ± 1a 1.16 mb 34.4 ± 1.2f 35.6 ± 1.1e 41.1 ± 1.4c 42.2 ± 1.1bc yb = young buffalo cow; mb = old buffalo cow; within rows different superscripts denote a significant difference (a ≠ b ≠ c ≠ d ≠ e ≠ f ≠ g: p < 0.05). table ii. heal measurements of forelimb claw of young buffalo cow (yb) and old buffalo cow (mb) before and after trimming. the sole thickness has been standardized. parameter before trimming after trimming bonferr test medial lateral medial lateral heel bulb length yb 46.1 ± 4.3c 49.2 ± 4bc 43.1 ± 4.1cd 46.1 ± 4.1c 3.9 mb 51.9 ± 3.8ab 55.3 ± 3.8a 46.3 ± 3.5c 50.5 ± 3.4b heel bulb height yb 39.6 ± 4.7cd 43.4 ± 4.7bc 36.4 ± 4.5d 39 ± 4.5d 4.1 mb 47.3 ± 3.5b 51.4 ± 3a 44.5 ± 4.3b 46 ± 2.7b heel bulb width yb 44.7 ± 3.9e 49.9 ± 3.8cd 47.7 ± 2.2de 50.1 ± 3.8cd 3.6 mb 51 ± 4.2bcd 54.8 ± 3.6a 51.8 ± 2.9abc 54.5 ± 4.2ab yb = young buffalo cow; mb = old buffalo cow; within rows different superscripts denote a significant difference (a ≠ b ≠ c ≠ d ≠ e: p < 0.05). table iii. sole measurements sole length, sole width and claw length measurement of forelimb claw of young buffalo cow and old buffalo cow before and after trimming. the sole thickness has been standardized. parameter before trimming after trimming bonferr test medial lateral medial lateral sole length yb 116.8 ± 5d 109.2 ± 4.9e 127.5 ± 3.9bc 122.6 ± 4.2cd 3.9 mb 118.1 ± 10.5d 112.5 ± 10.3e 135.2 ± 11a 130.3 ± 11.2ab sole width yb 45.1 ± 1.0c 47.6 ± 3.5abc 45.3 ± 1.5bc 47.8 ± 3.1ab 4.1 mb 47.4 ± 2.6abc 49.8 ± 3.2a 47.4 ± 2.6abc 48.6 ± 4.3a claw length yb 166.6 ± 2.6a 154.2 ± 2.6c 149.6 ± 0.8de 146.4 ± 1.2e 3.6 mb 170.5 ± 5.2a 160.4 ± 8.1b 155.2 ± 3.5c 152.8 ± 3.6cd yb = young buffalo cow; mb = old buffalo cow; within rows different superscripts denote a significant difference (a ≠ b ≠ c ≠ d ≠ e: p < 0.05). 339veterinaria italiana 2021, 57 (4), 335-340. doi: 10.12834/vetit.2172.13765.1 bonelli et al. claw dimensions in egyptian buffalo cows related to trimming et  al. 2016) who stated that the normal length of the dorsal wall (the distance from coronet to tip of claw) in buffalo after claw trimming is usually considered 90 mm. the ta of the horn capsule was similar to a study about cattle (fessl, 1974), but lower compared to other (nuss et  al. 2011). correct claw trimming resulted in a higher ta which could help the claws stability and to increase the height of the heel reducing some diseases conditions such as the digital dermatitis and the interdigital necrobacillosis (burgi and cook, 2008). in the front feet, the lateral claw was wider than that of the inner claw, and the bulb of the heel was also greater than in the medial claw. however, the front limb lateral claw is the first to hit the ground and the most stressed one, thus a large heel bulb might afford better cushioning (schmid et al. 2009). despite a smaller size and a lighter body weight, buffaloes might have larger toes compared to dairy cattle, due to an adaptation to their typical environmental conditions. larger feet may allow toes to spread apart and give them better footing in mud and water pedding area. before functional trimming, the lateral digit of the front limbs in buffalo cows was shorter than the medial one. moreover, the sole thickness of the lateral claws was larger than the medial claw. thus, the functional trimming should start with paring the sole of the lateral claw in order to achieve an appropriate sole thickness; then the paring of the medial claw sole at the level of the lateral claw need to be performed. these results agreed with those of nuss and paulus (nuss and paulus 2006) and muggli and colleagues (muggli et al. 2011) in cattle. after trimming the sole length was increased, this may be the consequence of cutting the horny part of heel bulb which lead to more exposure of the solar aspect. the trimming of the lateral heel bulb is challenging during a functional claw trimming because cutting the lateral heel bulb at the same level of the medial heel bulb might lead to excessively short heel bulbs, too short dorsal wall, and substantially thinner soles in the lateral claws. consequently, the animal can be predisposed to digital dermatitis, excessive sole thinning and heel horn erosion of the lateral claw. an over trimmed toe may lead to a thin sole, disruption of bearing weight, corium compression, sole hemorrhage and other claw horn lesions (tsuka et al. 2014, mahendran and bell 2015). in conclusion, the minimum recommended claw length for buffalo cow in functional trimming should be at least 90 mm in yb and 95 mm in mb, measured from the proximal limit of wall horn. these fixed length for buffalo cow's claw can be useful for standardize the functional trimming in this species. are the same (greenough et al. 1997). however, only reproductive disorders (toussaint raven 2002), and mastitis (nuss and paulus 2006) have been recognized as cause of important economic loss in buffalo, so far. foot diseases in bovines cause different degrees of lameness, which result in significant production losses (radišić et  al. 2012). corrective trimming leads to maintain the health of the herd if it is performed on a regular base (radišić et  al. 2012). hoof measurements in cows and the relationship between them have been well established (nuss and paulus 2006, radišić et al. 2012). on the other hand, literature about foot diseases and their potential role in loosing welfare and economic profit in buffaloes is poor and incomplete (giuccione et  al. 2016). thus, the present study aims to evaluate standard measures of the claws in buffaloes for corrective trimming. functional trimming in dairy cows aims to assure that both claws bear the same amount of weight (toussaint raven 2002). the dorsal wall of the hind medial claw in a normal holstein friesian was 75 mm that became 80 mm in total considering the thickness of the sole. the sole thickness appears to be the more critical measure than the length of the dorsal wall (toussaint raven 2002). for this reason, in the present study the standardized thickness of the sole horn was established before performing the measurements. the claw measurements in cows were established at a 5-8 mm sole thickness (nuss and paulus 2006). similarly, in present study the claw measurements in buffalo were determined at a defined sole thickness before and after functional claw trimming. in this study, the mean average of tl between the lateral and the medial claws at the defined sole thickness showed no significant differences. in the yb the mean average of tl of the lateral and medial claws were 88.7 and 89.9 mm, respectively while in the mb the mean were 93.8 and 95.7 mm, respectively. these findings are slightly different from other studies in which the cut length of the sole during trimming was 79 mm (tsuka et al. 2014), 80 mm (nuss and paulus 2006), and 90 mm (archer et  al. 2015). the tl has been used as guideline for the front feet trimming. in cattle, toussaint raven (toussaint raven 2002) established a 75 mm for the dorsal wall length plus 5 mm for the sole thickness. moreover, the noticeable difference in measurement between cattle and buffalo make the cattle measurements not acceptable in buffalo. in this study, the minimum recommended cut length for trimming the front feet in buffalo cows, starting from the proximal limit of wall horn, was assumed to be 90 mm in yb and 95 mm in mb. our results are similar to guccione and colleagues (guccione 340 veterinaria italiana 2021, 57 (4), 335-340. doi: 10.12834/vetit.2172.13765.1 claw dimensions in egyptian buffalo cows related to trimming bonelli et al. archer s.c., newsome r., dibble h., sturrock c.j., chagunda m.g.g., mason c.s. & huxley j.n. 2015. claw length recommendations for dairy cow foot trimming. vet rec, 177, 222. borghese a. & moioli b. 2011. buffalo: mediterranean region. in encyclopedia of dairy sciences, 2nd ed. (j.w. fuquay, p.f. fox & p.l.h. mcsweeney, eds). academic press, london, 780-784. burgi k. & cook n. 2008. three adaptations to the functional trimming method. proc. 15th international symposium & the 7th conference on lameness in ruminants, koupio, 9-13 june 2008. savonia university of applied sciences, 196. cramer g., lissemore k.d., guard c.l., leslie k.e. & kelton d.f. 2008. herd-and cow-level prevalence of foot lesions in ontario dairy cattle. j dairy sci, 91 (10), 3888-3895. de rosa g., grasso f., pacelli c., napolitano f. & winckler c. 2009. the welfare of dairy buffalo. ital j anim sci, 8 (1), 103-116. fessl l. 1974. changes in the form of interdigital space in cattle during locomotion. a contribution to the analysis of movement in cattle. zentralbl veterinarmed a, 21 (7), 592-602. fjeldaas t., nafstad o., fredriksen b., ringdal g. & sogstad a.m. 2007. claw and limb disorders in 12 norwegian beef-cow herds. acta vet scand, 49 (24), 1-11. greenough p.r., weaver a.d., broom d.m., esslemont r.j. & galindo f.a. 1997. basic concepts of bovine lameness. in lameness in cattle (p.r. greenough & a.d. weaver, eds). wb saunders, philadelphia, 3-13. guccione j., cosandey a., pesce a., di loria a., pascale m., piantedosi d., steiner a., graber h.u. & ciaramella p. 2014. clinical outcomes and molecular genotyping of staphylococcus aureus isolated from milk samples of dairy primiparous mediterranean buffaloes (bubalus bubalis). j dairy sci, 97, 7606-7613. guccione j., carcasole c., alsaaod m., d’andrea l., di loria a., de rosa a., ciaramella p. & steiner a. 2016. assessment of foot health and animal welfare: clinical findings in 229 dairy mediterranean buffaloes (bubalus bubalis) affected by foot disorders. bmc, 12 (1), 107. references khan a.a. & coskun m. 2018. water buffalo production in turkey part 1: global trend and geographical distribution. livestock, 23 (1), 32-38. kshandakar s., verma m.r., singh y.p., sharma v.b. & kumar s. 2017. effect of lameness on lactation curves of murrah buffaloes. int j agr stat sci, 13 (2), 693-703. mahendran s. & bell n. 2015. lameness in cattle 2. managing claw health throw appropriate trimming techniques. in pract, 37 (5), 274. muggli e., sauter-louis c., braun u. & nuss k. 2011. length asymmetry of the bovine digits. vet j, 188 (3), 295-300. nuss k. & paulus, n. 2006. measurements of claw dimensions in cows before and after functional trimming: a post-mortem study. vet j, 172 (2), 284-292. nuss k., sauter-louis c. & sigmund b. 2011. measurements of forelimb claw dimensions in cows using a standardised sole thickness: a post-mortem study. vet j, 190 (1), 84-9. perez-cabal m.a. & charfeddine n. 2016. short communication: association of foot and leg conformation and body weight with claw disorders in spanish holstein cows. j dairy sci, 99 (11), 9104-9108. radišić b., matičić d., vnuk d., lipar m., balić i.m., ditko b., smolec o., orak a., capak h. & kos j. 2012. measurements of healthy and pathologically altered hooves, their interrelation and correlation with body mass in simmental breeding bulls. vet arh, 82 (6), 531-544. schmid t., weishaupt m.a., meyer s.w., waldern n., von peinen k. & nuss k. 2009. high-speed cinematographic evaluation of claw-ground contact pattern of lactating cows. vet j, 181 (2), 151-157. toussaint raven e. 2002. cattle footcare and claw trimming (e. toussaint raven ed). farming press, ipswich. tsuka t., murahata y., azuma k., osaki t., ito n., okamoto y. & imagawa t. 2014. quantitative evaluation of the relationship between dorsal wall length, sole thickness, and rotation of the distal phalanx in the bovine claw using computed tomography. j dairy sci, 97 (10), 6271-6285. 307 veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 accepted: 20.05.2021 | available on line: 31.12.2022 1department of veterinary clinic, school of veterinary medicine and animal science, univ estadual paulista (unesp), botucatu, são paulo, brazil. 2departament of veterinary surgery and animal reproduction, school of veterinary medicine and animal science, univ estadual paulista (unesp), botucatu, são paulo, brazil. 3departament of physics and biophysics, institute of bioscience‑ ibb, univ estadual paulista (unesp), botucatu, são paulo, brazil. 4school of veterinary medicine and animal science, univ estadual paulista (unesp), botucatu, são paulo, brazil. *corresponding author at: department of veterinary clinic, school of veterinary medicine and animal science, univ estadual paulista (unesp), botucatu, são paulo, brazil. e‑mail: ful.bueno@gmail.com. fúlvia bueno de souza1*, natália volpi gonçalves1, shayra peruch bonatelli2, alexandra frey belotta2, silvano salgueiro geraldes1, maria jaqueline mamprim2, priscylla tatiana chalfun guimaraes-okamoto1, maria lúcia gomes lourenço1, paulo roberto rodrigues ramos3, sheila canevese rahal2 and alessandra melchert4 keywords body condition score, domestic cats, doppler, kidney, resistive index. summary this study aimed to compare renal function between obese and normal-weight healthy cats, using intrarenal resistive index (ri), serum symmetric dimethylarginine (sdma), and serum creatinine, and to identify the variables that might influence intrarenal ri. thirty crossbred client-owned cats met the inclusion criteria and were allocated into two groups: control and obese. body weight, body mass index (bmi), body condition score (bcs), sap, serum sdma, urea, and creatinine were evaluated. b-mode and doppler ultrasound of the kidneys were done. ri evaluation was in the interlobar artery. sdma and intrarenal ri were compared between groups, also considering the gender of the cats. a correlation analysis between intrarenal ri with the other parameters was performed. sdma was higher in the obese group. intrarenal ri was higher in females than males in the obese group. obese females presented higher ri and sdma than control females. a positive correlation was observed between ri, age, body weight, and bmi. six obese cats (40%) showed increased ri. the increase in body weight, bcs, and bmi resulted in a simultaneous increase in ri and sdma. the ri may assist in monitoring renal function, and may be associated with preclinical kidney changes in obese cats. renal resistive index in obese and non‑obese cats a factor for chronic kidney disease (ckd) (salama 2011). on the other hand, kidney diseases are important causes of morbidity and mortality in cats (rivers et al. 1996), but the association with obesity need to be investigated (pérez-lópez et al. 2020). renal biopsy classifies the etiology of the kidney disease and severity of kidney injury, but the procedure is invasive, requires anesthesia, and has some potential complications (vaden et  al. 2005). therefore, non-invasive methods that aid in the diagnosis and prognosis of kidney injuries are highly beneficial (rivers et al. 1996). b-mode and doppler ultrasound is an important non-invasive tool for assessing the urinary system in cats since the reduction in renal perfusion may be the first clinical finding pointing towards dysfunction introduction obesity represents an important health problem for people worldwide (mayer et  al. 2009). feline obesity is considered one of the most important chronic diseases, related to several comorbidities and reduced lifespans (larsen and villaverde 2016). excess body fat has been associated with kidney diseases in mice (deji et  al. 2009), as well as heart diseases, high arterial pressure and dyslipidemia in cats (rowe et al. 2015). obese people are susceptible to renal alterations (ninomiya et  al. 2006) due to comorbidities, such as diabetes mellitus and high blood pressure, risk factors for renal diseases (salama 2011). in humans, obesity induces complex metabolic abnormalities that may affect the kidneys (kovesdy et  al. 2017) and, consequently, represents 308 veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 renal resistive index in obese cats bueno de souza et al. evaluation methods all cats were weighed on the same digital weighing scale (kg). the body mass index (bmi) was calculated with the following equation (hawthorne and butterwick 2000): [(rib cage/0.7062)-lim/0.9156]-lim where: lim (leg index measurement)  =  length from top of the patella to end of calcaneus; rib cage  =  circumference of the rib cage at the 9th rib level (cm). the body fat content was considered ideal between 15 and 30%, overweight between 30 and 42%, and obesity above 42%. the systolic arterial pressure (sap) was measured by doppler ultrasound. the cats were gently restrained in a comfortable position in lateral recumbency. a cuff with adequate size (approximately 30-40% of the cuff site circumference) was positioned in the middle third of the antebrachium (brown et  al. 2007). five measurements were taken for each cat and the average values were recorded. outliers were excluded from the final calculation. for kidney function tests, blood samples were collected via jugular venipuncture in a silicone-coated tube containing no anticoagulant, and centrifuged immediately after removal. then, the serum samples were frozen at -  20  °c for later analysis. serum creatinine and urea were determined by enzymatic colorimetric method and kinetic method, respectively, using a semi-automatic analyzer (cobas mira plus, roche diagnostic systems, rotkreuz, switzerland). sdma concentrations were determined using liquid chromatography-mass spectroscopy (idexx laboratories) as previously described (hall et al. 2014). b-mode and doppler ultrasound of the kidneys were done with the cats fasted for 8 hours to minimize imaging artifacts. the non-sedated animals were placed in the right or left lateral recumbency for scanning the left and right kidneys, respectively. after renal echotexture, architecture, shape and size, and vessels have been assessed, the ri was measured using color doppler at the interlobar artery. doppler measurements were repeated three times (figure 1). intrarenal ri was calculated by the built-in software as follows: (peak systolic velocity − end-diastolic velocity)/peak systolic velocity (tipisca et  al. 2016). all ultrasonography examinations were performed by a doppler ultrasonography machine (mylab alpha, esaote healthcare do brasil, são paulo/sp, brazil) equipped with a 3-13 mhz linear transducer, and 5-8 mhz convex transducer. ultrasonography was carried out by an experienced evaluator. (carvalho and chamas 2011). the kidneys are highly vascularized organs, and several nephropathies have an important vascular component (novellas et  al. 2010). since the blood supply plays a role in renal function, the use of the doppler-derived intrarenal resistive index (ri) may be suitable in the diagnosis (nelson and pretorius 1988, tipisca et  al. 2016), treatment, and prognosis of kidney diseases (park et al. 2008). however, ri may be influenced by age, patency of the urinary tract, and the circulatory status of the cat (park et al. 2008). additionally, symmetric dimethylarginine (sdma) is a kidney biomarker and permits earlier diagnosis of kidney disease (reldford et  al. 2016). in cats, the renal function has been correlated with serum concentration of sdma, which compared to serum creatinine was considered more reliable and allowed earlier detection of ckd (hall et al. 2014). therefore, this study aimed to compare renal function between obese cats and normal-weight healthy cats by using intrarenal ri, serum sdma, and serum creatinine, and to identify the variables (systolic arterial pressure, body weight, body condition score, body mass index, serum sdma, and serum creatinine) that might influence intrarenal ri. materials and methods this study was approved by the institutional ethics committee on animal use (n. 0176/2016-ceua). permission for the participation of cats in the study was obtained from their owners by signing an informed consent form. animal selection thirty crossbred client-owned cats were evaluated and allocated into two groups based on a 9-point body condition score (bcs) (lund et  al. 2005): control group, cats with a bcs of 5; obese group, cats with a bcs of 8 or 9. in the 9-point bcs scale, cats with a bcs of 6 or 7 are overweight, and cats with a bcs of 8 or 9 are obese. the cats were selected aleatorily during a routine of a veterinary medical teaching hospital, over years. the inclusion criteria for the control group were healthy cats through complete physical examination, cbc, biochemical profile, and urinalysis. inclusion criteria for the obese group were obese cats with no other abnormalities on complete physical examination, or history of medication administration for at least two months. the exclusion criteria in both groups included a history of chronic diseases, previous kidney or heart diseases, changes in heart rhythm observed on the electrocardiogram, and proteinuria. 309veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 bueno de souza et al. renal resistive index in obese cats of 4.5  ±  2.3 years. the obese group (n  =  15) was composed of seven spayed females (46.7%) and eight neutered males (53.3%), with an average age of 6.5 ± 2.9 years. body weight, bcs, bmi, age, and sap were statistically higher in the obese group (table i) compared to the control group. based on the age of obese cats (adult: two to six years old; and senior: > seven years), there were no differences between adult (n = 8) and senior (n = 7) cats (p = 0.83), which had intrarenal ri values of 0.64 ± 0.09 and 0.63 ± 0.08, respectively. high statistical analysis to compare the intrarenal ri between groups, both right and left kidney values were considered, totalizing 30 ri values per group. both kidney values were also used to compare ri values between males and females within each group and between groups. the control group included 18 ri for nine males and 12 ri for six females. the obese group included 16 ri for eight males and 14 ri for seven females. the data were analyzed in commercially-available software (prism 5; graphpad software). the procmeans procedure of the sas system (sas system version  9.12, sas institute) was used to represent the box plot figures (figures 2a and 2b). the scatter plot (figure 3) was created using microsoft excel®. the normal distribution of variables was tested using the kolmogorov-smirnov test. non-paired student’s t-test for parametric data or mann-whitney test for non-parametric data were used to compare the data (age, ri, sdma, sap, body weight, bmi, bcs, serum urea and creatinine) between groups, as well as assess the influence of gender in the obese and control groups, and to compare intrarenal ri in adult (two to six years old) and more aged cats (seven to 12 years). fisher’s exact test was performed to assess increases in the intrarenal ri (right and left kidneys) when comparing the obese and control groups. pearson’s test was performed to establish a correlation between intrarenal ri with body weight, bmi, age, sdma, creatinine, and sap. the spearman’s test evaluated a correlation between ri and bcs. statistical significance was defined as p < 0.05. results a total of 30 crossbred cats met the inclusion criteria. the control group (n  =  15) was composed of six spayed females (40%) and nine males (60%), six neutered and three intact, with an average age figure 1. ultrasonographic representation of color doppler of renal vasculature (asterisk) and renal artery wave spectra to calculate resistivity index in the cat. the arrow indicates the systolic wave peak. 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 r i a ri control ri obese 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 r i b f control m control f obese m obese figure 2. a. box plot of resistive index distribution in control and obese cats. b. box plot of resistive index distribution in control and obese male (m) and female (f) cats. 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 10 bmi r = 0.42, p = 0.027 r i 20 30 40 50 60 70 figure 3. scatter plot and trend line with bmi versus ri, demonstrating a positive correlation between body mass index (bmi) and intrarenal resistive index (ri). 310 veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 renal resistive index in obese cats bueno de souza et al. a correlation between intrarenal ri and sap was not observed. six cats (30%) in the obese group (n = 6), and two (11.3%) in the control group (n = 2), presented high arterial pressure. among these animals, two obese cats (33.3%) showed a concurrent increase in intrarenal ri. no correlation occurred between intrarenal ri and serum concentrations of urea and creatinine. both groups had urea and creatinine levels within normal limits. on the other hand, a moderate correlation occurred between ri and serum sdma (table iii). discussion the mean values of intrarenal ri in the control group were 0.60 ± 0.03. the ri values and vessels evaluated to obtain ri in healthy non-sedated cats have shown variation among studies (carvalho and chammas 2011, pollard et  al. 1999, novellas et al. 2007). in healthy persian cats with a mean age of 30 months, the ri of the interlobar arteries was 0.54 ± 0.07 (carvalho and chammas 2011). neutered young adult domestic shorthair cats had renal ri values of 0.58 ± 0.06 and 0.55 ± 0.03 for right and left kidneys, respectively (pollard et  al. 1999). however, in healthy mixed-breed cats, mean age of 7.8 years, the ri measured from interlobar or arcuate arteries was 0.62 ± 0.04 (novellas et al. 2007), value relatively close to those obtained in the present study. blood pressure or hypertension (sap ≥ 150 mmhg) was observed in six cats (40%) of the obese group and two cats (13.3%) of the control group. the intrarenal ri of the obese group was higher than the control group, although not statistically significant (p = 0.115) (figure 2a). the sdma for the obese group was higher than the control group (p  <  0.01); however, all the values were within the normal range for cats. according to gender, ri (p = 0.026) and sdma (p = 0.007) were significantly higher in females than males in the obese group. also, females in the obese group presented a statistically significant increase in intrarenal ri compared to females in the control group. for males, no differences were observed between the groups (figure 2b). the control group did not present differences between males and females. the general ri values for males and females of both groups are in table ii. the intrarenal ri of at least one of the kidneys (right) presented a positive correlation with increased sdma, body weight, and bmi (table iii). a weak positive correlation between ri and bmi (r  =  0.42) showed that increases in bmi are accompanied by a concurrent increase in the intrarenal ri (figure 3). a weak positive ri correlation with age (r  =  0.37) was observed in the obese but not in the control group. based on the normality limits (ri < 0.70 for cats), six obese cats (40%) presented increased intrarenal ri in at least one kidney, while one animal (6.7%) in the control group presented ri above the normal ranges. considering the kidneys separately (right and left kidneys), eight kidneys of the obese group (26.6%) presented increased ri, while one kidney (3.3%) from the control group showed increased ri. a significant difference was observed when comparing the number of kidneys with increased intrarenal ris in the obese and control groups using fisher’s test, and considering each kidney separately (p = 0.027). despite the higher sap values in obese cats (table i), table i. demographic data for obese (n = 15) and control cats (n = 15). parameters groups p control obese age (years) 4.5 ± 2.3 6.5 ± 2.9 0.04 weight (kg) 4.2 ± 0.7 6.2 ± 1.5 < 0.001 bcs 5.0 ± 0.0 8.4 ± 0.5 < 0.001 bmi 25.7 ± 5.4 38.9 ± 11.0 < 0.001 sap (mmhg) 126.1 ± 19.7 151.2 ± 29.0 0.01 sdma (µg/dl) 6.7 ± 1.5 8.8 ± 2.6 0.001 urea (mg/dl) 53.9 ± 10.0 51.9 ± 8.2 0.57 creatinine (mg/dl) 1.10 ± 0.2 1.02 ± 0.3 0.45 bcs = body condition score; bmi = body mass index; sap = systolic arterial preassure; sdma = symmetric dimethylarginine. table ii. intrarenal resistive index in male cats, female cats and both (general) from control (normal-weight) (n = 15) and obese (n = 15) groups. resistive index groups p-valuescontrol (mean ± standard deviation) obese (mean ± standard deviation) general 0.60 ± 0.03 0.64 ± 0.08 0.12 males 0.60 ± 0.05 0.60 ± 0.08 0.91 females 0.60 ± 0.05 0.67 ± 0.06 0.008 table iii. correlations between intrarenal resistive index (ri), body weight, body condition score (bcs), body mass index (bmi), systolic arterial pressure (sap) and serum creatinine in control (normal-weight) (n = 15) and obese (n = 15) groups. correlated measures correlation coefficient (r) p-value ri x sdma 0.52 0.004* ri x body weight 0.38 0.04* ri x bcs 0.27 0.14 ri x bmi 0.42 0.03* ri x sap 0.03 0.86 ri x creatinine 0.04 0.82 *positive correlation in pearson test. 311veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 bueno de souza et al. renal resistive index in obese cats 2011). the association between increased bmi and risk factors for the end-stage of renal disease has already been shown in humans. furthermore, the increased bmi and waist-hip ratio in obese persons have been considered independent conditions for the increased ri in human patients with high blood pressure (trovato et al. 2012). although the present study did not find a correlation between sap and ri, two of the six obese cats with high arterial pressure presented a concurrent increase in ri (33.3%). one study found no correlation between intrarenal ri and sap in healthy dogs (novellas et al. 2007). it is important to consider the stress effects of sap in cats. a previous research evaluated physiologic parameters, such as blood pressure in cats in the home environment and the veterinary hospital, and a statistically significant increase in the veterinary hospital environment was reported (quimby et  al. 2011). increases in blood pressure occur because of the environment measurement process, caused by autonomic nervous system alterations that arise from the effects of stress or anxiety, also called “white coat” hypertension in human patients (acierno et al. 2018). there were no differences between serum creatinine of the obese and control groups. however, there was a significant difference between sdma of the obese group and control groups, despite the normal values in all the cats. increased circulating methylarginines have linked to metabolic syndrome, and serum concentrations of sdma is a more reliable and earlier marker for ckd compared with serum creatinine in human patients (marliss et  al. 2006). however, a study in normal-weight and overweight cats showed no differences in the concentrations of established markers of ckd, such as creatinine or sdma (pérez-lópez et al. 2020). the main limitation of this study was a non-homogenous reproduction status between the groups, since three cats were not neutered in the control group. however, this difference did not alter the results, because it corresponded to only 10% of the animals. in conclusion, the results obtained in this study suggest that the intrarenal ri may assist in monitoring renal function in obese cats. increases in body weight, bmi, and age resulted in concurrent increases in ri, indicating that obesity status is associated with preclinical hemodynamic changes at the kidney level. there was no significant difference between intrarenal ri of the obese group and control group, despite the mean ri value of 0.64 ± 0.08 in the obese group. however, considering the upper limit of ri measured in interlobar renal arteries in non-sedated cats, such as 0.68 (ostrowska et  al. 2016) or 0.70 (novellas et  al. 2007), 40% of the obese animals (n = 6) presented increased ri in at least one kidney. in hiv-1-infected patients with visceral obesity, it was observed higher intrarenal artery ri compared with those without visceral obesity, which was attributed to endothelial damage (grima et al. 2010). in addition, intrarenal ri in the obese group was significantly higher in females than in males, and obese females presented a significant increase concerning control females, not observed among males. the influence of gender on intrarenal ri has shown conflicting results in human patients (kaiser et al. 2007, toledo et al. 2015). in intrarenal ri between healthy persons and those with fatty liver disease, it was not observed influence of gender, but age dependency was observed (kaiser et al. 2007). on the other hand, higher renal ri was reported in women afflicted by ckd28 associated with a possible hormonal influence, but the exact reason was not established (toledo et  al. 2015). the hormonal influence in the present study may not play a role since all female cats were spayed. thus, the cause of higher ri in obese female cats needs further investigation. in the present study, age was related to increased intrarenal ri in obese cats, and senior obese cats showed a tendency to a higher ri. however, the intrarenal ri between adult and aged cats (seven to 12 years old) did not show significant differences. in a previous study with dogs, the intrarenal ri was influenced by age, with higher values in young and senior dogs (mamprim et al. 2018). however, none of the cats included in the present study was geriatric (> 12 years). a positive correlation occurred between intrarenal ri and bmi and body weight. a previous study with korean domestic short-hair cats showed a high correlation between body weight and renal dimensions and a weak correlation with ri (park et  al. 2008). on the other hand, human patients with or without hypertension showed a correlation between intrarenal ri and bmi by univariate analysis, and ri was considered an indicator of vascular damage caused by atherosclerosis (kawai et  al. 312 veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 renal resistive index in obese cats bueno de souza et al. acierno m.j., brown s., coleman a.e., jepson r.e., papich m., stepien r.l. & syme h.m. 2018. acvim consensus statement: guidelines for the identification, evaluation, and management of systemic hypertension in dogs and cats. vet intern med, 32 (6), 1803-1822. brown s., atkins c., bagley r., carr a., cowgil l., davidson m., egner 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serum concentrations of symmetric dimethylarginine and creatinine as kidney function biomarkers in cats with chronic kidney disease. j vet intern med, 28 (6), 1676-1683. hawthorne a. & butterwick r.f. 2000. predicting the body composition of cats: development of a zoometric measurement for estimation of percentage body fat in cats. j vet intern med, 14 (3), 349-365. kaiser c., götzberger m., landauer n., dieterle c., heldwein w. & schiemann u. 2007. age dependency of intrarenal resistance index (ri) in healthy adults and patients with fatty liver disease. eur j med res, 12 (5), 191-195. kawai t., kamide k., onishi m., yamamoto-hanasaki h., baba y., hongyo k., shimaoka i., tatara y., takeya y., ohishi m. & rakugi h. 2011. usefulness of the resistive index in renal doppler ultrasonography as an indicator of vascular damage in patients with risks of atherosclerosis. nephrol dial transplant, 26 (10), 3256-3262. kovesdy c.p., furth s.l. & zoccali c. 2017. obesity and kidney disease: hidden 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kim j., nam s.j., choi r., oh k.s., son c.h. & hyun c. 2008. ultrasonographic evaluation of renal dimension and resistive índex in clinically healthy korean domestic short-hair cats. j vet sci, 9 (4), 415-419. pérez-lópez l., boronat m., melián c., brito-casillas y. & wägner am. 2020. kidney function and glucose metabolism in overweight and obese cats. vet q, 40 (1), 132-139. pollard r., nyland t.g., bernsteen l., gregory c.r. & hornof w.j. 1999. ultrasonographic evaluation of renal autografts in normal cats. vet radiol ultrasound, 40 (4), 380-385. quimby j.m., smith m.l. & lunn k.f. 2011. evaluation of the effects of hospital visit stress on physiologic parameters in the cat. j feline med surg, 13 (10), 733-737. relford r., robertson j. & clements c. 2016. symmetric dimethylarginine: improving the diagnosis and staging of chronic kidney disease in small animals. vet clin north am small anim pract, 46 (6), 941-960. rivers b.j., walter p.a., o’brien t.d. & polzin d.j. 1996. duplex doppler estimation of pourcelot resistive index in arcuate arteries of sedated normal cats. j vet intern med, 10 (1), 28-33. rowe e., browne w., casey r., gruffydd-jones t. & murray j. 2015. risk factors identified for owner-reported feline obesity at around one year of age: dry diet and indoor lifestyle. prev vet med, 121 (3-4), 273-281. 313veterinaria italiana 2022, 58 (3), 307-313. doi: 10.12834/vetit.2294.15564.2 bueno de souza et al. renal resistive index in obese cats trovato g.m., martines g.f., trovato f.m., pirri c., pace p. & catalano d. 2012. renal resistive index and parathyroid hormone relationship with renal function in nondiabetic patients. endocr res, 37 (2), 47-58. vaden s.l., levine j.f., lees g.e., groman r.p., grauer g.f. & forrester s.d. 2005. renal biopsy: a retrospective study of methods and complications in 283 dogs and 65 cats. j vet intern med, 19 (6), 794-801. salama a.d. 2011. obesity and kidney disease: a call to action. nephr tim, 4, 8-9. tipisca v., murino c., cortese l., mennonna g., auletta l., vulpe v. & meomartino l. 2016. resistive índex for kidney evaluation in normal and disease cats. j feline med surg, 18 (6), 471-475. toledo c., thomas g., schold j.d., arrigain s., gornik h.l., nally j.v. & navaneethan s.d. 2015. renal resistive index and mortality in chronic kidney disease. hypertension, 66 (2), 382-388. 5 veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 accepted: 07.04.2020 | available on line: 27.07.2021 1department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of nigeria, nsukka, enugu state, nigeria. 2department of veterinary public health and preventive medicine, college of veterinary medicine, federal university of agriculture, abeokuta, ogun state, nigeria. 3department of veterinary public health and preventive medicine, university of abuja, federal capital territory, nigeria 4department of veterinary microbiology and pathology, university of jos, plateau state, nigeria. 5department of animal health and production, enugu state polytechnic, iwollo, enugu state, nigeria. 6department of veterinary medicine, university of nigeria, nsukka, enugu state, nigeria. *corresponding author at: department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of nigeria, nsukka, enugu state, nigeria. e‑mail: njoga.emmanuel@unn.edu.ng. emmanuel okechukwu njoga1*, akwoba joseph ogugua1, innocent okwundu nwankwo1, olajoju jokotola awoyomi2, chinwe elizabeth okoli3, deborah maigawu buba4, felix ayodele oyeleye1, festus ejike ajibo5, nichodemus azor5 and temitope mofoluso ogunniran6 keywords antibiotic stewardship, nigeria, poultry farmers, imprudent antimicrobial use, survey. summary good antimicrobial drug stewardship in food-producing animals boosts productivity and limits transmission of zoonotic pathogens, but the imprudent usage is counterproductive. a nation-wide survey to determine the pattern of antimicrobial drug usage in poultry was therefore conducted across 12 of the 36 states in nigeria. the survey was conducted using structured questionnaire designed to elicit information on socio-demographics, pattern of antimicrobial drug usage and awareness of the consequences of imprudent use of antimicrobials in food-producing animals, among 2,402 randomly selected poultry farmers. critically important antimicrobials, belonging to the who’s lists of ‘highest’ and ‘high’ priority antibiotics, were administered sub-therapeutically for prophylaxis and growth enhancement purposes. many diseases that warranted the antimicrobial administration were of viral etiology. only 64.2% of the farmers administered the drug based on veterinary doctors’ prescription. majority (62.3%) of the farmers did not observe recommended withdrawal period after the drug administration. awareness of the respondents on consequences of non-prudent agricultural use of antimicrobials was generally poor. there is need for enlightenment of the farmers on the benefits of prudent agricultural use of antimicrobials. immediate discontinuation of sale of essential antimicrobials as over-the-counter drugs is imperative to safeguard their therapeutic efficacy and curtail spread of antimicrobial-resistance organisms. antimicrobial drug usage pattern in poultry farms in nigeria: implications for food safety, public health and poultry disease management producing 650,000 tonnes of eggs and 300,000 tonnes of poultry meat (fao 2018). in nigeria, the poultry subsector is the most commercialized agribusinesses, and has been adjudged the most dynamic and fastest growing agribusiness, contributing significantly to job creation, poverty alleviation and production of animal protein (njoga et  al. 2019a). with about 13 million households introduction poultry production refers to the rearing of different species of domesticated birds, for production of meat, egg, poultry manure, feather or other poultry products. with estimated poultry population of 180 million in 2015 (heise et  al. 2015), nigeria is the second largest producer of poultry in africa, 6 intestinal flora in favour of pathogenic bacteria (olatoye and ehinmowo 2010), organ toxicity and carcinogenicity (njoga et al. 2018). apart from the health problems, non-prudent use of antimicrobials in poultry production also has negative economic implications. the accumulation of residual antimicrobials in poultry products is a serious food safety concern; and hence limits the marketability and monetary value of such products at the international and local markets. in addition, akinwumi and colleagues (akinwumi et  al. 2014) reported shrinkage, toughening and alteration in taste of cooked meat, as indications of reduced meat quality, due to presence of residual antimicrobials in meat. there is also drastic increase in the costs and durations of medical and veterinary treatments, due to non-susceptibility of bacterial pathogens to previously lethal antimicrobials. a report in nigeria revealed that poultry farmers incurred additional costs (up to 35% of their cost of production) on treatment of antibiotic-resistant bacterial, which amounted to about 75 million us dollars (adebowale et al. 2016). non-prudent agricultural use of antibiotics has deleterious effects on the environment and biodiversity. given that antibiotics can be excreted unchanged in poultry faeces, these drugs can contaminate the environment through the use of poultry manure as feed in fisheries and piggeries (njoga et  al. 2019a). the unchanged drugs or its metabolites in discarded poultry waste can also contaminate natural water bodies and aquatic lives therein (sarmah et al. 2006, rahmatallah et al. 2018). this may disrupt the microbial flora in the new environment, in favour of harmful organisms to the detriment of animals and humans inhabiting the environment. despite the negative health and economic impacts of imprudent use of antimicrobials in animal agriculture, dearth of information on the pattern of antimicrobial drug administration in poultry farms in nigeria continues to exist. this nation-wide study was therefore carried out to determine the antimicrobial drug usage pattern in poultry farmers in nigeria, and further characterize the socio-demographics of the farmers and their awareness on consequences of non-prudent administration of antimicrobials in food animals. these will guide policy formulation for preservation of the therapeutic efficacy of antimicrobials for sustainable poultry production and to safeguard human health. materials and methods study area nigeria is a west african country whose geographic involved in poultry production, the poultry industry also contributes about 10% of the agricultural gdp and 3.1% of the nation’s gdp (world bank 2017). the boost and viability in the poultry industry may be attributed to prudent use of antimicrobials in combating most infectious poultry diseases, inimical to poultry production in the tropics (njoga et  al. 2018). prudent use of antimicrobials in food-animal production has also curtailed spread of zoonotic pathogens, transmissible through the food chain. prudent use of antimicrobial drugs in animal agriculture involves administration of the appropriate drug for the right disease, at the appropriate dose, all through the recommended treatment period. chemotherapeutic and prophylactic uses of antibiotics in poultry production are well recognized worldwide (chowdhury et  al. 2015, njoga et al. 2018); but use of sub-therapeutic doses for growth promotion purposes is rapidly gaining popularity in nigeria. escalation in demand for foods of animal origin, occasioned by boom in human population and increase in middle class income in most developing countries, is driving the sub-therapeutic usage to cater for the shortfall in a record short time. however, sub-therapeutic use of antibiotics to fast-track poultry production is not without costs. selection of antimicrobial-resistant pathogens which may worsen the animal’s health problem or may be transmitted to humans via the food chain is perhaps the most important of the costs, from food safety and public health perspectives. misuse or overuse of antimicrobials in animal agriculture creates selective evolutionary pressure that facilitates amplification and dissemination of antimicrobial-resistant bacteria, far above the antimicrobial-susceptible organisms (adesokan et  al. 2013, chinwe et  al. 2014, njoga et  al. 2018). evidences of transfer of antibiotic-resistant bacteria from food animals to humans or antibiotic-resistant genes from non-zoonotic bacteria to zoonotic pathogens, for onward transmission to humans have been documented (van den bogaard and stobberingh 2000, martins da costa et al. 2013). although humans are non-target population for veterinary antimicrobials, the residues could reach the human populations through consumption of poultry products (nisha 2008, chowdhury et al. 2015). this is particularly true when poultry products are sourced from antibiotic-treated birds, before the end of the stipulated withdrawal period. consumption of antimicrobial residues in edible animal products predisposes to health problems including: allergy in sensitized individuals (woodward 1991, sassanya et al. 2008, njoga et al. 2018), bone marrow depletion or aplastic anemia (gassner and wuethrich 1994, young 2002, njoga et al. 2018), disruption of normal antimicrobial drug usage in nigerian poultry farms njoga et al. veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 7veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 njoga et al. antimicrobial drug usage in nigerian poultry farms association (p < 0.05) between educational levels and pattern of antimicrobial usage (pau), purpose of antimicrobial administration (paa) and aciuap. similarly, the same statistic was used to test for association (p < 0.05) between farming experience and pau and paa. all the analyses were done at five per cent probability level using graphpad prism® software, version 6.04 (graphpad® inc., san diego, california, usa). results socioeconomic characteristics majority of the farmers (67.5%) were males. fourteen per cent of the farmers had no formal education. however, 28.2% (678/2402), 35.8% (859/2402) and 21.9% (526/2402) attained primary, secondary and tertiary educational levels, respectively. broiler, layers, turkey, cockerel and nigerian indigenous chicken were the major types of poultry reared. some of the farmers reared other food animals, while 5.3% (134/2,402) kept dog or cat for security purposes or biological control of rodents. furthermore, some (36.5%) of the farmers were new in poultry farming business, having less than 5-years’ experience, while 21.9% (528/2,402) have been farming for over 10  years. similarly, majority of the farmers (72.4%) adopted intensive husbandry system of production, while only 23.1% and 4.4% practiced semi-extensive and extensive management systems, respectively. antimicrobial drug usage pattern in poultry the classes of antimicrobials administered and percentage of farmers involved were: tetracycline (25.4%), aminoglycosides (18.5%), macrolides (14.8%), quinolone and fluoroquinolone (14.8%), chloramphenicols (11.3%), sulfonamides (8.5%) and penicillin and betalactam (6.8%). only 64.2% of the farmers administered the drug based on veterinary doctors’ prescription. on the purpose of the drug administration, 36.2% used the drugs for treatment, while 36.7% and 27.1% administered it for prophylaxis and growth promotion purposes, respectively. majority (62.3%) of the farmers who administered these drugs did not observe the recommended withdrawal period. biosecurity practices and common poultry diseases found details on biosecurity practices adopted in farms surveyed are presented in table  i. routine vaccination against endemic diseases was the most practiced biosecurity measure, done in 81.8% of the center is located on latitude 9°4’55.2”n and longitude 8°40’31”e. it comprises of 36 states and the federal capital territory, grouped into six geopolitical zones – southeast, south-south, southwest, northeast, northwest and north-central. nigeria is bounded in the west by republic of benin, in the east by chad and cameroon and niger republic in the north. the coast lies on the gulf of guinea in the south and lake chad at the northeastern region. with a population currently estimated at 203 million, nigeria has been ranked the most populous african country and the 7th most populated country worldwide. the total land area is 910,770 km2 while the population density is 221 persons per km2. selection of farms surveyed a total of 12 states, 24 agricultural zones (az) and 2,402 poultry farms were randomly selected and surveyed. at first, four of the six geopolitical zones, namely southeast, southwest, north-central and northwest were purposively selected for the study, based on the history of high poultry production activities. in each selected zone, three states and two azs per state were selected by simple random sampling. the same sampling method was used to select at least 100 poultry farms from each selected az, based on the consent of the farm owners to participate in the study and accessibility of the farm. the questionnaire survey structured and pretested open-ended questionnaire was used to obtain data on socioeconomic characteristics, husbandry and biosecurity practices, types and pattern of antimicrobial drug use in poultry, diseases that warranted the drug administration, and awareness of consequences of imprudent use of antibiotics in food-producing animals. the respondents were the farm owners or managers, who were acquainted in the daily running of the farms, and had earlier expressed their willingness to participate in the survey. one respondent per farm was surveyed. the content of the questionnaire was translated in native language, in the form of interview, to respondents who were not proficient in the use of english language. thereafter, completed copies of the questionnaire were collected and the responses collated for statistical analysis. data analysis and presentation data bothering on socioeconomic characteristics and biosecurity measures adopted in the farms surveyed and awareness on consequences of imprudent use of antibiotics in poultry (aciuap) were analyzed descriptively and presented in tables. chi-square statistic was used to test for significant 8 antimicrobial drug usage in nigerian poultry farms njoga et al. veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 that non-observance of the stipulated withdrawal period may enhance accumulation of drug residues in edible poultry products; while 63.2% were oblivious of the fact that consumption of residual antimicrobials in animal tissues predisposes to health problems. effects of educational and farming experiences on pattern of antimicrobial usage effects of educational levels and farming experience on pattern of antimicrobial usage are summarized in tables iii and iv, respectively. the correct use of antibiotic (after veterinary diagnosis) and their source were strongly associated (p < 0.05) with farmers’ educational level. equally, statistical associations (p  <  0.05) were found between farming experience and antibiotic prescription by veterinarian and procuring antimicrobials from veterinary pharmacy but none existed between farming experience and diagnosis made by a veterinarian before antibiotic use at p = 0.453. discussion the use of antimicrobial drugs in all the farms surveyed is worrisome and may be due to a number of reasons. the quest to produce more poultry meat (chicken) and eggs to satisfy demand for animal protein, which currently lags the supply (njoga et  al. 2018), may be one of the reasons. apart from rapid population growth and increase in middle class income, that has increased demand for animal protein in developing countries, preference for ‘white meat’ over ‘red meat’ may have aided the increased demand for poultry meat worldwide. the farms surveyed. the study found that 31.9% of the respondents adopted good biosecurity practices i.e. practiced more than eight of the 15 biosecurity measures surveyed for. good biosecurity practices were found to be highest among farmers who attained tertiary education. in the same vain, 78.2% of the respondents who practiced good biosecurity measures were farmers having flock size greater than 500 birds. the level of good biosecurity according to husbandry management systems was intensive (69.7%), semi-intensive (29.7%) and extensive (0.6%). major poultry diseases that necessitated antibiotic treatment, as indicated by the farmers were: newcastle (31.7%), coccidiosis (21.3%), gumboro (26.4%), salmonellosis and colibacillosis (13.4%). awareness of the consequences of imprudent agricultural use of antimicrobial awareness of poultry farmers surveyed on consequences of imprudent use of antimicrobial in food animal production is generally poor as presented in table ii. some (40.1%) of the farmers were not aware that indiscriminate use of antimicrobials can worsen the health condition of their birds. majority (70.3%) of the respondents were ignorant of the fact that antibiotic-resistant bacteria, transmissible through the food chain, can emerge following imprudent use of the drug in poultry. similarly, 67.9% of the farmers did not know table i. biosecurity measures adopted in poultry farms (n = 2,402) surveyed in nigeria. biosecurity measures number of respondents (%) fencing 746 (31.1) netting 786 (32.7) sanitization of drinking water 716 (29.8) all-in-all-out stock replacement program 944 (39.3) quarantine of exposed or incoming birds 578 (24.1) traffic regulation and unidirectional movement in the farm 1,078 (44.8) use of protective clotting while on duty 710 (29.6) siting farms away from water bodies 1,942 (80.8 ) farm sited at least 200m away from residential areas 832 (34.6) periodic fumigation 1,108 (46.1) availability of rodents and migratory birds control programs 680 (28.3) availability and use of hand washing facilities at farm entrances 470 (19.6) use of only new or disinfected egg crates in the farm 464 (19.3) availability of foot dips at farm and pen entrances 890 (37.1) regular vaccination against endemic poultry diseases 1,966 (81.8) table ii. awareness of the consequences of imprudent agricultural use of antimicrobial on human and animal health among poultry farmers (n = 2,402) surveyed in nigeria. information required number of respondents (%) yes no aware that imprudent antibiotic administration in poultry may worsen the health condition 1,438 (59.9) 964 (40.1) aware that indiscriminate use of antibiotics in poultry can enhance development of antibiotic-resistant pathogens, transferable to humans via the poultry food chain 714 (29.7) 1,688 (70.3) know that non-observance of withdrawal period can aid accumulation of antibiotic residues in poultry products 769 (32.0) 1,633 (67.9) aware that consumption of residual antibiotics in edible animal tissues can cause health problems in humans 883 (36.8) 1,519 (63.2) 9 njoga et al. antimicrobial drug usage in nigerian poultry farms veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 a ‘quick fix’ or compensation for poor management practices in the farms surveyed. good farm management practice promotes strict biosecurity, routine vaccination and adequate nutrition and not antibiotic doping for prophylaxis or growth enhancement. antimicrobial-resistant organisms in poultry can spread directly to humans via the poultry food chain or less commonly by contact in occupationally exposed individuals such as poultry farmers, veterinarians and abattoir workers (ekere et  al. 2018). when an antibiotic-resistant organism is not zoonotic, it may transfer its resistance genes to zoonotic organisms for onward transmission to humans. cognizant of the fact that egg is sometimes consumed raw in nigeria (onyenweaku et al. 2018), these resistant-organisms can easily reach the human population via consumption of infected raw eggs or undercooked poultry products. the practice of administering un-prescribed antimicrobial drugs in poultry farms is unethical (njoga et al. 2019b). olatoye and ehinmowo (olatoye and ehinmowo 2010) had reported that farmers administer veterinary drugs without prescriptions to cut cost of production and probably maximize profit. this attempt to save cost of veterinary services, without recourse to the negative implications on human and animal health, is counterproductive. when people, who are not formally trained in veterinary clinical practice, administer un-prescribed antimicrobials to food animals, the likelihood of incorrect doses and other chemotherapeutic errors, that may adversely affect the pharmacokinetics of the drugs, is most probable. these, no doubt, facilitate emergence and spread of antimicrobial-resistant bacteria (alhaji et  al. 2018), and accumulation of synergy of increased demand and marketability of poultry products, due to its wide acceptance by all and sundry, may have fueled the quest for increased production of poultry and hence the wide spread use of antimicrobials in the farms surveyed. also, traditional husbandry systems (semi-intensive and extensive) adopted by 27.5% of the farmers surveyed may have predisposed birds to diseases, and therefore necessitated antibiotic treatments. unlike the intensive management system which has the advantage of low pathogen infectivity (abonyi and njoga 2019), the traditional system predisposes to disease, especially in the tropics. in these regions, most poultry diseases are endemic due to availability and interconnectivity of factors that enhance survival and proliferation of pathogens. this may explain why critically important antimicrobials, including third generation fluoroquinolone (ciprofloxacin), were used for prophylaxis in some farms. the high rate of antimicrobial administration (63.8%) for non-therapeutic purposes in poultry production in this study is in tandem with the 68.5% found in south africa (eagar et  al. 2013). farmers may have resorted to non-therapeutic use of antimicrobials as table iii. effect of educational levels on usage of antimicrobials and awareness of the consequences of imprudent usage among poultry farmers (n = 2, 402) surveyed in nigeria. information required number of yes respondents p-valueno formal educ. (n = 339) primary educ. (n = 678) secondary educ. (n = 859) tertiary educ. (n = 526) pattern of antimicrobial usage a 195 461 518 367 0.0005* b 181 394 255 61 0.0001* c 88 376 401 417 0.0001* d 136 229 328 213 0.0681 purpose of antimicrobial usage treatment 156 277 338 196 0.0708 prevention 119 308 332 223 0.0052* growth promotion 77 227 264 156 0.0054 * awareness of consequences of imprudent agricultural use of antimicrobials e 197 391 521 329 0.3092 f 123 149 291 151 0.0010* g 95 186 331 143 0.0001* h 179 191 215 298 0.0001* *denotes statistical significance, chi-square statistic, graphpad prism 6.04; a = antibiotic prescribed by veterinarian; b = diagnosis made by a veterinarian before antibiotic use; c = sourced antibiotics from veterinary pharmacy; d = observed the stipulated withdrawal period; e = aware that imprudent antibiotic administration in poultry may worsen the health condition; f = aware that indiscriminate use of antibiotics in poultry can enhance development and of antibiotic-resistant pathogens, transmissible via the food chain; g = know that non-observance of withdrawal period can aid accumulation of antibiotic residues in poultry products; h = aware that consumption of residual antibiotics in poultry products predisposes to health problems in humans. table iv. effect of farming experience on pattern and purpose of antimicrobial usage among poultry farmers (n = 2, 402) surveyed in nigeria. information required number of yes respondents p-valueless than 5 years (n = 877) 5-10 years (n = 997) more than 10 years (n = 528) pattern of antimicrobial usage a 587 669 285 0.0001* b 322 361 208 0.4526 c 488 501 293 0.0355* d 136 229 328 0.0681 purpose of antimicrobial usage treatment 393 411 163 0.0001* prevention 333 338 311 0.0001* growth promotion 199 293 232 0.0001* *denotes statistical significance, chi-square statistic, graphpad prism 6.04; a = antibiotic prescribed by veterinarian; b = diagnosis made by a veterinarian before antibiotic use; c = sourced antibiotics from veterinary pharmacy; d = observed the stipulated withdrawal period. 10 antimicrobial drug usage in nigerian poultry farms njoga et al. veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 the end of withdrawal period, are unsafe for human consumption and farmers may have jettisoned withdrawal period instruction for fear of economic losses. this fear of economic losses may explain why some educated farmers, who should know the negative implication of imprudent antimicrobial usage in food-producing animals, administered antimicrobials without valid diagnosis and or veterinary prescription. availability of medically important antimicrobials, even those in who list of “high” and “highest” priority antibiotics, as otc drugs may have sustained this unethical practice. similarly, farmers with less working experience cooperated with the veterinarians on the health needs of their birds more than those having more years of work experience, as the later believe that they have enough knowledge to use drugs without the advice of a veterinarian. the resultant effect of all these is abuse and misuse of antimicrobials at the detriment of human and animal health. conclusions there is extensive imprudent administration of antimicrobials in poultry and poor awareness of the consequences on animal and human health among the poultry farmers surveyed. these underscore the urgent need to enlighten the farmers on the benefits of prudent agricultural use of antimicrobials on food safety and poultry disease management. imprudent use of antimicrobials even among the “educated” and “experienced” farmers reiterates the needs for discontinuation of sale of essential antimicrobial as otc drugs and strict implementation of veterinary drug laws to safeguard the therapeutic efficacy for sustainable poultry production and to preserve public health. antibiotic residues beyond safe levels in poultry products or the environment at the detriment of human health. sometimes, human lives may be lost, especially in developing countries, where effective drugs for the treatment of antimicrobial-resistant pathogens may be unavailable or unaffordable (o’neill 2014, laxminarayan et al. 2016). the continued use of chloramphenicol in poultry after over two decades of banning the use in food-producing animal in nigeria by the national agency for food and drug administration and control (nafdac) in 1996, is regrettable. the prohibition was due to haematological problems, particularly aplastic anaemia, associated with chloramphenicol, especially in children and young adults (gassner and wuethrich 1991, young 2002). in nigeria, easy access to veterinary antimicrobials, including those classified as medically important antibiotics (who 2016), as examplified by their sale as over-the-counter (otc) may be contributory to the continued use of chloramphenicol for systemic administration. most importantly, lack of legislation against this practice and obvious inadequate implementation of the available veterinary drug laws in nigeria are to be blamed. the problems of non-adherence to the stipulated withdrawal period and poor awareness on the consequences of non-prudent use of antimicrobials found in this study, may be attributed partly to the level of education of some farmers. paltry level of education makes it difficult for farmers to read or follow  instructions on drug labels. additionally, excessive quest for economic gains may be contributory to non-observance of withdrawal period, given that majority of the farmers were educated enough to read and understand drug label instructions. animal products obtained from antibiotic-treated food animals prior to 11 njoga et al. antimicrobial drug usage in nigerian poultry farms veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 abonyi f.o. & njoga e.o. 2019. prevalence and determinants of gastrointestinal parasite infection in intensively managed pigs in nsukka agricultural zone, southeast, nigeria. j parasit dis, 44, 31-39. adebowale o.o., adeyemo o.k., awoyomi o., dada r. & adebowale o. 2016. antibiotic use and practices in commercial poultry laying hens in ogun state nigeria. rev elev med vet pays trop, 69, 41-45. adesokan h.k., agada c.a., adetunji v.o. & akanbi i.m. 2013. oxytetracycline and penicillin-g residues in cattle slaughtered in south-western nigeria: implications for livestock disease management and public health. j s afr vet assoc, 84, 1-5. akinwumi o.a., olatoye i.o., odunsi a.a., omojola a.b., olayeni t.b. & adedeji m.f. 2014. evaluation of antibiotic residue on the quality of pork sold for human consumption in oyo state, south west nigeria. j anim sci adv, 4, 772-776. alhaji n.b., haruna a.e., muhammad b., lawan m.k. & isola t.o. 2018. antimicrobial usage assesement in commercial poultry and local birds in north central nigeria: associated pathways and factors for resistance emergence and spread. prev vet med, 154, 139-147. chinwe n., ayogu t., iroha i., ejikeugwu c., nwakaeze e., oji a. & ilang d. 2014. cloacal feacal carriage and occurrence of antibiotic resistant escherichia coli in chicken grown with and without antibiotic supplemented feed. j vet med anim health, 6, 91-94. chowdhury s., hassan m.m., alam m., sattar s., bari m.s., saifuddin a.k.m. & hoque m.a. 2015. antibiotic residues in milk and egg of commercial and local farms at chittagoog, bangladesh. vet world, 8, 467-471. eagar h., swan g. & van-vuuren m.a. 2013. survey of antimicrobial usage in animals in south africa with specific reference to food animals. j s afr vet assoc, 83, 1-8. ekere s.o., njoga e.o., onunkwo j.i & njoga u.j. 2018 serosurveillance of brucella antibody in food animals and role of slaughterhouse workers in spread of brucella infection in southeast nigeria. vet world, 11, 1171-1178. food and agriculture organization of the united nations (fao). 2018. food and agriculture data. (www.fao.org/ faostat/en/#data/qa accessed on 7 november 2019). gassner b. & wuethrich a. 1994. pharmacokinetic and toxicological aspects of the medication of beef-type calves with an oral formulation of chloramphenicol palmitate. j vet pharmacol ther, 17, 279-283. heise h., crisan a. & theuvsen l. 2015. the poultry market in nigeria: market structures and potential for investment in the market. int food agribusiness manag rev, 18, 197-222. laxminarayan r., matsoso p., pant s., brower c., røttingen j.a., klugman k. & davies s. 2016. access to effective antimicrobials: a worldwide challenge. lancet, 387, 168-175. martins da costa p., loureiro l. & matos a.j.f. 2013. transfer of multidrug-resistant bacteria between intermingled references ecological niches: the transfer between humans, animals and the environment. int j environ res public health, 10, 278-294. nisha a.r. 2008. antibiotic residues a global health hazard. vet world, 4, 375-377. njoga e.o., ariyo o.e. & nwanta j.a. 2019a. ethics in veterinary practice in nigeria: challenges and the way-forward. niger vet j, 40, 85-93. njoga e.o., nwankwo i.o. & ugwunwarua j.c. 2019b. epidemiology of thermotolerant campylobacter infection in poultry in nsukka agricultural zone, nigeria. int j one health, 5, 92-98. njoga e.o., onunkwo j.i., chinwe c.e., ugwuoke w., nwanta j.a. & chah k.f. 2018. assessment of antimicrobial drug administration and antimicrobial residues in food animals in enugu state, nigeria. trop anim health prod, 50, 897-902. o’neill j. 2014. antimicrobial resistance: tackling a crisis for the health and wealth of nations. (http://www. jpiamr.eu/wp-content/uploads/2014/12/amr-review -paper-tackling-a-crisis-for-the-health-and-wealth-of -nations_1-2.pdf accessed on 12 april 2019). olatoye i.o. & ehinmowo a.a. 2010. oxytetracycline residues in edible tissues of cattle slaughtered in akure, nigeria. niger vet j, 31, 93-102. onyenweaku e., ene-obong h., williams i. & nwaehujor c. 2018. comparison of nutritional composition of bird egg varieties found in southern nigeria: a preliminary study. food sci nutr, 9, 868-879. rahmatallah n., el-rhaffouli h., amine i.l., sekhsokh y., fihri o.f. &, el-houadfi m. 2018. consumption of antibacterial molecules in broiler production in morocco. vet med sci, 4 (2), 80-90. sarmah a.k., meyer m.t. & alistair b.a.b. 2006. a global perspective on the use, sales, exposure pathways, fate and effects of veterinary antibiotics in the environment. chemosphere, 65, 729-759. sassanya j.j., ejobi f., enyaru j., olila d. & ssengoye g. 2008. public health perspective of penicillin-g residues in cow milk and edible bovine tissues collected form mbarara and masaka districts, uganda. afr j anim biomed sci, 3, 35-40. van den bogaard a.e. & stobberingh e.e. 2000. epidemiology of resistance to antibiotics links between animals and humans. int j antimicrob agents, 14, 327-335. van den bogaard a.e., london n., driessen c. & stobberingh e.e. 2001. antibiotic resistant eschericha coli in poultry, poultry farmers and poultry slaughterers. j antimicrob. chemother, 47, 763-771. world health organization (who). 2016. critically important antimicrobials (cia) list 5th review. (http:// who.int/foodsafety/publications/antimicrobials-fifth/ en/ accessed on 2 december 2019). woodward k.n. 1991. hypersensitivity in humans and exposure to veterinary drugs. vet hum toxicol, 33, 168-172. 12 antimicrobial drug usage in nigerian poultry farms njoga et al. veterinaria italiana 2021, 57 (1), 5-12. doi: 10.12834/vetit.2117.11956.1 world bank. 2017. better life farming unlocking the potentials of smallholder farmers to reach sustainable development goals. (https://live.worldbank.org/better life-farming accessed on 22 october 2019). young n.s 2002. acquired aplastic anemia. ann intern med, 136, 534-546 271 introduction pteridophytes are primitive vascular cryptogamic plants which occupy a systematic position between the lower non‑seed bearing and the higher seed‑bearing plants. they are generally a much neglected group of plants (madhusoodanan et  al. 2001). ferns are represented by about 305 genera, comprising more than 12,000 species all over the world. about 70 families, including 191 genera and 1,000 species presumably occur in india. in the southern western ghats region the current status of pteridophytes is 272 species belonging to 95 genera and 34 families. most of the pteridophytes are listed among endemic and rare species. the pteridophytes are little known for use since they are not as easily available as the flowering plants. therefore, economic and medicinal values of higher plants are being exhaustively investigated where as the pteridophytes have been largely ignored (vashishta et al. 2012). ethnobotanical approach to human and veterinary medicines is an emerging trend in the treatment of chronic diseases such as skin diseases, tumours, parasitic infections, etc. the drugs/therapies in this approach are least toxic and highly cost‑effective. however, there is little research in respect of the pteridophytic medicinal plants, which are on the decline and are being delimited due to habitat displacement, natural calamities, soil erosion, etc., and many of these plants are now in the list of threatened species, and some are in the list of endangered species in the red data book of international union for conservation of nature (iucn). there has been little effort to conserve them. the ferns were once popular in folk medicine especially practiced by the tribal communities (manickam and irudayaraj 1992). since these are smaller traditions of indian systems of medicine the uses have not been recorded, as the result of which there is only fewer literature pertaining to application/ utility of pteridophytes against endoparasitic 1a. veeriya vandayar memorial sri pushpam college, bharathidasan university, india. 2tamilnadu veterinary and animal sciences university, india. *corresponding author at: a. veeriya vandayar memorial sri pushpam college, bharathidasan university, india. e‑mail: kalpanafern@gmail.com. summary four ferns blechnum orientale linn. (bo), dicranopteris linearis (burm.f ) underw. (dl), marattia fraxinea sm. (mf), and microlepia speluncae (l.) moore (ms) were extracted in varied combination of organic solvents followed by the preparation of eluates and isolation of secondary metabolites using chromatography on a glass column with silica gel as the fixed phase. the chemical components were identified using hptlc and gc-ms analysis. the in vitro anti-trematodal activities of these eluates and compounds were evaluated against the sheep trematode worm gastrothylax crumenifer (plagiorrchiida: gastrothylacidae) at increasing concentrations (1 to 5 mg/ml), to find the relative motility (rm) values for 0  to  60  min of incubation as reflection of paralysis and death of the worms. hedon-fleig salt solution was used as negative control and oxyclozanide® 1% as standard control. in vitro incubation study showed dl and ms extracts had strong trematodicidal activity. bo extract (5 mg/ml) produced moderate trematodicidal activity and mf (5 mg/ml) showed the least trematodicidal activity. phytochemicals analysis revealed that the ferns are a potential source of trematodicidal compounds such as phytol isomers fern-8-ene and fern-9(11)-ene (terpenoid derivative), quercetin 7,3´,4´-trimethoxy (flavonoid derivative), etc., which offer scope for a more elaborate study for exploitation of ferns for human welfare. kalpana devi r.1, rajesh n.v.2, vasantha s.1 and jeyathilakan n.2 screening of in vitro antitrematodal activities of compounds and secondary metabolites isolated from selected pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 accepted: 07.11.2019 | available on line: 31.12.2020 keywords ferns, blechnum orientale, dicranopteris linearis, marattia fraxinea, microlepia speluncae, secondary metabolites, gastrothylax crumenifer. 272 veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. greatly to the discovery of novel compounds of pharmaceutical and biomedical importance. column chromatographic technique is especially useful in the isolation of bioactive compounds. the compounds can further be purified using high performance liquid chromatography (hplc), followed by nuclear magnetic resonance (nmr) spectral analyses. at present there are innumerable products in the market which inflict adverse side effects on one’s health. therefore, the use of secondary metabolites of plant origin could be an advantage and an effective solution to narrow down the use of unhealthy products. in the past, plant or microbial extracts, in crude or partially purified form, were the only sources of medication available for the treatment of human and animal diseases. the effect of a drug in human body is due to an interaction of the drug with the biological molecules. this opened up new vistas in pharmacology, whereupon isolated chemicals, rather than extracts, are practiced for treatment of diseases. therefore, the objective of this study has been to fractionate and isolate the biologically active secondary metabolites present in the ferns for in  vitro anti‑trematodal activity. phytochemical investigation of the ferns has been conducted only to a limited extent as compared to the higher plants. reporting the compounds in ferns responsible for the anti‑trematodal activity may be first of its kind in the field of ethnobotany. the phytochemical analysis of plants present in the wild and controlled endoparasitic drug trials along with contemporary knowledge of parasite control strategies may offer new opportunities for effective and economical control of parasitic diseases. applied to veterinary medicine, this would improve the economic conditions of the farmers who hold the livestock and, thus, improve the economic status of the country. in this study we investigated extracts of four species of ferns for in  vitro trematodicidal property using gastrothylax crumenifer (creplin, 1847) as the model trematode parasite, and attempt was also made to isolate the compounds with the potential therapeutic property. materials and methods the study was carried out during the period from october, 2014 to october, 2016, in the department of botany and microbiology, a. veeraiya vandayar memorial (a.v.v.m.), sri pushpam college, poondi, thanjavur district, tamil nadu, india. four species of ferns, blechnum orientale linn. (bo) (polypodiopsida, blenchnaceae), dicranopteris linearis (burm.f ) underw. (dl) (polypodiopsida, gleicheniaceae), marattia fraxinea (sm.) (mf) (polypodiopsida, marattiaceae) and microlepia speluncae (l.) (ms) (polypodiopsida, dennstaedtiaceae) were collected from kothayar hills (latitude, 8° 31´ 24.42´´ n, longitude, 77° 21´ infections and their antioxidant potential. around 300 b.c., theophrastus recommended oil extract of ferns to expel internal parasites (amritpal singh 2011). blechnum orientale linn. (blechnaceae) is used ethnomedicinally for the treatment of various skin diseases, stomach pain, urinary bladder complaints and sterilization of women (lai et  al. 2010). rhizomes of various shield ferns (e.g., polystichum and dryopteris) have been used as a cure for some intestinal worm infections since the 18th century. cyathea manniana hook., (cyatheaceae) from east africa, has been used by the chagga and german troops as an anthelmintic during the first world war. however, the pteridophytes find their use in the ancient systems of medicine, viz., homoeopathy, ayurveda, and unani, for worm infections. helminthiasis is the condition resulting from round worm, tape worm and/or flat worm infections, especially in ruminants, and is one of the major prevalent diseases around the world, particularly the tropical countries such as india, pakistan, bangladesh, sri lanka, etc (soulsby 1982). the worm infection is managed using anthelmintics but problems have emerged with their uses, especially i) development of resistance in the helminth parasites to the various anthelmintic compounds, ii) accumulation of residues of the compounds in the host, and iii) toxicity issues (waller and prichard 1985). in general, the recognition of antigenic complexity of the parasites has caused a slow‑down of vaccine development. the frequent use of anthelmintics over many years has inevitably led to the development of resistance to the varied anthelmintics used. it has been documented that helminths are most probably resistant to all three broad spectrum families of anthelmintics viz., benzimidazole, imidazothiazole and ivermectin. the medicinal plants in use for endoparasitic infection, on the other hand, produce fewer unanticipated side effects and apparently do not trigger parasitic chemo‑resistance. under these circumstances investigation of alternatives, such as medicinal plants, to commercially available anthelmintics is being emphasized. for centuries, medicinal plants have been used to combat parasitic infections, and in many parts of the world they are still in use. the use of medicinal plants for prevention and/ or therapy of gastro‑intestinal parasitic infections, in fact, originated as an aspect of ethno‑veterinary medicine. but, there are some problems concerning the use of herbal medicine the most important being lack of scientific evaluation and/or validation. the most effective approach to do such evaluation/ validation is ethnobotanical approach, which assumes that indigenous uses of plants indicate the presence of biologically active compounds in them. chromatographic techniques have significant place in natural products chemistry and contribute 273veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes total terpenoid content by the method described in ferguson (ferguson 1956). extraction using column chromatography in order to isolate the bioactive compounds from the crude extracts, the latter were further fractionated using column chromatography, with silica gel  g (40  g, 60‑120 #, loba chemie pvt. ltd.) as the stationary phase (https://www.shodhganga. inflibnet.ac.in). the dried crude extract was loaded onto a glass column (60 x 3.0 cm) packed with silica gel g. the extract was dissolved in a minimal quantity of ethanol and adsorbed on to silica gel g. when the sample had adsorbed to the silica gel, a small amount of sand was poured to cover sample. the mobile phase was poured continuously to the top of the column by aid of a funnel. the bottom outlet of the column was opened. the eluates (fractions) were collected in separate test tubes. the column was eluted with solvents of different polarities, first eluted with petroleum ether (100%), followed by graded mixtures of petroleum ether 80% and chloroform 20%, chloroform 100%, graded mixtures of chloroform 80% and acetone 20%, acetone 100%, graded mixtures of acetone 80% and ethyl acetate 20%, ethyl acetate 100%, graded mixtures of ethyl acetate 80% and ethanol 20%, and ethanol 100%, in that order. the test tubes were changed after 10 ml of each fraction was collected, and thereafter all fractions were analyzed. the eluted components were monitored using acetone, chloroform, ethanol (4:4:1, v/v/v) as the mobile phase, and visualized under uv light as well as after derivatization with anisaldehyde sulfuric acid reagent followed by heating in oven for 15 min at 80 °c. the fractions were collected and the solvent was removed, then dried, weighed and analyzed. high performance thin layer chromatography (hptlc) hptlc studies were carried out using ethanolic extracts of the four ferns, where one hundred milligrams of four extracts were dissolved in 1 ml of ethanol. a number of solvent systems were tried, for further extraction of specific classes of chemical compounds. ethyl acetate‑ butanone‑ formic acid‑ water (5:3:1:1, v/v/v/v) was the most satisfactory solvent for flavonoids; toluene, acetone, formic acid (4.5:4.5:1, v/v/v) proved to be good for phenols; toluene‑ethyl acetate‑ formic acid‑ methanol (3:3:0.8:0.2, v/v/v/v) did well for tannins and n‑hexane, ethylacetate (7.2:2.9, v/v) proved to be good for terpenoids. chromatography was performed on silica gel 60 f254 tlc precoated plates using hamilton syringe and camag 11.40´´ e and altitude 1,250 m a.s.l.), a portion of the southern‑most part of western ghats, the peninsular kanyakumari district. identification of ferns and preparation of extracts specimens of the selected ferns were species‑identified based on report of different flora (manickam and irudayaraj 1992, beddome 1868‑1874). the voucher specimens of blechnum orientale linn. (spch 1004), dicranopteris linearis (burm.f ) underw. (spch 1005), marattia fraxinea (sm.) (spch 1006) and microlepia speluncae (l.) (spch 1007) were deposited at the herbarium of a.v.v.m. sri pushpam college. extracts were prepared as per the method already described (kalpana devi et  al. 2015). briefly, 100 g of the dry powder of each of the four ferns was extracted successively (1:10,  w/v) in petroleum ether (polarity index = 0.1) (b.p., 60‑80 °c), chloroform (polarity index  =  4.1), acetone (polarity index = 5.1) and ethanol (polarity index = 5.1) by cold maceration for 24 hours. the extracts were filtered through whatman #1 filter paper and dried in ika‑rv 10 digital rotary evaporator until constant dry weight of each extract. the extracts were stored aseptically at 4  °c until further use. in two more separate batches, the powdered plant material was extracted by cold maceration for 48 hours using sterile distilled water (polarity index  =  10.2) and ethanol (polarity index = 5.1) in a ratio of 1:10 (w/v). the mixture was filtered and centrifuged at 3,500 rpm for 20 minutes. the supernatant was filtered through whatman #1 filter paper followed by 0.2 μm membrane filter. the extract thus obtained was evaporated to dryness in ika‑rv 10 digital rotary evaporator and preserved aseptically at 4 °c until further use. phytochemical screening phytochemical screening of the fern extracts was carried out using different solvents based on increasing polarity index viz., non‑polar to most polar solvents successively (petroleum ether, chloroform, acetone, ethanol and water) to identify the major natural chemical groups (harborne 1998) such as tannins, saponins, flavonoids, phenols, terpenoids, alkaloids, quinones, glycosides, cardiac glycosides, coumarins, steroids, anthocyanin and beta‑cyanin. general reactions in these analyses revealed the presence or absence of the respective compounds in the fern extracts. total flavonoid content in the fern extracts was determined by aluminium chloride colorimetric method (mervat et  al. 2009), total phenolic content by folinciocalteau colorimetric method (slinkard and singleton 1956), tannin content by the method of fagbemi and colleagues (fagbemi et  al. 2005), and 274 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 more than five million references, for searching and matching the spectrum. only those compounds which had spectral fit values equal to or greater than 700 were considered positive identification. in vitro trematodicidal activity collection of worms adult live amphistomes were collected from the rumen of infected kilakarsal sheep breed, 10 months old slaughtered at orathanadu and pattukottai abbatoir of thanjavur district, tamilnadu, washed thoroughly in physiological saline and maintained in hedon‑fleig (hf) salt solution (ph 7.0), an ideal medium for in vitro maintenance of the amphistomes (veerakumari 1996). in  vitro incubation was performed within 1 h of collection. the worms were identified based on morphology (soulsby 1982). in vitro incubation study twenty five milliliters of hf salt solution containing increasing concentrations (1, 2, 3, 4 and 5 mg/ml) of the four fern extracts in ethanol, standard control (oxyclozanide 1%, i.e., 0.25 g/25 ml) and negative control (only hf salt solution) were individually distributed to petri‑dishes. twenty five amphistomes were incubated in each of the petri‑dishes. the motility of the parasites was observed visually at 5, 15, 30 and 60 min intervals. the motility response of the parasites was categorized as very active (+++), moderately active (++), sluggish (+) and dead (‑). a score index was made for the motility criteria as follows (jiraunkoorskul et al. 2005). very active ‑ score 3 – moving the whole body moderately active – score 2 – moving only parts of the body sluggish – score 1 – immobile but alive dead – score 0 statistical analysis the efficacies of the tested drugs against adult g. crumenifer were inferred based on calculated relative motility (rm) values using the formula as below (kiuchi et al. 1987). rm value = motility index (mi) = mi control mi test × 100 n ∑nn n = motility score, n = number of flukes with the score of n. relative motility assay was conducted for estimation and comparison of mean mortality and drug concentration (saowakon et  al. 2009, saowakon et al. 2013). linomat 5 instrument. 2 μl of standard solution and 2 μl of the test solution (extract) were loaded as 5 mm band length in the 4” × 10” glass plates at the distance of 10 mm from the edge of the plates, with the help of a camag liwomat 5 sample applicator. after the application of sample, the chromatogram was developed in twin trough glass chamber (10 × 10 cm) saturated with previously equilibrated mobile phase for 30 min. the chromatographic conditions were previously optimized to achieve the best resolution and peak shape. the air‑dried plates were viewed in ultraviolet radiation to mid‑day light. the chromatograms were scanned by densitometer at 420 nm after spraying with specific reagents like dragendorff ’s reagent for terpenoids, ethanolic aluminium chloride for flavonoids, sodium carbonate solution and folin‑ciocalteu reagent for phenols and ferric chloride reagent for tannins, respectively. phytol, quercetin, resorcinol, catechol, glycallic acid, hydroquinone and tannic acid were used as the reference standards. the plates were kept in photo‑documentation chamber (camag reprostar 3) and images were captured in white light, uv 254 nm and uv 366 nm. after derivatization, the plates were fixed in scanner stage (camag tlc scanner 3) and scanning was done at uv 366 nm, 254 nm and 500 nm. the peak table, peak display and peak densitogram were recorded. the retention factors (rf ) and % area were calculated by the wincats software. the tlc was run under laboratory conditions. the spots were quantified using a camag tlc scanner model 3 equipped with camag wincats software. gas chromatography‑mass spectrometry (gc‑ms) analysis the ethanolic extract of the ferns was further used for identification of bioactive compounds by gc-ms analysis. the trace gc ultra and dsqii model ms from thermo fisher scientific limited was engaged for the analysis (renuka 2014). the instrument was set as follows, injector port temperature set to 250  °c; interface temperature set to 250  °c; and source kept at 200  °c. the oven temperature was programmed as a variable, 70 °c for 2 min, 150 °c at 8  °c/min, up to 260  °c at 10  °c/min. split ratio was set as 1.50 and the injector used was splitless mode. the db‑35 ms non‑polar column was used, the dimensions of which were 0.25 mm od  ×  0.25  μm id  ×  30 m long, procured from agilent co., usa. helium was used as the carrier gas at 1 ml/min. the ms was set to scan from 50 to 650 da. the source was maintained at 200 ºc and < 40 motor vacuum pressure. the ionization energy was ‑ 70 ev. the ms was also having an inbuilt pre‑filter which reduced the neutral particles. the data system had two in‑built libraries, nist4 and wiley9, each containing 275 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 fraction ix (89.6%) in ms extracts showed the highest antioxidant value under column chromatography when compared to the standard butylated hydroxytoluene (bht). the column‑separated fractions of ms showed the highest antioxidant activity, followed by dl, mf and bo extracts. high performance thin layer chromatography the results of hptlc analysis of terpenoid, phenol, flavonoid and tannin profiles for the four fern extracts are presented in figure 2. the chromatogram using finger print profile for uv derivatization was scanned under uv 254 nm/ uv 366 nm/visible light. the peak table, peak display and peak densitogram are presented in figure 3. the hptlc results for terpenoids were compared using standard phytol. similarly, four standards were used for analysis of phenols (s2.1‑resorcinol, s2.2‑catechol, s2.3‑glycallic acid; s2.4‑hydroquinone). flavonoids were determined using quercetin as the standard. tannin profile was determined using tannic acid as the standard. the bands of bo, dl and ms were matching with the standard phytol as similar to the case of uv 366 nm. no band was noticed in mf extracts. this could be correlated with the peak densitogram which depicted the peak in rf values of bo, dl and ms, matching the phytol standard. the most prominent peak in rf value was present in ms extract. the hptlc analysis of phenols revealed that the bands of the four fern extracts were matching the four standards (s2.1, s2.2, s2.3 and s2.4) used under uv 254 nm and the densitogram showing the peak in rf values of the four fern extracts which were matching the standards used. the hptlc results qualitative and quantitative phytochemistry qualitative phytochemical analysis of extracts of the four fern plants, using the respective solvents, revealed that ethanol extracted out most of the phytochemicals present in bo and dl. acetone did the best in respect of mf and ms. among the 13  classes of phytochemicals studied, anthocyanin was absent in the extracts of all four ferns. glycosides were absent in mf and ms extracts. selected secondary metabolites which might contribute to the anthelmintic property (total flavonoids, total phenol, total tannin and total terpenoids) were quantitatively analyzed. the highest amount of total phenol (28.27 ± 0.18 mg gallic acid equivalent/g) was present in dl extracts. terpenoids and flavonoids were present in the highest concentrations in ms [terpenoids, 136.78  ±  1.32  mg/g; flavonoids (quercetin), 19.00 ± 0.58 mg/g] extracts. total tannin content was the highest in bo (34.93  ±  0.41  mg tannic acid equivalent/g) extracts. to sum up, dl and ms extracts contain higher amounts of terpenoids, tannins, phenols and flavonoids as compared to bo and mf (table i). column chromatographic fractions a total of 9 fractions were separated as eluents from bo extracts 13 each from dl and mf extracts and 16 from ms extracts under column chromatography (figure  1). among the eluted fractions, fraction  vi (64.4%) in bo extracts, fraction v (79.5%) in dl extracts, fraction iv (74.01%) in mf extracts and table i. percentage yield, total terpenoids, total phenolics, total flavonoids and tannin content of various whole fern extracts. sample percentage yield (w/w) total terpenoids (mg/g) total phenolics (mg gae/g) total flavonoids (mg qe/g) tannins (mg tae/g) bo dl mf ms bo dl mf ms bo dl mf ms bo dl mf ms bo dl mf ms pe 3.23 6.67 3.05 4.08 6.32 ± 0.24 1.41 ± 0.26 3.23 ± 0.17 1.11 ± 0.29 2.44 ± 0.22 0.97 ± 0.03 2.74 ± 0.13 0.74 ± 0.14 1.10 ± 0.05 ch 4.45 6.97 2.60 5.28 1.22 ± 0.13 2.17 ± 0.09 7.70 ± 0.17 2.28 ± 0.36 1.89 ± 0.20 0.70 ± 0.05 2.13 ± 0.08 0.71 ± 0.15 0.77 ± 0.15 1.50 ± 0.25 ac 30.77 34.75 32.75 40.00 73.02 ± 0.10 90.87 ± 0.47 20.58 ± 0.42 138.18 ± 0.40 0.82 ± 0.12 20.75 ± 0.38 6.43 ± 0.23 18.40 ± 0.25 7.38 ± 0.19 7.72 ± 0.13 3.13 ± 0.07 21.23 ± 0.12 14.28 ± 0.17 18.42 ± 0.30 1.30 ± 0.15 8.13 ± 0.35 et 33.81 39.60 31.97 38.71 79.05 ± 0.33 97.66 ± 1.08 16.83 ± 0.83 136.78 ± 1.32 1.43 ± 0.16 28.27 ± 0.18 5.27 ± 0.13 17.23 ± 0.38 8.30 ± 0.36 8.33 ± 0.17 1.25 ± 0.14 19.00 ± 0.58 34.93 ± 0.41 30.27 ± 0.27 7.41 ± 0.26 19.97 ± 0.26 wa 12.08 13.18 9.60 10.03 6.28 ± 0.21 8.17 ± 0.09 0.69 ± 0.16 17.15 ± 0.15 3.45 ± 0.26 11.26 ± 0.14 4.72 ± 0.11 5.51 ± 0.26 1.15 ± 0.10 16.44 ± 0.17 2.88 ± 0.48 values were obtained in triplicates and represented as mean ± se. pe = petroleum ether; ch = chloroform; ac = acetone; et = ethanol; wa = water; bo = b. orientale; dl = d. linearis; mf = m. fraxinea; ms = m. speluncae. 276 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 with wiley 9.0 and national institute of standards and technology libraries. the major compounds detected were 1,2‑benzenedicarboxylic acid and 1,2,3‑propanetricarboxylic acid 2‑hydroxy‑triethyl ester (cas). the compounds responsible for anthelmintic activity in bo extract were quercetin 7´,3,´4´‑ trimethoxy, a flavonoid derivative (polyphenols group) (0.13%) and phytol isomer, a diterpenoid (0.53%). in dl extract, the compounds for anthelmintic activity were phytol, a diterpenoid (0.37%), quercetin 7´,3,´4´‑trimethoxy, a flavonoid derivative (polyphenols group) (0.20%), and fern‑8‑ene, a triterpenoid (0.40%). similarly, in mf extract the compounds responsible for anthelmintic activity were phytol (0.44%), xanthorrhizol, a terpenoid (0.69%), and fern‑9(11)‑ene, a fernane type triterpenoid (2.83%). in the case of ms extract the compounds responsible for anthelmintic activity were phytol (5.67%), quercetin 7´,3,´4´‑trimethoxy (0.23%), and fern‑8‑ene, a triterpenoid (2.55%). analysis for flavonoids showed the band width of fern extracts bo, dl and ms to match the standard quercetin. the densitogram peak in rf value was in accordance with the chromatogram studied under uv 254 nm. hptlc analysis of tannin profile as compared to standard tannic acid revealed close matching of band width in dl and bo extracts against the standard tannic acid and partial matching with ms extracts. however, for mf extract the band was negligible and not matching, and was in accordance with the rf value under densitogram graphical representation. gas chromatography ‑ mass spectrometry analysis the bioactive compounds present in the ethanolic extract of the four ferns were identified by gc‑ms analysis (figure 4). six compounds were detected in the extract of bo, seven in dl, eight in mf and ten in ms. the spectra of the compounds were matched a. blechnum orientale b. dicranopteris linearis c. marattia fraxinea d. microlepia speluncae 1 .2 7 9 8 .3 1 5 .1 1 5 .3 1 0 .4 2 5 .6 2 8 .3 6 4 .4 3 4 .4 4 7 .3 4 0 .7 0 10 20 30 40 50 60 70 80 90 100 c o n tr o l b h t s td fr ac ti o n i fr ac ti o n ii fr ac ti o n ii i fr ac ti o n iv fr ac ti o n v fr ac ti o n v i fr ac ti o n v ii fr ac ti o n v iii fr ac ti o n ix a n ti o xi d an t a ct iv it y (% ) eluted fractions 1 .2 7 9 8 .3 4 1 .5 6 4 .5 6 7 .5 6 9 .3 7 9 .5 6 6 .3 5 4 .4 5 8 .1 5 1 .8 3 2 .7 3 4 .7 4 3 .4 2 9 .4 0 10 20 30 40 50 60 70 80 90 100 c o n tr o l b h t s td fr ac ti o n i fr ac ti o n ii fr ac ti o n ii i fr ac ti o n iv fr ac ti o n v fr ac ti o n v i fr ac ti o n v ii fr ac ti o n v iii fr ac ti o n ix fr ac ti o n x fr ac ti o n x i fr ac ti o n x ii fr ac ti o n x iii a n ti o xi d an t a ct iv it y (% ) eluted fractions 1 .2 7 9 8 .3 4 8 .0 4 9 .6 5 4 .3 7 4 .0 5 8 .6 5 3 .2 4 1 .6 3 5 .3 4 4 .9 3 2 .4 4 6 .3 4 3 .4 3 8 .7 0 10 20 30 40 50 60 70 80 90 100 c o n tr o l b h t s td fr ac ti o n i fr ac ti o n ii fr ac ti o n ii i fr ac ti o n iv fr ac ti o n v fr ac ti o n v i fr ac ti o n v ii fr ac ti o n v iii fr ac ti o n ix fr ac ti o n x fr ac ti o n x i fr ac ti o n x ii fr ac ti o n x iii a n ti o xi d an t a ct iv it y (% ) eluted fractions 1 .2 7 9 8 .3 2 8 .0 3 0. 0 4 2 .6 3 5 .3 4 8 .2 4 0 .1 5 3 .1 6 1 .5 8 9 .6 3 8 .2 4 5 .6 4 6 .7 2 7 .4 2 4 .7 2 1 .6 1 8 .5 0 10 20 30 40 50 60 70 80 90 100 c o n tr o l b h t s td fr ac ti o n i fr ac ti o n ii fr ac ti o n ii i fr ac ti o n iv fr ac ti o n v fr ac ti o n v i fr ac ti o n v ii fr ac ti o n v iii fr ac ti o n ix fr ac ti o n x fr ac ti o n x i fr ac ti o n x ii fr ac ti o n x iii fr ac ti o n x iv fr ac ti o n x v fr ac ti o n x v i a n ti o xi d an t a ct iv it y (% ) eluted fractions figure 1. comparative analysis of various fractions of fern extracts eluted from column chromatography. 277 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 a. terpenoids b. phenols c. flavonoids d. tannins uv 254 nm uv 366 nm visible light bo mf dl ms s1.1 bo mf dl ms s1.1 bo mf dl ms s1.1 uv 254 nm uv 366 nm visible light uv 254 nm uv 366 nm visible light uv 254 nm uv 366 nm visible light bo mf dl ms s2.1 s2.2 s2.3 s2.4 bo mf dl ms s2.1 s2.2 s2.3 s2.4 bo mf dl ms s2.1 s2.2 s2.3 s2.4 bo bomf mfdl dlms mss3.1 s3.1 bo mf dl ms s3.1 bo bomf mfdl dlms mss4.1 s4.1 bo mf dl ms s4.1 a. terpenoids b. phenols c. flavonoids d. tannins no mortality during the entire 60 min incubation. dl extract (table iii) also, at 5 mg/ml concentration, produced drug effect comparable to the standard, killing all flukes at 10 min incubation; at the lesser concentrations (1,2,3,4 mg/ml) the extracts were equally effective killing all flukes at varied incubation times at rates proportional to drug concentration in a manner dose‑dependent. extract of mf (table  iv) revealed poor efficacy at 5 mg/ml concentration it took almost 60 min incubation to kill all the flukes, in vitro trematodicidal property of the pteridophyte extracts in  vitro incubation study of bo extract (table  ii) showed that 5 mg/ml concentration would kill all the worms at 10 min incubation, the same as the standard control (oxyclozanide). on the other hand 2, 3, 4 mg/ml concentrations caused only partial mortality even at 60 min incubation. when incubated in the extract at 1 mg/ml concentration, there was figure 2. high performance thin layer chromatography chromatogram using finger printing profile. bo = b. orientale; dl = d. linearis; mf = m. fraxinea; ms = m. speluncae; s1.1 = standard (phytol); s2.1 = standard (resorcinol); s2.2 = standard (catechol); s2.3 = standard (glycallic acid); s2.4 = standard (hydroquinone); s3.1 = standard (quercetin); s4.1 = standard (tannic acid). figure 3. densitograms of high performance thin layer chromatograms (hptlc). bo = b. orientale; dl = d. linearis; mf = m. fraxinea; ms = m. speluncae; s1.1 = standard (phytol); s2.1 = standard (resorcinol); s2.2 = standard (catechol); s2.3 = standard (glycallic acid); s2.4 = standard (hydroquinone); s3.1 = standard (quercetin); s4.1 = standard (tannic acid). 278 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 c. marattia fraxinea d. microlepia speluncae a. blechnum orientale a 1  = dodecane; a 2  = 1,2,3-propane-tricarboxylic acid; a 3  = 1,2-benzenedicarboxylic acid; a 4  = phytol isomer; a 5  = hexanedioic acid; a 6  = quercetin 7’3’4’-trimethoxy 3-benzoyl-4-methyl-6-ethyl-2(1h)-pyridone. b. dicranopteris linearis b 1  = furan (cas); b 2  = dodecane; b 3  = 1,2,3-propane-tricarboxylic acid b 4  = 1,2-benzenedicarboxylic acid; b 5  = phytol; b 6  = quercetin 7’3’4’-trimethoxy hexadecanoic acid; b 7  = fern-8-ene. c 1  = 1-hexanol; c 2  = dihydrothiophene; c 3  = 1,2,3-propane-tricarboxylic acid; c 4  = 2-methyl-5-(1,2,2-trimethylcyclopentyl)-,(s)(cas); c 5  = dibutyl phthalate; c 6  = 2-hexadecan-1-ol; c 7  = hexanedioic acid; c 8  = fern-9(11)-ene. d 1  = 1-hexanol; d 2  = 2,3-dihydro-benzofuran; d 3  = 1-h-indene-4-carboxylic acid; d 4  = 1,2,3-propane-tricarboxylic acid; d 5  = 4-naphthoquinone, iso-vellaral; d 6  = dibutyl phthalate; d 7  = phytol isomer; d 8  = hexanedioic acid; d 9  = quercetin 7,3’,4’-trimethoxy, fern-8-ene; d 10  = ergost-5.en-3-ol,(3a)-(cas) campesterol. figure 4. gas chromatographic and mass spectrometric analysis of pteridophytes. 279 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 table iii. motility index and relative motility values of g. crumenifer on various time points of exposure to d. linearis extract. conc (mg/l) 0 min 10 min 15 min 30 min 60 min replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sdmi rm mi rm mi rm mi rm mi rm 0 (nc) 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.003.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 1 3.00 1.00 1.00 ± 0.00 2.28 0.76 0.75 ± 0.019 1.96 0.65 0.62 ± 0.018 1.40 0.47 0.48 ± 0.058 0.00 0.00 0.003.00 1.00 2.12 0.71 1.84 0.61 1.44 0.48 0.00 0.00 3.00 1.00 2.32 0.77 1.76 0.59 1.48 0.49 0.00 0.00 2 3.00 1.00 1.00 ± 0.00 1.92 0.64 0.62 ± 0.017 1.44 0.48 0.48 ± 0.006 0.92 0.31 0.28 ± 0.018 0.00 0.00 0.003.00 1.00 1.92 0.64 1.40 0.47 0.80 0.27 0.00 0.00 3.00 1.00 1.76 0.59 1.48 0.49 0.76 0.25 0.00 0.00 3 3.00 1.00 1.00 ± 0.00 1.48 0.49 0.49 ± 0.012 0.72 0.24 0.21 ± 0.032 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 1.40 0.47 0.44 0.15 0.00 0.00 0.00 0.00 3.00 1.00 1,52 0.51 0.76 0.25 0.00 0.00 0.00 0.00 4 3.00 1.00 1.00 ± 0.00 0.40 0.13 0.12 ± 0.013 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.28 0.09 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.40 0.13 0.00 0.00 0.00 0.00 0.00 0.00 5 3.00 1.00 1.00 ± 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 oxy (sc) 3.00 1.00 1.00 ± 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 nc = negative control; sc = standard control; mi = motility index; rm = relative motility. table ii. motility index and relative motility values of g. crumenifer on various time points of exposure to b. orientale extract. conc (mg/l) 0 min 10 min 15 min 30 min 60 min replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sdmi rm mi rm mi rm mi rm mi rm 0 (nc) 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.003.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 1 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.003.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 2 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 2.16 0.72 0.74 ± 0.045 1.16 0.39 0.56 ± 0.0833.00 1.00 3.00 1.00 3.00 1.00 2.04 0.68 1.92 0.64 3.00 1.00 3.00 1.00 3.00 1.00 2.48 0.83 1.92 0.64 3 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 2.88 0.96 0.82 ± 0.143 2.16 0.72 0.69 ± 0.013 1.16 0.39 0.45 ± 0.0993.00 1.00 3.00 1.00 1.60 0.53 2.04 0.68 1.92 0.64 3.00 1.00 3.00 1.00 2.88 0.96 2.04 0.68 0.92 0.31 4 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 1.72 0.57 0.58 ± 0.032 0.92 0.31 0.27 ± 0.026 0.52 0.17 0.18 ± 0.0153.00 1.00 3.00 1.00 1.60 0.53 0.84 0.28 0.64 0.21 3.00 1.00 3.00 1.00 1.92 0.64 0.88 0.22 0.48 0.16 5 3.00 1.00 1.00 ± 0.00 0.80 0.28 0.25 ±0.017 0.36 0.12 0.12 ± 0.003 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.76 0.25 0.40 0.13 0.00 0.00 0.00 0.00 3.00 1.00 0.88 0.22 0.36 0.12 0.00 0.00 0.00 0.00 oxy (sc) 3.00 1.00 1.00 ± 0.00 0.16 0.05 0.04 ± 0.003 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.12 0.04 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.12 0.04 0.00 0.00 0.00 0.00 0.00 0.00 nc = negative control; sc = standard control; mi = motility index; rm = relative motility. 280 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 table v. motility index and relative motility values of g. crumenifer on various time modes of exposure to m. speluncae extract. conc (mg/l) 0 min 10 min 15 min 30 min 60 min replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sdmi rm mi rm mi rm mi rm mi rm 0 (nc) 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.003.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 1 3.00 1.00 1.00 ± 0.00 2.28 0.76 0.72 ± 0.021 2.00 0.67 0.63 ± 0.021 1.40 0.47 0.44 ± 0.020 0.00 0.00 0.003.00 1.00 2.14 0.71 1.80 0.60 1.20 0.40 0.00 0.00 3.00 1.00 2.06 0.69 1.86 0.62 1.32 0.44 0.00 0.00 2 3.00 1.00 1.00 ± 0.00 1.92 0.64 0.61± 0.015 1.44 0.48 0.47 ± 0.009 0.92 0.31 0.31 ± 0.00 0.00 0.00 0.003.00 1.00 1.76 0.59 1.34 0.45 0.94 0.31 0.00 0.00 3.00 1.00 1.80 0.60 1.44 0.48 0.92 0.31 0.00 0.00 3 3.00 1.00 1.00 ± 0.00 1.48 0.49 0.51 ± 0.012 0.72 0.24 0.22 ± 0.012 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 1.54 0.51 0.60 0.20 0.00 0.00 0.00 0.00 3.00 1.00 1.60 0.53 0.62 0.21 0.00 0.00 0.00 0.00 4 3.00 1.00 1.00 ± 0.00 0.52 0.17 0.17 ± 0.003 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.52 0.17 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.48 0.16 0.00 0.00 0.00 0.00 0.00 0.00 5 3.00 1.00 1.00 ± 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 oxy (sc) 3.00 1.00 1.00 ± 0.00 0.20 0.07 0.05 ± 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.12 0.04 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.12 0.04 0.00 0.00 0.00 0.00 0.00 0.00 nc = negative control; sc = standard control; mi = motility index; rm = relative motility. table iv. motility index and relative motility values of g. crumenifer on various time modes of exposure to m. fraxinea extract. conc (mg/l) 0 min 10 min 15 min 30 min 60 min replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sd replicates mean rm ± sdmi rm mi rm mi rm mi rm mi rm 0 (nc) 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.003.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 3.00 1.00 1 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 2.96 0.99 0.97 ± 0.009 1.96 0.65 0.64 ± 0.007 1.16 0.39 0.41 ± 0.0123.00 1.00 3.00 1.00 2.88 0.96 1.88 0.63 1.28 0.43 3.00 1.00 3.00 1.00 2.92 0.97 1.96 0.65 1.20 0.40 2 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 2.84 0.95 0.92 ± 0.024 1.56 0.52 0.51 ± 0.013 1.04 0.35 0.33 ± 0.0093.00 1.00 3.00 1.00 2.80 0.93 1.56 0.52 0.96 0.32 3.00 1.00 3.00 1.00 2.60 0.87 1.44 0.48 1.00 0.33 3 3.00 1.00 1.00 ± 0.00 3.00 1.00 1.00 ± 0.00 2.76 0.92 0.88 ± 0.021 1.16 0.39 0.37 ± 0.007 0.60 0.20 0.19 ± 0.0153.00 1.00 3.00 1.00 2.60 0.87 1.08 0.36 0.48 0.16 3.00 1.00 3.00 1.00 2.56 0.85 1.12 0.37 0.64 0.21 4 3.00 1.00 1.00 ± 0.00 1.84 0.61 0.59 ± 0.011 1.32 0.44 0.43 ± 0.003 0.56 0.19 0.17 ± 0.007 0.20 0.07 0.06 ± 0.0073.00 1.00 1.72 0.57 1.28 0.43 0.52 0.17 0.20 0.07 3.00 1.00 1.76 0.59 1.28 0.43 0.48 0.16 0.16 0.05 5 3.00 1.00 1.00 ± 0.00 1.44 0.48 0.46 ± 0.012 0.84 0.28 0.25 ± 0.017 0.12 0.04 0.04 ± 0.006 0.00 0.00 0.003.00 1.00 1.32 0.44 0.76 0.25 0.08 0.03 0.00 0.00 3.00 1.00 1.40 0.47 0.88 0.22 0.16 0.05 0.00 0.00 oxy (sc) 3.00 1.00 1.00 ± 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 nc = negative control; sc = standard control; mi = motility index; rm = relative motility. 281 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 thus, alcohol (ethanol/methanol) is proven as the best solvent for fern species to extract out the chemical compounds as a crude mixture. the abundance of chemical classes in our ferns is in the order ms > dl > bo > mf. all four pytochemical classes, selectively analyzed quantitatively, are present in all four fern species studied‑ terpenoids and flavonoids forming the predominant chemicals in ms, phenol in dl, and terpenoid and tannin in bo and ms. however, all four classes are the lowest in mf, indicating that mf would be poorly rewarding as a plant of pharmacological importance whereas the other three ferns would prove to be good (lacaille‑dubois and wagner 2000). irrespective of species, terpenoids are chemicals of greatest abundance, followed by tannins, flavonoids and phenols, in that order. an earlier study of quantitative analysis of the extract of pteris species showed flavonoid content to be the highest followed by alkaloids and phenolic compounds (gracelin et  al. 2013). terpenoids, also known as isoprenoids, are the most numerous and structurally diverse natural products found in plants. several studies, in  vitro, preclinical and clinical, have confirmed that this class of compounds possesses a wide array of very important pharmacological properties (ludwiczuk et al. 2017). saponins, as a group, include compounds that are glycosylated steroids, triterpenoids and steroidal alkaloids. many saponins are known antimicrobials, inhibit moulds, and protect plants from insect attack. saponins may constitute an aspect of defence systems of plants and, hence, they are phytoanticipins or phytoprotectants (ludwiczuk et  al. 2017). these structurally diverse compounds have also been observed to kill protozoans and helminths, to be antioxidants and also act as antifungals and antivirals (takechi et al. 1999). intake of a small quantity of the correct kind of tannin may be beneficial to human health by affecting the metabolic enzymes, immunomodulation or other functions (chung et  al. 1998). phenolic compounds also possess a diverse range of beneficial biological activities (huang and cai 2010). flavonoids are directly associated with human dietary ingredients and health. prevention and cure of diseases using phytochemicals, especially flavonoids, are well known (kumar and pandey 2013). ferns, available in plenty and have little if any economic value, are herein proven to be a rich source of terpenoids, tannins, phenolic compounds and flavonoids which can be applied in human and animal health. hptlc analysis of the four fern extracts showed evidence of matching with respective standards, phytol for terpenoids, tannic acid for tannins, quercetin for flavonoids and s1‑resorcinol, s2‑catechol, s3‑glycallic acid and s4‑hydroquinone for phenols. it was a noteworthy feature that terpenoids, phenols, flavonoids, and tannins as compared to the standard which killed all flukes at 10 min or less incubation, and at the lower concentrations (1,2,3,4 mg/ml) the extract was not effective at all. the efficacy of extract of ms (table v) was almost equivalent to that of dl extract, killing all flukes at 10 min or less at 5 mg/ml concentration. the efficacies of all other drug concentrations were proportionate to the durations of incubation, lesser the concentration more the time. discussion phytochemical analysis the first outcome of this study is understanding of efficiency of the five solvents, water, ethyl acetate, chloroform, petroleum ether and acetone, in the decreasing order of polarity index, used for extraction of the phytochemical classes wherein alcohol as the general organic solvent extracts out all phytochemicals from all the four ferns, followed by acetone, ethyl acetate, water, chloroform and petroleum ether, in that order. looking at the number of chemical classes or extent of extraction, ethanol performed well with respect to bo and dl whereas acetone performed well in the case of mf and ms. this differential efficiency of extraction greatly depends on polarity of the solvent, solubility of the phytochemical of interest, and the physiological/ biochemical status of the fern species reflecting on number and variety of phytochemicals. this has been the pointer of many earlier studies of phytochemical analysis of ferns. for e.g., phytochemical screening of acetone, benzene, chloroform, ethanol, petroleum ether and aqueous extracts of whole plants of blechnum orientale, ceratopteris thalictroides, drymoglossum heterophyllum, dicranopteris linearis, hemionitis arifolia, lindsaea ensifolia, nephrolepis multiflora, pityrogramma calomelanos, and pteris confuse, and leaves and rhizomes of drynaria quercifolia, in all 66  extracts, revealed presence or absence of specific classes of compounds (mithraja et  al. 2012a). exclusive studies of d.  quercifolia adopting extraction in all these media, though with emphasis on antimicrobial (mithraja et al. 2012b) and anti‑arthritic (saravanan et  al. 2013) applications, also support this inference. phytochemical analysis of the ferns actinopteris radiata, drynaria quercifolia, dryopteris cochleata and pityrogramma calomelanos indicated that ethanol was the most efficient solvent with strong positivity for the major phytochemicals tested (kalpana devi et  al. 2014). qualitative and quantitative phytochemical analysis in pteris argyreae, pteris confusa, pteris vittata, pteris biaurita and pteris multiaurita revealed methanol extract to prove to be positive for most phytochemicals of interest (de britto et  al. 2012, gracelin et  al. 2013). 282 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 antioxidants. it was indicated that the presence of terpenoids (phytol and fern‑8‑ene), and flavonoids (quercetin  7,3´,4´‑trimethoxy) in the fern extracts would account for action as antioxidant and anthelmintic. tannins interfere with energy metabolism of worms by uncoupling oxidative phosphorylation or they bind to free proteins of the gastrointestinal tract of the host animal or glycoprotein on the cuticles of the worms and lead to death (patel et  al. 2010). alkaloids might act on central nervous system and cause paralysis. steroidal alkaloids and oligoglycosides are present in alkaloids, which might suppress transfer of sucrose from stomach to small intestine. alkaloids act as antioxidants capable of reducing nitrate generation which would interfere in local homeostasis that is essential for the embryonic development of helminths. saponins affect permeability of the cell membrane of parasites and cause vacuolization and disintegration of teguments (melzig et al. 2001). isoflavonones inhibit the enzymes of glycolysis and glycogenolysis and disrupt ca2+ homeostasis and no activity in the parasites (stepek et  al. 2006). in the present study, the phytochemical components under hptlc analysis provided substantial evidence of presence of terpenoids, phenols, tannins and flavonoids in the ferns which leads to understanding of the pharmacological and/or toxicological actions of the plants of interest (mukherjee et al. 2007). plant secondary metabolites such as saponins, alkaloids, non‑protein amino acids, tannins and other polyphenols, that are present on our ferns, must be responsible for the antiparasitic effect. there is a report connecting anthelmintic and free radical scavenging potentials of glinus oppositifolius associating with the polyphenols (bhaskar et al. 2012). terpenoids are also secondary plant metabolites that hold both antioxidant and anthelmintic properties (grassmann 2005). deriving thereupon, the phenols and terpenoids present in our fern extracts correlate with the antioxidant potential and anthelmintic activity. gc‑ms analysis revealed the presence of 6  compounds in bo, 7 in dl, 8 in mf and 10 in ms extracts. these compounds belong to different classes and so would possess varied biological activities. the major groups of compounds include terpenoids, flavonoids (polyphenols) and fatty acid derivatives. the secondary metabolites that would be responsible for the anthelmintic activity in bo would be quercetin 7´,3,´4´ trimethoxy, a flavonoid derivative (polyphenols group), and phytol isomer, a diterpenoid. in dl the compounds with anthelmintic activity would be phytol, quercetin 7´,3,´4´‑trimethoxy, and fern‑8‑ene, a triterpenoid. the compounds in mf that are responsible for contribute to anti‑trematodal and antioxidant drug action either independently of each other or in a combined effect of phytoconstituents in the pteridophytes. the important phytoconstituents (terpenoids, phenols, flavonoids, and tannins) under quantitative phytochemical analysis were confirmed and proved to be effective trematodicidal and antioxidant compounds flavonoids are pigments present in almost all terrestrial plants with a high spectrum of positive effects on plant and mammalian cells in relation to therapeutic applications. their ability to inhibit specific enzymes to stimulate some hormones and neurotransmitters, and to scavenge free radicals, has been established. it is well known that some flavonoids inhibit or kill many bacterial strains. chemical analyses of plant extracts which showed high anthelmintic activity revealed the presence of flavonoids, along with other classes of phytochemicals. although toxicity of most isolated flavonoids to animal cells is very low (middleon et  al. 2000), several ubiquitous flavonoids such as genistein, kaemferol, rutin, quercetin, etc., produced deleterious effects on selected species of parasitic helminths. it is possible that different developmental stages (larva, juvenile or adult) of helminths might be differently susceptibile to selected flavonoids (hrckova and velebny 2013). the phenolic diketone curcumin and the flavonoid kaempferol exerted a strong adulticidal effect on schistosoma mansoni, but no activity against nematodes was demonstrated. quercetin, a flavonoid that belongs to polyphenolic derivatives, possesses both antioxidant and anti‑parasitic activities (dupuy et al. 2003). kaempferol (flavonol) and its three derivatives were isolated from two plant species of styrax camporum and styrax pohlii (braguine et al. 2012), and examined in vitro. kaempferol was most effective in separating s. mansoni male and female couples and killing adult worms at a concentration of 100 µg/ml. the selective toxicity of the flavones quercetin, chrysin, and 3‑hydroxyflavone toward the several cancer cell lines but not normal mammalian cells is worth mentioning (pilatova et  al. 2010). phenolic compounds interfere with energy generation metabolism by uncoupling the oxidative phosphorylation and also interfere with the glycoprotein of the cell surface of the parasites and cause death. polyphenols are potent antioxidants and very good anthelmintics. both phenols and terpenoids are promising anthelmintics; the mechanism of action of these phytoterapeutics is multi‑targeted and would act against the helminth parasites through diverse spectra of action (mukherjee et  al. 2016). they would also prevent and promote apoptosis and disturb redox status and therefore are potent 283 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 potential against intestinal nematodes, cestodes, and trematodes. the active compounds were extracted into either chloroform, water, or polyethylene glycol/propylene carbonate (peg/ pc) and were examined on the cestode models hymenolepis diminuta, hymenolepis microstoma, taenia taeniaeformis, and the trematode models fasciola hepatica and echinostoma caproni; most of the extracts were anthelmintic in  vivo (abdel‑ghaffar et  al. 2011). treatment of infected animals with a combination of onion and coconut extracts in peg/pc eliminated all cestodes. the same composition of extracts was effective against e. caproni but failed to kill the liver fluke f. hepatica in the final hosts. validation of antiparasitic activities of compounds obtained from plant extracts requires standardization of methodologies and this issue is discussed in a review, wherein the authors focussed on the strengths and weaknesses of the existing methodologies used in the control and also discussed issues like the seasonal variability of the plant composition and how this can influence their antiparasitic properties (athanasiadou et  al. 2001). therefore, it is important to isolate and identify the phytochemicals and decipher their mechanisms of action in the parasites with respect to unique molecular targets and physiological pathways. essential oils are volatile, natural, complex odoriferous secondary metabolites produced by aromatic plants. essential oils being very complex mixtures can contain 20-60 components at quite different concentrations. they are characterized by 2‑3 major compounds at fairly high concentrations (20‑70%) compared to others components that are present only in trace amounts. the major compounds are terpenes (e.g., geraniol, carvacrol, thymol, cymene, sabinene, alpha‑pinene, betapinene, citronellol, sesquiterpene farnesol) or terpenoids (menthol, ascaridole), and the other aromatic and aliphatic compounds, characterized by low molecular weight (e.g., cinnamyl alcohol, eugenol, safrole). in general, the many compounds determine the biological properties of the particular essential oil and it is likely that they interact with several targets in the cells. the tegument of cestodes and trematodes is morphologically and physiologically different from the cuticule of nematodes. therefore, the essential oils are likely to pass through the layers of tegument. oils of spices, such as nigella sativa (black cumin), allium sativum (garlic) and piper longum (indian long pepper) have been many a times shown to be trematodicidal, based on studies in schistosoma mansoni and fasciola gigantica (mahmoud et al. 2002, singh et al. 2009). this provides support for our inference that the oils of the ferns in this study also would possess trematodicidal property. anthelmintic action would be phytol, xanthorrhizol, a terpenoid, and fern‑9(11)‑ene, a fernane type triterpenoid. the compounds in ms responsible for anthelmintic action would be phytol, quercetin 7´,3,´4´‑trimethoxy, and fern‑8‑ene. hexadecanoic acid dioctyl ester (cas), one of the major compounds detected through gc‑ms analysis, is a palmitic acid derivative. it is known for various biological functions such as antioxidant, flavouring agent, pesticide, lubricant, antiandrogenic, haemolytic, alpha reductase inhibitor, anti‑inflammatory, hypocholesterolemic, nematicide, insectifuge and anti‑bacterial (cowan 1999). the abundant flavonoids might be responsible for their therapeutic effectiveness against a wide array of microorganisms, probably due to their ability to complex with extracellular and soluble proteins and to complex with the bacterial cell wall. flavonoids and other phenolic compounds are potent water soluble antioxidants and free radical scavengers, which prevent oxidative cell damage, have strong anticancer activity, and anthelmintic activity (talukdar et  al. 2011). quercetin 7, 3´, 4´ trimethoxy present in these ferns, as revealed in the gc‑ms analysis, might be attributed with significant antioxidant property (lee et al. 2005). phytol also would possess antioxidant property (dukes). recently, in  vitro larvicidal and ovicidal activity of leaf and seed extracts of solanum torvum was discovered. it is antiparasitic to some hematophagous parasites of cattle and goat and also against a digenean fluke of sheep, paramphistomum cervi. several studies have assigned antiparasitic effect of medicinal plants to the alkaloids (athanasiadou and kyriazakis 2004). similarly, anthelmintic effect in  vitro of tropical plants lysimachia ramosa, olea europaea, and satureja khuzestanic has been reported (challam et  al. 2010). adult trematode fasciolopsis buski, nematode ascaris suum and cestode raillietina echinobothrida were exposed to alcoholic extract of lysimachia ramosa wall. the treated parasites were completely inactivated through loss of motility/ flaccid paralysis and deformity of the surface architecture, ultimately leading to death. the pharmacologically active components responsible for these effects are triterpenoids, saponins, organic acids, and flavones, from different species of the genus lysimachia. the terpenoids, phenols, fatty acids and flavonoids revealed in the gc‑ms analysis could be responsible for the anti‑parasitic action and antioxidant property. these form a good combination of secondary metabolites showing efficient action against trematodes. this is to be viewed in the background that ingredients in human food, such as coconut, onion, garlic, fig, date, pineapple, chicory, etc., possess high anthelmintic 284 in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes kalpana et al. veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 means of entry of non‑nutrient and non‑electrolyte substances into nematode, cestode and trematode parasites as opposed to oral ingestion (alvarez et  al. 2007). possible explanation for the good anthelmintic activity of ethanolic extract could be easier transcuticular absorption of ethanolic extract into the body. our study has revealed for the first time, to the best of our knowledge, broad anthelmintic activity of all four ferns relative to the efficacy of oxyclozanide, a salicylanilide anthelmintic. naturally occurring combinations of plant compounds in crude extracts would act synergistically and often produce appreciable antimicrobial activity compared to purified individual compounds. such plant‑based treatments could be made part of an integrated management strategy for control of helminths in developing countries. further studies are required to isolate and identify the active compound contained in the crude extracts so as to establish the mechanism of action. the present study used oxyclozanide, which by mechanism of action uncouples oxidative phosphorylation, as the standard drug and compared the response in g. crumenifer, as the trematode model, and compared the effect of treatment with extracts of the four selected ferns. it was found that the worm succumbed to death following complete paralysis at all concentrations tested. but 100% mortality (rm value 0) was accomplished at the highest concentration with less incubation time, effect comparable to that of the standard control. it was proved that dl and ms extracts produce the strongest trematodicidal action, at the highest concentration, 5 mg/ml (rm value 0), killing all the worms in the group at 10 min incubation time. however, bo extracts produced only moderate trematodicidal action at the same dose since all worms in the group were killed at 30 min incubation time. mf produced the least trematodicidal action whereupon all worms in the group were killed at this concentration at 60 min incubation time. overall, the in  vitro incubation study for anti‑trematodal property of the ferns revealed dl and ms extracts to produce the strongest trematodicidal action followed by bo and mf extracts. looking at the statistical interpretation for assessment of mi and rm value obtained at 5 mg/ml concentration, the rm value 0 (100% mortality) was achieved at 30 min for bo, 10 min for dl, 60 min for mf and 10 min for ms. the statistical analysis further revealed the action of fern extracts as dose‑dependent and directly proportional to the dose. among the four trials of fern extracts, the standard control (oxyclozanide) produced 100% mortality (rm value 0) at 10 min incubation time. a drug, which would produce adverse effects in a parasite, sparing the host, is a potential anthelmintic drug. anthelmintic drugs impair the vital activities in vitro anti‑trematodal property of the pteridophytes various active components of plant extracts have been tested for anthelmintic activity wherein it has been shown that tegument of trematodes and cestodes represents a potential target site since the small molecules could be adsorbed on to it (athanasiadou and kyriazakis 2004). the secondary metabolites, especially plant alkaloids, act on adult stage of flukes producing anti‑trematodal effects in cattle, sheep and goat. several studies have shown that extracts of botanical foods and spices possess high anthelmintic potential against intestinal nematodes, cestodes and trematodes (abdel‑ghaffar et al. 2011). our study shows the ferns to be effective in dealing with flukes of ruminants. anthelmintics are drugs that cause adverse effects on the helminth parasites by affecting vital activities such as feeding, neuromuscular transmission, ion exchange or the tegument (shokier et al. 2013). the normal phytochemicals present in the plant extracts act in manner similar to the mechanisms exhibited by conventional anthelmintics. the saponins interact with the cell membranes, causing changes in the cell membranes, and subsequent changes in the cell wall (radwan et al. 2012). tannins have the capacity to bind to proteins and impair vital processes like feeding, and reproduction of the parasite and disrupt the integrity of the cuticle (priya et al. 2014). the condensed tannins interact with proline‑rich proteins on cuticle that will interfere with feeding, motility and other key metabolic processes like ex‑sheathment and moulting (bachaya et  al. 2009). the ferns in our study not only demonstrated this property, but they also caused early death of worms. thus, this study reveals that fern extracts are promising in  vitro anthelmintics. these plants are known to be rich in alkaloids, phenols, terpenoids, flavonoids, glycosides, tannins, etc. (hossain et  al. 2012). the anthelmintic property could be attributed to these bioactive compounds individually or in combination. the in  vitro trematodicidal and antioxidant properties as found in this study might be attributed due to the action of the abundant fern secondary metabolites. classes of secondary metabolites such as alkaloids and flavonoids are considered the source of chemical compounds responsible for wide therapeutic activities of several medicinal plants (wink 2012). anthelmintic activity of plant extracts on larvae and adults of gastrointestinal nematodes is attributed to the capacity of tannins to bind to proteins and operate via several mechanisms (athanasiadou et al. 2001). anthelmintic activity of fumaria parviflora might be due to alkaloids which have the ability to intercalate with replicating dna (maqbool et  al. 2004). transcuticular diffusion is a common 285 kalpana et al. in vitro antitrematodal activities of compounds and secondary metabolites from pteridophytes veterinaria italiana 2020, 56 (4), 271-287. doi: 10.12834/vetit.1845.9825.4 practiced in folklore medicine. the study leads to the conclusion that ferns are a potential source of novel anthelmintics for medicinal practice. nevertheless, the conclusion is based on an in  vitro study, so the outcome must be corroborated with an in  vivo controlled array of drug discovery research. the study suggests fern‑8‑ene, fern‑9(11)‑ene, phytol, quercetin 7´,3´,4´ trimethoxy and xanthorrhizol as the potential plant secondary metabolites to look single chemical compounds for exploration in in vivo study. to sum up, this controlled array of in vitro drug research has expounded that ferns could be a potent source of trematodicidal antibiotics. acknowledgements the authors are thankful to the directorate of collegiate education, government of tamil nadu, for the grant which supported the successful completion of this ph.d. research. of the worm and, consequently, lead to death of the worm. embarking upon drug discovery/treatment requires a thorough knowledge of life cycle of the parasite, and its physiology, biochemistry and surface morphology/composition (tegument or cuticle). surface of flatworms, mouth of trematodes and gut of nematodes are the key role players in drug absorption/uptake. however, no single drug available today is useful for the treatment or prevention of both nematode and trematode infections in humans. there are also differences in susceptibility of worm species to individual chemical derivates in the same class. conclusions herein we have demonstrated and compared the trematodicidal action of four species of ferns collected from the wild and substantiated the antiparasitic medicinal property of pteridophytes as abdel-ghaffar f., margit s., al-rasheid k.a.s., bianca s., katja f., gülendem a., sven k. & heinz m. 2011. the effects of different plant extracts on intestinal cestodes and on trematodes. parasitol res, 108, 979-984. alvarez l.i., mottier m.l. & lanusse c.e. 2007. drug transfer into target helminth parasites. trends parasitol, 23, 97-104. amritpal singh s. 2011. herbalism, phytochemistry and ethnopharmacology. crs press, taylor and francis group, science publishers, new hampshire. 1-411. athanasiadou s., githiori j. & kyriazakis i. 2001. medical plants for helminth parasite control: facts and fiction. animal, 1, 1392-1400. athanasiadou s., kyriazakis i., jackson f. & coop r.l. 2001. direct anthelmintic effects of condensed tannins towards different gastrointestinal 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*corresponding author at: scientific production center of microbiology and virology llp, bogenbai batyr str., 105, almaty, kazakhstan. tel.: +7 870 58732835, e‑mail: saule.daugalieva@mail.ru. keywords paratuberculosis, single nucleotide polymorphism, electrophoretic mobility shift assays, rna-seq. summary previous studies led to identify snps in putative regulatory regions of the slc11a1 and card15 genes with association to paratuberculosis in cattle. aim of this study was to investigate the role of these mutations at the regulatory level by dna-protein interaction analyses and transcriptome comparison between wild-type and mutated animals. gene regions carrying the snps of interest were analysed by bioinformatic tools to predict allele-dependent binding sites for transcription factors (tfbs). putative tfbs were in vitro explored by electrophoretic mobility shift assays (emsa). emsa did not show specific gel shifts for any allele indicating that these snps may eventually influence gene transcription without altering tfbs. whole transcriptome expression analysis was performed on intestinal tissues of wild-type and mutated cattle by rna-seq. differential regulation of five genes involved in innate immune system was detected. specifically, ulbp3 was down-regulated, while s100a8, s100a12, loc510860, and ifi27 were up-regulated. in previous studies, ulbp3, s100a8, and s100a12 resulted differentially expressed in cattle affected by paratuberculosis, suggesting a possible implication in the pathogen response. further investigations are needed to elucidate the functional role of these snps and to understand the gene network involved in the interactions between non-coding snps and other genome regions. berik aryngaziyev1#, chiara beltramo2#, alessandro dondo2, talgat karymsakov1, katia varello2, maria goria2, alessia di blasio2, sabrina nodari2, silvia colussi2, paola modesto2, aida daugaliyeva1, pier luigi acutis2, saule daugaliyeva3* and simone peletto2 polymorphisms associated to bovine paratuberculosis: investigation of their role in dna‑protein interactions and transcriptional regulation veterinaria italiana 2020, 56 (2), 109-114. doi: 10.12834/vetit.2325.13205.1 accepted: 30.06.2020 | available on line: 27.07.2020 from 1.5 to 70% (eltholth et  al. 2009). in a recent survey of 48 countries paratuberculosis was found to be very common in livestock. in about half the countries more than 20% of herds and flocks were infected with map (whittington et al. 2019). in italy, prevalence has been reported to be around 20-40% (pozzato et al. 2011). in kazakhstan, one seropositive animal to paratuberculosis was detected in saiga antelope (saiga tatarica tatarica) (orynbayev et  al. 2016), but the significance of this finding is uncertain. however, map infection was also detected in cattle imported from abroad in east kazakhstan region (aida daugaliyeva, unpublished data). introduction paratuberculosis, or johne's disease, is a chronic enteritis of ruminants, caused by mycobacterium avium subsp. paratuberculosis (map). it is one of the most important diseases of dairy and beef cattle, resulting in large economic losses. some authors suggested a possible role of map in the inflammation observed in crohn's disease, an inflammatory bowel disease of humans (olsen et al. 2009) although the causal role of map has not been proven yet (mendoza et al. 2009, mcnees et al. 2015). prevalence around the world was estimated 110 veterinaria italiana 2020, 56 (2), 109-114. doi: 10.12834/vetit.2325.13205.1 impact of polymorphisms on dna-protein interactions and transcriptional regulation in bovine ptb aryngaziyev et al. were prepared in 50 µl of final volume containing 1x buffer, 2 mm of clmg 2 , 1 u of platinum taq (invitrogen), 0.2 mm of dntps and 300 nm of each primer. for the genotyping of the polymorphisms of interest, the following primer pairs were used: c.1157-91 a > t in intron 11 of slc11a1 gene, primers n1_9_f (5'-ttcagcaatcagctgtaggg-3') and n1_9_r (5'-gcatcagtttttccatctgc-3'); c.-90 c  >  a in the putative slc11a1 promoter, primers n1_2_f (5'-ccagtgcctctttcttctgg-3') and n1_2_r (5'-tctggattgtggttgagatcc-3'); c.2886-14 a  >  g in intron 10 of card15 gene, card15_11_f (5'-gaattcattgggaatctcagacag-3') and card15_11_r (5'-caggactagaggtctgag ccataa-3'). target sequences were analysed by match ( h t t p : / / w w w . g e n e r e g u l a t i o n . c o m / p u b / programs.html#match), promo (http://alggen. lsi.upc.es/cgi-bin/promo_v3/promo/promoinit. cgi?dirdb=tf_8.3), tfind (http://tfbind.hgc.jp/), and jaspar (http://jaspar.genereg.net/) software for the prediction of putative tfbs. putative tfbs to be investigated were selected when all the software provided consensus outputs. sequence regions flanking the polymorphisms of interest were selected to design double-strand hybridization probes according the protocol described by holden and tacon (holden and tacon 2011). ne-per nuclear and cytoplasmic extraction kit (thermo fisher) were used to obtain nuclear extract from bovine intestine. electromobility shift assays (emsa) experiments were carried out using the lightshift chemiluminescent emsa kit (thermo fisher) on both alleles of each snp following the manufacturer’s protocol. total rna was isolated from the 8 bovine intestine samples using the ribopure rna purification kit (agilent technologies). rna-seq libraries were prepared employing the trueseq rna library prep (illumina) and analysed on a illumina hiseq platform. sequence data were compared by principal component analysis (pca) and clustering analysis. results the genotyping of the three polymorphic sites under study showed the presence of wild-type and mutated alleles in the analysed cattle. specifically, for the c.-90 a > c polymorphism in the slc11a1 promoter, the a/c genotype was found in three animals. as regards the c.1157-91 a  >  t polymorphism in intron 11 of the slc11a1 gene, the a/t genotype was found in two cases. finally, for the polymorphism c.2886-14 a  >  g of intron 10 of card15 gene, one animal carried the a/g genotype. an association between host genetics and paratuberculosis was identified by previous case-control studies based on the candidate gene approach. single nucleotide polymorphisms (snps) represent common mutations that may modulate the response to pathogen exposure. associated snps often occur in open reading frame, thus acting by changing the amino acid composition of the related protein, but they can also be located in non-coding regions (e.g., gene promoter, introns). the latter may act by a plethora of functional mechanisms, including direct alteration of transcription factor binding sites (tfbs) or gene transcriptional modifications, which lead to differential host response following exposure to pathogens. a strong genetic involvement with susceptibility to paratuberculosis was established for solute carrier family 11 member 1 (slc11a1) gene in mouse models (roupie et  al. 2012). moreover, polymorphisms associated with susceptibility were reported also in sheep and goats (reddacliff et  al. 2005, korou et  al. 2010). slc11a1 variants in cattle were first described by pinedo and colleagues (pinedo et  al. 2009) in holstein, jersey and brahman-angus cross breeds. ruiz-larrañaga and colleagues (ruiz-larrañaga et al. 2010) carried out a snp-based candidate gene study on holstein-friesian cattle. caspase recruitment domain 15 (card15) was described as the first susceptibility gene for crohn’s disease in humans (hugot et  al. 2001, ogura et  al. 2001). in cattle pinedo and colleagues (pinedo et al. 2009) screened adult cows for snps reported in humans, suggesting a role for card15 also in the susceptibility of cattle to johne’s disease. this study aimed to get further insight on the functional role of three snps of slc11a1 (c.-90 a  >  c in the putative promoter; c.1157-91 a  > t in intron  11) and card15 (c.2886-14 a  >  g in intron 10) genes, previously associated to resistance/ susceptibility to bovine paratuberculosis, by investigating dna/protein interactions and by comparing the transcriptomes of wild-type and mutated animals. materials and methods sampling was carried out from 8 friesian cattle with more than 2 years of age directly at the slaughterhouse. small intestine was sampled and an aliquot was washed with sterile water and immediately placed in rnalater. another aliquot of intestinal tissue from each animal was used for genotyping of the selected genetic polymorphisms. dna was extracted from 50 mg of tissue by the gentra puregene tissue kit (qiagen) and quantified with the qubit 3.0 fluorimeter (thermo fisher). pcrs 111veterinaria italiana 2020, 56 (2), 109-114. doi: 10.12834/vetit.2325.13205.1 aryngaziyev et al. impact of polymorphisms on dna-protein interactions and transcriptional regulation in bovine ptb the disease susceptibility, replaces adenine. these predictions were in vitro investigated by setting up emsa experiments. emsa is expected to display gel shifts in case of reaction between the putative binding sites and transcription factors contained in the nuclear protein extract (hellman and fried 2007). however, emsa assays carried out to investigate possible interactions between slc11a1 c.-90/sp1 and card15/gata3 did not show any shift, thus not confirming the hypotheses raised in silico. the analysis of the binding between slc11a1 c.1157-91 and myod instead showed a gel shift in the presence of the probe carrying adenine (figure 1). with regard to the comparison of transcriptomes of wild-type and mutated cattle, the optimization of tissue collection (i.e. stored in rnalater directly at the slaughterhouse) and extraction procedure allowed to obtain rna of good quality from intestine tissue. rna integrity number (rin) values of the samples ranged between 6.6 and 7.9, with the exception of one sample that had a rin of 5.2 and it was therefore excluded from the analyses. rna-seq analysis revealed high variability, as demonstrated by pca and cluster analyses (figures 2 and 3), which show that samples group independently from the mutations. as a consequence, we observed non significant modulations of the expression of several genes. this was not completely unexpected due to the limited number of animals enrolled in the study. nevertheless, a panel of genes were significantly modulated according to slc11a1 genotype, and they were: ul-16 binding protein 3 (ulbp3), which was down-regulated [log 2 (fc) = 3.81] in the status g.-90 a  >  c; calcium-binding protein s100a8 and s100a12 (s100a8, s100a12), c4b-binding protein alpha-like (loc510860) and interferon α inducible the analysis of polymorphic and wild-type regions, carried out in parallel using different software, allowed to identify three putative tfbss. for the slc11a1 gene, software predictions showed that the sequence of the promoter containing cytosine create a recognition site for the sp1 transcription factor, which instead would not be able to bind to dna in the presence of adenine. as regards the polymorphism localized in intron 11 of the same gene, bioinformatics predicted a recognition site of the transcription factor myod for the sequence containing thymine. also for card15, it could be identified a binding site for gata3, which can no longer be recognized when guanine, associated with low high correlation a > t; a > g #9 a > c #2 a > c #5 a > c #7 a > t #8 wt #10 wt #1 a > t; a > g # 9 a > c #2 a > c #5 a > c #7 a > t #8 w t # 10 w t # 1 figure 3. heatmap of sample-to-sample distances for bovine intestine samples analyzed by rna-seq. wt = wild type animal; a > t = mutant for snp g.1157-91; a > c = mutant for snp g.-90. condition wt a > c a > t pc1: 66% variance it004991268143 it004991179710 it004990689956 it004990719986 it004991268167 it004991074518 it004990851858 -5 0 5 10 -10 -5 0 5 10 pc 2: 2 1% v ar ia nc e figure 2. principal component analysis on data from bovine intestine samples analyzed by rna-seq. wild-type animals are in orange; mutants for snp g.-90 are in green; mutants for snp g.1157-91 are in blue. figure 1. electrophoretic mobility shift assay (emsa) for snp g.1057-91 in intron 11 of slc11a1. ne = nuclear extract from bovine intestine. the arrow indicates the detected gel shift. snp: 1 = slc11a1 g.1057-91_t_bio; 2 = slc11a1 g.1057-91_t_bio + ne; 3 = slc11a1 g.1057-91_t_bio + ne + competitor; 4 = slc11a1 g.1057-91_a_bio; 5 = slc11a1 g.1057-91_a_bio + ne; 6 = slc11a1 g.1057-91_a_bio + ne + competitor; ctrl: 7 = ebna dna bio; 8 = ctrl ebna dna bio + ebna ne; 9 = ebna dna bio + ebna ne + competitor. 1 2 3 4 5 6 7 8 9 112 veterinaria italiana 2020, 56 (2), 109-114. doi: 10.12834/vetit.2325.13205.1 impact of polymorphisms on dna-protein interactions and transcriptional regulation in bovine ptb aryngaziyev et al. ligand for the nkg2d receptor of nk cells. when the receptor is activated following the binding with the ligand, the nk cell is activated for the recognition of invasive cells (kasahara et al. 2012, larson et al. 2006, mou et al. 2014). verschoor and colleagues (verschoor et  al. 2010) conducted microarray and rt-qpcr analyses on whole-blood rna from healthy and map-infected cattle showing different expression levels for the s100a12 and s100a8 genes. holstein cattle showed up-regulation of these genes, as found in our animals, which were friesian cattle, a breed related to holstein. s100a8 encodes a calcium-binding protein involved in innate immunity reactions and antimicrobial activities. the protein is localized in the cytosol of neutrophils, monocytes, leukocytes, fibroblasts, tumor cells, bone marrow (donato et al. 2013). in humans, elevated protein levels have been reported in cases of rheumatoid arthritis and inflammatory bowel disease, as well as in cases of bronchitis and tuberculosis, suggesting that this protein may play a role in the inflammation reaction (ikhtaire et  al. 2016). in particular, s100a8 protein plays a role in the migration of neutrophils to inflammation sites (vandal et  al. 2003). in addition, park et al.and colleagues (park et al. 2016) conducted a study to find marker genes for the detection of map infections. the authors compared rna extracted from whole blood of healthy cattle and cattle affected by paratuberculosis by rt-qpcr and they showed an up-regulation of the gene in diseased animals. s100a12 encodes a calcium-binding protein with phagocytic and pro-inflammatory activity (donato et al. 2013). this protein is a ligand for rage and the rage-s100a12 complex forms a pro-inflammatory axis that participates in many inflammatory diseases and has a central role in innate immunity (chen et al. 2010). the loc510860 gene encodes an alpha-like c4b-binding protein that has also been identified in the bovine genome. in humans, c4b-bindin protein is able to inhibit the activation of the classic pathway of the complement system. moreover, it has been shown that this protein is able to bind to the hypervariable region of streptococcus pyogenes thus protecting the bacterium from complement-mediated opsonization (areschoug et al. 2004). the ifi27 gene codes for an interferon alpha inducible protein 27. ifi27 induces apoptosis by sending extracellular signals to activate other pro-apoptotic genes. it is a membrane protein induced by interferon alpha and beta. in humans, it is up-regulated in the blood of men with prostate cancer undergoing radiation therapy (hsiao et al. 2013). in conclusion, this study provided further insights into the role of snps associated with resistance/ protein 27 (ifi27), which were up-regulated in the status 1157 g.-91 a  > t compared to the wild-type genotype, with log 2 (fc) of 6.86, 6.63, 3.70 and 4.34, respectively. discussion bioinformatic analysis allowed the identification of three putative tfbs whose consensus sequence was predicted as conserved or disrupted depending on snp allele. with regard to slc11a1 gene, snps in the promoter and in the intron might constitute sites for sp1 and for myod, while a tfbs for gata3 was predicted for card15 intronic sequence. however, emsa experiments did not show specific gel shifts, meaning that there are not altered tfbs in correspondence of the polymorphisms of interest. the only exception was a gel shift for slc11a1 c.1157-91 in the presence of the probe carrying adenine, and not with the wild-type allele (thymine) as predicted in silico. this result suggests that likely another transcription factor than myod exists, capable of recognizing intron 11 of slc11a1. actually, studies conducted on the promoter activities showed that polymorphisms located within 500 bp upstream the transcription start site (as the g.-90 c  >  a in the slc11a1 putative promoter) can influence the transcription levels and concur positively to the promoter activity (chorley et  al. 2008, hudson 2003). moreover, non-coding polymorphisms might have a role on flexibility of dna strand and chromatin folding (elkon and agami 2017). therefore, the snps of interest could have an indirect influence in the regulation of gene expression and further studies are needed to elucidate their functional role. rna-seq analysis allowed to identify a significant expression difference for five genes even though the variability in the samples was quite high, likely because the animals have been compared based on snp allele, which does not determine a sharp separation condition (i.e. healthy/diseased). noteworthy, these genes are involved in the innate immune response and three of them (ulbp3, s100a8 and s100a12) were already shown to be differentially expressed in cattle affected by paratuberculosis. shin and colleagues (shin et al. 2015) carried out a hybridization study on microarray using rna obtained from whole blood of healthy and paratuberculosis-affected cattle, and they found ulbp3 to be down-regulated in cattle affected by the disease. we observed the same expression pattern for ulbp3 gene in slc11a1-mutated cattle: however, it should be taken into account that the mutated a allele in the slc11a1 promoter was previously associated with disease resistance. the ulbp3 gene encodes for a gpi-anchored protein and acts as a 113veterinaria italiana 2020, 56 (2), 109-114. doi: 10.12834/vetit.2325.13205.1 aryngaziyev et al. impact of polymorphisms on dna-protein interactions and transcriptional regulation in bovine ptb help to identify other genetic factors associated with the investigated polymorphisms. acknowledgements this work was supported by the italian ministry of health (ricerca corrente 2014 grant izs plv 10/14 rc) and by the budget program 267 “improving the availability of knowledge and research” project: “creation of model cattle breeding farms to improve meat productivity in various regions of kazakhstan”. susceptibility to bovine paratuberculosis. the studied snps did not show direct effects on the mechanisms of regulation 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and their association with infection by mycobacterium avium subspecies paratuberculosis. j dairy sci, 4, 1713-1721. 105 veterinaria italiana 2021, 57 (2), 105-109. doi: 10.12834/vetit.2225.12292.1 accepted: 30.03.2020 | available on line: 31.12.2021 1department of veterinary public health and preventive medicine, university of nigeria, nsukka, nigeria. 2department of veterinary obstetrics and reproductive diseases, university of nigeria, nsukka, nigeria. 3department of animal health and production, university of nigeria, nsukka, nigeria. 4department of animal science, university of nigeria, nsukka, nigeria. 5department of animal health and production, enugu state polytechnic, iwollo, enugu state, nigeria. *corresponding author at: department of veterinary obstetrics and reproductive diseases, university of nigeria, nsukka, nigeria. e-mail: ugochinyere.njoga@unn.edu.ng. emmanuel okechukwu njoga1, ugochinyere juliet njoga2*, festus otaka abonyi3, henry onyeji edeh4, festus ejike ajibo5 and nichodemus azor5 keywords cattle, foetal wastage, nigeria, prevalence, public health implications. summary slaughter of pregnant animals for meat is unethical, counterproductive and enhances zoonotic disease spread. this study determined the prevalence and reasons for slaughtering pregnant cows (spcs) for meat. pregnancy status of cows slaughtered was determined by evisceration and longitudinal incision of the uterus for presence of fetus. closed-ended questionnaire was used to elicit information on causes of spcs and disposal of eviscerated fetuses. of the 851 cows slaughtered, 17.4% (148/851) were pregnant. of the 148 pregnant cows, 87 (58.8) were slaughtered during dry season while 43.2% (64/148) of the recovered fetuses were in their third trimester. reasons adduced for spcs by the participants in the questionnaire were: ignorance of the animals’ pregnancy status, 69.7% (n  =  119), high demand for beef, 61.3% (n = 148), buyers preference for large-sized animals, 47.9 (n = 148), economic hardship, 52.1% (n  =  148) and disease conditions, 42.9% (n  =  148). fetuses or uterine contents were sold for human consumption, 17.6% (n  =  119) preparation of dog food, 27.7% (n = 119) or disposed by open refuse dump method, 54.6% (n = 119). the 17.4% spcs prevalence is unacceptably high. this warrants ante-mortem pregnancy diagnosis in the slaughterhouses and strict implementation of the animal welfare act (meat edict of 1968) to conserve livestock production and limit animal cruelty and the spread of zoonoses. bovine fetal wastages in nsukka nigeria: prevalence, causes and public health implications health problem associated with slaughter of pfas in developing countries. animal protein crisis may ensue through unwarranted off takes from the national herd, without replacement, as epitomized in spcs or via deletion of future herd by the resultant decrease in calf yield. although spcs and the health and economic problems thereof are preventable, it is regrettable that this practice has persisted over the years in nigeria where demand for edible animal products incidentally supersedes the supply (njoga et al. 2018a). apart from the reduction in availability of animal protein, spcs enhances dissemination of zoonotic pathogens, especially in the tropical climate, where favourable conditions for pathogens’ survival abound (okoli et al. 2018, ajibo et al. 2020). brucella, listeria and zoonotic staphylococcus species are transmissible to humans via wound contamination introduction slaughter of pregnant female animals (pfas) for meat is unethical, threatens food security and connotes animal cruelty (njoga et  al. 2019). it is counterproductive also, considering the monumental losses in revenue and livestock resources thereof. slaughter of pregnant cows (spcs) does not just depopulate productive female animals but represents deletion of the future herd through the consequential fetal wastages. this practice jeopardizes effort towards achieving food security and self-sufficiency in provision of edible animal protein, especially in developing countries. it may also lead to introduction of exotic zoonoses, through meat importation, as a result of deficit in animal protein due herd depletion. diminution of animal protein is a major public 106 veterinaria italiana 2021, 57 (2), 105-109. doi: 10.12834/vetit.2225.12292.1 bovine fetal wastages in nigeria njoga et al. determined by visual examination of the external genitalia at the inguinal region or below the base of the tail. thereafter, the gestational age of the recovered foetuses were categorized as first (< 90 days), second (90-180 days) or third (> 180 days) trimester. structured and pretested open-ended questionnaire was used to elicit information on possible causes of spcs, and method of disposal of eviscerated fetuses or gravid uterine contents, from 119 randomly selected slaughterhouse workers. data analysis information obtained from the study were collated, analyzed and presented in tables. fishers’ exact test was used to determine whether there is significant association (p ≤ 0.05) between spcs and seasons, age, breed, months of the year and slaughterhouse locations. the statistic was performed at 5% probability level using graphpad® prism software version 6.04 (graphpad inc., san diego, california, usa). results causes of slaughter of pregnant cows reasons adduced for slaughter of gravid cows are presented in table i. some of the causes for spcs and proportion of the respondents were: ignorance of the animals’ pregnancy status 69.7% (83/119), high demand for beef 61.3% (73/148), buyers preference for large-sized animals 47.9% (57/148), feed scarcity during dry seasons 26.9% (32/148), economic hardship 52.1% (62/148) and disease conditions 42.9% (51/148). distribution of pregnant cows slaughtered results on age, season and breed distribution of pregnant cows slaughtered for meat are shown in during fetal evisceration or processing of slaughtered gravid animals (ekere et al. 2018, njoga et  al. 2018b). besides, slaughter or processing of pregnant animals infected with zoonotic pathogen may result in contamination of the meat or the environment, for onward transmission to humans; thereby facilitating the cycle of zoonotic infections between man, animals and the environment. in order to curtail zoonoses and improve animal welfare, slaughter of pfas is prohibited in nigeria through the meat edict of 1968. the act forbids to slaughter gravid animals except for emergency to relieve animal suffering, as recommended by veterinarians. however, reports show that fetal wastage due to slaughter of pfas has continued over the years in some parts of the country (cadmus and adesokan 2010, mishelia et al. 2015, odeh et al. 2015, iliyasu et  al. 2017, okorie-kanu et  al. 2018). the immediate cause(s) of this menace have largely remained elusive but may be associated with the upsurge in demand for edible animal products in nigeria due to the fast growing human population, currently estimated at 202 million at 2.63% annual growth rate occuring in the country (abonyi and njoga 2019). since spcs counteracts the growth of the national herd, and the only available report on spcs in southeast nigeria was dated back to three decades ago (wosu 1988), it is imperative to determine the current status of spcs in this region, and also to unravel the drivers underpinning this unethical practice. this will highlight the public health and economic consequences thereof, and proffer cost-effective solutions against this problem. materials and methods data collection the study location, nsukka, southeast nigeria, has already been described by nwanta and colleagues (nwanta et  al. 2011). the study adopted a cross-sectional survey design to determine the prevalence and causes of spcs in the selected area. three major slaughterhouses in the study area were visited weekly, for six months, (three month during dry season and another three months during wet season) for data collection. pregnancy status of cows slaughtered was ascertained by visual inspection of the uterine horns, for presence or absence of fetus after evisceration and longitudinal incision of the organ. the ages of the dams were estimated by dentition method (pace and wakeman, 2003). for the fetuses, ageing was done by crown-rump length measurement (odeh et  al. 2015) while sex was table i. reasons for sale or slaughter of pregnant cows for meat among abattoir workers (n = 119) in southeast nigeria. reasons number (%) of respondents yes no no response high demand for beef 73 (61.3) 21 (17.6) 25 (21.0) economic hardship 62 (52.1) 57 (47.9) 0 ignorance of the pregnancy status of the animal 83 (69.7) 32 (26.9) 4 (3.4) preference for pregnant cows because of size 57 (47.9) 46 (38.7) 16 (13.4) feed scarcity during dry seasons 32 (26.9) 87 (73.1) 0 disease conditions 51 (42.9) 42 (35.3) 26 (21.8) 107veterinaria italiana 2021, 57 (2), 105-109. doi: 10.12834/vetit.2225.12292.1 njoga et al. bovine fetal wastages in nigeria disposal of eviscerated fetuses eviscerated fetuses or uterine contents were sold for human consumption 17.6% (21/119), preparation of dog food 27.7% (33/119) or disposed by open refuse dump method 54.6% (65/119). likewise, 23.5% (28/119) of the slaughterhouse surveyed workers sold fetuses or afterbirth materials for feeding of pigs or fishes (table v). only 5.9% (7/119) and 3.4% (4/119) of the respondents disposed the materials by burial and incineration, respectively (table v). discussion slaughter of 17.4% pregnant cows for meat is unacceptably high. this is probably due to the average annual cattle growth rate (1.85%) (dunka et al. 2017) which is too slow to cope with the beef and dairy needs of over 200 million nigerians with a population growth rate of 2.63% per annum (njoga and abonyi 2019). slaughter of pregnant females creates multiple problems including unwarranted table ii while the spatial and temporal distribution of pregnant cows slaughtered are presented in table iii. majority of the slaughtered pregnant cows (60.1%) were in their active reproductive age (4-8  years). about 59% (87/148) of the pregnant animals were slaughtered during the dry season while 80.4% (119/148) of the spcs were of the white fulani breed. similarly, highest prevalence of the slaughter (34.5%) was recorded in december while 38.5% (57/148) of the pregnant animals were slaughtered at akwata slaughterhouse, located in enugu state, nigeria. in addition, 55.4% (82/148) of fetuses recovered were male while 22.3% (33/148), 34.5% (51/148) and 43.2% (64/148) were in their first, second and third trimester, respectively (table iv). significant association (p ≤ 0.05) existed between spcs and age (p = 0.029) and season (p = 0.038). in the same vein, there was significant association (p ≤ 0.05) between spcs and months of the year, but no association was evidenced between spcs and slaughterhouse location (p = 0.406). table ii. age, season and breed distribution of pregnant cows (n = 148) slaughtered in southeast nigeria. variables number (%) of cows slaughtered number (%) of pregnant cows slaughtered prevalence p-value age < 4 years 126 (14.8) 30 (23.8) 20.3 0.029* 4-8 years 498 (58.5) 89 (17.8) 60.1 > 8 years 227 (26.7) 29 (12.8) 19.6 season wet 418 (49.1) 61 (14.6) 41.2 0.038* dry 433 (50.9) 87 (20.1) 58.8 breed white fulani 621 (72.9) 119 (19.2) 80.4 0.149 sokoto gudali 130 (15.3) 18 (13.8) 12.2 red bororo 63 (7.4) 7 (11.1) 4.7 mixed breeds 37 (4.3) 4 (10.8) 2.7 *significant statistical association (p ≤ 0.05), fisher’s exact test. table iii. spatial and temporal distribution of pregnant cows (n = 148) slaughtered in major slaughterhouses in southeast nigeria variables number (%) of cows slaughtered number (%) of pregnant cows slaughtered prevalence p-value months december 197 (23.1) 51 (25.9) 34.5 0.021* january 157 (18.4) 24 (15.3) 16.2 february 129 (15.2) 21 (16.3) 14.2 july 118 (13.9) 17 (14.4) 11.5 august 121 (14.2) 16 (13.2) 10.8 september 129 (15.2) 19 (14.7) 12.8 slaughter locations enugu 327 (38.4) 57 (17.4) 38.5 0.406 nsukka 315 (37.0) 49 (15.6) 33.1 awka 209 (24.6) 42 (20.1) 28.4 *significant statistical association (p ≤ 0.05), fisher’s exact test. table iv. age and sex distribution of fetuses (n = 148) recovered from pregnant cows slaughtered in southeast nigeria. variables number (%) of fetuses age first trimester 33 (22.3) second trimester 51 (34.5) third trimester 64 (43.2) sex male 82 (55.4) female 66 (44.5) table v. method of disposal of eviscerated fetuses among abattoir workers (n = 119) surveyed in southeast nigeria. method of fetus disposal* number (%) of yes respondents sold fetuses for human consumption 21 (17.6) sold fetuses for preparation of dog food 33 (27.7) sold eviscerated fetus for feeding of fishes or pigs 28 (23.5) dumped unsold fetus or gravid uterine contents in municipal refuse dump 65 (54.6) incinerated unsold fetus or gravid uterine tissues 4 (3.4) buried unsold fetus or gravid uterine contents 7 (5.9) *many respondents disposed fetuses by more than one method. 108 veterinaria italiana 2021, 57 (2), 105-109. doi: 10.12834/vetit.2225.12292.1 bovine fetal wastages in nigeria njoga et al. season or soon afterwards. significant loss of body condition, rampant during this period because of drought-related food and water scarcity, usually compels livestock owners to salvage their animals by selling them off. additionally, demand for meat is usually uttermost during the dry seasons due to socio-cultural and religious celebrations. for economic reasons, some farmers usually sell some of their animals, including females, irrespective of their pregnancy status during this period. these may explain the high prevalence of spcs noted during the dry season, particularly in december, when drought is at peak and festivities abound. slaughtering of pregnant animals in their second (34.5%) and third trimesters (43.2%), as revealed in this study, is worrisome as pregnancies at these stages can easily be detected, even by visual assessment. this finding is in tandem with that of wosu (wosu 1988) in which 74% of pregnant cows slaughtered were in their second or third trimesters. this destructive practice does not only worsen the already precarious animal protein shortfall in the country, but also threatens the development of its livestock industry through excessive offtakes and loss of good genetics in the future herds (fayemi and muchenje 2013). the spcs for meat is unprofitable in all ramifications. farmers, who may have erroneously culled their cows for infertility reasons, probably due to poor proficiency in pregnancy detection, are at loss. the butchers and beef sellers are equally at loss; as pregnant animals, especially those in late stage of pregnancy, yield less meat than the non-pregnant ones (wythes et al. 1990, okorie-kanu et al. 2018). the quality of meat from pregnant animals is doubtful. meats sourced from pregnant animals are watery, have high ph and peak shear force values, poor eye appeal and also smells and tastes abnormal due to high progesterone tissue level (wythes et al. 1990). therefore, spcs for meat should be prevented to grow the national herd size and limit spread of important livestock and zoonotic diseases. since veterinarians are proficiently trained on pregnancy and disease detection in animals to forestall rampart slaughter of gravid animals for meat, they should be engaged and kept in the realm of affairs in the day to day running of slaughterhouses in the southeast region. there is a need to step up animal welfare via strict implementation of the provisions of the meat edict of 1968, which may warrant incorporation of pregnancy diagnosis in ante-mortem inspection in slaughterhouses in southeast nigeria. offtakes from the already depopulated national herd, as well as deletion of the future herd, through the consequent foetal wastages. given that most developing countries are yet to achieve self-sufficiency in beef production (fao 2013), it is unacceptable to imagine the colossal loss of tons of beef that would have accrued if the foetuses were born alive and raised to maturity. this translates to loss of about 20 tons of beef (based on 52% carcass yield and average maturity live weight of 260 kg), in only one of the six geopolitical zones of the country, in just six months. this avoidable loss is a major setback to food security, in a country where meat supply grossly lags behind the demand (njoga et  al. 2018b), and protein malnutrition is glaring, especially in rural areas. apart from the losses and the inherent economic wastages, spcs facilities transmission and spread of zoonotic pathogens inhabiting the reproductive tract of cows. contamination of processed meat and infection of the slaughterhouse workers during carcass processing is almost inevitable, considering the fact that slaughterhouse workers in the study area usually do not wear personal protective equipment during routine operations (ekere et  al. 2018, njoga et  al. 2018a). additionally, infection of food animals grazing around abattoir environment contaminated with zoonotic organisms, for onward transmission to humans, via the food chain is most probable. these underscore the need to curtail spcs to curb the possible cycle of zoonotic transmission inherent in the practice. lack of man-power and proficiency in pregnancy diagnosis among slaughterhouses workers, may have contributed to high rate of spcs being reported (fayemi and muchenje 2013). in nigeria, the local and state governments have the responsibility to oversee and regulate the operations of slaughterhouses but most of these authorities have transferred this oversight and regulatory functions to privately owned organizations. as a result, ante-mortem and post-mortem inspections have been reduced to mere collection of revenue per slaughtered animal. as a cost saving strategy, veterinarians who by training are competent on pregnancy and disease diagnosis in animals, are hardly employed in slaughterhouses nowadays by the new owners. these may have given rise to indiscriminate slaughter of gravid cows, including those in their last stage of gestation. slaughter of gravid females and the resultant foetal wastages are usually higher during the dry 109veterinaria italiana 2021, 57 (2), 105-109. doi: 10.12834/vetit.2225.12292.1 njoga et al. bovine fetal wastages in nigeria abonyi f.o. & njoga e.o. 2019. prevalence and determinants of gastrointestinal parasite infection in intensively managed pigs in nsukka agricultural zone, southeast, nigeria. j parasit dis, 44, 31-39. ajibo f.e., njoga e.o., azor n., idika k.i. & nwanta j.a. 2010. epidemiology of infections with zoonotic pig parasites in enugu state, nigeria. j vet parasitol reg stud reports, 100397. https://doi.org/10.1016/j.vprsr.2020.100397. cadmus s.i.b. & adesokan h.k. 2010. bovine foetal wastage in southwestern nigeria: a survey of some abattoirs. trop anim health prod, 42, 617-621. dunka h.i., buba d.m., gurumyen y.g., oragwa a.o., oziegbe s.d. & patrobas m.n. 2017. economic losses associated with the slaughter of pregnant animals in jos abattoir. int j adv res, 5, 1047-1052. ekere s.o., njoga e.o., onunkwo j.i. & njoga u.j. 2018. serosurveillance of brucella antibody in food animals and role of slaughterhouse workers in spread of brucella infection in southeast nigeria. vet world, 11, 1171-1178. fayemi p.o. & muchenje v. 2013. maternal slaughter at abattoirs: history, causes, cases and the meat industry. springer plus, 2, 125. doi: 10.1186/2193-1801-2-125. food and agricultural organization (fao). 2013. current worldwide annual meat consumption, per capita, livestock and fish primary equivalent (http://faostat. fao.org accessed on 14 december 2018). iliyasu d., ogwu d., yelwa h., yaroro i., ajani j.a. auwal u., bugau j.s, kuzayed i.g. & jibrin u.r. 2015. incidence of bovine pregnancy wastage in maiduguri abattoir, borno state nigeria. int j livestock res, 5, 59-65. mishelia g.d., maina v.a. & aminu m.d. 2015. reproductive wastages in ruminant species slaughtered in north-eastern nigeria. j anim prod adv, 5, 645-653. njoga e.o., ariyo o.e. & nwanta j.a. 2019. ethics in veterinary practice in nigeria: challenges and the way-forward. nigerian vet j, 40, 85-93. njoga e.o., onunkwo j.i., ekere s.o., njoga u.j. & okoro w.n. 2018a. seroepidemiology of equine brucellosis references and role of horse carcass processors in spread of brucella infection in enugu state, nigeria. int j current res rev, 10, 39-45. njoga e.o., onunkwo j.i., chinwe c.e., ugwuoke w., nwanta j.a. & chah k.f. 2018b. assessment of antimicrobial drug administration and antimicrobial residues in food animals in enugu state, nigeria. trop anim health prod, 50, 897-902. nwanta j.a., shoyinka s.v.o., chah k.f., onunkwo j.i., onyenwe i.w., eze j.i., iheagwam c.n., njoga e.o., onyema i., ogbu k.i., mbegbu e.c., nnadozie p.n., ibe e.c. & oladimeji k.t. 2011. production characteristics, disease prevalence, and herd health management of pigs in southeast nigeria. j swine health prod, 19, 331-339. odeh s., dawuda p.m., oyedipe e.o. & obande g.e. 2015. incidence of foetal wastage in slaughtered cattle at wurukum abattoir, makurdi, benue state. vom j vet sci, 10, 41-50. okoli c.e., njoga e.o., enem s.i., godwin e.e., nwanta j.a. & chah k.f. 2018. prevalence, toxigenic potential and antimicrobial susceptibility profile of staphylococcus isolated from ready-to-eat meats. vet world, 11, 1214-1221. okorie-kanu o.j., ezenduka e.v., okorie-kanu c.o., anyaoha c.o., attah c.a., ejiofor t.e. & onwumere-idolor s.o. 2018. slaughter of pregnant goats for meat at nsukka slaughterhouse and its economic implications: a public health concern. vet world, 11, 1139-1144. pace j.e. & wakeman d.l. 2003. determining the age of cattle by their teeth, university of florida, ifas extension (https://ufdc.ufl.edu/ir00004537/00001 accessed on 11 april 2018). wosu l. 1988. calf wastage through slaughtering of pregnant cows in enugu abattoir, nigeria. rev elev med vet pays trop, 41, 97-98. wythes j.r., shorthose w.r., fordyce g. & underwood d.w. 1990. pregnancy effects on carcass and meat quality attributes of cows. anim prod sci, 51, 461-468. 325 veterinaria italiana 2022, 58 (3), 325-329. doi: 10.12834/vetit.2241.16176.1 accepted: 09.09.2021 | available on line: 31.12.2022 1department of pharmacology, faculty of veterinary medicine, university of murcia, campus de espinardo, 30.071‑murcia, spain. 2escuela de medicina veterinaria, universidad san francisco de quito, ec 170157, cumbayá, ecuador. *corresponding author at: department of pharmacology, faculty of veterinary medicine, university of murcia, campus de espinardo, 30.071‑murcia, spain tel.: +34 868 888404, fax: +34 868 884147, e‑mail: pmarin@um.es. elena badillo1, elisa escudero1, juan sebastian galecio1,2, verónica hernandis1 and pedro marín1* keywords cephalosporins, staphylococcus aureus, minimum inhibitory concentration (mic), goats, rabbits. summary antimicrobial drug resistance is an important problem that challenges veterinary clinicians to provide effective treatments without further spreading this resistance to other animals and people. the most commonly used pharmacodynamic parameter to define potency of antimicrobial drugs is minimum inhibitory concentration (mic). the aim of this study was to evaluate the antibiotic susceptibility of thirty-six strains of staphylococcus aureus isolated from dairy goats with mastitis and rabbits with chronic staphylococcosis. four cephalosporins were tested: cephalexin, cephalotin, cefonicid and ceftiofur. mic tests were performed according to the microdilution broth method. the calculated values of sensitivity in goats and rabbits were 66.67% and 72.22% for cephalexin, 72.22 % and 94.44% for cefonicid, 77.78% and 94.44% for cephalotin and 77.78% and 100% for ceftiofur, respectively. for all antibiotics, mic 90 of s. aureus from rabbits were lower than mic 90 from goats. these data suggest that more antibiotics are used in goat milk production than in rabbit farming. according to mic values obtained in this study, ceftiofur and cephalotin may be the best option for treating s. aureus infections in lactating goats. for rabbits, ceftiofur showed lowest mic values, but cephalosporins can produce fatal diarrhoea in this species, therefore additional studies are needed to evaluate the effects of repeated ceftiofur administration on microflora of rabbits before recommending the use of this antibiotic in this species. cephalosporin susceptibility of staphylococcus aureus strains isolated from commercial rabbit and goat farms in spain cephalothin) second (cefonicid), third (ceftiofur) and fourth generation compounds displaying a variable activity in  vitro, structural similarities and the time of their introduction into the market (caprile 1988). from first to fourth generation, the spectrum of activity against gram negative organisms and the stability against β-lactamase increase commonly together with the same or reduced spectrum of activity against gram positive organisms, except for fourth generation agents, which have enhanced activity against gram positive organisms (papich and lindeman 2018). cephalotin (cl) and cefonicid (cd) are developed for use in human medicine, cephalexin (cx) for use in human and veterinary medicine and ceftiofur (cr) for exclusive use in animals and approved for use in food animals in europe. staphylococcus aureus is one of the most important pathogenic staphylococcus species in veterinary medicine. in rabbits, it mainly causes skin infections, pododermatitis and mastitis with subsequent economic losses in industrial rabbitries (hermans et  al. 1999). in goats, staphylococcus aureus is probably the most important infectious agent (mørk et al. 2005), since it causes a chronic and deep infection in the mammary glands which implies an evident economic loss in relation to the decrease in milk production and health status of animals (seegers et al. 2003). cephalosporins are a broad group of antimicrobial agents with a bactericidal mechanism of action similar to all β-lactam antibiotics inhibiting cell-wall synthesis. in accordance with their antimicrobial spectrum, they are divided into first (cephalexin, short communication 326 veterinaria italiana 2022, 58 (3), 325-329. doi: 10.12834/vetit.2241.16176.1 cephalosporin susceptibility of staphylococcus aureus strains badillo et al. the antibiotics selected for the study were provided by manufacturers in this way: cr and cl by sigma-aldrich (st. louis, usa), cx by tokyo chemical industry (tokyo, japan) and cd by santa cruz biotechnology (heidelberg, germany). according to the clinical and laboratory standards institute’s recommendations, antibiotic stock solutions were prepared at concentrations of 1,280 mg/l by dissolution in suitable solvents and then diluted in sterile distilled water (clsi 2015). minimum inhibitory concentration (mic) tests were performed according to the microdilution broth method recommended by the clsi (clsi 2015) using u-bottomed, 96-well microtiter plates. serial two-fold dilutions of the antimicrobial agents were prepared starting from the stock solution of each drug (concentrations ranging from 0.03 to 128 mg/l). inocula were prepared by diluting an overnight mueller-hinton broth culture in buffered saline solution to a density of 0.5 mcfarland turbidity scale and finally diluting again 40-fold before testing. the mic was defined as the lowest concentration at which bacterial growth was completely inhibited, that is the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in a broth dilution susceptibility test (clsi 2015). the mic values of these drugs that inhibited 50% and 90% of the isolates were expressed as mic 50 and mic 90 , respectively. in relation to breakpoints of susceptibility and resistance, a strain was considered resistant when the mic was ≥ 8 mg/l for cd, cx, cl and cr (clsi 2018). reference strains of s. aureus (atcc 29213) and e. coli (atcc 25922) were used as controls for each plate. in this case, all results for s. aureus atcc 29213 and e.  coli atcc 25922 were within the accepted range (clsi 2018). the specific mic values for each selected antimicrobial agent are showed in table i, where also mic 50 and mic 90 , and the minimum and maximum mic values (range) are included. in goats, it was according to the european medicines agency (ema), antibiotics are classified based on the potential consequences for public health of increased antimicrobial resistance due to their use in animals and depending on the necessity to use them in veterinary medicine. in the ema categorization, there are four categories, a (avoid), b (restrict), c  (caution), and d (prudence), with the latter as the lowest risk category (ema 2019). antibiotics from the a category are reserved for human treatment only, and their use is not allowed in food-producing animals. b category antibiotics (as third and fourth-generation cephalosporins) are critically important antimicrobials, and should be used only as a last option after susceptibility testing has been conducted when no other antibiotic would be clinically effective. c category antibiotics (as first and second-generation cephalosporins) should be considered only when there are no antibiotics in d category that could be clinically effective. because of this, the antibiotic consumption in animal production should be progressively reduced in order to decrease the antimicrobial resistance against the antibiotics to which the bacteria have been exposed. thus, the purpose of this study was designed to evaluate the degree of in  vitro activity of cd, cx, cl and cr against 36 s.  aureus strains, isolated from rabbits with chronic staphylococcosis and goats with clinical mastitis in spain. a number of 18 s.  aureus strains were isolated from rabbit farms with mastitis, subcutaneous abscesses and pododermatitis in the southeast region of spain. similarly, 18 s.  aureus strains were isolated from milk of goats with clinical mastitis. the plates were incubated aerobically at 37 °c to be examined at 24, 48, and 72 h. bacteria with target hemolysis were identified (presumptive) by catalase test and gram staining. commercial latex agglutination kit (staphytec plus, oxoid) was used by specific identification of s. aureus. the strains were stored at - 80 °c in a nutrient broth enriched with 15% glycerol. table i. frequency of staphylococcus aureus strains in relation to their minimal inhibitory concentrations and mic 50 , mic 90 , and mic range values of cefonicid, ceftiofur, cephalotin and cephalexin for rabbits (n = 18) and goats (n = 18). antibiotics animals number of strains with mic (mg/l) mic 50 (mg/l) mic 90 (mg/l) mic range (mg/l) 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 cefonicid goats 1 3 3 6 1 4 4 128 0.5-128 rabbits 1 3 5 8 1 2 4 0.25-128 ceftiofur goats 3 8 3 2 1 1 1 32 0.5-64 rabbits 1 3 11 2 1 1 2 0.25-4 cephalotin goats 3 7 4 1 2 1 0.25 32 0.12-128 rabbits 2 10 4 1 1 0.25 4 0.12-16 cephalexin goats 1 3 8 2 2 2 4 128 1-128 rabbits 1 2 10 4 1 4 8 0.12-64 327veterinaria italiana 2022, 58 (3), 325-329. doi: 10.12834/vetit.2241.16176.1 badillo et al. cephalosporin susceptibility of staphylococcus aureus strains caused by gram-positive bacteria is becoming a difficult problem. the situation is particularly critical in infections caused by s.  aureus which is also frequently resistant to multiple groups of antibiotics (livermore 2000). one of the strategies proposed for the control of s. aureus infections is the re-evaluation of older antimicrobial agents and develop them for using against this type of microorganisms with high degrees of resistance (chopra et  al. 1997). therefore, first and second generation of cephalosporins and ceftiofur have become a good option for the treatment of infectious diseases in veterinary medicine. the ratio t > mic is the best parameter for predicting the antimicrobial effect of time-dependent antimicrobial agents as β-lactam antibiotics. it has been previously determined that the maximal bactericidal action for cephalosporins is reached when plasma concentrations are greater than the mic of the pathogen for 60-70% of the dosing interval and a bacteriostatic effect is observed when t > mic is 30-40% of the dosing interval (drusano 2004). distribution of cephalosporins to the mammary gland and the consequent access to milk is limited. pharmacokinetic parameters of these four cephalosphorins in lactating goats (lacchini et  al. 2007, doré et  al. 2011, fernández-varón et  al. 2016, badillo et  al. 2020) after different routes of administration showed a low elimination through the milk. therefore, the treatment of mastitis in goats with these cephalosporins should be performed by intramammary administrations. according to mic values obtained in the present study, cr and cl may be the best options for treating s. aureus infections in these species due to their lowest mic values. considering mic values obtained in this study and pharmacokinetic parameters in rabbits (dzierzanowska et  al. 1982, meneses et  al. 2013, gardhouse et  al. 2017) after different routes of administration, cr could be an option for treating s.  aureus infections on account to the lowest mic value, high plasma levels and long half-life showed in this species. however, cr (b category) should be used only as a last option after susceptibility testing has been conducted when no other antibiotic would be clinically effective. moreover, cephalosporins can disrupt the caecal microflora in rabbits (because of biliary elimination of some members of this group) resulting sometimes in fatal diarrhoea. although single-dose parenteral administration of cr resulted in minimal adverse effects (gardhouse et  al. 2017), additional studies are needed to evaluate the effects of repeated cr administration in rabbits and to develop and implement appropriate specific dosing regimens that can maximize its clinical efficacy for use in production animals and reduce the risk of selection of resistant pathogens. observed four resistant strains for cr and cl each, five for cd and six for cx. in contrast, the number of resistant strains in rabbits was considerably lower, one resistant strain for cd and cl each, zero for cr and five resistant strains for cx were found. the calculated values of sensitivity in goats and rabbits were 66.6% and 72.2% for cx, 72.2 % and 94.4% for cd, 77.7% and 94.4% for cl and 77.7% and 100% for cr, respectively. the lowest mic 90 were obtained in goats for cr and cl (mic 90 = 32 mg/l). instead in rabbits, cr showed a lower value (mic 90 = 2 mg/l) than cd (mic 90 = 4 mg/l), cl (mic 90 = 4 mg/l) and cx (mic 90 = 8 mg/l). indeed, for all antibiotics, mic 90 of s.  aureus from rabbits were lower than mic 90 from goats. these evidences suggest that more antibiotics are used in goat milk production than in rabbit farming. there are reduced reported data on cephalosporin susceptibility of s.  aureus isolated from milk of goats with subclinical or clinical mastitis. for cl, our data were higher than those obtained in a previous study (mic 50 = 0.25 mg/l, mic 90 = 0.25 mg/l, range 0.06-128 mg/l, virdis et  al. 2010). there are no reported data with cd, cr and cx in s.  aureus isolated from goats. however, published research papers on cephalosporin susceptibility of s.  aureus isolated from bovine milk are numerous. in these studies, reported mic values were lower than those obtained in this research for cr (mic 50 = 1 mg/l, mic 90 = 1 mg/l, range 0.06-64 mg/l, de  oliveira et  al. 2000; mic 50 = 1 mg/l, mic 90 = 1 mg/l range 0.25-8 mg/l, de jong et al. 2015), cx (mic 50  =2 mg/l, mic 90 = 4 mg/l, range 0.25-64 mg/l, de jong et  al. 2015) and cl (mic 50 = 0.25 mg/l, mic 90 = 0.5 mg/l, lindeman et  al. 2013). cefonicid has not been studied in veterinary medicine, but susceptibility tests for s.  aureus from human isolates showed values of mic 50 = 4 mg/l and mic 90 = 8 mg/l (barry et  al. 1986). there are no reported mics for four cephalosporins tested in this study against isolated bacteria from rabbits. however, the obtained values of mic 90 were high compared to the cefiofur and cephalexin breakpoints [≤  2 μg/ml (susceptible), 4  μg/ml (intermediate), and ≥ 8 μg/ml (resistant)] reported for other bacterial species isolated from other animals (de jong et  al. 2014, papich and lindeman 2018). s.  aureus affects rabbits producing important dermal lesions and even invading subcutaneous tissues, resulting in suppurative dermatitis, mastitis, multisystemic abscessation and pododermatitis (segura et al. 2007). in addition, s. aureus is probably the most significant infectious agent that can cause mastitis in cows, goats, and sheep, because it produces a chronic and deep infection in the mammary glands that is extremely difficult to treat (mørk et  al. 2005, aires de sousa et  al. 2007). due to antibiotic resistance, treatment of infections 328 veterinaria italiana 2022, 58 (3), 325-329. doi: 10.12834/vetit.2241.16176.1 cephalosporin susceptibility of staphylococcus aureus strains badillo et al. aires-de-sousa m., parente c.e., vieira-da-motta o., bonna i.c., silva d.a. & de lencastre h. 2007. characterization of staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in rio de janeiro state, brazil. appl environ microbiol, 73, 3845-3849. badillo e., escudero e., hernandis v., galecio j.s. & marín p. 2020. pharmacokinetics of cefonicid in lactating goats after intravenous, intramuscular and subcutaneous administration, and after a long-acting formulation for subcutaneous administration. j vet pharmacol therap, 43 (1), 50-56. barry a.l., jones r. & packer r.r. 1986. antistaphylococcal activity of ceforanide and cefonicid in the presence of human serum. antimicrob agents chemother, 29 (1), 147-149. caprile k.a. 1988. the cephalosporin antimicrobial agents: a comprehensive review. j vet pharmacol therap, 11, 1-32. chopra i., hodgson j., metcalf b. & poste g. 1997. the search for antimicrobial agents effective against bacteria resistant to multiple antibiotics. antimicrob agents chemother, 41 (3), 497-503. clinical and laboratory standards institute (clsi). 2015. methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. 10th edition. nccls document m07-a10. clinical and laboratory standards institute, pennsylvania. clinical and laboratory standards institute (clsi). 2018. performance standards for antimicrobial susceptibility testing. 28th edition. nccls document m100. clinical and laboratory standards institute, pennsylvania. de jong a., moyaert h., simjee s., el garch f., haag-diergarten s., klein u., ludwig c., butty p., richard-mazet a., rigaut d., thiry j. & thomas v. 2015. antimicrobial susceptibility monitoring of mastitis pathogens isolated from acute cases of clinical mastitis in dairy cows across europe: vetpath results. int j antimicrob agents, 46, 13-20. de jong a., thomas v., simjee s., moyaert h., el garch f., maher k., morrissey i., butty p., klein u., marion h., rigaut d. & vallé m. 2014. antimicrobial susceptibility monitoring of respiratory tractpathogens isolated from diseased cattle and pigs acrosseurope: the vetpath study. vet microbiol, 172, 202-215. de oliveira a.p., watts j.l., salmon s.a. & aarestrup f.m. 2000. antimicrobial susceptibility of staphylococcus aureus isolated from bovine mastitis in europe and the united states. j dairy sci, 83 (4), 855-862. doré e., angelos j.a., rowe j.d., carlson j.l., weizlich s.e., kieu h.t. & tell l.a. 2011. pharmacokinetics of ceftiofur crystalline free acid after single subcutaneous administration in lactating and nonlactating domestic goats (capra aegagrus hircus). j vet pharmacol therap, 34 (1), 25-30. drusano g.l. 2004. antimicrobial pharmacodynamics: critical interactions of “bug and drugs”. nat rev microbiol, 2 (4), 289-300. references dzierzanowska d., patzer j. & jeljaszewicz j. 1982. pharmacokinetic studies of azlocillin, mezlocillin, cephalothin and sisomicin in rabbits. infection, 10 (3), s217-20. european medicines agency (ema). 2019. categorisation of antibiotics in the european union. answer to the request from the european commission for updating the scientific advice on the impact on public health and animal health of the use of antibiotics in animals. european medicines agency, amsterdam, the netherlands. fernández-varón e., cárceles-garcía c., serrano-rodríguez j.m. & cárceles-rodríguez c.m. 2016. pharmacokinetics (pk), pharmacodynamics (pd), and pk-pd integration of ceftiofur after a single intravenous, subcutaneous and subcutaneous-la administration in lactating goats. bmc vet 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16, s3-10. meneses m.l., albarellos g. & landoni m.f. 2013. pharmacokinetics of cephalexin after intravenous and single and multiple intramuscular administration to rabbit. int j vet med: res rep, 2013, 898594. mørk t., tollersrud t., kvitle b., jørgensen h.j. & waage s. 2005. comparison of staphylococcus aureus genotypes recovered from cases of bovine, ovine, and caprine mastitis. j clin microbiol, 43, 3979-3984. papich m.g. & lindeman c. 2018. cephalexin susceptibility breakpoint for veterinary isoletes: clinical laboratory standars institute revision. j vet diagn invest, 30 (1), 113-120. seegers h., fourichon c. & beaudeau f. 2003. production effects related to mastitis and mastitis economics in dairy cattle herds. vet res, 34, 475-491. segura p., martínez j., peris b., selva l., viana d., penades j.r. & corpa j.m. 2007. staphylococcal infections in rabbits does on two industrial farms. vet record, 160 (25), 869-872. 329veterinaria italiana 2022, 58 (3), 325-329. doi: 10.12834/vetit.2241.16176.1 badillo et al. cephalosporin susceptibility of staphylococcus aureus strains virdis s., scarano c., cossu f., spanu v., spanu c. & de santis e.p.l. 2010. antibiotic resistance in staphylococcus aureus and coagulase negative staphylococci isolated from goats with subclinical mastitis. vet med int, 2010 (2), 517060. 127 veterinaria italiana 2021, 57 (2), 127-134. doi: 10.12834/vetit.2302.13130.1 accepted: 01.09.2021 | available on line: 31.12.2021 1department of veterinary medicine, university of bari ‘aldo moro’, s.p. casamassima km. 3, 70010 valenzano (ba), italy. 2department of infectious diseases, istituto superiore di sanità, viale regina elena 299, 00161 rome, italy. 3interdisciplinary department of medicine, section of occupational medicine, university of bari ‘aldo moro’, piazza giulio cesare, 11, 70124 bari, italy. *corresponding author at: department of veterinary medicine university of bari ‘aldo moro’, str. prov. per casamassima, km. 3 70010 valenzano (ba), italy tel.: +39 0804679833, fax: +39 0804679843, e-mail: adriana.trotta@uniba.it, trotta.adri@pec.it. marialaura corrente1, adriana trotta1*, mariarosaria marinaro2, alessandra cavalli1, piero lovreglio3, margie cirilli1 and domenico buonavoglia1 keywords antibiotic use, antimicrobial resistance, cross-sectional survey, questionnaire, students. summary misconceptions about the use and effectiveness of antibiotics contribute to the persistence of antimicrobial resistance. the aim of this study was to gather information on appropriate use of antibiotics in students from the veterinary medicine college (g1, n = 119) and from high school (g2, n = 220), from bari (italy) through a questionnaire. the response rate was 89% in g1 and 89.5% in g2. fifty-five % of college students and 79% of high-school students had taken antibiotics in the last 12 months. unsurprisingly, high-school students had more misconceptions about antibiotics than g1. the majority of misconceptions stated that i)  antibiotics kill viruses (or 8.4, ci 4.8-14.7, p < 0.001); ii) they are active against cold and flu (or 4.6, ci 2.6-8.1, p < 0.001); iii) it is possible to purchase antibiotics without a medical prescription (or 7.3, ci 4.3-12.5, p < 0.001). information campaigns among young people are urgently needed to reduce misuse and to improve knowledge on antibiotics. basic knowledge and misconceptions on antibiotic use: a comparative survey between veterinary college and high school students in bari (italy) may result from a complex interaction between several factors, such as the patient’s attitudes, beliefs, and knowledge about antibiotic use and effectiveness (cockburn and pit 1997, davey et  al. 2002, baquero and campos 2003, vanden eng et al. 2003, mcnulty et al. 2007). the european union has therefore identified a community strategy against amr, in both human and veterinary medicine (ec action plan 2016). this action plan is intended to: i) prevent the spread of microbial infections, ii) ensure the appropriate use of antimicrobials, iii) undertake effective research programs to combat amr. communication, education and training of people represent a relevant part of this strategy. since 2008, the european centre for disease prevention and control (ecdc) has launched the ‘european antibiotic awareness day’ (eaad), an initiative that provides a platform for national campaigns to raise introduction antimicrobial resistance (amr) is a natural phenomenon in the context of bacterial homeostasis (martínez 2012). in addition, the discovery of antibiotics and their use for therapeutic purposes have generated a selective pressure toward antimicrobial resistance (hawkey 2008, davies and davies 2010). the inappropriate use of antibiotics in human and veterinary medicine has spread this phenomenon (bronzwaer et  al. 2002, who 2012, who 2014, marshall and levy 2011, ecdc 2014, prestinaci et  al. 2015). amr is nowadays a threat to global public health since it is responsible for increased costs and mortality rates for bacterial diseases in humans (cosgrove 2006, michaelidis et  al. 2016, friedman et  al. 2016) and animals (bengtsson and greko 2014). the inappropriate use of antibiotics in the community 128 basic knowledge on antibiotic use in students corrente et al. veterinaria italiana 2021, xx (x), xxx-xxx. doi: 10.12834/vetit.2302.13130.1 pilot study to assure the accuracy and consistency of the questions, a pilot study was conducted. the questionnaire was given to a selected convenience sample of 20 young people from the apulia region. the pilot study was successful and the questionnaire was not modified for the current study. data from the pilot study were not included in the subsequent survey. the survey a cross-sectional survey was conducted. two main population samples were selected: group 1 (g1, n  =  119) second-year students of the veterinary medicine college. the questionnaire was given at the beginning of the microbiology course. group  2 (g2, n = 220) students of the high school, istituto deviti demarco, located in valenzano (bari, italy). the survey was made within the project ‘percorso alternanza scuola lavoro’ in 2016-2017. the questionnaire was given during the lessons, with a brief introduction on the aim of the survey made by the teachers. this study did not require local ethics committee approval since it did not require the collection of biological samples or execution of medical treatments/ investigations. all participants provided written consent to participate. the study was conducted in accordance with italian and institutional standards, with the principles set down in the declaration of helsinki and its revisions, and with local legislation. following the guidelines of the italian data protection act, only limited data elements (including gender and year of birth) were collected from students, in order to analyse the data anonymously (d.lgs. 196/2003). statistical analysis the data were analysed with excel 2010. descriptive data analyses were undertaken including frequencies and percentages, calculated for the categorical data. the answers to q3-q4 were dichotomised, i.e. ‘correct’ versus ‘incorrect/i don’t know’, while the answers to q5 (source of the information other than school/university) were dichotomised as ‘always/ often’ vs. ‘never/sometimes’. statistical analysis was performed for categorical data by using the chi-square test with a significance level p < 0.05. the data were analysed using sas software, version 8.2. odds ratio (or) with 95% confidence interval (ci) was calculated for measuring the risk. results table i summarizes the characteristics of the 2 sample populations. awareness on prudent use of antibiotics (ecdc/ eaad 2008). as a part of this strategy, the directorate-general for health and consumers of the european union commissioned to eurobarometer agency three wide surveys in 2010 (ec eurobarometer report 338, 2010), in 2013 (ec eurobarometer report 407) and, in 2016 (ec eurobarometer report 445), to test the level of public knowledge on antibiotics and the risks associated with their unnecessary use. the overall picture emerging from those surveys is that the general public has many preconceived notions on antimicrobial substances and their effects. for instance, only 43% of europeans know that antibiotics are ineffective against viruses, and just 56% know that they are ineffective against cold and flu (ec eurobarometer report 445, 2016). there is a substantial difference between northern and southern nations of europe, regarding misconceptions, with italy classified among the nations with the highest personal consumption of antibiotics and lowest perception of the problem (ec eurobarometer report 445, 2016; ecdc 2017). the age of people interviewed and the level of education also influence the use of antibiotics and perception of amr. europeans aged from 15 to 24 years were found to have the worst perceptions on amr and the consumption of antibiotics resulted higher among those with a low level of education (ec eurobarometer 445, 2016). the aim of this study was to gather information on antibiotic use and beliefs in students from the veterinary medicine college of the university of bari and a high school in bari, italy. a questionnaire was administered to test their level of knowledge. materials and methods questionnaire design the survey was developed based on the amr eurobarometer surveys, report numbers 338, 407 and 445 (ec eurobarometer report 338, 2010; ec eurobarometer report 407, 2013; ec eurobarometer report 445, 2016), with some modifications. the questionnaire was reduced to 10 questions (q) covering the personal use of antibiotics, the knowledge on the efficacy of antibiotics and the source of information about the topic (q1-5). the final part of the questionnaire investigated the socio-demographic characteristics of respondents (age and sex) and the presence of health workers in the family. (q6-9). all the questions were close-ended, except for the q9 (age of the respondents), and q10 (suggestions or comments to the questionnaire) (s1 questionnaire). 129 corrente et al. basic knowledge on antibiotic use in students veterinaria italiana 2021, 57 (2), 127-134. doi: 10.12834/vetit.2302.13130.1 had taken antibiotics for fever (68), flu (63), cold (52), sore throat (50) cough (42), headache (37), bronchitis (14), diarrhoea (11), skin infection (59), uti (3), or for other causes (7) (figure 1). knowledge on antibiotic action (q3, statements 3.1, 3.2, 3.3 and 3.4) g1: eighty-three respondents (78.3%) knew that antibiotics are not active against viruses and 45  of them (43%) were aware that antibiotics are not effective against cold and flu. ninety-two respondents (86.8%) knew that antibiotics often produce side-effects and 78 (73.6%) knew that abuse of antibiotics makes them ineffective. g2: 59 respondents (30%) knew that antibiotics are not active against viruses, while just 27 students (13.7%) knew that antibiotics are inactive against cold and flu. eighty-three of the respondents (42.1%) knew that antibiotics often produce side-effects and 130 (66%) that unnecessary use of antibiotics makes them ineffective. use of antibiotics and compliance with the prescription (q4, statements 4.1, 4.2, 4.3, 4.4) g1: seventy-four respondents (70%) said that it is necessary to not forget one or more doses, while 87 (82%) knew that it is not correct to stop taking antibiotics when feeling better and 64 (60%) that prescriptions are needed to purchase an antibiotic. regarding the choice of a new generation antibiotic rather than an old one, 43 respondents (40.6%) were aware that this is not a correct practice. g2: 105 respondents (53.3%) correctly answered ‘false’ to q 4.1 (you may forget one or more doses). seventy-three (37%) knew that it is not correct to stop taking antibiotics as soon as you feel better. only 34 students (17.3%) knew that it is not possible to purchase an antibiotic without a response rate one hundred six out of 119 g1 students answered the questionnaire (response rate 89%). one hundred ninety-seven out of 220 g2 students completed the questionnaire (response rate 89.5%). demographic characteristics of the groups (q 6‑9) g1: the mean age was 21 years (range 20-23 years). sixty-two were females (58%), 44 males (42%). thirty-seven out of 106 respondents lived with one or more health-workers (35%): 15 were nurses, 10 were physicians, 7 were veterinarians, 3 were pharmacists, 3 were biologists, 2 were lab workers. g2: the mean age was 17 years (range 13-18 years). one hundred thirty-seven were males (69.5%), 60 females (30.5%). fifty-four students lived with one or more health workers (27.4%). thirty-two were nurses, 20 were physicians, 5 were pharmacists, 4 were veterinarians, 1 was a lab worker. antibiotic use (q1‑2) g1: fifty-eight students (55 %) had taken antibiotics in the last 12 months, for bronchitis (3), flu (18), sore throat (28), cough (10), fever (15) headache (5), diarrhoea (3), urinary tract infections (uti) (5), or for other causes (15). g2: one hundred fifty-six students (79%) had taken antibiotics in the last 12 months. in most cases, they table i. main characteristics of the two study populations. g1 undergraduate students (frequency %) g2 high school students (frequency%) total 119 220 total of respondents (%) 106 (89) 197 (89.5) gender female 62 (58) 60 (30.5) male 44 (42) 137 (69.5) mean age (range) 21 (20-23) 17 (13-18) a health care worker in the family yes 37 (35) 54 (27.4) nurse 15 32 physician 10 20 veterinarian 7 4 pharmacist 3 5 biologist 3 0 lab worker 2 1 other 0 0 no 69 (65) 143 (72.6) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% other bronchitis flu cold cough fever headache uti diarrhoea skin infection sore throat g1 g2 37 5 2 3 7 5 24 14 44 9 27 27 32 33 17 40 3 9 14 4 figure 1. reasons for taking antibiotics in two study populations. g1 = undergraduate students; g2 = high school students; uti = urinary tract infection. 130 veterinaria italiana 2021, xx (x), xxx-xxx. doi: 10.12834/vetit.2302.13130.1 basic knowledge on antibiotic use in students corrente et al. table ii. comparison between g1 (undergraduate students) and g2 (high school students) groups regarding the use and basic knowledge of antibiotics. questions answer g1 n=106 (%) g2 n=197 (%) or (95% ci) p value q.1 have you taken any antibiotics in the last 12 months? yes 58 (55) 156 (79) 0.3 (0.2-0.5) < 0.001 no 48 (45) 41 (21) q.3.1 antibiotics kill viruses false 83 (78.3) 59 (30) 8.4 (4.8-14.7) < 0.001 true 23 (21.7) 138 (70) q.3.2 antibiotics are effective against cold and flu false 45 (43) 27 (13.7) 4.6 (2.6-8.1) < 0.001 true 61 (57%) 170 (86.3) q.3.3 taking antibiotics often causes side-effects true 92 (86.8) 83 (42.1) 9 (4.8-16.9) < 0.001 false 14 (13.2) 114 (57.9) q.3.4 unnecessary use of antibiotics contributes to make them ineffective true 78 (73.6) 130 (66) 1.4 (0.8-2.4) > 0.05 false 28 (22.4) 67 (34) q.4.1 you may forget one or more doses false 74 (70) 105 (53.3) 2 (1.2-3.3) < 0.01 true 32 (30) 92 (46.7) q.4.2 you can stop to take it as soon as you feel better false 87 (82) 73 (37) 7.8 (4.4-13.8) < 0.001 true 19 (18) 124 (63) q.4.3 you can take it without a prescription from a pharmacy false 64 (60) 34 (17.3) 7.3 (4.3-12.5) < 0.001 true 42 (40) 163 (82.7) q.4.4 a new antibiotic is preferable to an old one false 43 (40.6) 68 (34.5) 1.3 (0.8-2.1) > 0.05 true 63 (59.4) 129 (65.5) no significant difference between the groups was observed on the use of antibiotics in the last 12  months. on the opposite, a significant difference in the level of general knowledge on antibiotics was found. students belonging to the g2 group seemed to have more misconceptions about the use of the antibiotics. in particular, most g2 students believed that antibiotics kill viruses (or 8.4, ci 4.8-14.7, p  <  0.001) and that they are active against cold and flu (or 4.6, ci 2.6-8.1, p  <  0.001). students of the g2  group had also a worse perception of antibiotic side effects (or 9, ci 4.8-16.9, p  <  0.001), when compared to the g1 students. regarding the statement ‘unnecessary use of antibiotics contributes to make them ineffective’, the difference of knowledge between the groups was not statistically significant (or 1.4, ci 0.8-2.4, p > 0.05). a higher number of g2 students gave incorrect answers to the statement: ‘you may forget one or more doses’ (or 2, ci 1.2-3.3, p < 0.01) than g1 students. when asked if it is possible ‘to stop taking antibiotics as soon as you feel better’, a greater proportion of g2 respondents gave an incorrect answer (or 7.8, ci  4.4-13.8, p  <  0.001), than g1 students. a greater and significant number of g2  respondents believed that it is possible to purchase an antibiotic without a medical prescription (or 7.3, ci 4.3-12.5, p  <  0.001), while no significant difference was found regarding the answer to the statement ‘a new antibiotic is preferable to an old one’ (or 1.3, ci 0.8-2.1, p > 0.05). medical prescription. regarding the choice of a new generation antibiotic rather than an old one, 68 respondents (34.5%) were aware that this is an incorrect practice. interestingly, within the g1 group just 2 students answered correctly to all q3 questions, and 1 student to all q4 questions. in the g2 group, 3 students gave 4 correct answers to all q3 questions and 2 students to q4 questions. in both groups, no one answered correctly to all questions. source of information (q5) when the g1students were asked where they obtained the information from (other than university courses), they answered either internet (82%) and television (62%) while only 36% consulted books or newspapers. students of the g2 group gathered information from the internet (63%) or television (54.4%) while 38% of them consulted books or newspapers. statistical analysis differences between g1 and g2 the comparison between the two groups is shown in table ii. 131veterinaria italiana 2021, 57 (2), 127-134. doi: 10.12834/vetit.2302.13130.1 corrente et al. basic knowledge on antibiotic use in students 338, 2010; ec eurobarometer report 407, 2013; ec eurobarometer report 445, 2016). thus, it was possible to compare our results with those from the eurobarometer surveys. the administration of the questionnaire during the lessons allowed us to obtain a high response rate. the study populations were composed of two age groups, both studying scientific topics although specific information on the use of antimicrobials was not included yet in their curricula. regarding the personal consumption of antibiotics, the number of students that had taken antibiotics in the last 12 months was high, 55% in g1 and 79% in g2. these numbers appear very high, especially when compared with the frequencies reported in the last eurobarometer survey (2016), i.e. 34%. in italy, in 2016 this proportion was already high (43%) (ec eurobarometer report 445, 2016) and an increasing trend was noted when considering the percentage reported in 2013 (36%) (ec eurobarometer report 407, 2013). the 2016 eurosurvey report showed that respondents, whose education ended at or before the age of 15 were more likely to have taken antibiotics (39%) than those who ended studying at 16 (33%) or later (32%) (ec eurobarometer report 445, 2016). regarding the g1 students, the high number of antibiotic consumers could be explained by several factors, such as the influence does the presence of health workers in the family improve knowledge about the proper use of antibiotics? to test if the presence of one or more health workers in the family was related to a better knowledge of the use of antibiotics, a statistical analysis was performed in both groups. g1: students living with health workers did not take more or fewer antibiotics than other students, neither seemed to have a better knowledge about the antibiotics (table iii). g2: students living with health workers did not take fewer antibiotics, but knew more about the efficacy of antibiotics, than other students. in fact, they knew that antibiotics are not effective against cold and flu (or 2.7, ci 1.2-6.1, p < 0.05), that they may cause side effects (or 4, ci 2.1-7.9, p < 0.001) and that compliance with drug prescription is a relevant issue (or 2.5, ci 1.3-4.7, p < 0.01) (table iv). discussion the present study aimed at investigating both personal consumption of antibiotics in young people and their level of knowledge about antibiotic action and proper use. two age groups of students were compared by administering a questionnaire. the survey was based on previous eurobarometer surveys on amr (published in 2010, 2013 and 2016) (ec eurobarometer report table iii. presence of health workers in the family and different knowledge on the use of antibiotics: g1 (undergraduate students). questions answer students with health workers students without health workers or (95%ci) p value q.1 have you taken any antibiotics in the last 12 months? yes 23 36 1.05 (0.7-3.4) > 0.05 no 14 33 q.3.1 antibiotics kill viruses false 30 52 1.4 (0.5-3.8) > 0.05 true 7 17 q.3.2 antibiotics are effective against cold and flu false 11 31 0.5 (0.2-1.2) > 0.05 true 26 38 q.3.3 taking antibiotics often causes side-effects true 26 51 0.8 (0.3-2) > 0.05 false 11 18 q.3.4 unnecessary use of antibiotics contributes to make them ineffective true 33 56 1.9 (0.6-6.4) > 0.05 false 4 13 q.4.1 you may forget one or more doses false 24 52 0.6 (0.25-1.4) > 0.05 true 13 17 q.4.2 you can stop to take it as soon as you feel better false 31 56 1.2 (0.4-3.5) > 0.05 true 6 13 q.4.3 you can take it without a prescription from a pharmacy false 23 45 0.9 (0.4-2) > 0.05 true 14 24 q.4.4 a new antibiotic is preferable to an old one false 17 24 1.6 (0.7-3.5) > 0.05 true 20 45 132 veterinaria italiana 2021, xx (x), xxx-xxx. doi: 10.12834/vetit.2302.13130.1 basic knowledge on antibiotic use in students corrente et al. are active against cold and flu were high in both g1 and g2 groups. in 2013, 60% of european people thought that antibiotics are active against viruses, and 52% that are active against cold and flu, in 2016 the proportions were 57% and 56%, respectively (ec eurobarometer report 407, 2013; ec eurobarometer report 445, 2016). italy is reported among the countries with lower percentages of correct answers. other surveys conducted in italy show similar results (napolitano et  al. 2013, prigitano et al. 2018), suggesting that information campaigns should be planned to inform people about viruses and that they are a common cause for cold and flu (mcnulty et al. 2011). regarding the compliance to the antibiotic therapy, altogether a good knowledge in the g1  group was found. however, few persons (40.6%) correctly answered the question: a new antibiotic is preferable to an old one. another issue was the possibility to purchase an antibiotic without a medical prescription. students from both groups were not aware that a medical prescription is necessary, even if prescriptions in italy are mandatory (d.lgs. 219/2006). eurosurvey reports that the proportion of people that were not aware of a medical prescription was very low (ec eurobarometer report 445, 2016). the percentage of g2 students giving a correct answer was in general lower than that of g1 students. young people use social networks as information source (wong et  al. 2014), in fact, in this survey, both groups declared to use the internet as a main of the parents. some self-limiting diseases may be a cause of concern for parents and prescription of antibiotics may be influenced by their pressure to medical practitioners, or could be an autonomous decision (bauchner et al. 1999, cabral et al. 2015). for instance, in a survey conducted in italy on parents of public schools’ students, a third of them declared to use antibiotics for self-medication, and they either purchased them without prescription or used those available at home (napolitano et al. 2013). another possible explanation of this high percentage could be the lack of specific knowledge of antibiotics. in fact, most of the respondents indicated they had taken antibiotics for treating viral infections or non-infectious diseases, such as headache. in general, as eurobarometer surveys highlight, most of europeans use antibiotics for reasons other than bacterial diseases (ec eurobarometer report 338, 2010; ec eurobarometer report 407, 2013; ec eurobarometer report 445, 2016). flu and sore throat are the commonest examples. also, bronchitis, that it is not always caused by bacteria, was indicated among the reasons for taking antibiotics. an additional problem in south europe countries is the use of antibiotics without a specific diagnosis made by medical practitioners (grigoryan et  al. 2006, morgan et  al. 2011). a discrepancy of knowledge between the two groups was found about the activity against viruses, with a lower level of knowledge in the g2  group. frequencies of students that incorrectly stated that antibiotics table iv. presence of health workers in the family and different knowledge on the use of antibiotics: g2 (high school students). questions answer students with health workers students without health workers or (95%ci) p value q.1 have you taken any antibiotics in the last 12 months? yes 42 114 0.9 (0.4-1.9) > 0.05 no 12 29 q.3.1 antibiotics kill viruses false 21 37 1.8 (0.93-3.6) > 0.05 true 33 106 q.3.2 antibiotics are effective against cold and flu false 13 15 2.7 (1.2-6.1) < 0.05 true 41 128 q.3.3 taking antibiotics often causes side-effects true 37 50 4 (2.1-7.9) < 0.001 false 17 93 q.3.4 unnecessary use of antibiotics contributes to make them ineffective true 33 91 0.9 (0.5-1.7) > 0.05 false 21 52 q.4.1 you may forget one or more doses false 23 80 0.6 (0.3-1.1) > 0.05 true 31 63 q.4.2 you can stop to take it as soon as you feel better false 30 48 2.5 (1.3-4.7) < 0.01 true 24 95 q.4.3 you can take it without a prescription from a pharmacy false 7 27 0.6 (0.3-1.6) > 0.05 true 47 116 q.4.4 a new antibiotic is preferable to an old one false 18 51 0.9 (0.5-1.7) > 0.05 true 36 92 133veterinaria italiana 2021, 57 (2), 127-134. doi: 10.12834/vetit.2302.13130.1 corrente et al. basic knowledge on antibiotic use in students other methods of data collection, and with a larger sample size may be useful to expand the results reported here. in conclusion, despite scientific studies, students interviewed were not aware of the exact function of antibiotics. including specific topics in school curricula may provide a better understanding of the use of antibiotics (avezedo et  al. 2009). as far as concern for veterinary students, the perception on amr in different phases of their education is critical in guiding them for their future profession (hardefeldt et al. 2018). acknowledgments the authors would like to thank the students of the veterinary college of bari, the teacher paola ruggieri and the students of the high school de viti de marco of valenzano (ba) for the participation at this study. source of information, while books or newspapers were poorly consulted. the consequences of using unofficial sources of information are obvious and may explain some of the statements reported here. despite the use of the same sources of information, misconceptions in g1 may be compensated by scientific studies. regarding the presence of a health worker in the family, it was interesting to note that g1 students having a health worker in the family did not use fewer antibiotics, nor knew more about antibiotics in general. on the opposite, in the g2 group some differences were noted. in fact, students living with a health worker were found to be 3-4 times better informed on antibiotic activity, side effects and compliance. a cross-sectional survey represents a snapshot of participants’ view at a particular time (kelley et al. 2003). moreover, when data are collected by questionnaire, respondents may 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martínez j.l. 2012. natural antibiotic resistance and contamination by antibiotic resistance determinants: the two ages in the evolution of resistance to antimicrobials. front microbiol, 3, 1-3. mcnulty c.a, lecky d.m., farrell d., kostkova p., 241 osama b. mohammed1*, omar i. omar2, elgailani a. elamin3, hamid o. bushara4, sawsan a. omer5 and abdulaziz n. alagaili1 1ksu mammals research chair, department of zoology, college of science, king saud university, p.o. box 2455, riyadh 11451, saudi arabia. 2joon trading company, p.o. box 325511 riyadh 11371, saudi arabia. 3department of parasitology, faculty of veterinary medicine, university of khartoum, p.o. box 32, postal code 13314, khartoum north, sudan. 4idac labs, p.o. box 7133, al‑karij 11942, saudi arabia. 5department of zoology, college of science, king saud university, university centre for women students, p.o. box 22452, riyadh 11495, saudi arabia. *corresponding author at: ksu mammals research chair, department of zoology, college of science, king saud university, p.o. box 2455, riyadh 11451, saudi arabia tel.: +966 504140713, e‑mail: obmkkwrc@yahoo.co.uk. veterinaria italiana 2019, 55 (3), 241‑245. doi: 10.12834/vetit.221.695.4 accepted: 16.11.2015 | available on line: 30.09.2019 riassunto lo scopo dello studio è stato quello di determinare la sieroprevalenza di toxoplasma gondii in 200 campioni di siero prelevati da gatti domestici e randagi nella città di riyadh (arabia saudita). la sieroprevalenza complessiva è risultata pari al 26%; la prevalenza di anticorpi contro t. gondii è risultata significativamente più alta (p < 0,05) nei gatti randagi (39%) rispetto ai gatti domestici (13%). la prevalenza nei gatti maschi è stata del 31,4%, mentre nelle femmine del 20,4% ed è risultata più alta nei gatti di età superiore ai 6 anni (43%) rispetto ai gatti di età inferiore a 4 anni (33%). la sieropositività più alta è stata riscontrata nei gatti di razza americana (38,5%), seguita da quelli di razza persiana (27%), himalayana (21%), bengalese (11,5%) e siamese (2%). non sono stati riscontrati anticorpi nei gatti di razza turca. la prevalenza non ha mostrato significative variazioni stagionali né territoriali tra le aree all'interno della municipalità di riyadh. la flottazione dei campioni fecali ha rivelato una prevalenza complessiva di oocisti di t. gondii pari al 12%. parole chiave arabia saudita, gatti, sieroprevalenza, toxoplasmosi. sieroprevalenza di toxoplasma gondii in gatti domestici e randagi, riyadh, arabia saudita keywords cats, saudi arabia, seroprevalence, toxoplasmosis. summary this study was undertaken to determine the seroprevalence of toxoplasma gondii in cats in the area of riyadh, saudi arabia. we examined 200 serum samples collected from stray and household cats for t. gondii antibodies by elisa. the overall seroprevalence was  26%. seroprevalence was significantly higher (p < 0.05) in stray cats (39%) compared to household cats (13%). the prevalence in male and female cats was 31.4% and 20.4%, respectively. the seroprevalence increased with age and was higher in cats over 6 years of age (43%) as opposed to cats less than 4 years old (33%). seropositivity varied according to the breed. the highest was recorded among cats of american breed (38.5%), followed by persian (27%), himalayan (21%), bengali (11.5%), and siamese (2%). antibodies were not reported from the turkish breed. overall seroprevalence among cats did not vary significantly with season or with the localities within the riyadh municipality. we also examined 100 faecal samples from stray and household cats by flotation technique, which revealed an overall prevalence of 12% of t. gondii oocycts. seroprevalence of toxoplasma gondii in household and stray cats of riyadh, saudi arabia 242 toxoplasmosis in cats in saudi arabia mohammed et al. veterinaria italiana 2019, 55 (3), 241‑245. doi: 10.12834/vetit.221.695.4 collected from the animal without sedation or following sedation with a dose of xylzine hydrochloride (rompun, bayer, leverkusen, germany) at a dose of 2 mg/kg combined with ketamine hydrochloride (imalgene, rhone merieux) at adose of 5 mg/kg. stray cats were baited with food containing 10 mg of acepromazine and then were given the same sedative dose in order to enable blood collection. cats were bled from the jugular vein using 20 g x ½  inch needle into 5  ml syringes without anticoagulant. blood was transferred into clean tubes and allowed to clot overnight at room temperature, centrifuged for 10 minutes at 1,500 g. sera were transferred into clean 1.5  ml eppendorf tubes and stored at ‑ 20°c until they were used. a volume of 5 grams of rectal stool was collected from each cat enrolled in the study. faeces were collected into polythene containers and preserved in 2.5% potassium dichromate (k 2 cr 2 o 7 ) and used for coprological studies. serum samples were subjected to an elisa method for the detection of t. gondii antibodies. this test was performed by using the chekit‑toxotest elisa kit (idexx laboratories, bommeli diagnostics, ag, and bern. switzerland) according to the manufacturer’s instructions. faecal samples from cats were examined for the presence of oocysts by sugar flotation technique, as described by dubey and colleagues (dubey et al. 1970). statistical analysis we used the statistical program spss in order to analyse data. the data was analysed with chi‑square test and the level was considered significant at p ≤ 0.05. results serological examination serological examination showed that 52 (26%) out of 200 cats had antibodies to toxoplasma gondii by elisa (table i). serum samples with o.d. values > 40% were considered positive for the presence of t.  gondii antibodies. most (< 50%) of the cats that tested positive had o.d. over 100% and only 2 (3.8%) cats recorded an o.d. value of > 200 (high positive), as it is shown in table i. in stray cats, antibodies were found in 39 out of 100  (19.5%) tested cats. antibodies were found in 13 out of 100 tested samples of household cats (6.5%). a significant difference in the seroprevalence between stray and household cats (p  <  0.05) was introduction toxoplasma gondii is one of the most common protozoan infecting a wide range of animals, and humans (dubey and beattie 1988). the infection causes considerable economic losses in the sheep industry. cats play an important role in the epidemiology of t. gondii in humans and animals. cats and other felidae, definitive hosts of t.  gondii, are the only animals that pass oocysts in the faeces; although as intermediate hosts they can harbour infectious tissue cysts. most feline infections occur post‑natally through the ingestion of infected tissue cysts or rarely oocysts, although congenital infection can also occur (dubey and jones 2008). feline infections are typically subclinical; congenitally infected kittens are the most likely to have clinical signs of infection, but previously clinically healthy adult cats may also be affected (vollaire et al. 2005, dubey and jones 2008). common symptoms of t.  gondii infection in cats include fever, ocular inflammation, anorexia, lethargy, myosyitis, abdominal discomfort, and neurologic abnormalities (vollaire et al. 2005). most cats only shed oocysts once in their lives, following infection. however following experimental infection and immunosuppression, repeated shedding occurred in kittens 20‑21 days following the immunosuppressive event. in addition, oocysts shedding was induced in a non‑immunosuppressed kitten following a second inoculation with infected mouse brain homogenate (malmasi et al. 2009). materials and methods origin of animals the main source of household cats in this study was a private pet clinic located in riyadh, saudi arabia. cats were presented either with clinical signs that warranted veterinary care or for routine general health checks and vaccination. some cats were admitted for minor veterinary interventions such as spaying. information detailing age, sex, and area originated from individual cat registration cards. cats (100) belonged to 6 breeds including persian (27), himalayan (22), american (19), turkish (14), siamese (12), and bengali (6). stray cats (100) wandering in the streets, parks, and nearby slaughterhouses, restaurants, and residential compounds represented the stray cat sample of this study. sampling and testing blood samples from household cats were either 243 mohammed et al. toxoplasmosis in cats in saudi arabia veterinaria italiana 2019, 55 (3), 241‑245. doi: 10.12834/vetit.221.695.4 the prevalence of t. gondii antibodies was higher in females. other investigators have also reported differences in prevalence according to sex (miro et al. 2004, lee et al. 2010, esteves et al. 2014). moreover, seroprevalence rates also increased with the age of cats, similarly to previous studies. variation in prevalence in different breeds was observed in this study. however, further studies are warranted in order to establish a close association between t. gondii seroprevalence and cat breed. the prevalence of infection did not vary significantly according to the different collection sites, or origin, of cats in the city of riyadh. this could be the result of similar environmental conditions present in riyadh. there is no information yet on density of stray cats and their spatial distribution within riyadh city. in this regard, the spatial distribution of areas contaminated by t. gondii oocyst in lyon, france, was described to be highly heterogeneous (afonso et al. 2008). in rural areas of france, local meteorological conditions were also found to influence the spatial distribution of incidence risk in cats (afonso et  al. 2008). the results of the coprological examination revealed an overall prevalence of 12%. in conclusion, the results of the present study confirm the occurrence of t. gondii in cats in riyadh city. further studies in additional areas will be necessary to understand the overall epidemiological status of toxoplasmosis in household and stray cats in saudi arabia. acknowledgments funding for this study was provided by the deanship of scientific research at the king saud university through vice deanship of research chairs. demonstrated. a difference in seroprevalence was also revealed between male (20.4%, 95% ci 0.98‑1.37) and female (31.4%) cats screened in this study, however this difference was not statistically significant (table  ii). cats aged 6 years and more showed significantly high prevalence (p  <  0.05) compared with cats of other age groups. the highest number of cats (5) showing anti‑t. gondii antibodies belonged to the american breed, followed by the persian breed (4), himalayan breed  (2), bengali and siamese (1). no antibodies were detected in cats belonging to the turkish breed. examination of faecal samples revealed the presence of oocysts in 17 stray and 7 household cats, giving an overall prevalence of 12%. all shedders cats were also seropositive. there was no significant difference in shedding between stray and household cats (p > 0.05). discussion in this study, elisa was employed to determine the seroprevalence of toxoplasmosis in stray and household cats in the riyadh area. the results showed that the overall infection rate was 26%. the prevalence of t.  gondii antibodies in stray cats (39%) was significantly higher (p < 0.05) than that of household cats (13%), likely due to the higher chance of stray cats to get infected. this scenario is expected in light of the dispersion of the infectious stages of t. gondii in the environment of stray cats. moreover, the hunting behaviour of felines further facilitates infection through the consumption of intermediate hosts. this general pattern of infection has been reported for stray versus household cats in various cities of the world, as in seoul, south korea (lee et al. 2011); tahran and sari, iran (haddadzadeh et al. 2006, sharif et al. 2009); ankara, turkey (ozkan et al. 2008); lanzhou, china (wu et al. 2011); colombo, sri lanka (kulasena et al. 2011); and la rioja, spain (miro et  al. 2004). the seroprevalence among household cats further indicates the ubiquity of t.  gondii and the strong association with its definitive host. the parasite is able to access both cats who live outdoors and those who are kept solely on canned food, if not properly cooked (de creaye et  al. 2008, lopez et  al. 2008, lee et al. 2010). table i. elisa results from stray and household cats. cats no. negative (%) no. positive (%) class interval for o.d.% 41-80 (%) 81-120 (%) 121-160 (%) 161-200 (%) >200 (%) stray 61 (61) 39 (39) 9 (23.1) 10 (25.6) 15 (38.5) 5 (12.8) 0 (0) household 87 (87) 13 (13) 0 (0) 3 (23.1) 3 (23.1) 5 (38.4) 2 (15.4) total 148 (74) 52 (26) 9 (17.3) 13 (25) 18 (34.6) 10 (19.2) 2 (3.8) table ii. comparison between the seroprevalence of t. gondii in male and female cats. sex no. of positive cats (%) no. of negative cats (%) total male 20 (20.4) 78 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pseudintermedius, dog, antibiotic resistance, university veterinary hospitals. summary staphylococcus pseudintermedius represents one of the most frequently bacteria isolated on dog's skin and it was recently recognized as a zoonotic pathogen responsible for severe diseases also in humans. this study aimed to define the occurrence of canine methicillin-resistant and methicillin-susceptible s. pseudintermedius (mrsp and mssp) strains and to compare their antimicrobial profiles. the study was carried out at veterinary microbiology laboratories of two different italian veterinary teaching hospitals, milan and naples, from 2015 to 2017. the statistical comparison of the results revealed significant differences in mrsp occurrence (p-value = 0.0435) and mrsp and mssp antibiotic resistance profiles. in milan, mrsp strains displayed significantly higher antibiotic resistance percentages (p < 0.001) for some antibiotics, such as ceftriaxone and tobramycin, compared to those of naples. conversely, mssp strains of naples presented significantly higher rates (p < 0.001) of resistance to amoxicillin/clavulanic acid, kanamycin, erythromycin, and tetracycline than to milan isolates. in conclusion, the results highlighted relevant variances among region-specific antibiotic resistance profiles, probably due to different antimicrobial selection pressures. therefore, this study stands out the need for continuous monitoring of both mrsp and mssp linked to different geographical areas, also considering their impact and importance on animal and human health. francesca paola nocera1#, gabriele meroni2#, filomena fiorito1, luisa de martino1* and piera anna martino2 occurrence and antimicrobial susceptibility patterns of canine staphylococcus pseudintermedius strains isolated from two different italian university veterinary hospitals veterinaria italiana 2020, 56 (4), 263-269. doi: 10.12834/vetit.2195.12897.1 accepted: 07.07.2020 | available on line: 31.12.2020 in the past, s.  pseudintermedius isolates were generally susceptible to β-lactams, whose principal antimicrobial agent is penicillin. therefore, methicillin-susceptible s.  pseudintermedius (mssp) strains originally circulated in canine population. however, already in 2006, methicillin-resistant s.  pseudintermedius (mrsp) strains were isolated in europe, becoming a relevant problem in veterinary medicine. over the years, mrsp have been reported with an increasing frequency (loeffler et  al. 2007, van  duijkeren et  al. 2011, kasai et  al. 2016) and interestingly mrsp strains have often been showing multidrug resistance profiles worldwide, including resistance to several classes of antimicrobial drugs introduction staphylococcus pseudintermedius (s. pseudintermedius) has been described for the first time in 2005 in dogs and pigeons, thanks to the 16s rrna gene sequence analyses (devriese et al. 2005). s.  pseudintermedius can be isolated in healthy dogs from forehead, nares, oral mucosa, pharynx, groin, and anus (garbacz et al. 2013). however, in particular conditions, such as injuries and sickness, it can act as an opportunistic pathogen for dogs and cats and, mainly in dogs, s.  pseudintermedius is associated with skin and ear infections (de martino et al. 2016, fitzgerald 2009, weese and van duijkeren 2010, van duijkeren et al. 2011). 264 antimicrobial susceptbility of s. pseudintermedius nocera et al. veterinaria italiana 2020, 56 (4), 263-269. doi: 10.12834/vetit.2195.12897.1 morphology, β-haemolysis, cellular morphology (gram's staining), catalase test, and then sub-cultured on mannitol salt agar plates (msa, liofilchem, italy). each mannitol salt negative colony was also subjected to staphylocoagulase (tube coagulase) test (oxoid, ltd, uk) to confirm their capacity to produce coagulase enzymes. the identification was assessed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) analysis (bruker daltonics, germany), using fresh colonies. values from 2.3 to 1.9 indicated the best identification of genus and species of staphylococci (santos et  al. 2013, nisa et  al. 2019). all the strains were stored in 25% glycerol until use (carlo erba, italy) at -  20  °c. before use, samples were thawed at room temperature, and 10 μl were plated on tryptic soy agar + 5% sheep blood (microbiol, italy) and incubated aerobically at 37  °c for 24 h. three or four isolated colonies were picked up and used for the analyses. genetic identification of s. pseudintermedius for the molecular identification of the selected strains, dna was extracted from fresh cultures of each s. pseudintermedius isolates. the bacterial dna extraction was carried out by the boiling method (adwan et al. 2014). the quantity and quality of dna were determined by the spectrophotometric reading of a260/a280 ratio (eppendorf biophotometer 6131). then, dna samples were stored at 20 °c. molecular identification, using species-specific nuc gene (sasaki et  al. 2010) was performed by pcr to confirm the identification of s.  pseudintermedius strains. dna of s. pseudintermedius atcc® 49444tm was used as a positive control. antimicrobial susceptibility test the antimicrobial susceptibility test was performed on all s.  pseudintermedius strains using kirby-bauer disk diffusion method according to clinical laboratory and standards institute (clsi 2015) guidelines. strains were classified as susceptible, intermediate and resistant by comparison of the zone of inhibition as indicated by the same guidelines. table i reported the antibiotics used . amplification of antibiotic‑resistance genes (arg) to study the dissemination of genes coding for antimicrobial resistance, the amplification of the following genes was performed by qualitative pcrs: meca (chovanovà et  al. 2015), mecc (stegger et  al. 2012) and tetracyclines family genes (tetk, tetl, tetm, (perreten et al. 2010). this scenario limits the treatment options and represents a relevant threat to small animal therapy in veterinary medicine, challenging infection control measures (van duijkeren et  al. 2011, bannoehr and guardabassi 2012, bond and loeffler 2012). there are several reports on isolates resistant almost to all antimicrobials authorized in veterinary medicine (wettstein et  al. 2008, perreten et al. 2010), inducing clinicians to use antimicrobials authorized only for human medicine (weese and van duijkeren 2010). the s.  pseudintermedius virulence potential and its zoonotic transmission should not be underestimated, even though it is not often reported. s.  pseudintermedius, particularly mrsp, has also been isolated from humans, especially in pet owners (van hoovels et al. 2006, stegmann et al. 2010, somayaji et al. 2016, robb et al. 2017, lozano et  al. 2017). as a member of the staphylococcus genus, s.  pseudintermedius has an extensive panel of virulence factors (van duijkeren et  al. 2011). the majority of these are firstly involved in the colonization of host tissues and the dissemination in the colonized district. specifically, exfoliative toxins (siet, expa, and expb) are virulence factors involved in canine pyoderma (futagawa-saito et  al. 2009, iyori et al. 2010, yoon et al. 2010). this study aimed to collect canine s. pseudintermedius strains, in order to define the occurrence of mrsp and mssp isolated from dogs suffering from skin disorders in two different italian veterinary teaching hospitals; moreover, the antibiotic resistance profiles among both mrsp and mssp isolates were compared. materials and methods sample collection between 2015 and 2017, s.  pseudintermedius strains were isolated from routine bacteriological examinations of canine samples. more in detail, clinical samples, represented by auricular and cutaneous swabs, were collected from dogs suffering from skin disorders, visiting one of the two university veterinary hospitals, located in milan and naples. isolation and phenotypic identification of s. pseudintermedius upon arrival at the laboratories, canine auricular and skin samples were cultured and streaked on columbia cna agar plates (liofilchem, italy), then incubated aerobically at 37 °c for 24 h. suspected staphylococcal colonies were firstly identified by standard, rapid screening techniques: colony 265 nocera et al. antimicrobial susceptbility of s. pseudintermedius veterinaria italiana 2020, 56 (4), 263-269. doi: 10.12834/vetit.2195.12897.1 staphylocoagulase and catalase tests. the bacterial identification results obtained by maldi-tof ms were confirmed by genetic identification using the species-specific nuc gene, revealing a clear concordance between molecular and proteomic analyses in both laboratories. antimicrobial susceptibility testing the phenotypical oxacillin resistance, investigated by disk diffusion test, was confirmed by the amplification of meca gene and allowed to distinguish between mrsp and mssp strains. no detection of mecc gene was revealed among all screened strains. the prevalence of mrsp was significantly higher in milan 30% (35/116) than that reported in naples 18% (23/126), resulting in statistical significance (p-value = 0.0435). as shown in table i, in milan and naples, the antimicrobial susceptibility profiles of the mrsp strains showed high resistance to amoxicillin-clavulanate being 88.6% and 100%, respectively. however, the overall prevalence of antimicrobial resistance among the mrsp isolates detected in this study appeared high. in milan, all the mrsp strains were found to be multidrug resistant strains showing resistance to at least three different antibiotic classes, while in naples, 91% of mrsp strains with multidrug resistant profiles were observed. furthermore, in milan a significantly higher percentage of mrsp isolates were resistant to ceftriaxone and tobramycin compared to those in naples (p < 0.001). clindamycin and kanamycin resistances also appeared to be statistically higher in milan than in naples (p < 0.05 and p < 0.01, respectively). this investigation also revealed that mrsp strains showed markedly high resistance to enrofloxacin, erythromycin, tetracycline in both groups of mrsp strains. similar results were also observed for resistance to gentamicin (48.5% milan, 52.2% naples). in both laboratories, the phenotypic profiles of antibiotic resistance showed lower percentages of resistance to mssp strains than mrsp ones (table  i). the mssp strains isolated in naples harbored the highest prevalence of resistance to amoxicillin/clavulanic acid (56.3%), erythromycin (36.8%), kanamycin (43.6%) and tetracycline (50.5%). conversely, those isolated in milan showed statistically significant lower percentages (p < 0.001) of resistence to the same antibiotics, 7.4% (amoxicillin/clavulanic acid), 7.4% (erythromycin), 7.4% (kanamycin) and 19.7% (tetracycline). a significant difference in ceftriaxone resistance was also observed between milan and naples (p < 0.01) with a higher prevalence in the second and teto) (ullah et al. 2012). furthermore, other arg were searched in both laboratories; aaca-aphd (strommenger et  al. 2003) and blaz (kang et  al. 2014) in milan laboratory; while erma, ermb, ermc (sutcliffe et  al. 1996) in naples. both positive and negative appropriate controls were used in all pcr experiments. statistical analysis samples were grouped in mrsp and mssp, according to the results of oxacillin disk diffusion test and the presence of mec gene. the tested antibiotics (n = 10) and the genes of tetracycline resistance, studied both in milan and naples, were statistically analyzed with the chi-square test to verify the difference in the distribution of phenotypic and genotypic resistance profiles. chi-square test was performed with graphpad prism (version 8.01 for windows, graphpad software, la jolla california usa, www.graphpad.com). results s. pseudintermedius isolation and identification between 2015 and 2017, 242 s.  pseudintermedius strains were collected from canine skin disorders (pyoderma and otitis externa), particularly 116 strains in milan and 126 in naples. all the strains showed β-haemolytic patterns on colombia cna agar and inability to ferment mannitol on msa. furthermore, they showed positive results at the table i. tested antibiotics against methicillin‑resistant s. pseudintermedius (mrsp) and methicillin‑susceptible s. pseudintermedius (mssp) in milan and naples labs. group antibiotics (µg) mrsp (% r)* milan / naples mssp (% r) milan / naples penicillins (β-lactams) amc(20/10 µg) 88.6 / 100 7.4 / 56.3 *** penicillins (β-lactams) ox (1 µg) 100 / 100 0.0 / 0.0 cephalosporins (β-lactams) cro (30 µg) 91.4 / 47.8 *** 1.2 / 12.6** lincosamides da (2 µg) 97.1 / 65.2* 32.2 / 33.0 fluoroquinolones enr (5 µg) 85.7 / 60.9 17.2 / 16.5 macrolides e (15 µg) 100 / 91.3 7.4 / 36.8*** aminoglycosides cn (10 µg) 48.5 / 52.2 2.4 / 9.7 aminoglycosides k (30 µg) 100 / 78.3** 7.4 / 43.6*** aminoglycosides tob (10 µg) 80.0 / 13.0*** 6.1 / 3.8 tetracyclines te (30 µg) 71.4 / 87.0 19.7 / 50.5*** amc = amoxicillin + clavulanic acid; ox = oxacillin; cro = ceftriaxone; da = clindamycin; enr = enrofloxacin; e = erythromycin; cn = gentamicin; k = kanamycin; tob = tobramycin; te = tetracycline. (% r) = percentage of resistance. χ2: *0.05 < p < 0.01; **p < 0.01; *** p < 0.001. 266 antimicrobial susceptbility of s. pseudintermedius nocera et al. veterinaria italiana 2020, 56 (4), 263-269. doi: 10.12834/vetit.2195.12897.1 all classes of antibiotics approved in veterinary medicine and used for systemic treatment in dogs (tetracycline, aminoglycosides, macrolides, sulfamethoxazole-trimethoprim, lincosamides), confirming the multidrug resistance profiles reported for mrsp worldwide (osland et  al. 2012, haenni et  al. 2014, moodley et  al. 2014, stefanetti et al. 2017). it is known that mrsp originates from an animal reservoir; consequently, pet animals might act as potential reservoirs for the emergence of novel methicillin-resistant clones in humans. furthermore, in recent years, it has been reported an increase of the zoonotic transmission of mrsp, probably due to more appropriate identification of this strain (somayaji et al. 2016, robb et al. 2017, lozano et al. 2017). the veterinary environments seem to play an essential role in the dissemination of mrsp between pet animals and humans, particularly people who have constant contact with pets (veterinary personnel and pet owners) (paul et  al. 2011, van duijkeren et al. 2011). in this study, interesting antibiotic resistance profiles were also shown by mssp strains, although their resistance profiles were lower compared to those reported by mrsp. the mssp strains from naples displayed a significantly higher resistance rate compared to the strains from milan and also in comparison to the available literature (ganiere et  al. 2005, norström et  al. 2009). however, the reported increasing antibiotic resistance percentage of mssp strains from naples is also confirmed by more recent studies in other european countries (haenni et  al. 2014, moodley et  al. 2014). the mssp strains showed high resistance rates to antibiotics commonly used to treat canine infections, such as the penicillinase-labile penicillins, tetracycline, aminoglycosides, and macrolides. it is worth noting that in naples, 34% of mssp were multidrug resistant strains, while only 10% of mssp isolates were susceptible to all tested antibiotics. furthermore, in accordance with the literature, this study shows that tetk and tetm genes were hospital. moreover, in milan, 7% of mssp strains were multidrug resistant and 45% of mssp resulted susceptible to all tested antibiotics; in naples, 34% of mssp were multidrug resistant and, among them, 10% resulted susceptible to all tested antibiotics. the phenotypic tetracycline-resistant mrsp strains of milan (n = 25) and naples (n = 20) harbored tetk and tetm genes, alone or in combination, as well as the mssp, that were exactly 16 and 52  strains, respectively. genotypic characterization of tetracycline resistance for the samples from milan showed a higher rate of tetm gene for both mrsp (56%) and mssp (81.2%). similar results were also evidenced for the strains of naples, 52% and 46.1%, respectively (table ii). only among mssp strains, the differences in the frequency of tetm gene between milan and naples resulted in being statistically significant with a p-value < 0.05 (table ii). the tetracycline-resistant strains, carrying tetk and tetm together showed the lowest prevalence among the isolates of both laboratories and no significant differences between milan and naples results were observed (table ii). besides, in milan, all the isolates positive to meca gene harbored also blaz gene, and the gentamicin resistance was confirmed with the presence of the gene aaca/aphd (data not shown); while in naples, all the phenotypic erythromycin-resistant strains carried ermb gene (data not shown). discussion bacterial otitis externa and pyoderma are the most common canine skin diseases, and s.  pseudintermedius is the staphylococcal species most frequently isolated from dogs suffering from these infections. this bacterium, an opportunistic canine skin pathogen, is the major coagulase positive (cps) agent, that inhabits healthy dogs (gómez-sanz et  al. 2013). in particular conditions, such as dog injures or sickness, this species can take advantage of the weakened host immune defenses and cause infection and illness. antibiotic resistance is the most puzzling question in recent years, and the spread of multidrug resistant staphylococci of animal origin, principally mrsp strains, has increased its public health relevance (deurenberg et  al. 2007, corrente et  al. 2013). in this context, the emergence in dogs of mrsp, often associated with even broader drug resistance, has become a great veterinary challenge. in this study, of the 242 isolated s.  pseudintermedius strains, 30% (33/116 in milan) and 18% (23/126 in naples), were mrsp presenting almost all worrying multidrug resistant profiles. besides β-lactam antibiotics, the multidrug resistance profile of mrsp strains showed relevant resistance rates to table ii. molecular profiles of tetracycline resistance in canine s. pseudintermedius isolates. molecular profiles of tetracycline resistance mrspmilan mrspnaples msspmilan msspnaples tetk gene 10/25(40.0%) 6/20 (26%) 2/16 (12.5%) 17/52 (32.7%) tetm gene 14/25(56.0%) 12/20 (52%) 13/16 (81.2%) 24/52 (46.1%)* tetk and tetm genes 1/25(4.0%) 2/20 (9%) 1/16 (6.3%) 11/52 (21.2%) χ2: * p-value < 0.05; mssp = methicillin-susceptible s. pseudintermedius; mrsp = methicillin-resistant s. pseudintermedius. 267 nocera et al. antimicrobial susceptbility of s. pseudintermedius veterinaria italiana 2020, 56 (4), 263-269. doi: 10.12834/vetit.2195.12897.1 and mssp strains with worrying antibiotic resistance profiles and the lack of effective new antibiotics highlight the limited therapeutic possibilities and the need for new treatment approaches to prevent and control staphylococcal infections. another fundamental aspect of this study is the necessity to monitor the antibiotic resistance profiles in different territorial areas. understanding the differences in antimicrobial resistance profiles and specific resistance gene carriage by s.  pseudintermedius strains isolated in different geographic regions may improve antimicrobial drug selection overall for clinical therapy and, consequently, provide insights into how resistance develops in both mrsp and mssp. acknowledgement the authors are grateful to dr. joel filipe for the english revision of the manuscript. the most prevalent tetracycline resistance determinants in both mrsp and mssp (schmitz et  al. 2001, youn et  al. 2014). moreover, for both mrsp and mssp strains isolated in the laboratory of naples, all the erythromycin-resistant strains harbored only ermb gene, that usually appears to be responsible for erythromycin resistance in canine s. pseudintermedius strains (boerlin et al. 2001, lüthje and schwarz, 2007, youn et al. 2014). mrsp strains are known to be more resistant than mssp strains, and their high prevalence of multidrug resistance is probably related to the 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antibiotic resistance and biofilm formation of staphylococcus pseudintermedius strains isolated from canine pyoderma. vet ital, 53, 289-296. stegger m., andersen p.s., kearns a., pichon b., holmes m.a., edwards g., laurent f., teale c., skov r. & larsen 339 veterinaria italiana 2022, 58 (3), 339-345. doi: 10.12834/vetit.2433.16160.1 accepted: 15.11.2021 | available on line: 31.12.2022 icar‑national institute of veterinary epidemiology and disease informatics, india. *corresponding author at: : icar‑national institute of veterinary epidemiology and disease informatics, india. e‑mail: sharanspin13@gmail.com. sharanagouda s. patil*, kuralayanapalya puttahonnappa suresh, akshata velankar, c. shivaranjini, divakar hemadri, jagadish hiremath and siju susan jacob keywords bohv-1, buffaloes, cattle, elisa, ibr, india, prevalence. summary infectious bovine rhinotracheitis (ibr) is a highly contagious disease of bovines causing respiratory symptoms, abortions, and reduced milk yield, leading to huge economic losses. reports on seroprevalence in bovines in india are available and restricted to districts/ states. in the present study, a nationwide seroprevalence of ibr in bovines was conducted to provide a national ibr seroprevalence to the chief veterinarian who in turn can design the control strategies. a total of 15,592 cattle and buffalo serum samples from 25 states and 3 union territories viz., jammu and kashmir, puducherry, and andaman and nicobar islands were tested for ibr antibodies using avidin-biotin (ab) elisa. cumulative seropositivity was found to be 31.37%. maharashtra and rajasthan states , part of the west zone of the country, showed the highest and lowest seroprevalence, respectively. a total of 11,423 cattle and 4,169 buffalo serum samples were tested, which showed 33.91% and 24.39% seropositivity, respectively. india has the highest buffalo population. presently, no ibr vaccination programs are implemented in india. considering the high seroprevalence, the authorities should plan control strategies for vaccinating dairy cows and buffaloes in india. seroprevalence of infectious bovine rhinotracheitis (ibr) in india: a 5‑year study have reported ibr antibody prevalence either restricted to districts/states/zones (choudhury et  al. 2016, farooq et  al. 2019, goswami et  al. 2017, kathiriya et al. 2018, katoch et al. 2017, kollannur et al. 2014, krishnamoorthy et  al. 2015, saravanajayam et al. 2015, patil et al. 2012, patil et al. 2017, trangadia et  al. 2012, tresamol et  al. 2019, verma et  al. 2014). there are no reports on the seroprevalence of ibr in the bovine population covering the vast majority of states of the country which is reared under a smallholder production system. in india, dairy farming is not organized and farmers do hold a small number of cattle and buffaloes. similar husbandry practices are followed at the village level. the animals are grazed in pastures in the daytime and kept in their barnes during the night. therefore, there is a strong possibility that each animal has an equal opportunity of getting infected. an epidemiological study was conducted to estimate the frequency of zone-, and species-specific ibr. information, based on ibr serosurveillance about the disease burden within the defined populations is very useful to researchers and policymakers, thereby infectious bovine rhinotracheitis (ibr) is a highly contagious disease of cattle caused by the bovine alphaherpesvirus 1 (bohv-1) which belongs to the genus varicellovirus, subfamily alphaherpesvirinae, family herpesviridae (mc lachlan 2011). four subtypes of the virus are known: 1.1 and 1.2a (associated with infectious bovine rhinotracheitis), 1.2b (associated with infectious pustular vulvovaginitis and infectious balanoposthitis (ibp), and, 1.3 (encephalitis) (biswas et al. 2013). these serotypes cannot be differentiated by common serological tests, so most of the studies describe them as ibr viruses. latent and subclinical infections are common in ibr (ranganatha et  al. 2013) which can be identified through the detection of antibodies against bohv-1 in serum (lemaire et al. 2000). bohv-1 infection was first reported in india in 1976 (mehrotra et al. 1976). it causes huge economic losses due to a drop in milk production, repeat breeding, and abortions. screening, surveillance, and monitoring are important to maintain the herd’s health status and to decrease the economic losses caused by this disease (raizman et al. 2011). ibr is endemic in india and there is no systematic study on the seroprevalence of ibr though many short communication 340 veterinaria italiana 2022, 58 (3), 339-345. doi: 10.12834/vetit.2433.16160.1 prevalence of infectious bovine rhinotracheitis in india s. patil et al. washing of the plate as described above. later on, 100 μl of tmb (3,3', 5,5;-tetramethylbenzidine) was added to all wells, incubated at 37 °c for 6-8 min, and kept observed for color development. in the final step, 50  μl of 1m stop solution (h 2 so 4 ) were added to all wells and measured od at 450  nm (reference at 620 nm) (annual report 2018). the sensitivity and specificity of the assay were found to be 92% and 95%, respectively. there was no cross-reactivity between the samples as the antigen was precipitated and purified with polyethylene glycol (peg). results were interpreted as below: ‘x’= average od of strong positive x 0.64; a test sample is positive if its od values are greater than ‘x’; a test sample is negative if its od values are less than ‘x’. ms excel v2016 has been used for the entry, storing, and management of surveillance data and spss v22 has been used for statistical analysis. the chi-square test has been used to determine the significant difference in the distribution of positive results with the total sample size in a different zone. the significance was assessed at a 5% level. true prevalence (tp) has been computed to adjust for the imperfectness of the test used for screening the samples. tp was calculated with blaker’s confidence limit using values obtained from apparent prevalence (ap) with wilson’s confidence limit at 95% confidence interval (ci) in the epitool: https://epitools.ausvet. com.au/trueprevalence (seargent et  al. 2018) in which sample size, sensitivity (0.92), and specificity (0.95) of the diagnostic test were considered. the significance of the difference was calculated by supporting the process of identification of priorities in veterinary healthcare, prevention, and policy. the study aimed to screen the bovine serum samples for antibodies against bohv-1 selected randomly to understand the prevalence. a total of 25 states (out of 28 states) and three union territories (out of 8 union territories) of the country with cattle and buffalo populations were included in the study. backyard dairy farming is most common in india and husbandry practices remain the same in most of the households having bovines. a two-stage random sampling methodology was followed wherein the number of random and representative villages and the number of animals in each village were selected using a survey toolbox (seargent et  al. 2018). villages having a minimum of 100 bovines were selected. a total of 15,592 bovine serum samples from 1,828 villages in 369 districts from 25 states and 3 union territories viz., jammu and kashmir, puducherry, and andaman and nicobar islands were collected (table i). bovine serum samples were obtained from the national livestock serum repository (nlsr) maintained at icar-nivedi, bengaluru, which were stored at 20 °c. enzyme immunoassay for detection of ibr antibodies serum samples were subjected to an enzyme immunoassay for the detection of antibodies against ibr using developed home made avidin-biotin elisa (ab elisa). positive and negative serum controls were selected from the repository and were subjected to a serum neutralization test using known bohv-1. each serum sample showing a > 1.5 neutralization index (as per woah) was selected as a positive control and the serum showing the lowest neutralization index (<  1.5) was selected as a negative control. all the controls (positive, negative, and conjugate controls), test samples, and other reagents were used and dissolved in a blocking buffer (1% bovine gelatin and 0.05% tween  20) and dispensed in 100 μl of volume. the 1:100 diluted controls and test samples were dispensed to bohv-1 antigen-coated plates. later on, plates were incubated on a shaker at 37 °c for 1 hr. afterward, the plates were washed three times with washing buffer (1x pbs with 0.05% tween  20). then, biotinylated anti-bovine igg (1:10,000 diluted in blocking buffer) raised in goats was added to all wells and incubated on a shaker at 37 °c for 1 hr. again, plates were washed as described earlier. then, horseradish peroxidase (hrpo) conjugated avidin (1:10,000) was added to all wells, incubated at 37  °c for 20 min and followed by the figure 1. percent positivity of infectious bovine rhinotracheitis (ibr) in india. 341veterinaria italiana 2022, 58 (3), 339-345. doi: 10.12834/vetit.2433.16160.1 s. patil et al. prevalence of infectious bovine rhinotracheitis in india 20-80% in all age groups of dairy animals and 55.49% of national herd prevalence, respectively (brock et al. 2020, maresca et  al. 2018). kipyego and colleagues (kipyego et  al. 2020) reported 17.4% animal-level seroprevalence in dairy cattle of meru county, kenya. noaman and colleagues (noaman et  al. 2020) have reported a seroprevalence of 72.20% in crossbred dairy cattle in the isfahan province of iran. the overall seroprevalence study conducted on 176 serum samples obtained from cattle imported from sudan to egypt showed 99.75% bohv-1 antibodies the chi-square test and p < 0.001 was considered statistically significant. a total of 15,592 bovine serum samples were collected during 2015-2019 and tested for ibr antibodies which showed a percent positivity of 31.37 [30.31 (95% ci: 29.48-31.15)] (figure 1). maharashtra showed the highest percent positivity. maharastra is a geographically large state and is also known for dairy farming, whereas rajasthan showed the lowest percent positivity (table i). european countries like ireland and italy have reported seroprevalence of table i. infectious bovine rhinotracheitis seroprevalence in indian cattle according to states of origin between 2015-2019. sl no zone state /union territory no districts no villages no tested no positive % positivity true prevalence at 95% ci* 1 north himachal pradesh 11 116 935 204 21.82 19.33 (16.42-22.50) haryana 23 74 715 176 24.62 22.55 (19.08-26.33) jammu and kashmir 23 78 519 289 55.68 58.26 (53.31-63.10) punjab 18 60 974 372 38.19 38.15 (34.71-41.71) uttar pradesh 16 22 204 23 11.27 7.21 (3.02-13.05) 13 50 290 50 17.24 14.07 (9.57-19.55) total 104 400 3637 1114 30.63 29.46 (27.76-31.20) 2 east andaman & nicobar 2 59 570 266 46.67 47.89 (43.23-52.61) bihar 14 26 217 95 43.8 44.57 (37.18-52.22) odisha 33 80 898 242 26.95 25.23 (22.01-28.67) 3 11 217 88 40.55 40.87 (33.61-48.50) total 52 176 1902 691 36.33 36.01 (33.56-38.53) 3 north east assam 27 77 657 158 24.05 21.90 (18.32-25.82) manipur 8 55 874 300 34.32 33.71 (30.17-37.40) meghalaya 7 52 346 94 27.17 25.48 (20.40-31.13) mizoram 7 45 111 59 53.15 55.35 (44.74-65.72) nagaland 11 122 530 171 32.26 31.34 (26.92-36.05) sikkim 4 101 683 221 32.36 31.45 (27.54-35.58) 4 110 604 202 33.44 32.69 (28.50-37.13) total 68 562 3805 1205 31.67 30.65 (28.98-32.37) 4 central madhya pradesh 29 89 798 143 17.92 14.85 (11.97-18.08) total 29 89 798 143 17.92 14.85 (11.97-18.08) 5 south andhra pradesh 13 59 301 135 44.85 45.81 (39.46-52.30) karnataka 20 63 519 135 26.01 24.15 (20.03-28.68) kerala 14 92 925 166 17.95 14.88 (12.19-17.87) puducherry 2 64 387 166 42.89 43.56 (38.00-49.28) tamil nadu 2 58 279 159 56.99 59.76 (53.02-66.28) 8 70 333 155 46.55 47.75 (41.68-53.92) total 59 406 2744 916 33.38 32.62 (30.62-34.68) 6 west goa 2 14 251 56 22.31 19.90 (14.48-26.27) gujarat 26 104 1650 410 24.85 22.81 (20.49-25.28) maharashtra 25 55 530 326 61.51 64.95 (60.11-69.60) 4 22 275 30 10.91 6.79 (3.16-11.66) total 57 195 2706 822 30.38 29.17 (27.21-31.19) grand total 369 1828 15592 4891 31.37 30.31 (29.48-31.15) zone wise = χ2=96.3, p < 0.001, significant; *diagnostic sensitivity = 92% and diagnostic specificity = 95%. 342 veterinaria italiana 2022, 58 (3), 339-345. doi: 10.12834/vetit.2433.16160.1 prevalence of infectious bovine rhinotracheitis in india s. patil et al. meghalaya, respectively. sikkim showed a percent positivity of 32.35, which shares a border with china and there is a restricted movement of men and materials along the border. overall, nationwide seroprevalence was found to be 35.8% (481/1,344) in dairy cattle in china (yan et al. 2008). central zone. a total of 798 samples from 89 villages of 29 districts of madhya pradesh were tested, of which 143 were found positive (17.92%) [14.85 (95% ci: 11.97-18.08)]. a seroprevalence of 68.9% was recorded in madhya pradesh in 2011 (nandi et al. 2011). south zone. samples analyzed from this zone were from 5 states (andhra pradesh, karnataka, kerala, tamil nadu, telangana) and one union territory of puducherry. a total of 2,744 bovine serum samples were tested and this zone showed a seropositivity of 33.38%. patil and colleagues (patil et  al. 2017) showed a seroprevalence of 56.20% in bovines from the southern region of india comprising tamil nadu, andhra pradesh, and karnataka. in the present study, tamil nadu showed the highest seropositivity of 56.99% [56.99 (95% ci: 53.02-66.28)], while the lowest positivity of 17.95% [14.88 (95% ci: 12.19-17.87)] was recorded in kerala. during 2016-2017, kerala showed a seroprevalence of 12% (tresamol et  al. 2019). earlier, seropositivity of 65.88% was recorded in tamil nadu (saravanajayam et al. 2015). puducherry showed 42.89% positivity. west zone. goa, maharashtra, rajasthan, and gujarat were a part of this zone. a total of 2,706 bovine serum samples were tested and showed positivity of 30.38% [29.17 (95% ci: 27.21-31.19)]. maharashtra showed the highest seropositivity of 61.51% [64.95 (95% ci: 60.11-69.60)] and rajasthan showed the lowest seropositivity of 10.91% [6.79 (95% ci: 3.16-11.66)]. geographically, maharashtra is a very large state that has a greater number of dairy animals, including organized dairy farms. patil and colleagues (patil et  al. 2017) showed a seroprevalence of 76.5% in maharashtra, whereas a seropositivity of 0% was recorded in rajasthan earlier (tanwar et al. 2009). the chi-square test (χ2 = 96.3; p < 0.001) analysis of zone-wise seroprevalence of ibr antibodies was found significant (table i). seroprevalence of ibr, species‑wise a total of 11,423 cattle and 4,169 buffalo serum samples from 25 states and 3 union territories were tested for ibr antibodies. 33.91% [33.23 (95% ci: 32.24-34.24)] positivity was recorded in cattle samples, whereas buffalo samples showed 24.39% [22.29 (95% ci: 20.82-23.82)] seropositivity. india is the highest buffalo-populated country and more (hekal et al. 2019). the following six zones of india had a varied seroprevalence of the ibr (table i). north zone. six states viz., himachal pradesh, haryana, punjab, uttar pradesh, uttarakhand, and jammu and kashmir union territory formed this zone. cumulative percent positivity was found to be 30.63 [29.46% (95% ci: 27.76-31.20%)]. the samples collected from 78 villages of 23 districts of jammu and kashmir, which shares an international border with pakistan, showed the highest percent positivity of 55.68 [58.26 (95% ci: 53.31-63.10%)]. pakistan has shown an ibr seropositivity of 69% in dairy cattle of lahore (rehman et al. 2020). there are illegal movements of men and materials alongside the border though it is under strict vigilance. uttar pradesh (up) showed the lowest seropositivity of 11.27% [7.21 (95% ci: 3.02-13.05)] and it shares a border with nepal. the overall seroprevalence of ibr was 18.47% in nepal (tiwari et al. 2020). up is a very large state and is required to test more samples for the ibr antibodies. haryana and punjab are prosperous states having more high-yielding cattle and buffalo and have recorded 24.62% and 38.19% positivity, respectively. earlier reports have recorded the seroprevalence of ibr in haryana as 48.78% (farooq et  al. 2020) and in punjab as 29.78% (gill et  al. 1984), 42.85% (aradhana et  al. 2004), 34.16% (dhand et  al. 2002), 36.51% (kollannur et  al. 2014), 38.50 (goswami et  al. 2017), and 84.50% (farooq et al. 2019). east zone. this is comprised of west bengal, odisha, bihar, and the union territory of andaman and nicobar islands. a total of 1902 samples from 117  villages of 52 districts were tested for ibr antibodies. this zone showed 36.33% positivity [36.01 (95% ci: 33.56-38.53)]. andaman and nicobar islands located 1,350 km from the indian mainland showed the highest seropositivity of 46.67% [47.89 (95% ci: 43.23-52.61)]. these islands are located at the remotest which procure dairy animals from tamil nadu and west bengal whose ibr seropositivity is also high. the overall seroprevalence of the ibr in the andaman and nicobar islands was recorded as 17.68% in 2005 and 20.58% in 2014 (sunder et  al. 2005, sunder, 2014). odisha showed the lowest of 26.95% seropositivity against 12.22% seropositivity reported earlier (das et al. 2014). north east zone. samples collected were from 7  states of this region viz., assam, manipur, meghalaya, mizoram, nagaland, tripura, and sikkim. a total of 3805 samples were tested and 31.67% positivity was recorded. mizoram showed the highest seropositivity of 53.15%. meghalaya showed the lowest positivity of 27.17%. rajkhowa and colleagues (rajkhowa et  al. 2004) found a seroprevalence of 52% and 8% in mizoram and 343veterinaria italiana 2022, 58 (3), 339-345. doi: 10.12834/vetit.2433.16160.1 s. patil et al. prevalence of infectious bovine rhinotracheitis in india cows and buffalo, if not bulls. bulls should be tested for ibr antibodies periodically and each semen ejaculate must be tested for virus by polymerase chain reaction. acknowledgments the authors are grateful to dg and ddg, icar, new delhi, and the director, icar-nivedi, bengaluru for their financial support and guidance throughout the study. acknowledgments are due to the serum repository of the institute. the authors are highly thankful to all the principal/co-principal investigators of the all india coordinated research project on animal disease monitoring and surveillance (aicrp on admas). grant support this study no ixx10709 was funded by icar-nivedi under project no. 201/2012-19. samples need to be tested against ibr antibodies to obtain a more detailed picture of ibr prevalence. seroprevalence of ibr antibodies between cattle and buffalo was significant, as evidenced by the chi-square test (χ2 = 129, p < 0.001) (table ii). year‑wise seropositivity of ibr bovine serum samples were collected from 2015-2019 (five years) and tested for ibr antibodies. the highest seropositivity of 42.88% [43.55 (95% ci: 41.63-45.49)] during 2019 was recorded. the lowest seropositivity was 18.04% [15 (95% ci: 13.46-16.63)] (table iii). the positivity varied between years though the total samples tested were almost the same and was not able to attribute the reason since the samples did not have sufficient data available. presently, the bovine population in the country is not vaccinated against ibr. the seroprevalence of ibr in the bovine population appears to be very high as the bovines must have experienced the infection and are latently infected, such animals are a continuous source of infection whenever there is a reactivation of the virus. therefore, the authorities should plan control strategies for vaccinating dairy table ii. seroprevalence of ibr in india, species wise. species total tested no positive % positivity true prevalence at 95% ci cattle 11423 3874 33.91 33.23 (32.24-34.24) buffalo 4169 1017 24.39 22.29 (20.82-23.82) total 15592 4891 31.37 30.31 (29.48-31.15) χ2 = 129, p < 0.001, significant. table iii. details of year wise collection of bovine serum samples. sl no year no tested no positive % positivity true prevalence at 95% ci 1 2015 3003 542 18.04 15 (13.46-16.63) 2 2016 3130 1002 32.01 31.03 (29.20-32.95) 3 2017 3117 861 27.62 26 (24.23-27.84) 4 2018 3003 1054 35.09 34.60 (32.66-36.58) 5 2019 3339 1432 42.88 43.55 (41.63-45.49) total 15592 4891 31.37 30.31 (29.48-31.15) 344 veterinaria italiana 2022, 58 (3), 339-345. doi: 10.12834/vetit.2433.16160.1 prevalence of infectious bovine rhinotracheitis in india s. patil et al. biswas s., bandyopadyay s., dimri u. & patra p.h. 2013. bovine herpesvirus-1 (bohv-1) – a re-emerging concern in livestock: a revisit to its biology, epidemiology, diagnosis, and prophylaxis. vet quart, 33, 68-81. brock j., lange m., guelbenzu-gonzalo m., meunier n., margarida vaz a., tratalos 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microbiol, 127, 136-141. sergeant e.s.g. 2018. epitools epidemiological calculators. ausvet. http://epitools.ausvet.com.au. sunder j. 2014. status of livestock and poultry diseases in a & n islands: strategies to make island disease free. adv anim vet sci, 2, 42-47. sunder j., rai r.b., kundu a., chatterjee r.n., senanis s. & jeyakumar s. 2005. incidence and prevalence of livestock diseases of andaman and nicobsr islands. ind j anim sci, 75, 1041-1043. trangadia b.j., rana s.k., nagmani k. & srinivasan v.a. 2012. serological investigation of bovine brucellosis, johne’s disease and infectious bovine rhinotracheitis in two states of india. j adv vet res, 2, 38-41. tiwari m.r., ghimire r.p. & shrestha s.p. 2019. proceedings 451 veterinaria italiana 2022, 58 (4), 451‑455. doi: 10.12834/vetit.2590.16869.2 accepted: 09.02.2022 | available on line: 31.12.2022 1tamil nadu veterinary and animal sciences university, department of veterinary microbiology, veterinary college and research institute, namakkal, india. 2central university laboratory, tamil nadu veterinary and animal sciences university, chennai. 3tamil nadu veterinary and animal sciences university, avian disease laboratory, thalaivasal, india. *corresponding author at: tamil nadu veterinary and animal sciences university. e‑mail: balasubramaniam.a@tanuvas.ac.in. annamalai balasubramaniam1*, s. jaisree2, thandavan vembuvizhivendan tamilam3 keywords duck hepatitis a virus, duckling, one‑step rt‑pcr. summary during winter of the year 2020, a flock of 9 day‑old 5000 non‑descript ducklings was affected with huge daily mortality, dullness, depression and opisthotonus. clinically, there was severe depression, spasmodic paddling and opisthotonus. on post‑mortem, liver was enlarged and pale with patchy ecchymoses. presence of perihepatitis and pericardititis during post‑mortem examination of one duckling might be attributed to secondary bacterial infection. upon completion of disease episode, there was 80 percent mortality in eight days and only less than 20 percent weak ducklings survived. liver homogenate which was subjected for molecular confirmation through one‑step reverse transcriptase polymerase chain reaction (rt‑pcr) using primers for rna dependent rna polymerase (3d) gene yielded positivity for duck hepatitis a virus (dhav‑1). histological observation of liver revealed hepatocyte degeneration and necrosis. it is clear that dhav‑1 which is epornitic in nature causes a major devastating disease endangering duck farming. please refer to the forthcoming article as: annamalai balasubramaniam et al. 2022. an outbreak of duck hepatitis a virus infection in nomadic ducklings. vet ital. doi: 10.12834/vetit.2590.16869.2 an outbreak of duck hepatitis a virus infection in nomadic ducklings presence of duck hepatitis a virus (dhav–1) is usually confirmed either by inoculating liver sample on to susceptible duckling, embryonated duck eggs wherein embryos die 24 to 72 hours post‑ inoculation via allantoic sac route with stunting and haemorrhages or by the inoculation of duck embryo liver cells primary culture of which produce rounding and necrosis of cells (woah terrestrial manual 2018). different serological tests which are time consuming and labour intensive were useful in diagnosis (murty & hanson 1961, hwang 1969, zaho et al. 1991) and epidemiological study. although the sudden onset, rapid spread and acute mortality are characteristic of dhav infection, the virus must be isolated or demonstrated by rt‑pcr. other causes of acute mortality in duckling include salmonellae and aflatoxin both of which do not cause liver lesions suggestive of dhav infection but cause rapid onset of mortality, ataxia, convulsions and introduction duck hepatitis, a highly fatal, acute and contagious disease affecting 1 to 3 week‑old ducklings with rapid onset and huge mortality (50 to 90%) is caused by duck hepatitis virus which is classified as a sole member of avihepatovirus of picornaviridae family. duck hepatitis is of paramount significance in duck farming ever since it was first described (levine & fabricant 1950). it is also reported that duckling flock of less than 6 week‑old may suffer 100% mortality leading to severe economic impact on duck farming (yugo et al. 2016). in india, the report of duck hepatitis caused by virus distinct from dhav‑1 (rao and gupta 1967) and presence of duck hepatitis virus b (sridhar et al. 1993) were documented. the disease can be caused by three different viruses namely dhav type 1, 2 and 3. it is not possible to distinguish among dhav types on the basis of clinical signs and pathology. the case report duck virus hepatitis a in ducklings balasubramaniam et al. 452 veterinaria italiana 2022, 58 (4), 451-455. doi: 10.12834/vetit.2590.16869.2 gel containing ethidium bromide (0.5 µg/ml) and the gel was visualized using gel doctm xrt imager with image labtm software. histopathology liver samples were collected from dead ducklings and fixed in 10% formalin. sections of 4 µm were cut and mounted on microscope slides that were stained with haemotoxylin and eosin (bancroft and gamble 2008). result and discussion ailing ducklings exhibited spasmodic paddling and opisthotonus and one of the dead ducklings opisthotonos in aflatoxicosis (https://www.cabi. org/isc/datasheet/84184). furthermore, the virus is known to evolve as a new recombinant one through recombination (rohaim et al. 2021). alternatively, dhav rna may be detected by a one‑step reverse transcriptase polymerase chain (rt‑pcr) reaction. further, rt‑pcr is the method recommended (+++) for detection of antigen in individual animal, contribution to eradication policies and confirmation of clinical cases of duck virus hepatitis by wold organisation for animal health (woah terrestrial manual 2018). therefore, one‑step rt‑pcr which is proved to be a rapid and sensitive tool (kim et al. 2007) is applied for diagnosis of dhav‑1 directly from the clinical specimen (fu et. al. 2008) in the present report. materials and methods four dead ducklings from a nine day‑old non‑ descript (nomadic type) flock of 5000 were brought for post‑mortem to avian disease laboratory, thalaivasal, south india. the flock was reported to be affected with 50% mortality in 5 days showing symptoms like severe depression, opisthotonus and spasmodic paddling. sampling was done from liver (2 numbers) for molecular detection of dhav‑1 and histopathology. molecular identification of dhav-1 rna isolation: liver homogenates were subjected for rna isolation (qiagen, usa) following manufacturer’s instruction. primers: for the detection of dhav‑1 nucleic acid, primers for rna dependent rna polymerase (3d) gene (kim et al. 2007) comf‑ 5’‑aag aag gag aaa atc aag gaa gg‑3’) and comr‑ 5’‑ttg atg tca tag ccc aa g aca gc‑3’ flanking a 467 bp dna sequence in the 3d gene were used. one‑step rt‑pcr: reverse transcription was carried out using verso cdna synthesis kit (thermo scientific # ab 1453/a). it was performed at 42oc for 30 minutes followed by 95ºc for 2 minutes to inactivate the enzyme. pcr was conducted in a 25 µl reaction mix where ampliqon red dye master mix 12.5 µl, forward primer (10 pmol/µl) 1µl, reverse primer (10 pmol/ µl) 1 µl, nuclease free water 8.5 µl and template dna 2 µl were added. reaction was conducted (biorad c1000 touch) for 35 cycles with following conditions: denaturation for 45 seconds at 94ºc, annealing for 45 seconds at 52ºc, elongation for 45 seconds at 72ºc and final elongation for 7 minutes at 72ºc. amplified pcr products were run on 1.5% agarose figure 1. typical opisthotonus in a dead duckling. presented typical posture (fig. 1). at necropsy, major changes were observed in liver (fig. 2) which was pale, enlarged with haemorrhagic spots in all the ducklings brought for post‑mortem examination. the above findings were similar to the reports of dhav–1 infection in ducklings made by other workers (kozydrun et al. 2014, kamomae et al. 2017, hisham et al. 2020). one duckling showed pericarditis and perihepatitis which might be attributed to secondary invasive bacterial infection culture of which revealed escherichia coli. especially when the immune system of the host is compromised due to other infections like dhav‑1 infection in this case, it is axiomatic that bacteria can easily invade resulting subsequent damage. in other reports, salmonella enteritidis was isolated from the liver obtained from liver of dead ducklings in dhav‑1 outbreak (kamomae et al. 2017). additionally, there was haemorrhagic line in exo‑occipital region (fig. 3). severe meningial hyperemia and haemorrhage were reported (niu et al. 2019) in dead ducklings experimentally infected with either dhav‑ 1 or 3. balasubramaniam et al. duck virus hepatitis a in ducklings veterinaria italiana 2022, 58 (4), 451-455. doi: 10.12834/vetit.2590.16869.2 453 figure 2. a dead ducking showing pale liver with ecchymoses. figure 3. haemorraghic line in exo-occipital region. both collected samples were positive for 3d gene of dhav‑1 amplifying 467 bp (fig. 4) products in one‑ step rt‑pcr. figure 4. electrophoresis of rt-pcr products in 1.5% agarose gel. lane 1 & 2: field samples amplifying 467 bp product (3d gene); lane 3: negative control; lane 4: 100 bp ladder. figure 5. histological section of the liver showing diffuse degeneration and necrosis of hepatocytes with proliferation of ductular cells around portal area. hematoxylin and eosin. 1000x. and kamomae et al. 2017. therefore, application of one‑step rt‑pcr in diagnosis of dhav is justifiable as it not only saves time and labour but also facilitates rapid reporting to the concerned authorities. histologically, there were severe congestion of hepatic blood vessels, diffuse degeneration and necrosis of hepatocytes which is congruous to the findings of kamomae et al. 2017 in natural outbreak and hisham et. al., 2020 in experimentally infected ducklings. additionally, proliferation of basophilic ductular cells around the portal areas which is in line with the findings of kamamae et al. 2017 and niu et al. 2019 was also observed (fig. 5). it reaffirmed the usefulness of one‑step rt‑pcr for detecting dhv‑1 genome directly from specimens and this observation is in line with the findings of kim et al. 2007, fu et al. 2008, kozydrun et al. 2014 duck virus hepatitis a in ducklings balasubramaniam et al. 454 veterinaria italiana 2022, 58 (4), 451-455. doi: 10.12834/vetit.2590.16869.2 dhav–1 infection in ducks attains importance as there is possibility of vertical transmission (zhang et al. 2021), apart from epornitic spread of the virus. although the disease occurred in non‑intensive duck rearing area, suitable guidelines were given to animal husbandry department in order to curtail further spread of infection to duck rearing areas. conclusion molecular identification of dhav‑1 in one week‑ old duckling flock which suffered 80% mortality within eight days was carried out for the first time in india. it could be concluded that one‑step rt‑pcr is suitable tool for detection of dhav‑1 genome directly from clinical specimens. it is believed that the role played by wild birds in the spread of dhav‑ 1 is one of the important factors to be considered while designing the disease preventive strategies. furthermore, recent occurrence of dhav‑1 infection spells a doom over duck farming. acknowledgement he facilities provided by the director, centre for animal health studies, tamil nadu veterinary and animal university, chennai, tamil nadu, india are gratefully acknowledged. by the end of the epidemic, there was 80% mortality within eight days and the 20% surviving weaklings were disposed taking sufficient care not to spill the infection. exposure to dhav‑1 either through contamination in hatchery or through wild bird (asplin 1961, kamomae et al. 2017) might have led to the disease epidemic. it is noted that the place of disease occurrence is a non‑intensive duck farming area and far from other duck rearing vicinity. severity of the disease could have been enhanced by stress factors arising due to travel of long distance combined with poor nutrition, as in this case, ducklings were transported for nearly 150 km on road in search of grazing field. in india, rao & gupta 1967 reported the incidence of duck virus hepatitis which is known to be distinct from dhav–1 but its relationship to other dhav types is not known (kim et al. 2008). sridhar et al. 1993 found duck viral hepatitis b antigen to the extent of less than 10% in an organised duck farm of south india. current scenario emphasizes the need for molecular epidemiological study of the virus in duck farming area including wild birds to understand the propensity and kinetics of the virus in order to formulate a fruitful strategy for prevention and control of duck virus hepatitis. prevention of balasubramaniam et al. duck virus hepatitis a in ducklings veterinaria italiana 2022, 58 (4), 451-455. doi: 10.12834/vetit.2590.16869.2 455 asplin, f.d. 1961. notes on epidemiology and vaccination for virua hepatitis of ducks. off int epizoot bull, 56, 793‑800. bancroft, j.d. and gamble, m. 2008. theory and practice of histological techniques. 6th edn. churchill livingstone. elsiever, london. fu, y., pan, m., wang, x., xu, y., yang, h. & zhang., d. 2008. molecular detection and typing of duck hepatitis a virus directly from clinical specimens. vet microbiol, 131, 247–257. gazdzinski, p. 1979. attenuation of duck hepatitis virus and evaluation of its usefulness for duckling immunization. i. studies on attenuation of the virus. bull vet inst pulawy, 23, 80–89. kamomae, m., kameyama, m., ishi, j., nabe, m., ogura, y., iseki, h., yamamoto, y. & mase, m. 2017. an outbreak of duck hepatitis a virus type 1 infection in japan. j vet med sci, 79, 917‑920. kim, m., kwan, y., joh, s., kwon, j., kim, j. & kim, s. 2007. development of one‑step rt‑pcr to detect duck hepatitis virus type 1. avian dis, 51, 540‑545. kim, m., kwon, y., joh, s., kwon, j., & lindberg, a. m. 2008. differential diagnosis between type‑specific duck hepatitis virus type 1 (dhv‑1) and recent korean dhv‑1‑like isolates using a multiplex polymerase chain reaction. avian pathol, 37, 171‑ 177, doi: 10.1080/03079450801918670. kozydrun, w., czekaj, h. & lorek, m. 2014. outbreak of duck viral hepatitis in duckling flocks in poland. bull vet inst pulawy, 58, 513 ‑ 515 hwang, j. 1969. duck hepatitis virus‑neutralization in chicken embryo. am j vet res, 30, 861‑864. hisham, i., ellakany, h.f., selim, a.a., abdella, m.a.m., zain el‑abideen, m.a., kilany, w.h., ali, a. & elbestawy, a.r. 2020. comparative pathogenicity of duck hepatitis virus type‑1 isolates in experimentally infected pekin and muscovy ducks. frontiers in vet sci, 7, doi: 10.3389/ fvets.2020.00234 levine, p. p., & fabricant, j. 1950. a hitherto‑ undescribed virus disease of ducks in north america. cornell vet, 40, 71‑86. references murty, d. k., & hanson, l.e. 1961. a modified microgel diffusion method and its application in the study of the virus of duck hepatitis. am j vet res, 22, 274‑278. niu, y., ma, h., ding, y., li, z., sun, y., li, m. & shi, y. 2019. the pathogenicity of duck hepatitis a virus types 1 and 3 on ducklings. poult sci, 98, 6333‑ 6339. doi: 10.3382/ps/pez455. rao, s.b.v. & gupta, b.r. 1967. studies on a filterable agent causing hepatitis in ducklings, and biliary cirrhosis and blood dyscrasia in adults. indian j poult sci, 2, 18–30 rohaim, m.a., naggar, r.f.e., abdelsabour, m.a., ahmed, b.a., hamoud, m.m., ahmed, k.a., zahran, o.k. & munir, m. 2021. insights into the genetic evolution of duck hepatitis a virus in egypt.  animals,  11, issn 2076‑2615, doi: org/10.3390/ani11092741. sridhar, g., valliammai, t., varalakshmi, c.s., udayasankar, k., panchanadam, m., ramakrishna, j., gopal, k.v., jayaraman, k. & thyagarajan, s.p. 1993. duck hepatitis b virus (dhbv) infection in indian domestic ducks: a pilot study. trop anim health prod, 25, 229‑233. wold organisation for animal health (oie) terrestrial manual. 2018. duck virus hepatitis. chapter 3.3.8. yugo, d.m., hauck, r., shivaprasad, h.l. & meng, x.j. 2016. hepatitis virus infections in poultry. avian dis, 60, 576‑88, doi: 10.1637/11229‑070515. zaho, x., phillips, m.r., li, g. & zhong, a. 1991. studies on the detection of antibody to duck hepatitis virus by enzyme‑linked immuno‑sorbent assay. avian dis, 35, 778‑782. zanaty, a., hagag, n., samy, m., abdel‑halim, a., soliman, m.a., arafa, a. & nasif, s. 2017. molecular and pathological studies of duck hepatitis virus in egypt. j of vet med res. 24, 374‑384. zhang, r., yang, y., lan, j., xie, z., zhang, x. & jiang, s. 2021. evidence of possible vertical transmission of duck hepatitis a virus type 1 in ducks. transboundary and emerging diseases, 68: 67‑75, doi: org/10.1111/tbed.13708. 163 1department of veterinary medicine, united arab emirates university, united arab emirates. 2department of data analytics in the digital era, college of business and economics, united arab emirates university, p.o. box 15551, al ain, united arab emirates. *corresponding author at: department of veterinary medicine, united arab emirates university, united arab emirates. e‑mail: rbarigye@uaeu.ac.ae. keywords abortion, cattle, coxiella burnetii, leptospira hardjo, neospora caninum, bovine viral diarrhoea virus. summary the serostatus of five abortigenic agents and the association between abortion history and coxiella burnetii seropositivity were assessed in 350 dairy cattle from al ain, uae. the bovine sera were elisa-screened for c. burnetii, leptospira hardjo, neospora caninum, and brucella abortus antibodies, plus bovine pestivirus (bvdv) antigen. the serology data were collated and the level of significance between the proportions of c. burnetii-seropositive cattle with and without abortion history assessed by the z test of two proportions. of the 350 cattle, 41.4%, 1.7%, 1.4%, 0.3%, and 0.0% were seropositive to the above pathogens, respectively. besides, 61.9%, 2.9%, 1.0%, 0.0%, and 0.0% of the 105 cattle with history of abortion and 32.7%, 1.2%, 1.6%, 0.0% and 0.0% of the 245 seropositive cattle with no history of abortion were also seropositive for the above pathogens respectively. moreover, the proportion of c. burnetii-seropositive cattle with history of abortion were significantly higher than the c.  burnetii-seropositive ones without abortion history (p-value < 0.01). apparent c. burnetii infections were relatively higher than the other four pathogens suggesting this bacterium contributed to abortion in the herd. additional research on the public and bovine health implications of c. burnetii and leptospira in the uae are urgently needed. robert barigye1*, nabeeha abdelgaleel hassan1, dhabia mohamed naser alqubaisi1 and ibrahim mohamed abdalla-alfaki2 serological evidence of coxiella burnetii, leptospira interrogans hardjo, neospora caninum and bovine pestivirus infections in a dairy cattle herd from the united arab emirates veterinaria italiana 2020, 56 (3), 163-168. doi: 10.12834/vetit.2257.12932.1 accepted: 10.06.2020 | available on line: 31.12.2020 like neospora caninum (okumu et al. 2019, shabbir et  al. 2011, yildiz et  al. 2017), trichomonas foetus (michi et al. 2016), toxoplasma gondii (pagmadulam et  al. 2018), and sarcocystis spp. (rassouli et  al. 2014) have also been reported. similarly, a number of abortigenic viruses like bovine viral diarrhoea virus (bvdv ) (aslan et  al. 2015, asmare et  al. 2018) and bovine herpesvirus-1 (chastant-maillard, 2015) have also been reported. periodically, a number of abortigenic vectorborne viruses like rift valley fever virus (ali et  al. 2012), bluetongue virus (ali et  al. 2012, nusinovici et  al. 2012), and akabane virus (kirkland 2015) have been particularly reported in tropical and subtropical regions of the world. finally yet importantly, a number of abortigenic fungal pathogens including, but not limited to, aspergillus sp. and mortierella wolfii have also been reported (mccausland et al. 1987). introduction bovine abortion is widely recognized as a cause of significant economic losses in dairy cattle worldwide (knudtson and kirkbride 1992, thurmond et al. 1990). even though non-infectious factors may cause reproductive failure, abortigenic infectious agents are likely to cause more epidemiologically dynamic forms of abortion in dairy cattle (kaveh et  al. 2017, knudtson and kirkbride 1992). universally recognized abortigenic bacterial pathogens of cattle include, but are not limited, to brucella abortus (okumu et  al. 2019, shabbir et  al. 2011), coxiella burnetii (bildfell et  al. 2000, cabassi et  al. 2006), campylobacter foetus (michi et  al. 2016), leptospira spp. (delooz et  al. 2018) and listeria monocytogenes (yağcı-yücel et al. 2014). in addition, a few abortigenic protozoans 164 veterinaria italiana 2020, 56 (3), 163-168. doi: 10.12834/vetit.2257.12932.1 serostatus of five abortigenic agents of cattle in the united arab emirates barigye et al. separated by centrifugation at 4,000 rpm, 5 min and then kept at - 20 °c until testing. immunoenzymatic assays the screening for c.  burnetii antibodies was done using an indirect c. burnetii elisa (q fever c. burnetii antibody test kit, idexx laboratories, switzerland) according to the manufacturer’s instructions. after diluting test sera and positive and negative controls to 1:400 in the kit wash buffer solution, 100 µl/well of each were dispensed into 96 microtiter plate wells pre-coated with inactivated c. burnetii antigen. after incubating the plates at 37 °c for 60 min, they were washed three times with the kit wash buffer, 100 µl/ well of a peroxidase labelled anti-ruminant igg conjugate added and the plates then incubated for 60 min at 37 °c. the plates were then washed three times with the kit wash buffer like before and 100 µl/ well of the 3,3’,5,5’-tetramethylbenzidine (tmb) substrate added. after stopping the reaction, the elisa plates were read at rt in a spectrophotometer (bio tek instruments. inc. highland park, usa) at 450  nm and the results expressed as a percentage of the ratio of the test sample od 450 to the positive control od 450 (s/p%). in line with recommendations of the test kit manufacturer, the test samples with s/p% ≥ 40% were interpreted as positive while those with s/p% < 30% were deemed negative. for the indirect elisa for b.  abortus antibodies (brucella abortus antibody test kit, idexx laboratories, switzerland), 100 µl/well of test sera, as well as the positive and negative controls, were diluted 1:10 in the kit sample diluent, placed in the 96 microtitre wells and the plates tightly sealed. after incubating at 37 °c for 60 min, the plates were washed three times using 300 µl/well of the kit wash solution and 100 µl/well of peroxidase labeled anti-ruminant igg conjugate added. after tightly sealing the plates, they were further incubated at 37 °c for 60 min. following the incubation step, the plates were washed three times with 300 µl/well of the kit wash buffer and 100 µl/well of tmb substrate then added. after stopping the reaction, the plates were read at 450 nm using a spectrophotometer (bio tek instruments. inc. highland park, usa). in line with the test kit manufacturer recommendations, samples having s/p% < 80% were deemed as negative while those with s/p% values ≥ 80% were considered as positive. the screening of test sera for l.  hardjo antibodies was done using indirect elisa (leptospira hardjo ab bovine elisa, demeditec diagnostics, germany) according to manufacturer’s instructions. after washing the plates five times with 100 µl/well of the kit wash buffer, 100 µl/well of test sera diluted at 1:100 in the kit sample diluent as well as the despite anecdotal reports on incidents of abortion problems at a number of dairy farms in the united arab emirates (uae), there is complete absence of country-specific peer-reviewed literature on bovine abortion. such paucity of uae-specific literature is a hindrance to development of evidence-based methods for the control and management of reproductive failure in dairy cattle herds in the country. it is noteworthy that biosecurity guidelines exist for the control of brucellosis, coxiellosis, and leptospirosis in the uae but not for neosporosis or bvd (adafsa 2011). while such biosecurity guidelines exist for the former three diseases, however, only brucellosis has previously had an active surveillance program. the broader aim of the present pilot study was to collect baseline data on the serostatus of selected abortigenic pathogens of dairy cattle in the al ain region, uae. specific study objectives were to determine the serostatus of c.  burnetii, b.  abortus, l.  hardjo, n.  caninum, and bvdv in an intensively managed dairy cattle herd from the periurban dairy production system of al ain region, uae. the study also evaluated the association between the c.  burnetii-seropositivity and history of abortion. materials and methods study dairy farm and sample size calculation blood samples were collected by venipuncture from 350 randomly selected dairy cattle that belonged to a herd of 6,000 holstein-friesian cattle. the study farm is located in the al ain region, emirate of abu dhabi, uae and up until the time of the research, the dairy farm had been experiencing abortion problems. the sample size was determined using the formula: n = z2 pq/l2α where, n = sample size, z α  = normal deviate (1.96) at 5% level of significance, p = estimated prevalence, q = 1 – p and l = precision of estimate usually at 5% (thrusfield 2007). for the sample size calculation, a priori bovine coxiellosis prevalence of 22.3% reported in iran was used (azizzadeh et al. 2012). the sample size was therefore derived as follows: n = (1.96)2 × 0.223 × 1 − 0.223 = ≈ 266 (0.05)2 to adjust for potential non-compliance and design effect, the calculated sample size was increased to 350. following collection, the blood samples were allowed to clot at room temperature (rt), the sera 165veterinaria italiana 2020, 56 (3), 163-168. doi: 10.12834/vetit.2257.12932.1 barigye et al. serostatus of five abortigenic agents of cattle in the united arab emirates results were expressed as corrected od values (s-n) for each sample using negative control. in line with the test kit manufacturer’s recommendations, the test samples with (s-n) ≤ 0.300 were considered as negative, while those with (s-n) ≥ 0.300 were considered as positive. statistical data analysis the serology data were graphically presented and descriptive statistics done to demonstrate the proportions of cattle that were seropositive to the five abortigenic agents. in addition, the z score test for two proportions was applied to evaluate the level of significance in the difference between the proportion of c.  burnetii-seropositive cattle that had a history of abortion and the proportion of c. burnetii-seropositive cattle that did not have a history of abortion (p value 0.01). refer to the formula below relating to the test hypothesis: null hypothesis h₀: p₁ – p₂ = 0 alternative hypothesis h₁: p₁ p₂ > 0 (p 1 – p 2 ) – 0– – p(1 – p)– – +n 1 n 2 1 1 owing to the few samples that were seropositive for b. abortus, l. hardjo, n. caninum, and bvdv, this test was not performed for these agents. results descriptive statistics and data analysis of the 350 bovine serum samples that were tested by elisa, 41.4% (145/350) were seropositive for c.  burnetii, 0.0% (0/350) for b.  abortus, 1.7% (6/350) for l.  hardjo, 1.4% (5/350) for n.  caninum, and 0.3% (1/350) for bvdv (figure 1). by comparison 58.6% (205/350) were seronegative for c.  burnetii, 100.0% (350/350) were seronegative for b.  abortus, 98.3% (344/350) seronegative to l. hardjo, 98.6% (345/350) negative to n.  caninum, and 99.7% (349/350) were negative for bvdv antigen (figure 1). furthermore, of the dairy cattle that were screened by elisa in the present study, 30.0% (105/350) had history of abortion while 70.0% (245/350) did not. interestingly, of the 105 cattle that had history of abortion, 61.9% (65/105) were seropositive for c.  burnetii, 2.9% (3/105) for l.  hardjo, 1.0% (1/105) for n.  caninum, 0.0% (0/105) for bvdv, and 0.0% (0/105) for b. abortus. when the number of seropositive cattle without history of abortion are considered, 32.7% (80/245) were positive for antibodies against c. burnetii, 0.0% negative and positive controls diluted at 1:50, were separately dispensed into the microtiter plates coated with leptospira antigen. the plates were incubated at 37 °c for 60 min and then washed five times with the kit wash buffer. next, 100 µl/well of horseradish peroxidase labelled anti-bovine igg conjugate were added, and the plates incubated for another 60 min at 37  °c. after five washes, 100  µl/ well of tmb substrate were added and the plates incubated at rt in darkness for 15 min. after stopping the reaction, the plates were read at 450  nm in a spectrophotometer (bio tek instruments, inc. usa). in line with recommendations of the test kit manufacturer, test samples with s/p% ≥ 34% were considered as positive for l. hardjo antibodies while samples with s/p% < 34% deemed as negative. the screening for n.  caninum antibodies was done using an indirect elisa (neospora caninum antibody test kit, idexx laboratories, usa) following the kit manufacturer’s instructions. briefly, 100 µl/well of test samples diluted at 1:100 in the kit sample diluent as well as undiluted negative and positive controls were dispensed into microtiter plates coated with n. caninum antigen. the plates were then incubated at rt for 30 min after which they were washed four times with kit wash buffer. 100 µl/well of peroxidase labelled anti-ruminant igg conjugate were added and the plates incubated at rt for 30 min. the plates were then washed four times with kit wash buffer, 100  µl/well of tmb substrate added to each well, and the plates further incubated at rt in darkness for 15  min. after stopping the reaction, the plates were read using a spectrophotometer (bio tek instruments. inc. highland park, usa) at 630 nm. in line with the test kit manufacturer recommendations, the test samples with s/p% ≥ 50% were considered as positive while those with s/p% < 50% were deemed as negative. the detection of bvdv antigen was done using an antigen capture elisa (bovine viral diarrhoea virus antigen test kit/serum plus, idexx laboratories, switzerland) according to the manufacturer’s kit instructions. briefly, 50 µl of the detection antibodies were dispensed into a microtiter plate wells pre-coated with monoclonal antibodies specific for bvdv antigen. this was followed by 50 µl of the test sera alongside the negative and positive controls. the plates were then tightly sealed and incubated at 37 °c for 2 h. the plates were then washed five times with the kit wash buffer. after incubating at rt with 100 µl/well of peroxidase labelled anti-ruminant igg conjugate for 30 min, the plates were washed five times with the kit wash buffer, 100  µl/well of tmb substrate were then added to each well and the plates incubated at rt for 10 min. after stopping the reaction, the plates were read at 450  nm using spectrophotometer (bio tek instruments. inc. highland park, usa). the 166 veterinaria italiana 2020, 56 (3), 163-168. doi: 10.12834/vetit.2257.12932.1 serostatus of five abortigenic agents of cattle in the united arab emirates barigye et al. reported in a number of animal species on uae territory (afzal et al. 1994, chaber et al. 2012, hassan et  al. 2018, lloyd et  al. 2010), this is the first time research data suggests coxiellosis infection in dairy cattle. as serological evidence of c. burnetii infection was previously reported in racing dromedary camels (afzal et  al. 1994), a dama gazelle that had aborted (lloyd et  al. 2010), as well as semi-free ranging wild ungulates (chaber et  al. 2012), and sheep and goats (hassan et  al. 2018), a complex coxiellosis epidemiology that arguably involves cross-species c.  burnetii transmission cannot be ruled out. this can only be clarified through undertaking more comprehensive epidemiological studies in the country. it is noteworthy that of the five abortigenic agents evaluated in the present pilot study, the proportion of c.  burnetii-seropositive cattle was comparatively greater than for the other four agents for which the proportions of seropositive cattle were 0.0%, 0.3%, 1.4%, and 1.7% for b.  abortus, bvdv, n.  caninum, and l.  hardjo, respectively. to further evaluate the abortigenic significance of c. burnetii, the z test was applied to the proportion of c.  burnetii-seropositive cattle that had history of abortion and seropositive animals that did not have such a history. as it turned out, the data demonstrated there was statistically significant difference between the two groups (z test of two proportions; p < 0.01) further implicating c. burnetii causing abortion in the affected herd. indeed, while the list of abortigenic agents screened for was not exhaustive, future study protocols will need to further delineate the role of c.  burnetii in bovine abortions in the study region of al ain and beyond. elsewhere, detection of c.  burnetii in the foetal membranes and other biological specimens taken from aborted or stillborn foetuses (agerholm 2013, muskens et  al. 2012) has been reported. moreover, c. burnetii-induced placentitis was demonstrated in aborting cattle (bildfell et al. 2000; cabassi et al. 2006). (0/245) for b.  abortus, 1.2% (3/245) for l.  hardjo, 1.6% (4/245) for n. caninum, while 0.0% (0/245) were negative for bvdv antigen (figure 2). the z-test shows that the proportion of c. burnetii seropositive cattle with a history of abortion was significantly higher than the c. burnetii seropositive cattle that did not (p-value < 0.01) with the difference between the proportions of 29.3% (95% ci: 18.2% to 40.2%). discussion and conclusions the present pilot study evaluated the serostatus of five abortifacient pathogens in an intensively managed dairy cattle herd from al ain, uae. as the study herd had a perennial history of abortion, and since the proportion of seropositive animals was only high for c.  burnetii, the present survey further assessed if history of abortion was significantly associated with being seropositive for this pathogen. according to the present data, the dairy cattle herd under study demonstrated variable serostatus in respect to the five abortigenic agents. to the author’s knowledge, this is the first time serological evidence is adduced on apparent c.  burnetii, l.  hardjo, n.  caninum, and bvdv infections in dairy cattle in the uae. as anecdotal reports have previously suggested high prevalence of animal brucellosis, it was rather unexpected that all the 350-screened cattle were seronegative to b.  abortus antibodies. it should be noted that biosecurity guidelines exist for the control of brucellosis (adafsa 2011), and the surveillance interventions for the disease have previously been instituted in the country (m.e.h mohamed, personal communication). it is possible that the biosecurity measures adopted in response to previous concerns over animal brucellosis may have been effective. while c.  burnetii antibodies have previously been p ro p o rt io n s (% ) abortigenic agents history of abortion no history of abortion c. burnetii b. abortus n. caninuml. hardjo bvdv 0 10 20 30 40 60 50 61.9 2.9 1.0 00 0 32.7 1.2 1.6 0.4 figure 2. bar graph showing the comparative proportions of dairy cattle with or without history of abortion that were seropositive for five abortigenic agents including coxiella burnetii, brucella abortus, leptospira hardjo, neospora caninum, and bovine virus diarrhoea virus (bvdv). p ro p o rt io n s (% ) abortigenic agents seropositive seronegative c. burnetii b. abortus n. caninuml. hardjo bvdv 0 20 40 60 80 100 41.4 1.7 1.4 0.30 58.6 100 98.3 98.6 99.7 figure 1. bar graph showing the comparative proportions of cattle that were seropositive and seronegative for five abortigenic agents including coxiella burnetii, brucella abortus, leptospira hardjo, neospora caninum, and bovine virus diarrhoea virus (bvdv). 167veterinaria italiana 2020, 56 (3), 163-168. doi: 10.12834/vetit.2257.12932.1 barigye et al. serostatus of five abortigenic agents of cattle in the united arab emirates guidelines exist for the control of brucellosis, coxiellosis, and leptospirosis in the uae (adafsa 2011) there are currently no active surveillance programs for these diseases. considering the present data therefore, initiating an active surveillance program for coxiellosis while further investigating the epidemiology of all the studied diseases is strongly warranted. in particular, further research focused on delineating the animal and public health implications of c. burnetii infection is recommended. by continuing to generate baseline data on infectious causes of bovine abortion, such studies will, in future, ultimately pave way to more evidence-based disease control and management strategies for bovine abortion in the country. in the meantime, the present data should inform diagnostic investigation protocols whenever investigating cases of bovine abortion in the country. acknowledgements this study was funded by financial support provided by the united arab emirates university research office through a startup grant no. 31f099. it should be noted that while biosecurity guidelines exist for the control of coxiellosis (adafsa 2011), no surveillance interventions for the disease exist in the country and as such this gap needs to be urgently addressed. it should be noted that the proportions of l.  hardjo, n.  caninum and bvdv-seropositive cattle were comparatively lower than for c.  burnetii. as the former three organisms are primary abortigenic agents of cattle (asmare et  al. 2018, delooz et  al. 2018, yildiz et  al. 2017), their animal health implications in cattle in the uae needs to be further investigated. this is more so important since this is the first time they are being reported in the country. further still, as leptospira is also an important zoonotic pathogen (garshasbi et  al. 2018), the potential public health significance of this bacterium should also be evaluated in the uae. it should be noted that biosecurity guidelines exist for the control of leptosiprosis (adafsa 2011), even when no surveillance program for the disease exists in the country. after additional 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seegers h., joly a., beaudeau f. & fourichon c. 2012. increase in the occurrence of abortions associated with exposure to the bluetongue virus serotype 8 in naïve dairy herds. theriogenol, 78, 1140-1151. 405 1department of veterinary pharmacology and toxicology, federal university of agriculture, makurdi, nigeria. 2gan-rovet animal hospital, warri, nigeria. 3department of veterinary physiology and biochemistry, federal university of agriculture, makurdi, nigeria. 4department of veterinary medicine, federal university of agriculture, makurdi, nigeria. *corresponding author at: gan-rovet animal hospital, warri, nigeria. e-mail: kshimaelyx@gmail.com. fidelis aondover gberindyer1, felix kundu shima2*, victor masekaven ahur3, solomon tsekohol agu3, thaddaeus ternenge apaa4 and matthew terzungwe tion4* keywords dog, environmental pollutants, exposure, health risk, potentially toxic metals. veterinaria italiana 2022, 58 (4), 405‑411. doi: 10.12834/vetit.2464.17442.3 accepted: 20.04.2022 | available on line: 31.12.2022 summary environmental pollutants pose a health risk to animals and humans. we evaluated levels of some potentially toxic metals in environmental dust, blood, and hair samples of apparently healthy security dogs from a crude oil well drilling site (a) and liquefied natural gas production site (b) industrial environments in nigeria. these samples were routinely digested and analyzed for lead, cadmium, nickel, chromium, and zinc using atomic absorption spectrophotometry assay. mann‑ whitney u test was used to compare concentrations of the metals in different samples. dust samples contained a high amount of the metals considered. there was no significant difference between levels of heavy metals in blood and hair samples from dogs guarding both sites, except for blood (p=0.034) and hair (p=0.015) chromium which were higher in those securing site a compared with site b. higher nickel (p=0.001) and zinc (p=0.001) with lower chromium (p=0.004) levels occurred in the hair samples than in the blood. lead was not detected in blood and hair samples suggesting safety. there was no correlation between the same metal in blood and hair. hair chromium and nickel levels were above the reference suggesting toxic exposure. there is a need for regular monitoring and decontamination of air pollutants within similar facilities for environmental safety. please refer to the forthcoming article as: gberindyer f.a. et al. 2022. potentially toxic metals in dust, blood, and hair in exposed security dogs in an oil and gas industry. vet ital. doi: 10.12834/vetit.2464.17442.3 potentially toxic metals in dust, blood, and hairs from exposed security dogs in an oil and gas industry introduction heavy metals are defined as metals and metalloids having densities >5 gcm‑3 (hawkes 1997). heavy metals and metalloids are often used directly or indirectly at home, in agriculture, medicine, technology, and industries (hawkes 1997). they are found naturally in the environment, certain foods, medicines, and water (hawkes 1997; zhuang et  al. 2009; renner 2010; vodyanitskii 2016; nworu et al. 2019). their wide distribution in the environment has potential effects on the environment, plant, animal, and human health (slotnick et al. 2000; mcbride et al. 2003; zhuang et  al. 2009, orisakwe et  al. 2012). some heavy metals considered to be potentially toxic are required by the body in trace quantities for biochemical and physiological processes (singh et al. 2018; pourret and hursthouse, 2019). however, at higher concentrations and depending on the dose, route of exposure, chemical speciation, age, gender, and nutritional status of the exposed animal or human being, they can have deleterious effects (jaishankar et al. 2014; pourret and hursthouse, 2019). animals and humans get exposed through ingestion of contaminated food, drinking of levels of potentially toxic metals in occupationally exposed dogs gberindyer et al. 406 veterinaria italiana 2022, 58 (4), 405-411. doi: 10.12834/vetit.2464.17442.3 materials and methods sampling and sample preparation this study was carried out on dogs used for security purposes in crude oil well drilling (site a) and liquified natural gas production (site b) in nigeria. blood (5 ml) and hair (2 g) samples opportunistically collected from 13 dogs (in july 2016) from blood used for health parameters evaluation, i.e., for routine clinical and laboratory screening were used for some heavy metal analysis. technically, no animal was specifically sampled for this study. ethical approval from the university of ibadan animal care use and research ethical committee (ui‑acurec/ app/2015/027) was obtained for this study after reviewing the panel and the listed guidelines and principles of animal handling and care were strictly adhered to during and after sampling. the dogs sampled were all males, with a mean age of 9.7±1.4 years (range 8.0 ‑ 13.0), and a mean body weight of 33.2±5.3 kg (range 18.0 ‑ 38.0) which have spent at least 7 years in that environment. also, environmental dust within the vicinity of the kennels (sites a and b) was collected for heavy metal analysis. dust was collected manually by using clean dry cotton wool and plain paper to gather dust on windows and dry hard surfaces in the kennels. dust samples were collected from each of the 16 and 10 kennel partitions from site a and site b, respectively. subsequently, the dust samples from each site were pooled together into two sets before the levels of metals were quantified. all the samples collected were labeled appropriately and stored for further processing. blood samples collected in heparinized bottles were stored at ‑20°c until heavy metals were quantified. the hair samples were repeatedly washed in the laboratory with metal‑free distilled water, properly rinsed and dried in a special drying oven, and kept in a humid‑free plastic container at room temperature before digestion and heavy metals analysis. the samples were digested following a standard procedure (metals and others, method 971.21, chapter 9) as described by the association of official analytical chemists (aoac, 2000) in a laboratory shared by veterinary physiology, biochemistry, pharmacology, and toxicology units of the federal university of agriculture, makurdi, nigeria. we measured 1ml of blood, 1g of hair, and 1g of the dust samples and digested them in three separate reactions with 2 ml of hno 3 , hclo4 , and h 2 so 4 mixture in a ratio of 3:1:1 (v/v/v), and microwaved in a closed container until fumes became clear. subsequently, the volume was made up to 50 ml with deionized water and stored in clean plastic test tubes for metal analysis. contaminated water, inhalation of contaminated air, exposure to contaminated soils and industrial wastes, and absorption through the skin (airey 1983; alexander and davidson 2006; babalola et al. 2007; renner 2010; sharma et al. 2016). high blood levels of heavy metals imply high exposure levels, while lower levels of trace elements and essential minerals represent insufficient intake which may be a sign of nutritional deficiency (flora et al. 2008). a whole blood level of heavy metals is representative of extracellular and intracellular metal concentrations, while plasma and serum concentrations depict extracellular concentrations (flora et al. 2008). heavy metal toxicity is primarily due to oxidative damage to the biological macromolecules following the binding of metals to the dna and nuclear proteins (flora et al. 2008). consequently, this distorts the normal functions of the brain, lungs, kidneys, liver, and hematopoietic organs (broun et al. 1990; järup 2003; dorne et  al. 2011). some of the degenerative conditions associated with heavy metal toxicity are parkinson’s and alzheimer’s diseases among others (flora et al. 2012). chronic exposure to some of these potentially toxic metals could lead to varying types of cancers, while some metallic elements induce multiple organ damage even at lower levels of exposure (aquino et al. 2012; chervona et al. 2012). some common heavy metals with potentially high occupational and environmental adverse effects are mercury (hg), cadmium (cd), lead (pb), copper (cu), vanadium (v), chromium (cr), cobalt (co), nickel (ni), selenium (se), arsenic (as), manganese (mn), silver (ag), zinc (zn), and uranium (u) (jaishankar et al. 2014; vodyanitskii 2016). in nigeria, crude oil and gas exploration, exploitation, and production, as well as gas flaring are common sources of environmental pollution (nworu et al. 2016). crude oil and gas pollution occurs in form of spillage from accidental discharges, corrosion of pipelines, oil well blowout, oil pipeline vandalism, and gas flaring. dogs are used to support securing many of the crude oil and gas production facilities in nigeria and are exposed to the environmental pollutants associated with these anthropogenic activities just as human beings working or living within those locations. also, the pathophysiology of the adverse effects of these toxic metals in animals and human beings are similar (airey 1983; jaishankar et al. 2014; santin et al. 2005). animals are valuable sentinel for environmental contamination; however, they are often ignored. consequently, this study was designed to assess the levels of some potentially toxic heavy metal elements in environmental dust, blood, and hair of dogs used for securing a crude oil well drilling and liquefied natural gas production facility, generally regarded as one of the environments at higher risk of elemental pollutants. gberindyer et al. levels of potentially toxic metals in occupationally exposed dogs veterinaria italiana 2022, 58 (4), 405-411. doi: 10.12834/vetit.2464.17442.3 407 tissue samples to check if any relationship existed between blood and hair metals concentrations was applied. a statistical test of significance was set at p<0.05. results results (table i) showed a higher level of pb, cr, and ni in the dust samples from the kennel located within site b compared to site a where crude oil well drilling activities take place. on the other hand, dust samples from the kennel in site a contained a higher level of cd and zn as compared with dust samples from site b. furthermore, there was no significant difference between the mean amount of heavy metals in blood and hair samples from dogs securing sites a and b, except for blood (p=0.034) and hair (p=0.015) cr levels which were significantly higher in dogs guarding site a compared with those in site b. the level of cr in the hair and blood of the dogs guarding site a were 1.6‑ and 1.7‑folds, respectively higher than the mean level in those operating within site b. regarding the body tissues, significantly higher levels of ni (p=0.001) and zn (p=0.001) were observed in the hair than in the blood samples. no amount of pb was detected in the hair and blood samples from the investigated dogs. correlation analysis between blood and hair metal levels showed no connection between the same metal in blood and hair. however, some interesting correlations observed were that between blood cr and hair cd (r= 0.64; p=0.045); and that between cr and zn in the blood (r= 0.57; p=0.041). analyses of heavy metal concentrations in the samples the levels of pb, cd, cr, ni, and zn in the dust, hair, and blood samples were determined employing atomic absorption spectrophotometer (buck scientific‑210vgp aas, norwalk, ct, united states) in the department of soil science, university of ibadan, nigeria. standard salt preparations for each metal were used to calibrate the aas machine. limits of detection for the analyzed samples, expressed as a wet weight (w/w) were ‑ cd 0.00024 mg/kg, cr 0.0015 mg/kg, pb 0.013 mg/kg, ni 0.004 mg/kg, and zn 0.014 mg/kg for both hair and dust. those of blood were ‑ cd 0.00013 mg/l, cr 0.0021 mg/l, pb 0.002mg/l, ni 0.0041mg/l, and zn 0.053mg/l. data analysis the reading obtained from the aas was multiplied by the dilution factor (50) to obtain the actual amount of heavy metal in each of the samples. statistical analysis was performed with spss v20 program. the normality of data was assessed by applying the kolmogorov‑smirnov test. as the data were not normally distributed, non‑parametric statistics were applied. mann‑whitney u test statistic by ranks on blood and hair results of the sampling sites (a and b) was considered. because the data were not normally distributed, the geometric mean of the quantified metals was computed to take care of outliers in order to avoid overestimating the means or skewness of the mean values towards higher values. furthermore, a correlation test within the table i. heavy metals mean values ± sd (ppm) in kennel dust, hair and blood of dogs from crude oil well drilling (site a) and liquified natural gas production (site b). different letters in the same column means statistical difference {a,b(p=.015), a,c(p=.034) for site; a,d(p=.001), a,e(p=.004), a,f(p=.001)}. statistical comparison was not applied to the dust samples since they were pooled together before elements quantification; lod denotes limit of detection. site mean + sd pb cd ni cr zn dust (ppm) a 109.0 1.7 89.3 128.6 4620.0 b 300.0 0.1 304.3 248.1 3095.0 hair (ppm) a (n = 7) < lod <lod a 18.68 ± 1.51a 4.53 ± 1.45a 113.31 ± 1.81a b (n = 3) < lod 0.02 ± 0.03a 26.04 ± 1.58a 2.79 ± 1.36b 103.69 ± 2.57a blood (ppm) a (n = 8) < lod 0.01 ± 0.02a 11.83 ± 1.36a 12.08 ± 1.31a 51.08 ± 1.28a b (n = 5) < lod 0.02 ± 0.04a 11.12 ± 1.12a 7.01 ± 1.49c 48.62 ± 1.20a body tissue (ppm) hair (n = 10) < lod 0.01 ± 0.03a 20.64 ± 1.54a 3.92 ± 1.51a 110.33 ± 1.93a blood (n=13) < lod 0.01 ± 0.03a 11.55 ±1.26d 9.80 ± 1.51e 50.12 ± 1.22f levels of potentially toxic metals in occupationally exposed dogs gberindyer et al. 408 veterinaria italiana 2022, 58 (4), 405-411. doi: 10.12834/vetit.2464.17442.3 physiological reducing agents (thiols and ascorbate) to form reactive oxygen species (superoxide, hydrogen peroxide, and hydroxyl radicals) resulting in oxidative stress and consequent damage to important biological macromolecules, such as the dna and proteins (stohs and bagchi 1995). again, ni and cr are among the carcinogenic metals that are environmental and occupational pollutants (aquino et al. 2012, chervona et al. 2012). even though ni is an essential physiological mineral, chronic exposure to it has been associated with an increased risk of breast and pulmonary cancers, and cardiovascular, kidney, liver, and neurological diseases due to the release of free radicals that cause oxidative damage (aquino et al. 2012, chervona et al. 2012, das et al. 2008, zhao et al. 2009). zinc is an “essential trace element” because very small amounts are required for human health, e.g., immune and thyroid functions as well as vision (girodon et al. 1999). hair metal analysis is a toxicological screening that detects and measures essential minerals and metals reflecting tissue stores; however, the body does not store excess zn hence detection of its high levels in hair samples obtained from the investigated dogs may not necessarily be predictive of its high tissue concentration. nevertheless, it could suggest that these dogs were previously exposed to high levels of zn. one of the toxic effects of zn following inhalation by animals and humans is anosmia which is one of the very important and valuable defensive characteristics of security dogs. anosmia following intranasal intake of zn is a result of damage to the olfactory epithelium, inducing overt intracellular edema (alexander and davidson 2006; mcbride et al. 2003; slotnick et al. 2000). exposure of security dogs to toxic levels of zn would adversely affect their defending and guarding capabilities. this study also showed no significant (p>0.05) difference in the levels of heavy metals in blood and hair samples of dogs from the two sites investigated, except for cr which was observed to be significantly higher in the hair (p=0.015) and blood (p=0.034) of dogs guarding site a compared with those in site b. the high levels of cr in the hair and blood of dogs from site a could be attributed to the higher exposure level due to anthropogenic or crude oil well drilling activities which release these pollutants into the atmosphere and consequently settle as dust and precipitations. naturally, cr is found in high concentrations in activities involving the burning of oil, coal, and crude oil well drilling (jaishankar et al. 2014). this result also revealed a significant difference in the levels of ni (p=0.001), cr (p=0.004), and zn (p=0.001) (all trace elements) in hair and blood samples from dogs kept in the two units of the company. the levels of ni and zn were about 1.8‑ and 2.2‑fold, discussion heavy metals are environmental pollutants that cause harmful effects in animals, and humans following absorption into the body through soft tissues above the specified permissible levels (flora et al. 2012; who, 2020). the most common metals that the human body could absorb in toxic amounts are hg, v, as, pb, and cd (jaishankar et al. 2014; renner 2010; flora et al. 2012). high levels of exposure to these heavy metals could be from contaminated food, air, water, medicine, food containers with improper coating, and industrial exposures (zhao et al. 2009; zhuang et al. 2009; renner 2010; flora et al. 2012; wei et al. 2015; sharma et al. 2016; singh et al. 2018). in this study, we evaluated levels of cd, cr, ni, pb, and zn in the dust from the kennels as well as blood and hairs of some dogs used for security purposes in the oil and gas industry which are zones with potentially high levels of elemental pollutions. this study revealed that dust, hair, and whole blood samples obtained from the dog kennels and dogs guarding the crude oil drilling and liquified natural gas‑producing areas contained varying amounts of cd, ni, cr, and zn. however, pb was not detected in any of the blood and hair samples that were analyzed, except for dust samples. the high enrichment of dust with heavy metals observed in this study could be explained by the anthropogenic activities, gas flaring, and non‑biodegradability of these metals in the dust medium (wei 2015; nworu et al. 2016). there is no reported safe or beneficial level of pb in blood and other biological tissues for animals or humans (renner 2010; who 2020; flora et al. 2012). therefore, the non‑detection of pb in the blood and hair of the investigated dogs also suggests that they could be safe from pb poisoning and perhaps those working in that environment at the period within which the samples were obtained. the hair tissue mineral analysis showed that the amount of cr (3.9 ± 1.5 ppm), ni (20.6 ± 1.5 ppm), and zn (110.3 ± 1.9 ppm) recovered from the blood and hairs of the dogs investigated were well above the reference ranges of 0.02 ‑ 0.08 ppm, 0 ‑ 0.1 ppm, and 10 ‑ 21 ppm, respectively considering animal and human reference values (puls 1994; renner 2010; flora et al. 2012). natural sources of cr are the burning of oil and coal as well as crude oil well drilling activities (jaishankar et al. 2014). the later natural source is one of the major activities in the study area which probably explains the basis for the high level of cr recorded in the hair samples. two forms of cr exist, cr (iii) and cr (vi) also referred to as hexavalent cr which is highly soluble, extremely toxic to animals and humans and predominates in the environment (cervantes et al. 2001). even though cr is essential for many biological functions, when the concentrations in the body are above the safe level, it reacts with the gberindyer et al. levels of potentially toxic metals in occupationally exposed dogs veterinaria italiana 2022, 58 (4), 405-411. doi: 10.12834/vetit.2464.17442.3 409 of these binding sites following chronic exposure, leading to a plateau in metals concentrations and consequently the lack of correlation with blood levels (patra et al. 2007). this study was limited by small sample size as only 13 dogs were kept in these facilities. conclusions this study showed that non‑invasive analysis of ni and zn using hair samples from animals could be used to predict previous exposures, environmental pollution, and the nutritional metabolic activity that has occurred within a given period. non‑detection of pb and a negligible amount of cd in hair and blood samples from dogs in the study area suggest environmental safety from these metals in this location within the period considered. the observed higher levels of cr and ni in hair samples above the reference values instead suggest the high level of exposure of security dogs and by extension, workers in this industrial area to these environmental pollutants. consequently, there is a need for routine monitoring and decontamination of potentially toxic environmental metal pollutants for the safety of aquatic lives, security dogs, and workers within the vicinity of related industries. acknowledgements the authors appreciate gan‑rovet animal hospital, warri for the opportunity given to one of the leading authors that resulted in this study. respectively higher in the hair samples than what was obtained in the blood samples. inversely, no significant difference (p>.05) was observed for cd levels in blood and hair samples. mineral and heavy metal contents of the hair are considered as the spillover from what is in the body. essential elements and toxic heavy metals are sequestered into the hair from the follicular cells and their blood supply as part of the detoxification mechanism to prevent the expression of their adverse biological effect, thus implying that hair element analysis is a valid means for screening mineral deficiencies and toxic element exposures (airey 1983). correlation analysis between blood and hair metal levels showed no connection between the same metal in blood and hair. this could suggest differences in the kinetics of the metals considered (zaccaroni et al. 2014). also, it is established that mineral and heavy metal contents of the hair are the spillover from what is in the body (airey 1983). the existing study recorded a correlation between the same metal (ni and cr) in hair and blood (zaccaroni et al. 2014). two remarkable correlations were that between blood cr and hair cd (r=0.64, p=0.045), and that between cr and zn in the blood (r=0.57, p=0.041). however, the explanation for these connections remains elusive and warrants further investigation. the lack of correlation between blood and hair cd and pb is consistent with existing reports (gyori et al. 2005; patra et al. 2007; zaccaroni et al. 2014). hair is a keratin‑rich tissue, with abundant sulfhydryl groups which bind divalent cations such as pb and cd, leading to their persistence in hair (hasan et al. 2004). this lack of correlation between blood and hair pb and cd may be linked to the saturation levels of potentially toxic 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letter to the editor 66 manes et al. veterinaria italiana 2020, 56 (2-3), 65-66. doi: 10.12834/vetit.2375.13627.1 gallagher j. & clifton-hadley r. 2000. tuberculosis in badgers; a review of the disease and its significance for other animals. res vet sci, 69, 203-217. kollias g.v. & fernandez-moran j. 2015. mustelidae. in fowler's zoo and wild animal medicine. (r.e. miller & m.e. fowler, eds). elsevier, 8, 476-491. rupprecht c., smith j., fekadu m. & childs j. 1995. the references ascension of wildlife rabies: a cause for public health concern or intervention? emerg infect dis, 1 (4), 107-114. shi j., wen z., yang h., wang c., huang b., liu r., he x., sun z., zhao y., liu p., liang l., cui p., wang j., zhang x., guan y., tan w., we g., chen h. & bu z. 2020. susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2. science, 368, 1016-1020. 331 veterinaria italiana 2022, 58 (3), 331-337. doi: 10.12834/vetit.2596.16323.2 accepted: 15.11.2021 | available on line: 31.12.2022 1department of veterinary public health and preventive medicine, university of abuja, nigeria. 2the nigerian police force, department of operations, veterinary unit, presidential state house, abuja, nigeria. *corresponding author at: : department of veterinary public health and preventive medicine, university of abuja, nigeria. e‑mail: andrew.adamu@uniabuja.edu.ng. rukaiya musa-gobe1,2, gabriel omeiza1, wesley nafarnda1 and andrew adamu1* keywords west nile virus, elisa, horses, domestic chickens, nigeria. summary west nile virus (wnv) is an emerging arbovirus which affects humans and horses. a cross sectional study was carried out on 106 local horses in kaduna and 78 domestic chickens in federal capital territory. a total of 184 sera were screened for west nile virus anti pr-e antibodies using id screen® west nile competitive enzyme linked immunosorbent assay. for the horses, an overall prevalence of 92.45% was recorded while domestic chickens had a preponderance of 7.69%. from our study, there was a statistical significant difference between the occurrences of wnv in stallions than mares with p < 0.05. comparing the occurrence of west nile virus between species, horses were more likely to be infected by west nile virus than domestic chickens (or 147). this is the first seroprevalence study investigating west nile virus infection in domestic chickens in nigeria. the presence of the antibodies indicates the widespread circulation and the potential risk of infection in humans and animals. in order to understand the epidemiology of west nile virus infection in nigeria, there is need for surveillance to be implemented in human and animal sectors. evidence of west nile virus in chickens and horses in nigeria: results from a serosurvey disease, which can be characterized by acute flaccid paralysis, encephalitis, and meningoencephalitis (de fillet et al. 2012, vilibic-cavlek et al. 2014). from the 1990s, west nile disease outbreaks have emerged across various continents and wnv is now recognized as one of the most prevalent flaviviruses worldwide (vilibic-cavlek et al. 2014, cox et al. 2015, fall et  al. 2016). in nigeria, culex species like culex pipens, culex quinquefasciatus have been reported in south west nigeria (motayo et  al. 2016). this is a clear indicator that most of the vectors in nigeria are present and may predispose both humans and animal population to the wnv infection. west nile virus has been reported in horses and humans by different authors in nigeria. olaleye and colleagues (olaleye et  al. 1989) reported a prevalence of 71% from horses in southwest nigeria, sule and colleagues (sule et  al. 2015) recorded 90.3% in the same geopolitical zone while baba and colleagues (baba et al. 2013) documented a prevalence of 25% from humans in maiduguri north east nigeria. avian species have been categorized into four groups for the purpose of wnv surveillance: dead birds, trapped birds, captive and sentinel birds or domestic birds (chintoutis et al. 2015). in the united introduction west nile virus (wnv) is a vector-borne virus belonging to the japenese encephalitis complex within the flaviviridae family. the complex includes japanese encephalitis virus (jev), saint louis encephalitis virus (slev), murray valley encephalitis virus (mve) and usutu virus (usuv) (fall et al. 2016). most of these flaviviruses, such as wnv, tick-borne encephalitis virus (tbev), or jev, are considered as emerging zoonoses that pose serious public health threats to animals and man (cardinale et al. 2017). west nile virus was first isolated from a febrile woman in omogo region in the west nile district of uganda in 1937, from a set out survey to define the endemicity of yellow fever (smithburn et al. 1940). wnv is chiefly transmitted by bite of infected culex sp. mosquitoes. birds are considered as a main reservoir host and migratory birds can play an important role in long distance viral dissemination (hubalek and halouzka 1999, hayes et al. 2005, ergunay et al. 2015). humans and horses are known to be incidental hosts of wnv (murray et  al. 2010). clinical symptoms of wnv infection may range from asymptomatic or mild influenza-like illness to severe neurological short communication 332 west nile virus in nigerian horses and chicken musa-gobe et al. veterinaria italiana 2022, 58 (3), 331-337. doi: 10.12834/vetit.2596.16323.2 study area, design and sample collection a cross sectional study was conducted with blood samples taken in 2019 from a total of 106 randomly selected local horses in three local government areas of kaduna state with coordinate (10°30'36.7"n 7°25'01.6"e). the three local government areas were selected based on the concentration (mshelia et  al. 2012) of local horse presence of polo fields, and their unvaccinated status: zaria lga (11°03'40.6"n 7°42'20.6"e) (n = 40), sabon gari lga (11°08'01.0"n 7°43'02.0"e) (n = 30) and igabi lga (11°08'01.0"n 7°43'02.0"e) (n = 36) figure 1 and table  i. similarly, blood samples from domestic chickens (n = 78) were collected from federal capital territory (9°04'26.3"n 7°28'43.1"e) in commercially reared chickens (isa brown) (n = 40) and free range chickens (n = 38) in kuje area council (8°52'49.0"n 7°13'37.7"e) to determine the spread of wnv. blood samples were aseptically taken by jugular venipuncture and were allowed to clot and centrifuged at 10,000 g for fifteen minutes to allow for proper separation of serum from the clotted blood. sera were harvested using a sterile pipette into 2 ml cryovial tubes, labeled and stored at -  80  °c at the national veterinary research institute laboratory, vom, nigeria for sample analysis. states of america and italy, for decades, domestic birds have been used as living sentinels in arbovirus programs aimed at monitoring virus transmission (komar et  al. 2003b). west nile virus surveillance using domestic birds has been documented in america, europe and australia (chintoutis et  al. 2015) and africa (fall et al. 2016, amdouni et al. 2020). chickens are frequently being used as sentinels for the surveillance of bird-transmitted arboviral encephalitides (langevin et  al. 2005, blackmore et  al. 2003). furthermore, arbovirus surveillance systems based on testing of sentinel chickens provided evidence of wnv circulation prior to the occurrence of human cases (healy et al. 2012). this study was therefore carried out to determine the presence of wnv infection serologically in domestic chickens and local horses in nigeria. materials and methods ethical statement this study was approved by the university of abuja ethical committee and animal care committee uaecau/2019/01. horses and domestic chickens were selected with the consent of their owners after they were briefed on the objective of the study. figure 1. map of nigeria with kaduna state highlighted and sampling local government areas in red. 333 musa-gobe et al. west nile virus in nigerian horses and chicken veterinaria italiana 2022, 58 (3), 331-337. doi: 10.12834/vetit.2596.16323.2 area recorded the highest prevalence (38/40; 95%), followed by igabi local government area (34/36; 94.44%) and sabon gari lga (26/40; 86.67%) (table i). horses in the sampled areas were divided into three age groups. the first group included animals 1-5 year old, the second included animals between 6 and 10 years, and the third animals aged between 11 and 15 years. the prevealence found in the first group was 80% (12/15), 96.83% (61/63) was the prevalence found in the second group while in the third group it was 89.29% (25/28). all males (100%; n = 94) were found positive to the wnv c-elisa, while in only 66.67% of the sampled mares (n = 12) wnv antibodies were detected. there was a statistically significant difference (p < 0.05) between the occurrences of wnv in stallions and in mares (table i). prevalence of wnv antibodies in chickens in kuje local government area a total of 78 chickens were sampled in kuje local government area of federal capital territory with an overall wnv prevalence of 7.69%. free range chickens recorded a prevalence of 5.26% (2/38) while in exotic chickens (isa brown hybrid chicken) the prevalence found was 10% (4/40/) (table ii). a total of thirty eight (38) free range birds where sampled. all cocks (n = 4) were negative. a prevalence of 5.88% was detected in the 34 hens (table ii). comparison of wnv antibodies among local horses and chickens in kaduna and fct from the sampled horses, 92.45% (98/106) prevalence was recorded in kaduna state while 7.69% (6/78) was recorded from chickens in the federal capital territory. in this study the wnv prevalence found in horses was much (p < 0.05) than that found in chicken. also, horses were 147 times more likely to be infected by wnv than chicken (or 147) (table iii). discussion the present study highlighted the circulation of west nile specific igg antibodies using competitive enzyme linked immunosorbent assay (c-elisa) in nigerian local horses and chickens (exotic and free range chickens) in kaduna and federal capital territory abuja, respectively. a prevalence of 92.45% was recorded in horses. the result of this study was similar to other reports of wnv in horses in tchad 90.3% or senegal 92.5%. conversely it was much higher than that found in cote d’ivoire 28%, congo democratic republic 30%, gabon 3% and djibouti 9% (cabre et  al. 2006, sule et  al. 2015). the high detection of wnv specific antibodies serum samples (n = 184) were screened for the presence of anti-wnv igg antibodies using the id screen® west nile competitive elisa kit (idvet, grabels, france) that detects wnv anti-pr-e antibodies, according to the manufacturer’s instructions. absorbance values were read at 450 nm wavelength, using a microplate spectrophotometer (thermo scientific multiskan® ex; thermo fisher scientific, waltham, massachusetts, usa). a presumptive positive diagnosis was made when the test samples produced an optical density (od) less than or equal to 40%, they were considered as doubtful when the od was between 40%-50%; and negative when the od was greater than 50%. data analysis results obtained from serological tests were subjected to analysis by spss version 20.0 statistical packages for descriptive statistics. chi square test and fisher’s exact test were used to test for association between categorical variables; p value of less than < 0.05 was considered significant. odd ratio was used to test for strength of association. results prevalence of wnv antibodies in local horses in selected local government areas of kaduna state a total of 106 local horses were sampled with an overall prevalence of 92.45%. zaria local government table i. distribution of west nile virus antibodies in local horses in three local government areas of kaduna state according to demographic characteristics. variables no. sampled no. positive % prevalence (95% ci) p value sex male 94 94 100 (nan infinity) 0.0001* female 12 8 66.67 (37.69-88.39) age (years) 1-5 15 12 80 (54.65-94.65) 0.065 6-10 63 61 96.83 (89.91-99.46) 11-15 28 25 89.29 (73.55-97.20) location (lga) zaria 40 38 95 (84.45-99.15) 86.67 (70.90-95.62) 0.365 sabon gari 30 26 94.44 (82.84-99.06) igabi 36 34 *nan-infinity = not a number. 334 west nile virus in nigerian horses and chicken musa-gobe et al. veterinaria italiana 2022, 58 (3), 331-337. doi: 10.12834/vetit.2596.16323.2 be attributed to the fact that they must have spent longer time in their stables thereby been exposed to several facilitating risk factors associated with the disease. this is contrary to the studies conducted by zohaib and colleagues (zohaib et  al. 2015) in pakistan where antibodies to wnv decreased with age. the significant difference observed between sexes in our study where stallions recorded more seropositivity may be attributed to the availability of more stallions found in the location during sampling than mares and their use in ceremonial and sporting activities in northern part of the country. this is contrary to the studies of sule and colleagues (sule et al. 2015) in southwest nigeria where mares recorded a higher seropositivity than stallions. birds are generally known to be host of wnv and other flaviviruses (furlong et  al. 2023). overall prevalence of 7.69% was recorded among domestic chickens in our study. a similar study was carried out by yapici and colleagues (yapici et  al. 2012) among domestic chickens with no seropositivity recorded. several authors have worked on wnv infection in chickens with varying prevalence 0.63% by komar and colleagues (komar et al. 2003a) while le francois and colleagues (le francois et  al. 2006) recorded 1.7% and 0.6%, respectively, which were all lower than our study. however, jozan and colleagues (jozan et al. 2003) had 69% preponderance which is way higher compared to the recorded prevalence. from our study, exotic chickens had a higher preponderance than the free range chickens. the low preponderance of wnv antibodies observed in free range chickens as compared to exotic chickens could be linked to location. most of the poultry farms are sited far away from human settlements with only farm workers residing in the farm. this encroachment of the natural habitat of mosquitoes may have played a role in the difference between breed preponderance of the wnv infection. exotic chickens are exposed several vectors such as mosquitoes and soft ticks which can be found in the crevices of walls and fruit bats that roost on trees and roofs of poultry pens and farm houses. thereby, predisposing poultry workers in poultry farms and markets (those that sell and dress chickens) to wnv infection. on the other hand, free range chickens wander occasionally into nearby bush to scavenge for feed and return home for shelter around their prevalence of wnv recorded in this study could be due to factors such as tranboundary movement of horses from neighboring african countries into nigeria and climate change. the vegetation of kaduna is sudan savannah with short trees, shrubs and grasses and relatively low rainfall that provide conducive conditions for the proliferation and adaptations of vectors that are capable of transmitting the virus to horses which corroborate with the studies conducted by hubalek and halouzka and chancey and colleagues (hubalek and halouzka 1999, chancey et al. 2015). kaduna state is characterized by high temperature which can cause an upsurge in growth rates of vector populations, decrease the interval between blood meals, shorten the incubation time from infection to infectiousness in mosquitoes, accelerate the virus evolution rate and increase virus transmission efficiency (paz 2015). local horses are used for different purposes ranging from sports (horse racing or polo), religious festivals and some are slaughtered for meat. horses are transported to different part of the country during competitions with the possibility of of introducing vectors along with them into naïve population further creating a complex dynamic in the transmission of wnv. also, less attention is given to the health and management of local horses as they are believed to be resistant to disease pathogens. this is in sharp contrast with the exotic horses which are well groomed and vaccinated or treated against various infectious diseases including wnv from their country of purchased. studies have documented that wnv infection risk increases during peak mosquito activity seasons (august-october) in temperate zones of the northern hemisphere, however, this observation is different in tropical and subtropical regions where disease epizootic patterns does not conform to what is obtainable in temperate regions (zohaib et al. 2015). horses in the age range of 6-10  years were found to record the highest prevalence of wnv antibodies. this could table ii. distribution of west nile virus antibodies among domestic chickens in kuje area council, federal capital territory. variables no. sampled no. positive % prevalence (95% ci) p value breed local 38 2 5.26 (0.89-16.32) 0.4326 exotic 40 4 10 (3.26-22.38) sex (local) male 4 0 0 0.618 female 34 2 5.88 (1.00-18.10) sex (exotic) male 0 0 0 not determinedfemale 40 4 10 (3.26-22.38) table iii. comparison of wnv antibodies among local horses and domestic chickens in the study areas. species of animal no. sampled no. positive proportion (%) (ci) 95% ci p value horses 106 98 92.45 86.17-96.43 0.0001 chickens 78 6 7.69 3.18-15.31 total 184 104 56.52 49.99-63.24 335 musa-gobe et al. west nile virus in nigerian horses and chicken veterinaria italiana 2022, 58 (3), 331-337. doi: 10.12834/vetit.2596.16323.2 in conclusion, this study was able to demonstrate the presence of wnv antibodies in local horses and domestic chickens (exotic and free range) in nigeria, to the best of our knowledge; this is the first report in chickens. since avian species play an important role in wnv life cycle, its close interaction with humans poses a risk of zoonosis as some of the febrile illness in man can be misjudged as malaria in communities with poor diagnostic capabilities. there is need to assess the clinical impact of wnv infection on humans, equine and domestic chicken populations. further molecular studies are needed in order to characterize the wnv in various livestock and human populations. this will be able to rule out any cases of cross reactions with other arboviruses. acknowledgements we will like to thank all the animal owners and the livestock attendants that gave us their cooperation during the sample collection. our gratitude goes to mr. dan marwa agom of viral division, national veterinary research institute vom, plateau state for his support during the laboratory work. pens or some sleep outside by perching on trees, thereby exposed to adverse weather conditions and mosquitoes. at least, there is an effective mosquito control around homes where these chickens reside. from the free range chickens, hens were the only chickens that tested positive. hens are mostly kept in rural areas with only few cocks that have good reproductive pedigrees are left to stay longer in the population. cocks are either sold to meet immediate economic challenges or consumed during festivals and ceremonies. horses and chickens seropositivity were compared. horses were 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pakistan. epidemiol infect, 143, 1931-1935. neurotropic virus isolated from the blood of a native of uganda. american j trop med hyg, 20, 471-492. sule w.f., oluwayelu d.o. & adedokun r.a. 2015. high seroprevelance of west nile virus antibodies observed in horses from southwestern nigeria. vector borne zoonotic dis, 15 (3), 218-220. vilibic-cavlek t., kaic b., barbic l., pem-novosel i., slavic-vrzic v., lesnikar v., kurecic-filipovic s., babic-erceg a., listes e., stevanovic v., gjenero-margan i. & savini g. 2014. first evidence of simultaneous occurrence of west nile virus and usutu virus neuroinvasive disease in humans in croatia during the 2013 outbreak. infection, 42 (4), 689-695. 311 veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 accepted: 15.04.2020 | available on line: 31.12.2021 #giuseppe aprea and ilaria di bartolo have contributed equally to this work. 1istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, teramo, italy. 2istituto superiore di sanità, rome, italy. 3polis società cooperativa sociale onlus, perugia, italy. 4university of teramo, faculty of bioscience, teramo, italy. 5local health unit of teramo, teramo, italy. *corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, teramo, italy. e-mail: g.aprea@izs.it. giuseppe aprea1*#, ilaria di bartolo2#, marina monini2, daniela d’angelantonio1, silvia scattolini1, arianna boni2, gennaro truglio3, silvia di giacobbe1, annalisa serio4, salvatore antoci5, violeta di marzio1, giacomo migliorati1, nicola d’alterio1 and francesco pomilio1 keywords long term care facility, norovirus gii.4, outbreak, surface contamination. summary some residents and people from the staff in an italian geriatric health care facility, developed acute gastroenteritis from march 8th to march 21st 2017, in teramo province. a prompt epidemiological investigation was conducted to identify the etiological agent of the outbreak and the potential mode of transmission. the cases (n = 50) were investigated according to an epidemiological questionnaire. samples from all the cases (faeces) and highly transmissible environmental surfaces (swabs) were collected for analysis. among faecal samples, 34 out of 50 were positive for norovirus (nov) with no other pathogen detected. in particular, 2  (2/34) were positive only to nov genogroup i (gi), 31 (31/34) were positive only to nov genogroup ii (gii), and one sample (1/34) was positive to both nov gi and gii. moreover, people from the canteen were also tested and they resulted negative to nov detection in faeces. among the positive samples, 12 nov strains were subtyped as nov gii.4 sydney_2012 variant. person-to-person close contact and contaminated environmental surfaces were the probable transmission route among the people of the health care facility. the members of the staff were considered to play an important role in transmission of nov. a proper disinfection procedure applied during the outbreak could have been critically important to limit the dissemination of the viral infection. the role of staff and contaminated environmental surfaces in spreading of norovirus infection in a long-term health care facility in italy genotypes. of the 5  identified genogroups (gi to gv), only 3 have been shown to be pathogenic to humans; amongst them, gii.4 sydney_2012 is the most widespread genotype since the mid-1990s (ahlfeld et al. 2015, de graaf et al. 2015). nov is the leading cause of acute gastroenteritis in people of all ages worldwide. it is estimated to cause 12-24% of community-based or clinic-based cases of acute gastroenteritis, 11-17% of emergency room or hospital cases and approximately 70,000-200,000 human deaths of all ages, annually (bányai et  al. 2018). novs are highly contagious, and 10-100 viral particles may be sufficient to infect an individual (zarb et al. 2012). in immunocompetent people, the introduction norovirus (nov) is a single-stranded positive-rna virus of the family caliciviridae. being resistant to several stressors (e.g. high temperatures and desiccation), including disinfectants (atmar and estes 2006), noroviruses are able to persist in the environment for several days. however, its presence is strongly linked to human population density and inadequacy of wastewater treatments (bonadonna et  al. 2019, fusco et  al. 2019, purpari et al. 2019). novs are classified into ten genogroups (g) based on the variation of the major capsid protein (vp1), and are further divided into 49 312 spreading of norovirus infection in a long-term health care facility aprea et al. veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 environmental investigation an environmental investigation was carried out in order to collect information related to the layout of buildings, the disinfection procedures and staff organization within the premises. the ltcf was organised in 2 different buildings: the building a and the building b. the building a had 4 floors, each with 1 living room. in this structure, there were 76 single rooms and 12 double rooms, each with a toilette. the building a was reserved to partially or completely self-sufficient elderly people, generally with minor health problems, and able to leave the residence, independently. the second building (b), was divided into 2 floors, each with a refectory, infirmary and living room. the building b consisted of 5 units with a total of 42 double and triple rooms, equipped with bathrooms. it housed elderly people with serious health problems and senile pathologies, some of them with movement difficulties and the need of continuous assistance. inside the 2 buildings, each nursing assistant was in charge of about 25 residents and there was no precise distinction between staff from the building a and the building b. the most part of nursing assistants did not have systematic professional nursing training. the canteen was located at the first floor of the building a; a private company managed the cooking and distribution with its own staff. sample collection between the 8th and the 21st of march, 180 samples were collected. they include 58 faecal and 122 environmental surface samples. of the 58 faecal samples, 50 were individually collected from all cases (n  =  50), while 8 were from people of the canteen staff with no gastrointestinal symptoms. one-hundred-and-two samples were collected from highly touched environmental surfaces between the 10th and the 11th of march, to check the effectiveness of the ongoing cleaning/disinfection procedures. these surface samples were taken from all the different areas of buildings a and b. the protocol for cleaning/disinfection described in table i was applied from the end of day 16th of march 2017, which differed from the protocol commonly used before in the following parts: the usage of disposable cloth when changing surfaces; the application of sodium hypochlorite for 5  minutes at minimum. soon after the application of the new protocol, 20 further samples were taken (on the 17th of march) from the same previously positive surfaces, in order to verify the success of this new cleaning/disinfection procedure. all samples were transported to the laboratory disease is self-limited with recovery within 2-5 days while in those who are immunecompromised, it can cause severe dehydrating diarrhoea. the main clinical signs of viral gastroenteritis also include vomiting accompanied by nausea, abdominal cramps, and fever. there are a number of different routes through which nov transmission could occur. the main transmission mode is person to person through faecal-oral route. other possible way include contaminated food, water or surfaces (lin et  al. 2011, parrón et  al. 2020). several nov outbreaks have been recorded in cruise ships, hospitals and long term care facilities (ltcfs), where infections spread rapidly, have high attack rates and are difficult to control (centers for disease control and prevention 2003, hofmann et al. 2020). in particular, in ltcfs most residents are bedridden and elderly, and the nov spread is facilitated by enclosed living quarters and reduced levels of personal hygiene, because of faecal incontinence, immobility, dementia or need of assistance (yang et  al. 2010, ali et  al. 2014). in these settings, environmental surfaces and health care assistants can play a key role in the transmission of nov. from the 8th to the 21st of march 2017, a gastroenteritis outbreak occurred in a geriatric nursing facility in teramo province (abruzzo, italy) and involved 38 elderly patients, 8 health care workers, 2  nurses, 1 animator and 1 maintenance technician. the outbreak was investigated, the causative pathogen identified, the main routes of transmission hypothesized and the risk factors analysed. in particular, our investigation highlighted the role of health care workers and environmental surfaces in the spread of nov infection. moreover, a new protocol for cleaning and disinfection of the healthcare environments that helped to control the viral infection in the ltcfs has been presented. methods case definition and data collection the total number of residents in the ltcf was 177 people; 72 of these were health care assistants. in this study a norovirus case was defined as residents or staff in the geriatric nursing facility with at least 1 of the following symptoms: (1) diarrhoea (more than 3 times in a 24-hour period), (2) vomiting, (3) nausea, and (4) abdominal pain, occurring from the 8th to the 21st of march 2017. the investigation was based on an epidemiological questionnaire where information on the onset of symptoms, history of contact with infected persons and personal hygiene habits was collected. 313 aprea et al. spreading of norovirus infection in a long-term health care facility veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 using the primer sets cog1f/g1skr for gi and cog2f/ g2skr for gii. dna amplicons were purified by the qiaquick pcr purification kit (qiagen, milan, italy) or by the exosap (affimetrix, usa) enzyme and sequences were elaborated by eurofins (milan, italy). sequencing data were edited and aligned using mega6. the genotypes were assigned using the public database noronet typing tool (http://www.rivm.nl/mpf/ norovirus/typingtool). results detection of pathogens involved in the outbreak of the 180 samples tested in this survey, 62 (34.44%) [34 (54.83%) faecal samples and 28 (45.16%) surface swabs] were positive for nov. no other pathogens (intestinal bacteria and enteric viruses) were detected. genotype determination of nov viral rna from 12 faecal and 5 surface samples was successfully characterized (blastn and noronet typing tool database) as nov gii.4 sydney_2012 variant (sequence submitted to genbank database; accession numbers mn581063 mn581079). for the other positive samples it was not possible to identify the genotype. epidemiological investigation the epidemiological investigation started only from the 8th of march 2017, when it appeared clear to public authority that the spreading of the disease was worsening. all residents in this ltcf were old (average 84.64  years old) and nursing assistants provided most of their daily living care. from the 8th to the 21st of march, 34 out of 50  cases were confirmed to be infected with nov (25 residents, 7 social health operators, 1 nurse and 1 maintenance technician) (table  ii). in particular, 2 (2/34) were positive to nov gi, 31 (31/34) to nov gii and one sample (1/34) was positive to both nov  gi and gii. twelve nov gii positive samples were genotyped as nov gii.4 sydney_2012 strain (99% nucleotide identity with the reference gii.4 sydney_2012 strain accession number jx459908). all obtained sequences showed 100% nucleotide identity. faecal samples from 8 people of the canteen staff with no gastrointestinal symptoms were negative to nov. as being the total number of to detect escherichia coli (iso/ts 13136: 2012), salmonella  spp. (uni en iso 6579-1: 2017), shigella spp. (iso 21567: 2004), yersinia enterocolitica (iso 10273:2017), vibrio  spp. (iso 8914: 1990), rotavirus and adenovirus (van maarseveen et  al. 2010), nov and hepatitis a virus (iso 15216-2: 2013). sample processing and viral rna extraction for the viral concentration step, approximately 1 gram (gr) of each faecal sample was dissolved into 1 millilitre (ml) of phosphate buffered saline (pbs) at ph 7.2, added with gentamicin, penicillin, nystatin and streptomycin. in the case of environmental samples, each cotton swab from the surfaces was absorbed in 2 ml of pbs at ph 7.2, immediately after sampling. ten ± 0.1 microliters (µl) of mengovirus (process control virus, national reference laboratory for foodborne viruses, istituto superiore di sanità, rome, italy) were added to each sample at the final concentration as reported in iso 15216-2: 2013. after vortexing for 1 minute (min), faecal samples were clarified by centrifugation at 10,000 gravity (g) for 10 min at room temperature. rna was extracted from 500 µl suspension using the nuclisens minimag platform with the nuclisens magnetic extraction kit (biomeırieux, marcy-l'étoile, france) according to the manufacturer’s instruction. real time rt‑pcr detection of nov rna samples were amplified by real time reverse transcriptase polymerase chain reaction (rt-pcr) for nov gi and gii, as described by the iso 15216-2: 2013, using ultrasensetm one-step quantitative rt-pcr system kit (invitrogen, germany). sequence analysis of nov real time rt-pcr positive samples were also analysed by end-point rt-pcr, with primer sets g1skf/g1skr and g2skf/g2skr annealing to open reading frame (orf) 2 and specific for gi and gii, respectively (kojima et al. 2002). the rt-pcr was followed by a semi-nested pcr, table i. cleaning and disinfection procedure applied during the outbreak. step action 1 surface cleaning with a common detergent 2 sanitization with sodium hypochlorite 50,000 ppm (5%), left on surfaces for 5 minutes 3 removal with a disposable wet cloth 4 cleaning with disposable cloth moistened with alcohol 90° 314 spreading of norovirus infection in a long-term health care facility aprea et al. veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 table ii. details of confirmed human cases showing clinical manifestations of nov infection. id number date age role gender symptoms setting 1 08.03.2017 56 health care worker female vomiting and diarrhea 2 08.03.2017 52 health care worker female vomiting and diarrhea 3 08.03.2017 75 resident female vomiting and diarrhea building a, 2nd floor 4 08.03.2017 93 resident female vomiting, diarrhea, dehydration, sensory deprivation building a, 2nd floor 5 08.03.2017 90 resident male vomiting, diarrhea, dehydration, sensory deprivation building a, 1st floor 6 08.03.2017 78 resident male vomiting and diarrhea building a, 3rd floor 7 08.03.2017 66 health care worker female vomiting and diarrhea 8 08.03.2017 93 resident female vomiting, diarrhea, dehydration, sensory deprivation building a, 3rd floor 9 08.03.2017 89 resident male vomiting and diarrhea building a, 4th floor 10 08.03.2017 92 resident female vomiting, diarrhea, dehydration, sensory deprivation building a, 3rd floor 11 08.03.2017 94 resident male vomiting, diarrhea, dehydration, sensory deprivation building a, 2nd floor 12 08.03.2017 95 resident female vomiting, diarrhea, dehydration, sensory deprivation building a, 1st floor 13 08.03.2017 88 resident female vomiting, diarrhea, dehydration, sensory deprivation building a, 1st floor 14 11.03.2017 54 health care worker female vomiting and diarrhea 15 11.03.2017 92 resident male vomiting, diarrhea, dehydration, sensory deprivation building a, 4th floor 16 11.03.2017 85 resident male vomiting and diarrhea building a, 4th floor 17 13.03.2017 36 nurse female vomiting and diarrhea 18 13.03.2017 80 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 2 19 13.03.2017 87 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 2 20 14.03.2017 85 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 5 21 14.03.2017 90 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 5 22 14.03.2017 91 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 5 23 15.03.2017 87 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 1 24 15.03.2017 81 resident female vomiting and diarrhea building b, unit 2 25 15.03.2017 75 resident female vomiting and diarrhea building a, 1st floor 26 16.03.2017 43 health care worker female vomiting and diarrhea 27 16.03.2017 73 resident female vomiting and diarrhea building b, unit 1 28 20.03.2017 90 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 3 29 20.03.2017 63 health care worker female vomiting and diarrhea 30 20.03.2017 88 resident female vomiting and diarrhea building b, unit 4 31 20.03.2017 68 resident male vomiting and diarrhea building b, unit 4 32 20.03.2017 84 resident female vomiting, diarrhea, dehydration, sensory deprivation building b, unit 4 33 21.03.2017 30 health care worker female vomiting, diarrhea, dehydration, sensory deprivation 34 21.03.2017 24 maintenance technician male vomiting and diarrhea the outbreak involved only residents from building a (n = 12) and health care workers (n = 4) (table  ii). the first cases among the residents in building  b appeared on the 13th of march. nevertheless, the evidence of the nov presence in surface samples in building b was detected on march 10th and 11th (figure 1), demonstrating that the virus was already present in those premises before the onset of the first cases. in total, 13 residents from the building a and 12 from the building b were found infected with nov between march 8th and 21st, 2017. in the remaining 13/38 residents, though falling into case definitions (gastrointestinal symptoms), nov was not detected and so they were excluded from the nov cases. residents 177, and the total number of employees 72, the attack rate was respectively of 21.47% (38/177) and 13.89% (10/72). in addition, 28/122 environmental samples resulted positive to nov gii. the viral strains from the contaminated surfaces (5/28) were identified as nov gii.4 sydney_2012 (table iii). sequence alignment of the strains detected in human and environmental samples showed 100% nt identity. environmental investigation at the beginning (from the 8th to the 11th of march), 315 aprea et al. spreading of norovirus infection in a long-term health care facility veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 health care settings by visitors and staff who may be asymptomatic, pre-symptomatic or symptomatic. also, contaminated food can be the source of nov introduction in these settings and could be responsible for the beginning of outbreaks (rushton et al. 2019). a previous nov outbreak occurred in the same ltcf in 2009 (di giannatale et  al. 2013). as at that time, also in the present study no role was supposed to be played by food and canteen staff, since the canteen itself was located in a separate area of building a. no members of the staff fell within case definitions and the infections showed a progressive involvement of different areas of the two buildings. if the outbreak originated from contaminated food, a simultaneous involvement of all areas of both the ltcf buildings since the food prepared in the canteen was the same distributed in the whole facility, every day. in this outbreak, the source of nov and the identification of the first case were not defined, but the epidemiological investigations suggested that health care staff assistants have played a crucial role in the secondary spread of the infection, as frequently reported in literature (danzmann et  al. 2013, lai et al. 2013, di giannatale et al. 2013, ho et al. 2015, zheng et al. 2015, chong and atmar 2019). the detection of the same, unique nov strain, gii.4 sydney_2012 variant, during the whole period of surveillance, indicated that person-to-person transmission has significantly contributed in the spreading of the virus infection. the same virus was also detected in the environmental swabs, implying the occurrence of cross-contaminations, which may have also contributed to the spread of the infection. the outbreak started from building a and then progressively involved the building b, from march 13. in this scenario, the personnel of the ltcf provided continuous assistance to the residents, being in control measures after the application of the cleaning/disinfection procedure showed in table i, only 1 out of the 20  surface samples taken was still positive for nov gii to real time rt-pcr screening. nevertheless, the genome sequence was not achieved and subtype was not possible to be identified, due probably to the low amount of the virus particles. moreover, it was also suggested to reduce the staff movements among different units of the buildings as much as possible. other actions that were taken included cases isolation, health education on hand hygiene habits, more frequent cleaning and disinfection of bathrooms and toilets in the rooms. eight new cases were observed on march 20. five of them were due to nov gii. on the 21st of march, 2 further cases were confirmed. gii.4 subtype was identified as responsible for these cases. no more cases were observed from the 22nd of march and the outbreak was considered officially closed. discussion and conclusions nov is characterized by a low infectious dose and a strong stability in the environment (atmar and estes 2006). however, experimental studies estimated the 50% human infectious dose measured was similar to that of other rna viruses (atmar et al. 2014). this virus can be transmitted to the residents of table iii. list of environmental surfaces resulted positive to the detection of nov gii during the outbreak. environmental surfaces number of surfaces tested number of surfaces positive to nov gii (%) building a building b bed rails 10/122 6/10 (60) 3 3 (unit 5) elevator push-button panel 10/122 2/10 (20) 0 1 (unit 3) 1 (unit 2) cleaning trolley handles 12/122 2/12 (16,7) 1 1 (unit 3) handrails 6/122 1/6 (16,7) 0 1 (unit 4) laundry trolley clamps 6/122 1/6 (16,7) 0 1 (unit 3) bathroom taps 31/122 8/31 (25,8) 3 1 (unit 4) 1 (unit 3) 1 (unit 2) 1 (unit 1) 1 (unit 5) door handles 39/122 6/39 (15,4) 3 1 (unit 3) 2 (unit 5) sink pedals 5/122 1/5 (20) 0 1 (unit 3) radio buttons (from a shared radio in one of the living rooms) 3/122 1/3 (33,3) 0 1 (unit 5) n u m b er o f p o si ti ve s am p le s sampling dates 08 /0 3/ 20 17 09 /0 3/ 20 17 10 /0 3/ 20 17 11 /0 3/ 20 17 12 /0 3/ 20 17 13 /0 3/ 20 17 14 /0 3/ 20 17 15 /0 3/ 20 17 16 /0 3/ 20 17 17 /0 3/ 20 17 18 /0 3/ 20 17 19 /0 3/ 20 17 20 /0 3/ 20 17 21 /0 3/ 20 17 20 18 16 14 12 10 8 6 4 2 0 faeces environmental surfaces figure 1. positive samples (faeces and environmental swabs) collected and analysed during the outbreak period. 316 spreading of norovirus infection in a long-term health care facility aprea et al. veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 shortage of staff as well as the time-consuming activities for an effective disinfection, may have been the most critical factors that contributed to the spreading of nov infection. to date, no methods have been demonstrated efficacious for the inactivation of human nov. the development of real time rt-pcr protocols for nov rna detection improved the possibility to identify viral fragments in different matrices, but the inability to assess the viability of viral particles is still an important limit. murine nov is often used as human nov surrogate, but there are deep differences between the 2 viruses including the susceptibility to inactivation methods (cromeans et  al. 2014). there are some indications about the efficacy of environmental cleaning against nov using sodium hypochlorite at concentrations of 1,000-5,000  ppm (keswick et al. 1985, cdc 2011). nevertheless, some kind of faecal and soil may render 5,000  ppm of sodium hypochlorite not effective against infective particles and longer exposure time could be needed (barker et  al. 2004). for these reasons, in our study, we decided to use a more concentrated sodium hypochlorite solution (50,000 ppm), that was applied in the protocol described in table  i, and that probably helped in reducing the number of new cases up to the 19th of march. however, 8  new cases were identified on day march 20th, with 62.5% of confirmed diagnostic positivities to nov gii (5/8), and 2 more confirmed cases on day march 21st. nevertheless, the outbreak was considered officially closed the 22nd of march, with no more cases. in conclusion, in this work we reported a nov associated acute gastroenteritis outbreak that occurred in a long-term care facility in italy in 2017. nov gii.4 was the genotype associated with this outbreak. person-to-person close contact and contaminated environmental surfaces were the probable transmission routes, with health care assistants playing the key role in virus spreading. in particular, for nov outbreaks, the implementation of infection control measures is fundamental. from our point of view, an effective environmental cleaning and disinfection, accurate and frequent hand washing and limited circulation of the staff among different areas of the premises, are the most important actions to prevent viral infections spreading when residents from ltcfs are involved. these measures could be important also as general principals, in order to mitigate the burden related to the environmental nov contamination from infectious people (bonadonna et  al. 2019, fusco et al. 2019, purpari et al. 2019). acknowledgements we thank all the staff of the ltcf and, in particular, dr constant contact with them. however, the finding of nov on the surfaces of building b detected on the 10th and the 11th of march let us suggests that the virus was already present in these premises before the occurrence of the first cases. none of the residents were transferred from building a to b and no contacts among residents were recorded. health care staff was promiscuous between the buildings, and the contact with residents was continuous. from the results of this survey, it appears that health care assistants were responsible for spreading the infection not only by direct contact but alos by contaminating the surfaces of the health care facility (lin et  al. 2011). environmental surfaces not well disinfected have been demonstrated to be source of infection for residents and nursing assistants (wu et al. 2005). in our study, 28 out of 122 (22.95 %) surface samples were positive for nov gii (table iii). highly touched surfaces from the 2 buildings were chosen for sampling (swabbed). our results corroborated what is already reported in the literature regarding the most common surfaces involved in spreading the virus in close and semi closed settings (rico et  al. 2020) (table  iii). the sample from 1 bed rail continued to test positive for nov gii also after accurate cleaning and disinfection (1 sample over 20). this paper also reports for the first time the application of an iso method (iso 15216-2: 2013) for the detection of nov from environmental surfaces, while the laboratory methods used in the previously cited papers were developed in-house. in particular, we successfully applied the same part of the standard procedure that describes the detection of nov rna from food contact surfaces. no validation activities were carried out to verify the quality parameters in relation to this modification to the standard method adopted; therefore, more in-depth studies should be carried out in the future at this regard. although the source of nov was not clearly identified in this outbreak, nov gii.4 can indeed be considered as the cause of infection. this variant has been reported as the most frequent cause (70-80%) of nov associated gastroenteritis outbreaks worldwide since the mid-1990s (de graaf et al. 2015). apart from 3 faecal samples positive to nov gi, all the other samples collected in this outbreak were positive to nov gii. nov gii.4 was detected on all contaminated surfaces. the main control measures to be implemented during an outbreak include: quarantine of infected individuals, enhanced environmental decontamination and enhanced hand hygiene (arias et  al. 2013). however, the application of these procedures can be difficult for many reasons; in our case scenario, in particular, 317 aprea et al. spreading of norovirus infection in a long-term health care facility veterinaria italiana 2021, 57 (4), 311-318. doi: 10.12834/vetit.2479.15150.1 health with the ccm programme ccm 2018-2020 ‘sorveglianza delle infezioni gastroenteriche da norovirus e rotavirus associate all’assistenza sanitaria’. francesco sbraccia and dr sandra di domenico for the kind support given in this work. funding the study was partially funded by the ministry of ahlfeld b., li y., boulaaba a., binder a., schotte u., zimmermann j.l., morfill g. & klein g.2015. inactivation of a foodborne norovirus outbreak strain with nonthermal atmospheric pressure plasma. mbio, 6 (1), e02300-14. ali m.m., aburowes a.h., albakush a.m., rzeg m.m., alrtail a. & ghenghesh ks.2014. identification of multidrug-resistant bacteria and bacillus cereus from healthcare workers and environmental surfaces in a hospital. libyan j med, 9 (1), 25794. arias a., emmott e., vashist s. & goodfellow i. 2013. progress towards the prevention and treatment of norovirus infections. future microbiol, 8 (11), 1475-1487. atmar r.l. & estes m.k. 2006. the epidemiologic and clinical importance of 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norovirus outbreaks in closed and semi-closed settings. j hosp infect, 105 (1), 3-9. rushton s.p., sanderson r.a., reid w.d.k., shirley m.d.f., harris j.p., hunter p.r. & o'brien s.j. 2019. transmission routes of rare seasonal diseases: the case of norovirus infections. philos trans r soc lond b biol sci, 374 (1776), 20180267. wu h.m., fornek m., schwab k.j., chapin a.r., gibson k., schwab e., spencer c. & henning k. 2005. a norovirus outbreak at a long-term-care facility: the role of environmental surface contamination. infect control hosp epidemiol, 26 (10), 802-810. yang l.c., chiang p.c., huang t.h., chi s.f., chiu y.p., lin c.s., chou y.f., hsu s.c., zhang x.s., huang c.g., kao 315 veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 accepted: 07.03.2022 | available on line: 31.12.2022 1university manouba, institution de la recherche et de l'enseignement supérieur agricoles, service de microbiologie et d’immunologie, ecole nationale de médecine vétérinaire, 2020 sidi thabet, tunisie. 2university manouba, institution de la recherche et de l'enseignement supérieur agricoles, laboratoire de parasitologie, école nationale de médecine vétérinaire, 2020 sidi thabet, tunisie. *corresponding author at: service de microbiologie et d’immunologie, ecole nationale de médecine vétérinaire, 2020 sidi thabet, ariana, tunisie. tel.: +216 71 552 200, mobile: +216 97003060 , fax: +216 71 552 441, e‑mail: lilia_messadi@yahoo.fr. ghassan tayh1, asma ben haj yahia1, rachid selmi1, sarrah landolsi1, faten ben chehida1, aymen mamlouk1, mohamed habib jemli2, monia dâaloul-jedidi1 and lilia messadi1* keywords e. coli o157:h7, camels, shiga-like toxin genes, enterohemorrhagic escherichia coli, enterohaemolysin (ehxa), antimicrobial resistance. summary shiga-toxin-producing e. coli (stec) is a foodborne pathogen associated with outbreaks worldwide that can be identified in the feces and in the meat of food-producing animals. our study aimed to evaluate the incidence of e. coli o157:h7 in the feces of diarrheic camels (camelus dromedarius) in tunisia. from january 2018 to april 2019, 120 unduplicated fecal samples were obtained from diarrheic camels located in southern tunisia. non-sorbitol-fermenting colonies were confirmed as e. coli o157 via latex agglutination test and were screened for the presence of rfbeo157, flich7, stx1, stx2, eaea, and ehxa genes by pcr. all isolates were examined for their susceptibility to 21 antibiotics. of the 70 e. coli isolates that were recovered from 120 diarrheic camels, 4 (5.7%) were identified as stec o157:h7. all isolates harbored ehxa and eae genes. shiga toxin genes stx2 and stx1 were present in 50% and 25% of isolates, respectively. all e. coli o157:h7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole-trimethoprim. all isolates belonged to the phylogroup e. this is the first report of e. coli o157:h7 isolates from diarrheic camels in tunisia with a prevalence of 4 isolates (3.3%) amongst 120 fecal samples. this study supports the necessity for a platform purposed for regular screening and surveillance programs in food-producing animals and meat products, to perform early and rapid identification of food-borne pathogens. prevalence of escherichia coli o157:h7 isolated from fecal samples of diarrheic camels in tunisia (hus), and/or hospitalization (al-ajmi et  al. 2019, falup-pecurariu et al. 2019). these bacteria represent a significant public health concern and have the efficiency to produce shiga toxin type 1 (stx1) and shiga toxin type 2 (stx2), which are very potent toxins and are the main virulence determinants of this pathogen. importantly, stx2-producing strains cause more severe infections than stx1-producing strains (ogura et  al. 2015). in fact, purified stx2 is 1,000 times more toxic to human renal endothelial cells than stx1. the other main virulence factors produced by these serotypes are enterohaemolysin (ehxa) and intimin (eae) (sperandio and nguyen 2012). stec o157 is the most clinically important serogroup but some serogroups non-o157 are also clinically significant foodborne pathogens, including stec  o26, o45, o103, o111, o121, and o145 (al-ajmi et al. 2019, hegde et al. 2012). introduction escherichia coli is a gram-negative, rod-shaped bacterium, that normally colonizes the intestine of human and most animals and is considered an opportunistic pathogen (tayh et  al. 2016). some strains of e.  coli are capable to cause severe diseases in the human gut. they are recognized as enterohemorrhagic e.  coli (ehec) belonging to intestinal pathogenic (diarrheagenic) strains that cause gastroenteritis. these strains cause illness in humans by producing an effective toxin known as shiga toxin (meng et al. 2012). shiga-toxin-producing e.  coli (stec) still known as vtec (verotoxin-producing escherichia coli) brings together several serotypes like ehec pathogenic to humans, as food-borne bacteria linked with outbreaks worldwide. they can be associated with severe bloody diarrhea, hemolytic uremic syndrome 316 veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 e. coli o157:h7 in diarrheic camels tayh et al. bacterial isolation and identification fecal samples were enriched in buffered peptone water overnight at 37  °c, and then 10 µl were plated on macconkey agar for 18-24 hrs at 37  °c. the e.  coli isolates were identified by conventional biochemical tests. e.  coli isolates were stored at -  20  °c in brain heart infusion broth supplemented with 20% glycerol. identification of e. coli o157:h7 one isolated strain was streaked on a plate of sorbitol macconkey with cefixime tellurite (smac-ct) agar. the bacterial plate was incubated overnight at 37 °c. after incubation, all colonies which are not able to ferment sorbitol (colorless/white colonies) were selected as probably e. coli o157. all e. coli colonies of non-sorbitol-fermenting on smac-ct medium were tested for the presence of the o157 antigen by agglutination test (dryspot™ e.  coli o157 latex agglutination test, thermo fischer scientific). dna extraction of bacteria genome the extraction of the e.  coli genome was carried out by boiling method. bacterial colonies were suspended in 1 ml of sterile distilled water. after centrifugation at 13,000 rpm for 5 min, the supernatant was removed, replaced by 100 ml of sterile distilled water, heated at 95 °c for 10 minutes, and then kept at 20 °c to be used for amplification by polymerase chain reaction (pcr). the pcr was carried out in this study including positive (e.  coli o157:h7 isolated from cattle in our laboratory) and negative (dna-free water) controls. detection of o157 and flich7 genes by pcr pcr amplification was used to detect the rfbe and flich7 genes encoding for o157 and h7 antigens of e.  coli o157:h7 strains, respectively, using oligonucleotide primers listed in table i. dna amplification reactions were carried out using a dna thermal cycler (2720 thermal cycler, applied biosystem by life technologies, singapore) with the following program: one cycle of denaturation at 94 °c for 5 min; 35 cycles of denaturation at 94 °c for 45  sec, annealing for 45 sec at 52  °c and 60  °c for o157 for detecting rfbe and extension at 72  °c for 45  sec; and a final extension at 72  °c for 10 min for detecting flich7. the pcr amplification products were separated by gel electrophoresis with 1.5% agarose and visualized under ultraviolet (uv) light using ethidium bromide staining. according to the centers for disease control and prevention (cdc), the dissemination of the stec to humans might take place through contaminated food such as beef meat, fruits and vegetables, contaminated water, or via contact with contaminated animals or persons (cdc 2019). it is found in the intestines of healthy cattle, goats, and sheep which are considered natural reservoirs and feed may be contaminated with livestock manure (ferens and hovde 2011). according to el-gallas and colleagues (el-gallas et  al. 2006), in tunisia, 3.4% of isolates from human stool samples were e. coli o157:h7. however, the incidence reports of e.  coli o157:h7 in camels (camelus dromedaries) are rare. studies from the united arab emirates (uae) (moore et  al. 2002, al-ajmi et al. 2019), iran (rahimi 2012), kenya (baschera et al. 2019) and iraq (mohammed hamzah et  al. 2013) failed to isolate these bacteria among camel fecal samples. furthermore, the same failure to detect this pathogen was reported in african countries (egypt, somalia, djibouti, kenya, and sudan) (el-sayed et  al. 2008). however, in some countries, the prevalence of stec o157:h7 isolates among healthy camel feces, has been reported, as in uae was 4.3 (6/140) (al-ajmi et  al. 2020), saudi arabia (2.4% and 11.5%) (al humam 2016, bosilevac et  al. 2015) and iran (2%) (sami and adeli 2013), as well as from diarrheic camel feces in egypt (17.9%) (el-hewairy et al. 2009). no data are available on the prevalence of e.  coli o157:h7 among camels in tunisia. we selected diarrheic camels to determine whether e.  coli o157 is an agent of diarrhea as this has not been studied in tunisia and can potentially pose a risk to people in contact with diarrheic animals. therefore, this study aimed to determine the prevalence, virulence factors, and antimicrobial resistance profiles of stec o157:h7 among fecal samples of diarrheic camels. to the best of our knowledge, this is the first report of stec o157:h7 in camels in tunisia. materials and methods samples collection one hundred and twenty fecal samples were collected from diarrheic camels of southern tunisian regions (douz, tozeur, gabes and ben guerden) from january 2018 to april 2019. only one specimen per animal was included. these samples were transported appropriately to the microbiology laboratory at the national school of veterinary medicine of sidi thabet for bacterial isolation and further investigations. 317veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 tayh et al. e. coli o157:h7 in diarrheic camels antimicrobial sensitivity test the antimicrobial sensitivity was studied by the disk-diffusion method on mueller-hinton agar plates according to the guidelines and clinical breakpoints of the antibiogram committee of the french society (casfm-vétérinaire 2018) using twenty-one antibiotics discs belonging to eight classes comprising μg/disk (bio rad, france): twelve β-lactams [(amoxicillin  (25), amoxicillin/ clavulanic acid (20/10), ticarcillin/clavulanic acid (75/10), cefotaxime  (30), ceftazidime (30), cefepime (30), cefoxitin (30), aztreonam (30) ertapenem (10), piperacillin (30), cefalotine (30), cefuroxime (30)], and nine non-β-lactams antibiotics [chloramphenicol (30), gentamicin (15), nalidixic acid (30), enrofloxacin detection of virulence factors the presence of stx1, stx2, and ehxa virulence genes with uida gene was screened in e.  coli o157:h7 isolates by the multiplex pcr method and eae by the simplex method using primers listed in table  i. the pcr thermocycler apparatus was used to perform pcr reactions. the reaction consists of 25  cycles of dna denaturation for 1 min at 95  °c, primer annealing for 1 min at 56 °c, and first extension for 1 min at 72  °c. the last step is the final extension for 5 min at 72 °c. the separation of pcr bands was performed by electrophoresis using 2% agarose gel, the pcr products were watched under uv light by ethidium bromide stain. table i. primers for pcr amplification of e. coli o157:h7. pcr reaction gene primer sequence (5’-3’) size of pcr product (bp) annealing temperature (°c) reference phylogenetic genes quadruplex chua chua.1b: atggtaccggacgaaccaac 288 60 clermont et al. 2013 chua.2: tgccgccagtaccaaagaca yjaa yjaa.1b: caaacgtgaagtgtcaggag 211 60 clermont et al. 2013 yjaa.2b: aatgcgttcctcaacctgtg tspe4c2 tspe4c2.1b: cactattcgtaaggtcatcc 152 60 clermont et al. 2013 tspe4c2.2b: agtttatcgctgcgggtcgc arpa acek.f: aacgctattcgccagcttgc 400 60 clermont et al. 2013 acek.r: tctccccataccgtacgcta group e arpa arpagpe.f: gattccatcttgtcaaaatatgcc 301 57 clermont et al. 2013 arpagpe.r: gaaaagaaaaagaattcccaagag group c trpa trpagpc.1: agttttatgcccagtgcgag 219 59 clermont et al. 2013 trpagpc.2: tctgcgccggtcacgccc internal control trpa trpba.f: cggcgataaagacatcttcac 489 57 clermont et al. 2013 trpba.r: gcaacgcggcctggcggaag virulence factors shiga toxin type 1 stx1 f: cagttaatgtggtggcgaagg 348 bp 56 sjöling et al. 2015 r: caccagacaatgtaaccgctg shiga toxin type 2 stx2 f: atcctattcccgggagtttacg 584 bp 56 sjöling et al. 2015 r: gcgtcatcgtatacacaggagc enterohaemolysin ehxa f: gcatcatcaagcgtacgttcc 534 bp 56 grispoldi et al. 2017 r: aatgagccaagctggttaagct enteropathogenical attachment and effacement eae f: tgcggcacaacaggcggcga 629 pb 58 ranjbar et al. 2017 r: cggtcgccgcaccaggattc others part of o-antigen 157 o157 f: cggacatccatgtgatatgg 259 bp 52 paton and paton 1998 r: ttgcctatgtacagctaatcc encoding h7 flagellar antigens flich7 f: gcgctgtcgagttctatcgagc 625 bp 60 al-ajmi et al. 2020 r: caacggtgactttatcgccattcc beta-glucuronidase uida f: atcaccgtggtgacgcatgtcgc 486 bp 56 heininger et al. 1999 r: caccacgatgccatgttcatctgc 318 veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 e. coli o157:h7 in diarrheic camels tayh et al. biochemical tests as e. coli. testing of e. coli isolates on smac-ct and by agglutination test, revealed four isolates (5.71%) considered as presumptive e.  coli o157:h7 isolates. thus, 4 e. coli o157 were identified out of 120 camels (3.33%). the frequency of isolation in this study was different between males and females with statistical significance (p = 0.045) (table  ii). the difference between the rate of isolation according to the age of the animals was not statistically significant (p = 0.657) (table  ii). furthermore, there was no statistically significant difference between the isolation frequency of o157 according to animal’s origin (p = 0.231) (table ii). the confirmation of e.  coli o157 by specific genes showed that uida, o157 and flich7 were present in all isolates. the virulence genes eae and ehxa were present in the four isolates; stx1 and stx2 were present in one and two isolates, respectively (table iii). all isolates belong to the phylogroup e. all isolates were susceptible to cefoxitin, enrofloxacin, florfenicol, colistin, and sulfamethoxazole-trimethoprim. furthermore, 75% of the isolates were susceptible to piperacillin, cephalotin, cefotaxime, nalidixic acid and tetracycline. however, 50% of the isolates were (5), tetracycline (30), sulfamethoxazole/trimethoprim (1.25/23.75), streptomycin (10), florfenicol (30) and colispot]. detection of phylogenetic groups the phylogenetic groups (a, b1, b2 and d) were identified in o157 isolates by quadruplex pcr using chua, yjaa genes and the tspe4‑c2 fragment. the phylogroups c and e were detected by pcr using arpa and trpa genes. the primers and the method were previously described by clermont and colleagues (clermont et al. 2013). data analysis the data of the camels; age, gender and origin were analyzed with the frequency of e.  coli o157 by the statistical package for the social sciences (spss) version 26 software (ibm corporation, somers, ny). the comparison of data was performed using pearson's chi-square with a p  <  0.05 value of statistical significance. results a total of 70 non-duplicate isolates isolated from 120 diarrheic camel samples from four major cities in southern tunisia were identified by conventional table ii. prevalence of e. coli o157:h7 in tunisian camels according to gender, age groups and region. factors no. (%) samples no. e. coli o157 rate gender male 61 (50.8) 4 6.56% female 59 (49.2) 0 0 total 120 (100) 4 3.33% p-value = 0.045 age (years) < 1 70 (58.3) 2 1.67% 1-5 28 (23.3) 2 1.67% 6-10 6 (5.0) 0 0 ≥ 10 16 (13.4) 0 0 total 120 (100) 4 3.34% p = 0.657 origin (city) gabes 51 (42.5) 4 7.84% douz 34 (28.3) 0 0 tozeur 19 (15.8) 0 0 ben guerden 16 (13.4) 0 0 total 120 (100) 4 3.34% p = 0.231 table iii. characteristics of the 4 e. coli o157:h7 isolated in tunisian camels. bacterial code specific genes virulence genes o157 flich7 uida stx-1 stx-2 eae ehxa d5 + + + + + + d10 + + + + + + d25 + + + + + d42 + + + + + + figure 1. pcr for detection of virulence genes. lane m = ladder 100 bp; lane 1, 2, 3, 4 = positive samples; agarose gel concentration: 2%. 600 bp m 1 2 3 4 500 bp 400 bp stx2; 584 bp ehxa; 534 bp iuda; 486 bp stx1; 348 bp 319veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 tayh et al. e. coli o157:h7 in diarrheic camels interestingly, in iran, salehi and colleagues (salehi et  al. 2011) detected the serotype o157:h7 in two dromedaries with haemorrhagic diarrhea. the occurrence of e.  coli o157 was investigated among 70 e. coli isolated from feces of 120 diarrheic animals in the main southern tunisian cities. this is the first report of e. coli o157 isolation from camels in tunisia and also the first report in animals in tunisia. the incidence of e.  coli o157:h7 in camel’s fecal samples was 5.71%, with four isolates amongst 70 strains. the occurrence studies of this bacterium in camels are limited; a study conducted in the uae was unsuccessful in detecting e.  coli o157:h7 in fresh feces of racing camels (moore et  al. 2002). the same failure to identify e. coli o157 was among 400 camel fecal samples obtained from african countries (egypt, somalia, djibouti, kenya, and sudan) (el-sayed et al. 2008) and also from 40 camel samples in iran (rahimi 2012). more recently, in a study in kenya, none of 163 fecal samples of grazing dromedaries contained stec o157:h7 (baschera et al. 2019). an iraqi study was performed on a zoo animals in baghdad; they detected 24 e.  coli o157:h7 out of 174  fecal samples from bear, deer, pony, horses, zebra, ostrich, hyena, llama, goat, and jaguar. however, no e.  coli o157:h7 was identified among camels (mohammed hamzah et al. 2013). our methods used for the detection and isolation of o157:h7 are considered appropriate but non-selective, so the results might be an underestimate of the actual prevalence of o157:h7 among our samples. the iso 16654:2001 method is the standard for detection of e.  coli o157 but it requires separation and concentration using immunomagnetic beads coated (tozzoli et al. 2019). our findings were compatible with the results of a study in the uae (al-ajmi et  al. 2020) in which the prevalence of e.  coli o157 was 4.3%. they used an isolation method like the iso 16654 method. the demonstration of e.  coli o157 in camels was also reported in saudi arabia among 200 camel fecal samples in six isolates (11.5%) from 52 e. coli isolates (al humam 2016). in iran, they reported 2% of 150 healthy camel feces were e.  coli o157 (sami and adeli 2013). another report from iran demonstrated that one (1.3%) strain was e.  coli o157 out of 75 camel meat samples using the protocol of selection of sorbitol negative colonies and pcr amplification of the o-antigen encoding region of the o157 gene and flagellar h7 gene (flic) (hajian et al. 2011). a high prevalence (17.9%) was recorded among 60 egyptian camel calves; half of them suffered from diarrhea (el-hewairy et  al. 2009). in iraq, al-gburi (al-gburi 2016) detected e. coli o157:h7 with 19% in healthy camels by the same protocol as our study. resistant to cefuroxime, ticarcillin-clavulanic acid and streptomycin (table iv). discussion escherichia coli o157:h7 infections in humans originally came from food of animal or vegetable origin via food contamination with feces containing this pathogen. this particularly concerns ruminants such as cattle, sheep and goats which are considered the major natural reservoirs for this serovar and mainly implicated in human infections (jo et  al. 2004). due to the consumption of camel meat in tunisia, specifically in the southern part, and given the absence of studies on this subject, it was an established decision to study the prevalence of e.  coli o157:h7 in camels. the choice of diarrheic dromedaries is related to the lack of data on this pathology in tunisia and on the zoonotic risk of serotype o157:h7, in order to raise awareness of this risk among individuals responsible for the maintenance and care of dromedaries (breeders, veterinarians, butchers, slaughterhouse employees etc). to our knowledge, in tunisia, only one study has focused on e.  coli isolated from diarrhoeic camels but has not targeted stec (bessalah et  al. 2016). table iv. antimicrobial susceptibility of e. coli o157:h7 tunisian camel isolates. antimicrobial susceptible intermediate resistant ampicllin 25% 75% 0 piperacillin 75% 25% 0 cephalotin 75% 25% 0 cefuroxime 50% 0 50% cefoxitin 100% 0 0 cefotaxime 75% 25% 0 ceftazidime 25% 50% 25% cefepime 50% 50% 0 aztreonam 25% 75% 0 ertapenem 50% 50% 0 amoxicillin/ clavulanic acid 25% 75% 0 ticarcillin/ clavulanic acid 50% 0 50% gentamicin 50% 50% 0 streptomycin 25% 25% 50% nalidixic acid 75% 25% 0 enrofloxacin 100% 0 0 chloramphenicol 50% 50% 0 florfenicol 100% 0 0 colistin 100% 0 0 sulfamethoxazole/ trimethoprim 100% 0% 0% tetracycline 75% 0 25% 320 veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 e. coli o157:h7 in diarrheic camels tayh et al. important epidemiological marker for the rapid detection of stec strains (schwidder et  al. 2019). another important virulence factor is intimin protein (eae) which plays an important role in the attaching of host intestinal mucosa within the colonization and causes severe infections (sperandio and nguyen 2012). in the present study, the stec o157:h7 was confirmed by the identification of specific genes rfb‑o157 and flich‑7 using pcr as well as assessment of shiga toxins genes (stx1, stx2) and enterohaemolysin (ehxa). our finding revealed that eae and ehxa genes were present in all isolates; stx2 and stx1 were present in two and one isolates, respectively. in a recent study, out of the 12 e.  coli o157 isolates from fecal samples of healthy camels, stx2 was found in all isolates, eae in 11 (91.7%), hlya in 11 samples (75%) and stx1 was absent in all isolates (al-ajmi et al. 2020). in a study conducted in saudi arabia, they found one isolate carrying both shiga toxins genes (stx1 and stx2), and like to our results, all 6  isolates of e. coli o157:h7 possessed the eae gene (bosilevac et  al. 2015). rahimi and colleagues (rahimi et  al. 2012) confirmed 2.0% of 50 camel samples as e. coli o157:h7 but they failed to identify any virulence gene (stx1, stx2, hlya and eae). antimicrobials have saved millions of human lives and their use has made a significant contribution to the improvement of the health of animals and humans. antimicrobial resistance development is known as a global health threat. antimicrobial agents are used in animals intended for human consumption to make animals healthier, reducing diseases and mortality (oliver et  al. 2011). the increased quantity of antibiotic residues in food-producing animals participates in the increase of antimicrobial resistance problems. misuse and overuse of antimicrobials constitute a serious health enigma in many countries, such as using them for growth progression and infection prevention; furthermore, in several regions, the antibiotic quantities used in animals are four times larger than the quantities used in humans (hudson et al. 2017). the results of this study revealed that all isolates were susceptible to most tested antibiotics except cefuroxime, ticarcillin/clavulanic acid and streptomycin with resistance detected in 50% of the isolates and some isolates were intermediate to antibiotics which might progress to resistance. the results of an iraqi study showed resistance of e.  coli o157:h7 isolates to all the antibiotics tested in the study (doxycycline, cephalexin, erythromycin, clarithromycin, ceftriaxone, ampicillin, cloxacillin, rifampin and carbenicillin) except to trimethoprim (al-gburi 2016). a report in iran revealed the resistance of e. coli o157:h7 to most tested antibiotics (ampicillin, erythromycin, gentamycin, nalidixic acid, many reports confirmed that the prevalence of e.  coli o157 isolated from camel feces was lower than that of those reported in cattle (bosilevac et al. 2015, sami and adeli 2013) suggesting that camels cannot be considered the major natural reservoir for this pathogen in comparison with cattle. considering the low number of e.  coli o157:h7 in tunisia and few positive reports on camels in other studies, it can be concluded that the camel is not a significant source of stec o157:h7 infection; practically, the studied camels share environments or pastures or pens with cattle, goats, and sheep. these ruminants can therefore be the source of stec contamination of dromedaries. surveillance and screening programs on camel products remain necessary for the prevention of outbreaks, particularly for food-borne infections, especially if those camels share spaces with ruminants. the frequency of e.  coli o157 was greater in males than in females with statistical significance, while there was no significant difference according to age or region. our results correlate with another study in saudi arabia which reported that the prevalence of e.  coli o157:h7 was significantly greater in young males than among females (al-ajmi et  al. 2020). on the contrary, baschera and colleagues (baschera et al. 2019) reported from a kenyan study a prevalence of 14.5% (17/117) of female camels and 6.7% (3/45) of male camels. another study has found no statistically significant difference according to gender and age among camel samples (al-ajmi et al. 2020, al-gburi 2016). in the present study, no statistically significant relationship was found between e.  coli o157:h7 prevalence and the camel’s origin. all e. coli o157:h7 were isolated from animals sampled in gabes and these results highlight the high risk of the spread of this pathogen among animals from that region. moreover, this finding strongly encourages us to carry out investigations on a larger scale of camels from gabes and to try to find possible particularities of the camel farms of this region, and further investigations are necessary to study e. coli o157:h7 portage in cattle, sheep and goats from gabes. serotype o157:h7 might carry stx1 and stx2 genes encoding shiga toxins. strains that possess stx2 are associated with human illness more frequently than strains carrying stx1 (bosilevac et  al. 2015). both factors play important roles in the progress of hemolytic uremic syndrome. epidemiological evidence of stec o157 has revealed that strains carrying stx2 are associated with more severe human infections than strains carrying stx1 (manning et  al. 2008). furthermore, stec possesses hemolysin encoded by plasmid-carried enterohaemolysin gene (ehxa) which is associated with diarrheal disease and hus (fu et  al. 2018). this factor is an 321veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 tayh et al. e. coli o157:h7 in diarrheic camels the region suggest that camels might be considered as the natural reservoir and play an important role in the infection of humans with this pathogen. our results supply some baseline data concerning the occurrence of e.  coli o157:h7 in camels and their antimicrobial resistance that could be used in future studies. this work leads to highly recommended surveillance and screening programs on camel products to prevent outbreaks. furthermore, it supports the necessity for a platform purposed to regular screening and surveillance programs in food-producing animals and meat products, to allow early and rapid identification of food-borne pathogens. more studies should be performed to characterize the prevalence of stec o157 and other non-o157 pathogens in animals and meat food products with a large number of isolates. funding this work was supported by the research project peer 7-349 funded by the usaid “monitoring of antimicrobial resistance of bacteria for a better health of animals in tunisia”. doxycycline, streptomycin, kanamycin, tetracycline, chloramphenicol and amoxicillin) and susceptible to cefuroxime (tanzifi et al. 2015). in a uae study, e. coli o157 isolated from the fecal samples of camels were 100% susceptible to cefotaxime, chloramphenicol, ciprofloxacin, norfloxacin, and polymyxin b (al-ajmi et al. 2020). the results of our study showed that the e.  coli o157 isolates belonged to the phylogroup  e, which is in agreement with other studies (coura et al. 2015, tenaillon et al. 2010). antibiotic resistance in these isolates can be used as an indicator of antibiotic use in the animals or their environment. these resistances can be useful for studying the isolates, but o157:h7 infections in humans are not treated with antibiotics as this can induce expression of shiga toxins and worsen the disease. conclusions this is the first report of e. coli o157:h7 isolated from diarrheic camels in tunisia. the presence of this pathogen in our study and some positive reports in 322 veterinaria italiana 2022, 58 (3), 315-323. doi: 10.12834/vetit.2555.16997.2 e. coli o157:h7 in diarrheic camels tayh et al. al-ajmi d., rahman s. & banu s. 2019. complete genome sequence of escherichia coli o157: h7 strain al ain, isolated from camel feces in the united arab emirates. microbiol res announc, 8, e01171-19. al-ajmi d., rahman s. & banu s. 2020. occurrence, virulence genes, and antimicrobial profiles of escherichia coli o157 isolated from ruminants slaughtered in al ain, united arab emirates. bmc microbiol, 20, 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10.12834/vetit.2390.14842.1 accepted: 15.04.2021 | available on line: 31.12.2021 1department of veterinary medicine, university of nigeria nsukka, enugu state, nigeria. 2istituto zooprofilattico sperimentale della sicilia ‘a. mirri’, palermo, italy. 3department of animal health, federal college of animal health and production technology, national veterinary research institute vom, plateau state, nigeria. 4transboundary animal disease laboratory, agricultural research council-onderstepoort veterinary institute, onderstepoort, south africa. 5applied biotechnology division, national veterinary research institute vom, plateau state, nigeria. 6department of veterinary pathology and microbiology, university of nigeria nsukka, enugu state, nigeria. *corresponding author at: istituto zooprofilattico sperimentale della sicilia ‘a.mirri’, palermo, italy. e-mail: dottoremira@gmail.com. ijeoma chekwube chukwudi1, francesco mira2*, kenneth ikejiofor ogbu3, refiloe petunia malesa4, giuseppa purpari2, pam dachung luka5, emmanuel ikenna ugochukwu1, livio edward heath4, annalisa guercio2 and kennedy foinkfu chah6 keywords genetic characterization, goat, lineage ii, lineage iv, nigeria, peste-des-petitruminants virus, sheep, small ruminant morbillivirus. summary peste des petits ruminants virus (pprv) is the aetiologic agent of peste des petits ruminants (ppr), an important viral disease of sheep and goats. ppr is endemic in nigeria and leads to social and economic losses. the objective of this study was to determine the prevalence of ppr infection and genetically characterize pprv strains obtained from sheep and goats in three states of southeast nigeria. a total of 285 nasal swab samples collected in 2017-2018 were processed for pprv genome detection by rt-pcr. sixty-five (22.81%) of the samples were positive for pprv. sequence and phylogenetic analyses revealed that the pprv belonged to lineages ii (11/38, 28.9%) and iv (27/38, 71.1%). the n gene fragment sequence showed a 99.77%-100% and 99.98%-100% identity among the strains of lineages ii and iv, respectively. fourteen amino acid substitutions, previously unreported in pprv strains from nigeria, were recorded. this study confirms the circulation of pprv lineages ii and iv in southeast nigeria, the dominance of strains belonging to lineage iv in recent years, and their close genetic relationship with those previously reported in other parts of nigeria and neighboring countries. peste des petits ruminants in nigeria: identification and variations of lineage ii and lineage iv in sheep and goats spread and has been reported in several countries across african, the near and middle east, asia and, more recently, europe (parida et  al. 2015, donduashvili et al. 2018). ppr is caused by peste des petits ruminants virus (pprv ), taxonomically classified by the international committee on taxonomy of viruses (ictv ) as specie small ruminant morbillivirus (srmv ) (amarasinghe et  al. 2019), a member of the morbillivirus genus, belonging to the family paramyxoviridae, subfamily orthoparamyxovirinae (gibbs et al. 1979). pprv is a single-stranded negative-sense rna virus, whose genome consists of approximately 15-16 kb in size, encoding for six structural proteins, namely introduction peste des petits ruminants (ppr) is a transboundary viral disease of small ruminants, whose clinical signs are characterized by pneumonia, ocular and nasal discharges, conjunctivitis, necrotic stomatitis and diarrhoea (zahur et  al. 2011, gibbs et  al. 1979). the disease associated with high morbidity (100%) and mortality (up to 90%), particularly among naïve populations of sheep and goats, might cause severe social and economic losses amongst rural households/small ruminant farmers (abu elzein et al. 1990). following its first report in côte d'ivoire (gargadennec and lalanne 1942), the disease has 190 veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 pprv in southeast nigeria chukwudi et al. institute (ovi), south africa, for processing, analysis, and sequencing. rna extraction and rt‑pcr sterile phosphate-buffered saline (pbs) (500 µl) was added into each tube containing the swab sample. this was centrifuged at 10,000 x g for 3-5  minutes (+  4  °c) and supernatants (swab extract) was decanted into a sterile tube. viral rna was extracted from 140 µl of swab homogenate using the commercial viral rna kit, qiaamp viral rna mini kit (qiagen, hilden, germany), according to the manufacturer’s instruction. presence of pprv rna was evaluated using the forward primer np3 and reverse primer np4 amplifying a 351-bp fragment of the nucleocapsid (n) gene (couacy-hymann et al. 2002). reverse transcription-polymerase chain reaction was carried out in 25 µl volume reaction mix using the qiagen® onestep ahead rt-pcr kit (qiagen) consisting of 10 µl of 2.5x onestep ahead rt-pcr master mix, 1 µl of 25x onestep ahead rt-mix, 1.5 µl of 10 pmol (each) of forward and reverse primers, 5 µl of 5x q solution, 3.8 µl dh 2 o, 0.2  µl of 125x onestep ahead master mix tracer and 2 µl of template rna. aliquot of molecular grade water was used as the negative control while the positive control consisted of an established positive sample (a clone of the live-attenuated pprv vaccine derived from the nigeria/75/1 strain, grown on vero cell, and imported from the pirbright institute, pirbright, united kingdom). amplification was conducted under the following thermal conditions: 50 °c for 10 min to activate the reverse transcriptases, 95 °c for 5 min to activate the dna polymerases followed by 40 cycles of 95 °c for 10 sec, 55°c for 10 sec, 72  °c for 10 sec and a final extension of 72  °c for 2 min. each amplicon (5 µl) nucleocapsid (n), phosphoprotein (p), matrix (m), fusion (f), haemagglutinin (h) and large (l), and two nonstructural (v and c) proteins (bailey et al. 2005). although pprv has only one serotype, pprv isolates are genetically grouped into four distinct lineages (i, ii, iii and iv), based on sequence analysis of the fusion (f), nucleocapsid (n) or haemagglutinin (h) protein genes (forsyth and barrett 1995, dhar et al. 2002, couacy-hymann et al. 2002, kumar et  al. 2014). lineage i, which was circulating in west african countries in 1988-1994 (kwiatek et  al. 2007), has not been identified at molecular level in africa since 2001 (tounkara et  al. 2018). lineage ii has been identified in central and western africa; lineage iii in eastern africa and southern part of the middle east while lineage iv circulates across the middle east, including israel and turkey, part of asian continent and northern, western, central and eastern africa (kwiatek et al. 2007, banyard et al. 2010, munir et al. 2013, cosseddu et al. 2013, parida et  al. 2015, mantip et  al. 2016, woma et  al. 2016, baazizi et al. 2017). lineages iii and iv co-circulation in the united arab emirates and qatar (kwiatek et al. 2007, libeau et al. 2014) has been reported. because of these previous reports, it has become necessary to provide further epidemiological and genetic data on the spread of pprv among sheep and goats in nigeria. this study was therefore conducted to determine the prevalence of pprv infection and genetic characteristics of pprv strains circulating in south-east nigeria. materials and methods samples collection a multistage sampling method was performed with three hierarchical stages and the size of the sample used in this study was estimated using the formula according to thrusfield (thrusfield 2005), as described by chukwudi and colleagues (chukwudi et al. 2020). nasal swabs were collected within a period of seven months (from december 2017 to june 2018) from 285 randomly selected sheep (n =  172) and goats (n = 113) at local markets, households, abattoirs and veterinary clinics in three randomly selected states: anambra, ebonyi and enugu (latitudes 7°07’n and 3°90’n and longitudes 6°51’e and 8°30’e), located in south-east nigeria (figure 1). the animals had no history of vaccination and the sick animals showed one or a combination of the following clinical signs: nasal and ocular discharges, coughing, diarrhoea, ulcerative stomatitis, weakness, emaciation (supplementary table). the samples were properly packaged in dry ice and sent to the transboundary animal disease laboratory, agricultural research council (arc) ondersteport veterinary research figure 1. map of nigeria showing the three states sampled (marked with gray background). 191veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 chukwudi et al. pprv in southeast nigeria results detection of pprv viral genome by rt‑pcr sixty-five (22.81%) of the 285 nasal swab samples were positive for pprv by rt‐pcr assay. the highest infection rate of 27.73% was obtained from enugu state while 21.73% and 16.21% were recorded for anambra and ebonyi states, respectively. infection rate was higher in sheep (23.26%) than in goats (22.12%) while more females (24.5%) than males (20.8%) tested positive for pprv. based on breed distribution, uda, yankass and west african dwarf breeds of sheep had infection prevalence of 33.3%, 13.3% and 23.5%, respectively, while red sokoto and west african dwarf breeds of goats had 25% and 20.8%, respectively. each analysed parameter is presented in table i. there were no significant associations (p > 0.05) between the infection prevalence and state/location, species, sex or breed of animals sampled. based on the month of sampling, the highest infection prevalence (68.8%) was obtained in march while the least (5.6%) was obtained in june. there was a significant association (p < 0.05) between the infection prevalence and the months of sampling (figure 2). sequence analysis among the 65 samples that tested positive for pprv by rt-pcr, 38 good quality sequences (303  nts was analysed by electrophoresis at 120 volt/80 ma for 45 min on a 1.5% agarose gel stained with ethidium bromide, examined and photographed under ultraviolet light using a gel documentation system (biorad gel doctm xrt model no. universal hoodii, biorad, usa). sequencing amplicons were purified with the commercial kit qiaquick® gel extraction kit (qiagen), according to manufacturer’s instruction. the concentration and purity of the dnas were quantified using spectrophotometer (nanodrop nd-1000, peqlab biotech gmbh), according to manufacturer’s protocol. amplicon that had more than 20 ng dna load were stored at -  20  °c and then submitted to inqaba biotech™ (pretoria, south africa) for direct sanger sequencing in both forward and reverse directions with primer pair np3/np4. sequences were viewed and reversed using chromas ver 2.6.4 (technelysium pty ltd, south brisbane, australia) and low-quality sequences were not included in the further analyses. sequences were then assembled and analysed using bioedit ver 7.2.5 software and submitted to the nblast program (https://blast.ncbi. nlm.nih.gov/blast.cgi) to search related sequences in public domain databases. these sequence data have been submitted to the ddbj/embl/genbank databases under accession number as presented in supplementary table. phylogenetic analysis to elucidate the genetic relationships of the analysed pprv strains, a phylogenetic tree based on the partial n gene sequences was constructed with the mega x software. the model selection was performed using the best-fit model of nucleotide substitution with mega x software. the phylogenetic tree was constructed using the maximum-likelihood method, according to the kimura 2-parameter (k2) model with invariant sites (i) and bootstrap analysis with 1,000 replicates. statistical analysis the infection prevalence (total positive/total sample) was calculated. pearson’s chi-squared test was used to determine if there were associations in the number of positive and negative test results within each variable (area/location, species, sex, breeds, and months of sampling). significance was accepted at p < 0.05. table i. presence of the pestes des petit ruminants virus in sheep and goats in south-east nigeria according to state, species, breed and sex. state no. positive no. negative total samples enugu 33 86 119 anambra 20 72 92 ebonyi 12 62 74 total 65 220 285 species no. positive no. negative total samples sheep 40 132 172 goat 25 88 113 total 65 220 285 breed no. positive no. negative total samples uda 4 8 12 yankassa 2 13 15 wad sheep 34 111 145 red sokoto 9 27 36 wad goat 16 61 77 total 65 220 285 sex no. positive no. negative total samples male 27 103 130 female 38 117 155 total 65 220 285 192 veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 pprv in southeast nigeria chukwudi et al. n gene fragments, by excluding the primers sequences; sequences ranged from nucleotide 1205 to 1507 of the n gene sequence acc. no. kr828813) were obtained by sanger sequencing. based on the amino acid (aa) residues, the 38 sequences clustered into lineage ii (11/38, 28.9%) and iv (27/38, 71.1%). the n gene fragment showed 99.77%-100% and 99.98%-100% nt identities among the strains of the lineages ii and iv, respectively. when compared with the reference strain ngkw2012-msln (acc. no. kr828814), a number of amino acid substitutions previously reported among nigerian pprv strains belonging to lineage ii (mantip et al. 2016, woma et al. 2016), were also found among the 11 strains in this study. however, two of these strains varied from previous lineage  ii strains from nigeria by additional substitutions iv and pa at table ii. amino acid changes of pprv lineage ii strains collected in nigeria. strain/isolate id. acc. no. year amino acid residue 403 424 425 435 442 451 456 459 469 473 477 482 ngkw2012-mslnb kr828814 2012 e i g k i i p p l p v g n67-127-gym31kd-oyob kj124767 2011 d t d r i t s p p q l s vaccine nigeria/75/1b ky628761 1975 d t d r i t s p p q l s ngen_038 ca 2018 mt038903 2018 d t d r i t s p p q l s ngen_228 ov 2018 mt038904 2018 d t d r i t s a p q l s ngen_003 ov 2017 mt038905 2017 d t d r v t s p p q l s otherc reference 2010-2013 e/d i/t g/d k/r i i/t p/s p l p v g/s breference strain; cother previous pprv (peste des petit ruminants virus) sequences from nigeria (mantip et al. 2016, woma et al. 2016); bolded are amino acid changes reported for the first time among all the pprv sequences collected in nigeria in fe ct io n p re va le n ce 0 dec jan feb mar apr may jun 10 20 30 40 50 60 70 80 months pearson chi square value, df 32.571a, 6 p value 0.00 figure 2. trend of ppr virus infection prevalence in small ruminants in south-east nigeria. table iii. amino acid changes of pprv lineage iv strains collected in nigeria. strain/isolate id. acc. no. year amino acid residue 415 423 429 432 434 437 442 443 454 462 470 472 478 483 ngyo2013-2162d kr828813 2013 a r s p t g i s t e e m s q pprv/cameroon/ cmrs6/2017d mh447983 2017 a k s p t g i p t e e t s q srmv/niger/12/2017d mk673131 2017 k l p t g i p t e e t s q nig12:im21d kf479428 2012 a k s p t g i p t e e t s q nig12:tr10d kf479417 2012 a k s p t g i s t e e m s q ngan_150 ca 2018 mn271590 2018 a k s p t g i p t e e t s q ngan_084 ov 2018 mn271599 2018 a k s p a g i p t e e t s q ngen_137 ov 2018 mn271587 2018 a k s p t g i p a e e t s q ngen_255 ov 2018 mn271601 2018 a k s p t e i p t e e t p q ngan_217 ov 2018 mn271594 2018 a k s p t g i p t e e t s k ngen_146 ca 2018 mn271593 2018 a k s p t g t s t e d m s q ngan_087 ov 2018 mt038906 2018 a k s p t g t s t k d m s q ngan_156 ca 2018 mn271592 2018 v k l l t g i p t e e t s q othere reference 2010-2013 a/t r/k s p t/i g/e i s/p t/p e e m/t s/l q dreference strain; eother previous pprv (peste des petit ruminants virus) sequences from nigeria (mantip et al. 2016, woma et al. 2016); bolded are amino acid changes reported for the first time among all the pprv sequences collected in nigeria 193veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 chukwudi et al. pprv in southeast nigeria figure 3. unrooted maximum‐likelihood tree based on 59 partial n gene sequences of peste des petits ruminants virus strains (bootstrap 1,000 replicates; bootstrap values greater than 65 are shown). black squares () and black dots () markings indicate pprv strains of lineage ii and iv, respectively, analysed in this study. each sequence is indicated with strain/isolate name, country and year of collection, and accession number. clustered with lineage ii were 99% related to nigeria 75/1 vaccine strain and all were obtained from enugu state. discussion peste des petits ruminants is a transboundary viral disease of small ruminants, associated with high morbidity and mortality rates, leading to social and economic losses amongst rural households/small ruminant farmers (abu-elzein et al. 1990). because of the dramatic economic consequences it may cause, particularly in developing countries, the food and agriculture organization of the united nations (fao) and the world organisation for animal health (oie) supported a campaign for global eradication of ppr (https://www.oie.int/eng/ppr2015/background. html http://www.fao.org/ag/againfo/programmes/ en/empres/news_271014.html). positions 442 and 459, respectively (table ii). when compared with pprv sequences previously analysed and available in public domain databases, the n gene sequences of the analysed lineage ii strains showed the highest nt identities (99.00%-100%) with the vaccine strain 75/1, collected in 1975 in nigeria (acc.  no. dq840160 and ky628761), and with the strain n67-127-gym31kd-oyo, collected in 2011 in oyo state (nigeria) (acc. no. kj124767, woma et al. 2016). a number of amino acid substitutions were equally observed among the sequences of the 27 pprv strains belonging to lineage iv. based on the deduced amino acid profiles, the strains were assigned into eight haplotypes or groups. group  1 (n  =  2; strain  no.  9,  53) which consisted of strains with amino acid profiles similar to those of reference strains (acc.  no. mh447983 and kf479428) or those previously reported in nigeria (mantip et  al. 2016, woma et  al. 2016) while groups 2 (n  =  5; strain no. 3, 19, 20, 48, 49), 3 (n = 2; strain no. 46, 180), 4  (n  =  1; strain  no.  55) and 5 (n = 4; strain no. 12, 14, 16, 52) consisted of strains with single amino acid substitutions t434a, t454a, s478p and q483k, respectively. amino acid profiles of the representative of each of the eight haplotypes are shown in table iii. the amino acid changes recorded for the seven haplotypes (groups  2-8) have not been reported in pprv strains from nigeria. sequences clustered in haplotypes 1, 3, 4 and 8 showed 99.33%-97.35% nt identities with the strain pprv/cameroon/ cmrs6/2017 (acc.  no. mh447983), collected in cameroon in june 2017, and 99.21%-97.63% with the isolate srmv/niger/12/2017 (acc. no. mk673131), collected in niger in january 2017. while sequences clustered in haplotypes 2, 5 and 6, 7 showed 99.66% and 99.34%-98.01% nt identities with the strain nig12:im21 and with the strain nig12:tr10 (acc. no. kf479428 and kf479417, woma et al. 2016), collected in nigeria in 2012, respectively. phylogenetic analysis the n gene nucleotide sequence of the pprv strain representing each of the haplotypes was used for phylogenetic analysis. a phylogenetic tree constructed based on the n gene sequences of the representative pprv strains in our study and other pprv viruses representing the four lineages is shown in figure 3. the pprv strains obtained in this study clustered with the pprv strains of the lineage ii (11 strains) and iv (27 strains). the 27 pprv strains that clustered with the asian lineage iv were collected from all the states sampled (enugu, anambra, and ebonyi). these strains were 76%-85% related to pprv isolates from imo, anambra, plateau, taraba and ondo states of nigeria and isolates from cameroon and niger. the 11 pprv strains that 194 veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 pprv in southeast nigeria chukwudi et al. genetic characterization of the circulating pprv strains has largely been based on the comparison of partial or complete n or, less frequently, f gene sequences. according to the sequence analysis, pprv strains could be clustered in four different lineages (i-iv). due to the divergence in the n gene, the analysis of this gene highlights the relationship of the circulating strains more clearly (kwiatek et al. 2011). woma and colleagues (woma et  al. 2016) and mantip and colleagues (mantip et al. 2016) first described the spreading of the lineage iv in the years 2010 to 2013 in nigeria, where the lineage ii was, for a long time, the prevalent genetic type. in the present study both lineages ii and iv were detected in samples collected from sheep and goats in south-east nigeria, with lineage iv being the dominant genotype. thus, this study further confirms the wide circulation in nigeria of ppr viruses belonging to the asian lineage iv. this study allowed to depict the prevalence of the lineages and their genetic features in selected areas. mantip and colleagues (mantip et al. 2016) obtained negative results from the samples collected in enugu state while woma and colleagues (woma et al. 2016) obtained only two positive samples from anambra state, both belonging to the lineage  iv. genetically, the pprv strains in this study had a close relationship with previous strains collected in other states of nigeria and cameroon. however, amino acid changes reported for the first time were found in strains belonging to both lineage ii and lineage iv. lack of recent further genetic analyses in nigeria or neighboring countries makes it difficult to ascertain if these changes are due to the recent introduction of different but closely related strains from other territories or if they represent potential local adaptative genetic changes. the genetic close connection of the most prevalent haplotype of the lineage iv with pprv strains collected in the same years in neighboring cameroon and niger suggests the potential introduction of the related strains from other countries. indeed, in the phylogenetic tree depicted in figure 3, the lineage iv viruses described in this study cluster in a sub-clade together with strains collected in cameroon in 2017 (unpublished) and niger in 2015/2017 (souley et al. 2019). this close genetic relationship between pprv strains from neighboring countries (nigeria, niger, cameroon) suggests a common origin, probably connected to the transboundary movements of animals due to the absence of natural barriers or the commercial routes of animal trade (tounkara et  al. 2017, souley et  al. 2019). the varying amino acid changes observed among the lineage iv strains in this study indicate the strains circulating in nigeria are genetically diverse and reflect the geographical differences between the different parts of africa (dundon et al. 2020). although ppr was first described in nigeria in 1975 (taylor and abegunde 1979), it is only in the last decade that molecular analyses on pprv strains circulating in nigeria were provided. these studies, based on the n gene (mantip et  al. 2016, woma et al. 2016) or the f gene (luka et al. 2011) sequence analyses, characterized strains collected from 2007 to 2013 in many but not all states of nigeria, highlighting the circulation of the lineage ii and, since 2010, of the lineage iv of pprv (mantip et  al. 2016, woma et al. 2016). in the present study the molecular characteristics of pprv strains circulating in nigeria in 2017-2018 were determined and compared with those previuosly collected in nigeria and other countries. the analysis was based on the n gene sequence of pprv strains collected from states of nigeria not included in previous studies (ebonyi state) or with none or few tested positive samples (anambra and enugu states). pprv was detected by rt-pcr in 65 (22.8%) of the 285 animals tested, confirming the current circulation of pprv in south-east nigeria (anambra, ebonyi and enugu states). different infection rates from clinical samples (9.7%, mantip et al. 2016; 42%, woma et  al. 2016; 51.52%, luka et  al. 2016) were reported in previous studies conducted in other states of nigeria. the differences in detection rates recorded may be related to the sample size, sampling method, season of the year, management practice and detection method (couacy-hymann et al. 2005, devi et  al. 2016) as well as the clinical status of the animals at the period of sampling. the analysis of the results revealed that there were no significant associations between pprv detection rate and location/states, species, sex and breeds of the animal sampled. these results suggest that pprv affects equally both sheep and goats, males and females, and also all breeds of the animals studied. however, our findings differ with those of other investigators such as anderson and colleagues (anderson et  al. 1991) and al-majali and colleagues (al-majali et  al. 2008) in northern jordan. additionally, lefevre and diallo (lefevre and diallo 1990) also reported that goats are more sensitive to pprv. previous studies reported the variability on susceptibility among domestic species but the pprv spread is thought to be related also to the relationship of virus sequence and host species (banyard et al. 2010). the highest ppr infection rate observed in march (68.8%) coincides with the harmattan period. similar observation has been reported (ezeibe et  al. 2008, okoli 2003, wosu 1994, wosu et  al. 1990, ezeokoli et al. 1986, obi et al. 1983). this higher infection rate during the harmattan period has been attributed to the dry, cold and dusty weather, together with the poor nutrition as a result of inadequate pasture (ezeibe et al. 2008, wosu et al. 1990). 195veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 chukwudi et al. pprv in southeast nigeria of good laboratory practices, may contribute to clarify these hypotheses. indeed, the partial n gene sequence was commonly used as an effective target for ppr detection and lineage identification (kwiatek et  al. 2011, tounkara et  al. 2019), aiming mainly to understand the epidemiology and transmission dynamics of ppr (tounkara et  al. 2019), as in the present study. this study reports vast diversity in the amino acid data among the pprv lineages iv and ii circulating in south-east nigeria,and confirms the predominance of the lineage iv in nigeria. these data support the need for further molecular epidemiological investigation in order to better comprehend the spread of the pprv strains and their transboundary circulation, to support the future control strategies. acknowledgments the authors would like to thank dr. webster okonkwo and dr. adaeze nwabueze of the tropical veterinary centre ekwulobia anambra state, dr. sunday ali and mr. ndubuisi of ebonyi state, dr. ikechukwu udeani of veterinary teaching hospital university of nigeria, nsukka, for their assistance with sample collection. grant support this work was supported by the tertiary education trust fund (tetfund) of the nigerian government through university of nigeria nsukka tetfund (needs aassessment) bench space intervention. chukwudi i.c. is the only recipient of the grant. the funder had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. the high degree of sequence similarity in pprv lineage ii with the nigeria 75/1 vaccine strain recorded in our study has also been reported by other authors in nigeria (mantip et  al. 2016, woma et  al. 2016), sierra leone (munir et  al. 2012), tanzania (misinzo et  al. 2015, mahapatra et  al. 2015), china (zhou et  al. 2018), india (unpublished, acc  no. dq176750) and iran (unpublished; acc.  no. kc534492), although dundon and colleagues (dundon et  al. 2020) posited that some of these previously reported cases of sequence similarity are most likely as a result of laboratory contamination during sample processing or due to circulating strains that are very similar to or variants of the vaccine strain nigeria 75/1. although homologous ppr vaccine available in nigeria for immunization of small ruminant is of nigeria pprv 75/1 origin (sen et  al. 2010), a recent study reported lack of awareness of ppr vaccination among small ruminant farmers and non-availability of ppr vaccine at veterinary establishments in southeast nigeria (chukwudi et  al. 2020). there is not a documented evidence that animals tested in this study have been vaccinated against pprv but the molecular analysis of the 11 lineage ii strains collected from enugu state in this study shows their close relationship with a vaccine strain. therefore, albeit hypothesized, a clear origin of these viruses cannot be pointed out and necessarily this limitation deserves further evaluation or specific assays for discrimination between nigeria pprv 75/1-based vaccines and field strains of pprv. the limited data available makes this recent debate still open and requires further studies, in the direction of which this study goes, to be definitively documented. moreover, a sequence study based on multiple genes or the total genome, together with the pursuit 196 veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 pprv in southeast nigeria chukwudi et al. abu elzein e.m., hassanien m.m., al-afaleq a.i., abd elhadi m.a. & housawi f.m. 1990. isolation of peste des petits ruminants from goats in saudi arabia. vet rec, 127, 309-310. al-majali a.m., hussain n.o., amarin n.m. & majok a.a. 2008. seroprevalence of, and risk factors for, peste des petits ruminants in sheep and goats in northern jordan. prev vet med, 85, 1-8. amarasinghe g.k., ayllón m.a., bào y., basler c.f., bavari s. & blasdell k.r. 2019. taxonomy of the order mononegavirales: update 2019. arch 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& munir m. 2015. peste des petits ruminants virus lineage ii and iv from goats in southern tanzania during an outbreak in 2011. british j virol, 2, 1-4. munir m., zohari s., suluku r., leblanc n., kanu s., sankoh f.a., berg m., barrie m.l. & ståhl k. 2012. genetic characterization of peste des petits ruminants virus, sierra leone. emerging infect dis, 18, 193-195. obi t.u., ojo m.o., taylor w.p. & rowe l.w. 1983. studies on the epidemiology of peste des petits ruminants in southern nigeria. tropical veterinarian, 1, 209-217. okoli i.c. 2003. incidence and modulating effects of environmental factors on tripanosomosis, peste des petits ruminants (ppr) and bronchopneumonia of west african dwarf goats in imo state, nigeria. livestock research for rural development, 15, 9-11. parida s., muniraju m., mahapatra m., muthuchelvan d., buczkowski h. & banyard a.c. 2015. peste des petits ruminants. vet microbiol, 181, 90-106. taylor w.p. & abegunde a. 1979. the isolation of peste des petits ruminants virus from nigerian sheep and goats. res vet sci, 26, 94-96. thrusfield m. 2005. veterinary epidemiology. 2nd ed. blackwell science, oxford, 117-198. 198 veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 pprv in southeast nigeria chukwudi et al. supplementary table. details of tested positive samples. —cont’d annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 57; issue: 3; year: 2021; doi: 10.12834/vetit.2390.14842.1 s/no st/no state species breed sex vaccination status anamnesis date of collection accession number and sequence id 1 1 en ovi uda f unk nd, od, cou. dia, du 21/12/2017 2 3 an ovi wad m nv nd, cou. dia, du, rhc, wk, us 25/01/2018 mn271599, ngan_084 ov 2018 3 7 en cap wad f nv nd, od, cou. du, ema, rhc, wk 13/03/2018 4 8 en cap wad f nv nd, od 14/03/2018 5 9 an cap wad f nv nd, od, dia, us 19/03/2018 mn271590, ngan_150 ca 2018 6 10 an cap wad f nv nd, od, cou. dia, du, rhc, wk, se 26/03/2018 mn271592, ngan_156 ca 2018 7 12 an ovi wad f nv nd, dia, cou 16/04/2018 mn271594, ngan_217 ov 2018 8 13 an ovi wad f nv nd, od, cou. du, wk 16/04/2018 9 14 an cap wad m nv nd, cou. du, us, rhc 16/04/2018 10 16 en ovi uda f nv apparently healthy 22/12/2017 11 17 en cap rs f unk nd 09/01/2018 12 19 en ovi wad m nv severe mucoprulent nd, du 17/01/2018 13 20 an ovi wad f nv nd, dia, us, cou. du, rhc 25/01/2018 14 26 en cap wad m nv nd, cou. dia, du, rhc, ema, wk, se 12/03/2018 mn271593, ngen_146 ca 2018 15 27 en cap wad f nv nd, cou. du, rhc, ema 19/03/2018 16 28 an cap wad f nv nd, od, cou. du, rhc, ema 26/03/2018 17 29 an cap wad f nv nd, cou. dia, us, du, rhc, ema 26/03/2018 18 32 eb ovi wad f nv nd, od, cou. du, rhc 11/04/2018 19 33 en ovi wad m nv mucopurulent nd, us, cou, du 21/04/2018 20 35 eb ovi wad m nv mucopurulent nd, us, cou, du 23/05/2018 21 46 en ovi wad m nv apparently healthy 17/02/2018 mn271587, ngen_137 ov 2018 22 48 eb ovi wad f nv mucopurulent nd, us, cou, du 06/04/2018 23 49 eb ovi wad f nv mucopurulent nd, du 11/04/2018 24 52 an ovi wad m nv nd, dia, cou 16/04/2018 25 53 en ovi wad m nv mucopurulent nd, cou, du 21/04/2018 26 55 en ovi wad m nv mucopurulent nd, du 28/05/2018 mn271601, ngen_255 ov 2018 27 60 an ovi wad f nv nd rhc, du 25/01/2018 mt038906, ngan_087 ov 2018 28 61 en cap rs f unk apparently healthy 09/01/2018 mt038903, ngen_038 ca 2018 29 64 an ovi wad m nv apparently healthy 30/05/2018 30 66 en ovi wad m nv nd 21/04/2018 31 70 en cap rs m unk nd, du wk 17/01/2018 32 71 an ovi wad m nv mucopurulent nd, cou 25/01/2018 33 75 en cap wad m nv mucopurulent nd, dia, od, cou. du, rhc, us, ema 17/01/2018 34 85 an ovi yan m nv nd, cou 30/05/2018 continued s/no = sample number; st/no = strain number; state of collection: en = enugu, an = anambra, eb = ebonyi; specie of collection: ovi = ovine, cap = caprine; breed: wad = west african dwarf, rs = red sokoto, yan = yankassa; sex: m = male, f = female; vaccination status: nv = not vaccinated, unk = unknown; anamnesis: nd = nasal discharge, od = ocular discharge, cou = coughing, ema = emaciated, rhc = rough hair coat, wk = weak, du = dull, dia = diarrhea, se = sunken eyeball, us = ulcerative stomatitis. 199veterinaria italiana 2021, 57 (3), 189-199. doi: 10.12834/vetit.2390.14842.1 chukwudi et al. pprv in southeast nigeria supplementary table. details of tested positive samples. —cont’d annex 1 publisher: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’ | journal: veterinaria italiana | article type: reasearch article | volume: 57; issue: 3; year: 2021; doi: 10.12834/vetit.2390.14842.1 s/no st/no state species breed sex vaccination status anamnesis date of collection accession number and sequence id 35 90 an cap wad m nv nd, du, cou 16/04/2018 36 93 en ovi wad f nv nd, cou, du 23/01/2018 37 97 en cap rs f unk nd, rhc, du, 21/12/2017 38 108 en ovi wad f nv nd, rhc, du 23/01/2018 39 116 en ovi wad m nv nd, du 28/05/2018 40 118 en ovi wad m nv nd 21/04/2018 mt038904, ngen_228 ov 2018 41 122 an cap wad f nv nd, cou, du 26/03/2018 42 123 an cap wad f nv nd du, cou 26/03/2018 43 127 en cap rs f unk apparently healthy 28/12/2017 44 131 en ovi uda f unk nd 09/01/2018 45 132 eb ovi wad f nv nd, cou, du 23/05/2018 46 137 eb ovi wad m nv nd du, cou 23/05/2018 47 141 eb ovi wad m nv nd, cou 05/06/2018 48 143 en cap rs f unk nd 09/01/2018 49 144 eb ovi wad f nv nd 11/04/2018 50 149 an ovi wad f nv nd, du, wk 30/05/2018 51 150 an ovi wad m nv nd, cou, rhc 30/05/2018 52 159 en ovi wad m nv nd, cou, du 17/01/2018 53 160 en cap wad f nv mucopurulent nd, cou 22/05/2018 54 161 en ovi yan m unk apparently healthy 17/02/2018 55 165 an ovi wad m nv apparently healthy 30/05/2018 56 168 en cap wad f nv nd, du 13/03/2018 57 169 en cap rs f unk apparently healthy 28/12/2017 58 170 en ovi uda f nv apparently healthy 20/12/2017 mt038905, ngen_003 ov 2017 59 172 eb ovi wad f nv nd 06/04/2018 60 174 eb cap wad f nv apparently healthy 06/04/2018 61 175 eb ovi wad m nv apparently healthy 06/04/2018 62 177 en cap rs f unk apparently healthy 28/12//2017 63 178 en cap rs f unk apparently healthy 22/12/1017 64 179 en ovi wad m nv du 21/04/2018 65 180 eb ovi wad f nv apparently healthy 17/02/2018 s/no = sample number; st/no = strain number; state of collection: en = enugu, an = anambra, eb = ebonyi; specie of collection: ovi = ovine, cap = caprine; breed: wad = west african dwarf, rs = red sokoto, yan = yankassa; sex: m = male, f = female; vaccination status: nv = not vaccinated, unk = unknown; anamnesis: nd = nasal discharge, od = ocular discharge, cou = coughing, ema = emaciated, rhc = rough hair coat, wk = weak, du = dull, dia = diarrhea, se = sunken eyeball, us = ulcerative stomatitis. 133 1faculty of veterinary medicine, university of teramo, località piano d’accio, 64100 teramo, italy. 2istituto zooprofilattico sperimentale dell'abruzzo e del molise ’g. caporale’, campo boario, 64100 teramo, italy. *corresponding author at: faculty of veterinary medicine, university of teramo, località piano d’accio, 64100 teramo. tel.: +39 0861 xxxxx, e‑mail: ideamicis@unite.it. keywords chronic orchitis, calcified seminiferous tubules, stallion. summary orchitis and epididymo-orchitis are inflammatory lesions of the testicle. we herein describe a case of monolateral chronic orchitis which occurred in a tiro pesante rapido (tpr) stallion, born in 2002, with a history of good fertility. the stallion was healthy and asymptomatic although the left testis was found to be smaller as compared with the right one and was hard in consistency. histopathology examination revealed tubular atrophy and parenchymal sclerosis. scattered foci of calcification and chronic inflammation, the latter dominated by macrophages and lymphocytes, were also observed. although lesions were clearly present, the semen was demonstraed to be of good quality. this study highlights the need for periodic clinical and ultrasound evaluation of stallions, in order to preserve their reproductive performance. ippolito de amicis1*, roberta bucci1, brunella a. giangaspero1, nicola d'alterio2, abigail r. tracht1 and domenico robbe1 monolateral chronic orchitis in a stallion veterinaria italiana 2020, 56 (2), 133-135. doi: 10.12834/vetit.2330.13219.1 accepted: 27.04.2020 | available on line: 30.06.2020 the left testicle was reduced in size and hardened, although no pain was observed at palpation. ultrasound examination revealed the presence of scattered, hyperechogenic areas (figure 2). thereafter, the semen was collected and evaluated, showing no evidence of infertility. due to the hard consistency of the testis, the collection of a diagnostic biopsy was not feasible. therefore, the surgical removal of the entire left testicle was chosen. to this aim, the stallion was premedicated with 0.05 mg/kg of acepromazine (i.m); 20 min later, detomidine (0.01 mg/kg) and butorphanol (0.02 mg/ kg) were administered (i.v). anesthesia was induced introduction orchitis can occur in stallions as an asymptomatic inflammatory reaction, not necessarily associated with infertility (edwards 2008). diagnosis of chronic asymptomatic orchitis requires a testicular biopsy, as no negative influence of the condition on sperm quality has been demonstrated. orchitis can be caused by infectious pathogens, both bacterial and viral, arising from the ascending route such as chlamydia spp., e. coli and other enterobacteriaceae, or through the hematogenous route, such as mycobacterium spp. and brucella spp. (carmen ferreras et al. 2007) sterile orchitis and granuloma formation are also common events, occurring after trauma or obstruction of the genital tract. overall, the etiology of chronic orchitis often remains unknown. case description a 17-year-old tpr stallion was presented to the breeding program of the university of teramo. as per routine, the stallion underwent a clinical evaluation with particular focus on the reproductive apparatus. the stallion appeared clinically healthy, showing no relevant clinical signs. however, at visual evaluation of the scrotum a significant asymmetry of the testicles was observed (figure 1). in particular, case report figure 1. clinical examination. the left testis clearly appeared smaller as compared with the right one. 134 veterinaria italiana 2020, 56 (2), 133-135. doi: 10.12834/vetit.2330.13219.1 monolateral chronic orchitis in a stallion de amicis et al. the semen was reexamined and confirmed to be of satisfactory quality (table i). discussion macroscopically, hypoplasia of the testicle is distinguished from degeneration/atrophy as it with diazepam (0.05 mg/kg) and ketamine (2.2 mg/ kg) (i.v), and then maintained with thiopental (0.5 mg/kg). the testicle was infiltrated with 5 ml of 2% lidocaine before the incision, and the surgery was performed with the use of emasculator using an ‘open approach’. the direct inspection confirmed that the testicle was reduced in size and very hard. in the cut section, the parenchyma appeared fibrotic and calcified. the epididymis was proportionally increased both in volume and consistency. there was a small hemorrhage on the surface of the testicle, probably due to biopsy sampling or vaginal incision during orchiectomy (figure 3). the testicle was promptly fixed in 10% neutral buffered formalin and routinely processed for microscopic investigations. histopathology (hematoxylin and eosin stain) examination revealed tubular atrophy and parenchymal sclerosis. scattered foci of calcification and chronic inflammation, the latter dominated by macrophages and lymphocytes, were also observed (figure 4). based on the collected evidences, a unilateral, chronic orchitis of unknown origin was diagnosed. two months post-surgery, figure 3. left testicle. the epididimys appears relatively larger as compared with the small testis. figure 4. histological evaluation of the testicle. histologically, tubular atrophy, parenchymal sclerosis, scattered foci of calcification and an inflammatory infiltrate dominated by macrophages and lymphocytes are visible, confirming a diagnosis of chronic orchitis. figure 2. ultrasonographic images. a. right testicle: the ecostructure of the normal testicular parenchyma is characterized by fine and thickened echoes, arranged with high homogeneity. portions of the vaginal cavity are visible as anechoic borders whereas the parietal vaginal tunic is observed as a thin white line outside the vaginal cavity. b. left testicle: the testicular parenchyma is heterogeneous with a granular aspect. a b 135veterinaria italiana 2020, 56 (2), 133-135. doi: 10.12834/vetit.2330.13219.1 de amicis et al. monolateral chronic orchitis in a stallion to the testis (edwards 2008). in fact, the testicle in the case herein described was reduced in size, whereas the dimensions of the epididymis appeared normal, leading the case to be considered as a testicular degeneration arising from chronic orchitis. this hypothesis was confirmed by the histological evaluation after surgery. the collection of seminal material allowed for the description of the macro and microscopic characteristics. the urethral pulse characteristics were also evaluated two months post-surgery and were demonstrated to be within the norm. such findings further confirm that chronic orchitis can, in some cases, occur asymptomatically, making a correct clinical evaluation very difficult. in fact, several grades of testicular fibrosis did not influence the seminal quality in a case of salmonellosis in a bull, described by pinho and colleagues (pinho et al. 2012, pinho et al. 2013). in terms of other causes of testicular calcification, in the case herein reported, the etiology is unknown and further analyses are currently ongoing. in conclusion, the case herein described underlines the usefulness of periodic clinical and ultrasound evaluation for stallions, in order to preserve their reproductive performances. has a proportionally smaller epididymis relative to the hypoplastic testis and occurs in cases of cryptorchidism and intersexuality. on the other hand, a degenerate testis is associated with an epididymis that is relatively large in size as compared table i. semen collection. two months after surgery, the semen was proved to be of good quality both in terms of concentration and vitality. subject 1° collection 2° collection reaction time (min.) 10 minutes 15 minutes volume before filtration (ml) 100 80 volume after filtration (ml) 85 70 color milky milky smell heated metal heated metal ph 7.3 7.3 motility (%) 85 90 vitality (%) 90 95 concentration (x 106/ml) 210 145 total number of spermatozoa for ejaculate (x 109) 17 10.150 motility at environmental temperature (h) 2 2.30 primary morphological abnormalities (%) 10 7 secondary morphological abnormalities (%) 12 9 edwards j.f. 2008. pathologic conditions of the stallion reproductive tract. anim reprod sci, 107 (3-4), 197-207. ferreras mdel c., muñioz m., pérez v., benavides j., garcía-pariente c., fuertes m., adúriz g. & garcía-marín j.f. 2007. unilateral orchitis and epididymitis caused by salmonella enterica subspecies diarizonae infection in a ram. j vet diagn invest, 19,194-197. pinho r.o., costa d.s., siqueira j.b., martins l.f., neto t.m., references tavares do nascimento pereira j.v., facioni-guimarães s.e. & guimarães j.d. 2013. testicular fibrotic lesions and semen quality in adult montana tropical compound bulls. rev bras med vet, 35 (2), 105-110. pinho r.o., martins l.f., siqueira j.b., domeneck f., miranda neto t. & guimarães j.d. 2012. avaliação da qualidade do sêmen fresco de touros jovens da raça composto tropical montana e suas correlações com o teste hiposmótico. acta vet bras, 6 (3), 192-198. 265 veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 accepted: 24.11.2021 | available on line: 31.12.2022 university of sadat city, egypt *corresponding author at: university of sadat city, egypt. e-mail: amal.abdelhakam@vet.usc.edu.eg. amal hammad*, shaaban gadallah, tarek misk, ahmed sharshar, nahed thabet and ahmed mourad keywords horses, nalbuphine, pharmacodynamics, pharmacokinetics, xylazine. summary this study describes the selected pharmacodynamics and pharmacokinetics of nalbuphine (nal) in xylazine (xyl)-sedated horses. five adult healthy horses were randomly received 2 treatments at a 1-week interval; xyl treatment (0.55 mg/kg iv) and xyl/nal treatment (xyl, 0.55 mg/kg iv; nal, 0.3 mg/kg iv). the measured pharmacodynamic variables were sedative and analgesic effects and the effect on ataxia and some physiological parameters. for the pharmacokinetics of nal, its plasma concentrations were measured using hplc and a 2-compartment analysis was performed. greater and prolonged sedation was evident after xyl/nal treatment compared with xyl treatment. slightly improved and prolonged analgesia was demonstrated after xyl/nal treatment. significant changes in blood pressure and respiratory rate lasted for a shorter duration with xyl/nal treatment than with xyl treatment. after xyl treatment, rectal temperature was significantly different from baseline and xyl/nal treatment. elimination half-life of nal was 3.47 ± 1.39 hours and total body clearance was 2.88 ± 0.73 l/kg/hour. in conclusion, addition of nal to xyl resulted in remarkable advantages on the measured parameters. the obtained pharmacokinetics of nal could be useful in determining the effective nal infusion rate, which could be further evaluated as an adjunctive agent to xyl for prolonged sedation in horses. pharmacodynamics and pharmacokinetics of nalbuphine in xylazine‑sedated horses given in combination with xyl to horses (brunson and majors 1987). in another study (taylor et  al. 1990), the sedative and cardiorespiratory effects of detomidine/nal and acepromazine/nal combinations were also assessed. however, nal seems to be insufficiently studied in horses. considering limited studies targeting the assessment of nal in horses and the lack of data regarding its pharmacokinetics along with the previously mentioned merits of α 2 -agonists/opioids combinations over α 2 -agonists alone in terms of concomitant sedation and analgesia, this study aimed to describe the selected pharmacodynamics (involving sedative and analgesic effects and the effect on ataxia and some physiological parameters) and pharmacokinetics of nal in xyl-sedated horses. we hypothesized that the addition of nal to xyl might produce better sedative and analgesic effects than xyl alone, without adversely affecting physiological parameters. the used pharmacokinetic model was also hypothesized to be efficient in providing nal pharmacokinetics. introduction the sedative and analgesic effects of α 2 -agonists facilitate various diagnostic and surgical procedures in standing horses (rezende et  al. 2015). xylazine (xyl) is a commonly used α 2 -agonist in horses. however, it only confers a short duration of sedation and analgesia (queiroz-neto et  al. 1998, bueno et  al. 1999). because sedated horses may respond unexpectedly to imposed stimuli, which can be risky to both horses and practitioners, the administration of xyl or other α 2 -agonists alone was also reported to produce less reliable sedation (alitalo 1986, england et al. 1992, corletto et al. 2005). consequently, these agents can be combined with opioids to enhance and prolong the sedative and the analgesic effects of α 2 -agonists (clarke et  al.1991, seo et  al. 2011, gozalo-marcilla et al. 2017). nalbuphine (nal) is a semisynthetic agonist-antagonist opioid analgesic with agonistic activity at κ-receptors and antagonistic action at μ-receptors (kulkarni et al. 2015). the nal-associated analgesia was previously evaluated when it was 266 veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 effects of nalbuphine in xylazine-sedated horses hammad et al. intestinal motility was also assessed at baseline and at 8, 15, 30, 60, 90, and 120 minutes after xyl administration. evaluation of sedative effect (degree and duration of sedation) a multifactorial sedation scale (mfss) was designed to evaluate the degree of sedation and involved a summation of the scores of head drop percentage and the scores of horse response to auditory, visual, and tactile stimuli (table i). the head drop percentage was estimated by measuring the change in head height (distance from the most ventral bony portion of the chin to the ground in centimeters) with respect to the baseline head height using a measuring tape attached to the chin region (solano et  al. 2009, rezende et  al. 2014). to determine the baseline head height, the horses were observed for 15 minutes before drug administration, and the most frequently observed head position was recorded as the baseline head height. a numerical rating scale (nrs) modified from solano and colleagues (solano et al. 2009) was used to score the head drop percentage. for auditory stimulation of the studied horses, hand clapping was conducted 60 centimeters from the horses’ head (love et  al. 2011), and their response was scored using a scale modified from wagner and colleagues (wagner et  al. 2011). visual stimulation was done by waving a piece of cloth toward the horses’ head (peştean et  al. 2010) with scoring of their reaction using a scale adopted from ringer and colleagues (ringer et  al. 2013). tactile stimulation was done by touching inside the pinna of the ear using a pen (gozalo-marcilla et  al. 2017) with subsequent scoring for the resultant response using a scale modified from clarke and colleagues (clarke et al. 1991). the duration of sedation was considered as the period over which significant changes in sedation scores were determined. evaluation of analgesic effect (degree and duration of analgesia) the degree of analgesia was assessed using 2  nociceptive stimuli, namely, needle prick and electrical stimulation. for the needle prick, a 22-gauge, 1.25-inch needle was thrusted to its whole length just once at the right and left flank regions, alternatively. the horses’ response to this stimulus was graded using the nrs adapted from wagner and colleagues (wagner et al. 2011) (table ii). for electrical stimulation, an electrical stimulus (100 hz and 1 ms) was applied to the horses’ skin at the materials and methods animals five adult research horses (4 stallions and 1 mare) of mixed breeds, aged 14.00 ± 4.95 years and weighing 299.80 ± 16.87 kg, were enrolled in this study. the horses were considered healthy based on physical examination, complete blood count, and serum biochemistry analyses. this study was approved by the institutional animal care and use committee of the faculty of veterinary medicine, university of sadat city, egypt. during the study period, horses were kept in their stall at the university of sadat city where they were fed hay and allowed free access to water. study design this was a randomized, blinded, and crossover study. at the end of the day before the experiment, horses were weighed and the hair over the right and left jugular veins was shaved for placement of intravenous (iv) catheters. food but not water was withheld for 12 hours before the experiment. at the day of the study, horses were kept in a stock in a quiet room and allowed at least 2 hours to acclimatize to their surroundings. to decrease external stimulation, a fly repellent was also applied to the skin of the horses. the right and left jugular veins were catheterized (using a 16-gauge catheter) under local anesthesia with subcutaneous lidocaine 2% (2  ml) for the administration of drugs and collection of blood samples (in case of xyl/nal treatment), respectively. horses were randomly subjected to 2  treatments with 1-week washout period in between. assigned treatments were as follows: xyl treatment: horses received an iv dose of 0.55 mg/kg of xyl (xyla-ject 20 mg/ml, adwia co., 10th of ramadan city, egypt) alone over 1 minute (fernandes de souza et al. 2012). xyl/nal treatment: horses received an iv dose of 0.55 mg/kg of xyl over 1 minute followed 5  minutes later by an iv dose of 0.3 mg/kg of nal (nalufin 20 mg/ml, amoun pharmaceutical co., cairo, egypt) (taylor et  al. 1990). nal was administered slowly over 2 minutes. assessments during the study period, sedative and analgesic effects, ataxia, and physiological parameters such as heart rate (hr), respiratory rate (rr), blood pressure, and rectal temperature (rt) were evaluated before (baseline) and at 8, 15, 30, 45, 60, 75, 90, 105, and 120 minutes after xyl administration. 267veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 hammad et al. effects of nalbuphine in xylazine-sedated horses nociceptive threshold and the scores of animal response to the needle prick. evaluation of ataxia the degree of ataxia was assessed using the nrs described by fernandes de souza and colleagues (fernandes de souza et al. 2012) (table iii). throughout the study period, a single blinded investigator evaluated sedation, analgesia, and ataxia to enhance the reliability of the adopted assessment scales. over the entire observation period, the horses were also monitored for any central nervous system excitatory signs such as muscle tremors. evaluation of physiological parameters intestinal motility, hr, rr, blood pressure, and rt shoulder region using 2 adhesive electrodes of an electric muscle stimulator device (healthtronic muscle stimulator jh4207, china). the electrodes were placed 7 cm apart and secured in position using a sheet of adhesive and transparent medical tape. the intensity of electrical stimulus was gradually increased until the animal turned its head back toward the stimulus (avoidance response), and this intensity was considered the electrical nociceptive threshold. the electrodes of the unit were connected to an oscilloscope (fluke pm3394a, science park eindhoven 5110, son, 5692 ec netherlands) to determine the exact voltage of the recorded electrical stimuli (electrical nociceptive thresholds) throughout the study. the duration of analgesia was the period over which there were significant changes in electrical table i. the multifactorial sedation scale (mfss) used to score degree of sedation (scores of head drop% and the horse response to auditory, visual, and tactile stimuli). variable score description head drop % 0 no head lowering (head height was equal or higher than baseline) 1 head slightly lowered (<40%) 2 head moderately lowered (40–60%) 3 head markedly lowered >60% response to auditory stimulation 0 horse raised its head briskly and ears were erected or laid back 1 horse calmly raised its head 2 ear twitched or horse moved slightly 3 no observable response response to visual stimulation 0 undiminished response, animal move away vigorously 1 muted response or subdued reaction and movements 2 reaction significantly subdued (elevates head slightly) 3 no signs of visual arousal. response to tactile stimulation 0 rapid and marked response (head shake) to stimulation. it was also awarded when test was extremely difficult to be performed. 1 the response was easily elucidated but was slower. 2 the response was sluggish and only elicited by prolonged stimulation. 3 no response. total score of sedation degree 0-3 no sedation 4-6 mild sedation 7-9 moderate sedation 10-12 intense sedation table ii. numerical rating scale (nrs) used to assess analgesic effect in needle prick method. nrs response to pinprick 1 dramatic response (the horse was alert and moved, kicked, or bit) 2 moderate response (the horse raised its head briskly and ears were erect or laid back) 3 mild response (the horse calmly raised its head) 4 slight or barely perceptible response (the ear twitched or horse moved slightly) 5 no observable response table iii. numerical rating scale used to assess degree of ataxia. nrs degree of ataxia 0 no ataxia. 1 mild ataxia. the horse was stable but slightly swaying 2 moderate ataxia. the horse was swaying and leaning against the stock 3 intense ataxia. the horse was leaning against the stock and swaying with its hind limbs crossed and its forelimbs buckling at the carpal joints 268 veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 effects of nalbuphine in xylazine-sedated horses hammad et al. extraction of nal from plasma samples was performed using the method previously published by huang and colleagues (huang et  al. 2013) using the agilent 1200 series liquid chromatography system (agilent community, santa clara, united states). pharmacokinetic analysis plasma concentrations of nal versus time curve were generated and best fitted by 2-compartment open model system with the aid of computer polyexponential curve stripping program (rstrip, micromath scientific software, salt lake city, ut, usa). data from each horse were fitted individually, and the pharmacokinetic variables were computed using the software programs. the hybrid rate constants of the distribution and elimination phases (α and β) and elimination rate constant (k el ) and corresponding extrapolated zero-time intercepts of distribution and elimination phases (a and b) were calculated. distribution and elimination half-lives (t 0.5α , t 0.5β ), and k 12 and k 21 (distribution rate constants from central to peripheral and from peripheral to central compartment, respectively) were also calculated. the area under the curve from zero to infinite time (auc 0-∞ ) and mean residence time were calculated. other pharmacokinetic parameters such as total body clearance (cl tot ), the volume of the central compartment (v c ), apparent volume of distribution (v d area ), volume of distribution in terminal elimination phase (v dβ ), and the volume of distribution at steady state (v dss ) were calculated by standard methods (baggot 1978, dhanjal et al. 2009). statistical analysis statistical analysis was performed with spss 16.0 software (spss, usa). the parametric variables such as the head drop percentage, electrical nociceptive threshold, and physiological parameters (hr, rr, sap, dap, map, and rt) were analyzed using a one-way analysis of variance (anova) with dunnett’s post-test for comparisons of means within each group in relation to baseline. comparisons between groups at each time were performed with one-way anova followed by tukey’s test. the friedman test was used for the nonparametric variables such as the response to needle prick and auditory, visual, and tactile stimuli and sedation, ataxia, and intestinal motility. the results of different parametric variables were expressed as mean ± standard deviation with exception of head drop percentage and plasma concentration of nalbuphine which were expressed as mean. the results of non-parametric variables were expressed as median (range), and p < 0.05 was considered statistically significant. were evaluated. for intestinal motility, each of the right upper, right lower, left upper, and left lower abdominal quadrants were auscultated for 30  seconds and intestinal sounds were scored according to dhanjal and colleagues (dhanjal et  al. 2009) in which > 2 sounds in 30 seconds scored  2, 1-2  sounds scored 1, and no sounds scored 0. therefore, the cumulative score from all 4 quadrants ranged from 0 to 8. hr was measured by auscultation with a stethoscope for over 1 minute. rr was monitored by counting the number of thoracic excursions for over 1 minute. noninvasive measurement of arterial pressures [systolic (sap), diastolic (dap), and mean (map)] was performed via an oscillometric technique (contec patient monitor, contec medical systems co., qinhuangdao, china) with the cuff positioned at the tail base (adult noninvasive blood pressure cuff, 19-24 cm). the recorded blood pressure values were further corrected according to the height difference between the cuff and shoulder joint (1 cm h 2 o = 0.7355 mmhg) with the latter location representing the level of the right atrium of the heart (gozalo-marcilla et al. 2017). rt was measured in degrees celsius (°c) using a digital thermometer. pharmacokinetic study of nalbuphine sample collection during the evaluation of xyl/nal treatment, venous blood samples (10 ml) were collected at baseline and at 1, 3, 8, 15, 23, 38, 53, 68, 83, 98, 113, 240, 360, 480, and 600 minutes after nal administration. before the collection of the 10-ml blood sample, 2  ml of blood was withdrawn and discarded. the blood samples were transferred immediately into heparinized tubes and centrifuged for 10 minutes at 2,400 g (tabletop centrifuge, gemmy industrial corp, min chuan west road, taipei, taiwan). plasma was harvested and stored at - 80 °c until assayed. determination of plasma nalbuphine concentration the concentrations of nal in equine plasma were quantitated by high-performance liquid chromatography analysis with an ultraviolet detection. stock solution was prepared by dissolving 10 mg of nal hydrochloride in 10-ml water. working solutions were prepared by serially diluting nal stock solution. plasma calibrators were prepared by diluting working solutions with drug-free equine plasma to concentrations of 1 (the limit of quantitation), 5, 10, 25, 50, 100, 250, 500, 1,000, and 2,500 ng/ml. 269veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 hammad et al. effects of nalbuphine in xylazine-sedated horses was highest at 15 minutes, which was evidenced by moderate to intense sedation in the treated horses (3 of 5 and 2 of 5, respectively). in addition, at 15  minutes, sedation was significantly greater with xyl/nal than with xyl. concerning the duration of sedation (significant increase in sedation scores), sedation persisted for 15 and 45 minutes with xyl and xyl/nal, respectively (table iv). analgesia (for needle prick and electrical stimulation) persisted for 8 minutes with xyl and 15 minutes with xyl/nal. occasionally, analgesia was slightly better (not significant) with xyl/nal than with xyl (table v). ataxia scores were significantly higher than baseline only at 8 minutes with xyl and from 8 to 30 minutes with xyl/nal. ataxia was significantly higher with xyl/nal than with xyl at 15 and 30 minutes; however, moderate ataxia was the highest degree exhibited by the studied horses (1 of 5 with xyl and 3 of 5 with xyl/nal) after either treatment (table v). a significant difference in intestinal motility scores and hr was not detected from baseline with both treatments or in between at any observation time (tables v and vi). a significant decrease in sap, dap, map, and rr was detected for a shorter duration with xyl/nal than with xyl compared with baseline without being significantly different among treatments (table vi). a significant decrease in rt from baseline was only detected with xyl treatment at 8 and 15 minutes with being significantly lower with xyl than xyl/nal at 8 minutes only (table vi). the observation of the horses throughout the study period revealed an occurrence of muzzle tremors in 1 horse with xyl and 2 horses with xyl/nal. regarding the pharmacokinetic study of nal, a peak plasma concentration of 79.64 ng/ml was attained at 8 minutes after nal administration. after 8 minutes, nal concentration declined gradually over time, and its level was below the limit of quantification results a significant head drop was evident after both treatments compared with baseline (30 minutes with xyl and 45 minutes with xyl/nal). the deepest drop (59.65%) was recorded at 8 minutes with xyl, whereas the greatest drop (71.21%) was evident at 15 minutes with xyl/nal. the head was significantly dropped with xyl/nal compared with xyl at 15 and 30 minutes (figure 1). the horses’ reaction to auditory and tactile stimuli was significantly reduced from baseline at 8  minutes with xyl and at 8 and 15 minutes with xyl/nal. the horses’ response to both stimuli was significantly lower with xyl/nal than with xyl at 15 minutes. the horses’ response to visual stimulation was significantly reduced from baseline at 8 and 15 minutes with both treatments without a significant difference in between (table iv). the highest degree of sedation (based on sedation scores) with xyl was evident at 8 minutes, which was evidenced by moderate sedation in all studied horses (5 of 5), whereas sedation with xyl/nal 80 70 60 50 40 30 baseline 8 15 a b a b* a b* b 30 45 60 75 90 105 120 20 10 0 -10 h ea d d ro p % times (minutes) xyl xyl/nal figure 1. the mean values of head drop % in horses after receiving xylazine (xyl) and xylazine/nalbuphine (xyl/nal) treatments. a,bsignificant difference (p<0.05) from the respective baseline value for xyla and xyl/nalb. *significant difference (p<0.05) from the value of xyl tratment. table iv. median (range) of response scores to auditory, visual, and tactile stimuli and sedation scores in horses after receiving xylazine (xyl) and xylazine/nalbuphine (xyl/nal) treatments. variable treatment baseline time (minutes) 8 15 30 45 60 75 90 105 120 response scores to auditory stimulation xyl 1 (1-2) 2 (2-3)* 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) xyl/nal 1 (1-2) 2 (2-3)* 3 (2-3)*‡ 2 (1-3) 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (1-2) 1 (0-2) response scores to visual stimulation xyl 0 (0-1) 1 (1-2)* 1 (1-1)* 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) xyl/nal 0 (0-1) 1 (1-3)* 1 (1-3)* 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) response scores totactile xyl 0 (0-1) 1 (1-2)* 1 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) xyl/nal 0 (0-1) 2 (1-2)* 2 (1-2)*‡ 1 (0-1) 1 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) 0 (0-1) sedation scores xyl 2 (1-3) 7 (7-8)* 5 (4-5)* 3 (1-4) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) xyl/nal 2 (1-3) 8 (7-11)* 8 (7-11)*‡ 4 (3-5)* 3 (2-4)* 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) *significant difference (p < 0.05) from the respective baseline value; ‡significant difference (p < 0.05) from the value of xyl treatment. 270 veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 effects of nalbuphine in xylazine-sedated horses hammad et al. effect of α 2 -agonists and α 2 -agonists/opioids combinations (solano et  al. 2009, guilhen et  al. 2015). accordingly, in this study, it was involved in the designed mfss. a significant head drop was evidenced after xyl (0.55 mg/kg) administration (xyl treatment) to the studied horses. consistently, in a previous report (santonastaso et  al. 2014), a significant head lowering was demonstrated after an iv administration of 0.6 mg/kg of xyl to horses. even if, in the current study, xyl produced a shorter duration of head drop than the later in 2 horses (horse number 3 and 5) at 600 minutes whereas still being detected in others (figure 2). all pharmacokinetic parameters obtained from the 2-compartment open model system and the used pharmacokinetic equations are presented in table vii. discussion head drop is an objective indicator for sedation in horses (ringer et  al. 2013), which was previously judged to be satisfactory in assessing the sedative table v. the median (range) of response scores to needle prick, mean ± sd values of electrical nociceptive threshold and median (range) of ataxia and intestinal motility scores in horses after receiving xylazine (xyl) and xylazine/nalbuphine (xyl/nal) treatments. variable treatment baseline time (minutes) 8 15 30 45 60 75 90 105 120 response scores to needle prick xyl 2 (1-3) 3 (3-4)* 3 (1-4) 2 (1-4) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) xyl/nal 2 (1-3) 3 (3-4)* 3 (3-4)* 3 (2-4) 2 (2-4) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) 2 (1-3) electrical nociceptive threshold xyl 1.92 ± 0.35 2.58 ±0.47* 2.30 ± 0.27 2.24 ± 0.02 1.92 ± 0.35 1.92 ± 0.35 1.92 ± 0.35 1.92 ± 0.35 1.92 ± 0.35 1.92 ± 0.35 xyl/nal 1.85 ± 0.26 2.81 ±0.39* 2.97 ± 0.53* 2.43 ± 0.36 1.98 ± 0.46 1.85 ± 0.26 1.85 ± 0.26 1.85 ± 0.26 1.85 ± 0.26 1.85 ± 0.26 ataxia scores xyl 0 (0-0) 1 (1-2)* 0 (0-1) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) xyl/nal 0 (0-0) 2 (1-2)* 2 (1-2)*‡ 1 (0-1)*‡ 0 (0-1) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) intestinal motility scores xyl 4 (4-5) 4 (4-4) 4 (4-4) 4 (4-4) nd 4 (4-4) nd 4 (4-5) nd 4 (4-5) xyl/nal 4 (4-6) 4 (3-5) 4 (3-5) 4 (4-5) nd 4 (4-6) nd 4 (4-6) nd 4 (4-6) *significant difference (p < 0.05) from the respective baseline value; ‡significant difference (p < 0.05) from the value of xyl treatment; nd = not determined. table vi. mean ± sd values of heart rate (hr), arterial pressures (systolic (sap), diastolic (dap) and mean (map)), respiratory rate (rr) and rectal temperature (rt ) in horses after receiving xylazine (xyl) and xylazine/nalbuphine (xyl/nal) treatments. variable treatment baseline time (minutes) 8 15 30 45 60 75 90 105 120 hr (beats/minute) xyl 35.67 ± 4.04 30.67 ±2.52 31.67 ± 3.06 32.33 ± 3.51 33.33 ± 2.52 33.67 ± 3.06 34.33 ± 3.21 35.00 ± 5.29 35.33 ± 5.86 35.67 ± 4.04 xyl/nal 36.67 ± 3.06 35.00 ±3.61 35.33 ± 3.51 35.67 ± 3.06 35.67 ± 3.06 36.00 ± 3.00 36.67 ± 3.06 36.67 ± 3.06 36.67 ± 3.06 34.00 ± 4.58 sap (mmhg) xyl 119.67 ± 6.03 99.00 ±14.11* 97.33 ± 10.07* 92.00 ± 7.21* 98.67 ± 11.68* 109.00 ± 5.29 114.33 ± 1.53 110.67 ± 9.71 116.33 ± 2.52 117.67 ± 4.73 1 xyl/nal 123.33 ± 5.51 111.00 ±3.61* 108.67 ± 2.08* 105.67 ± 4.93* 116.67 ± 2.08 117.33 ± 2.52 119.00 ± 1.00 120.33 ± 2.52 121.33 ± 3.51 123.00 ± 5.00 dap (mmhg) xyl 77.33 ± 2.52 64.67 ±4.73* 61.67 ± 1.53* 64.00 ± 1.00* 70.67 ± 1.53 71.67 ± 2.52 73.67 ± 4.51 71.33 ± 2.52 74.00 ± 4.58 72.67 ± 4.93 xyl/nal 78.00 ± 3.61 65.00 ±4.58* 63.67 ± 4.04* 65.33 ± 6.11* 71.00 ± 6.25 75.00 ± 7.00 74.33 ± 4.04 75.00 ± 4.58 73.00 ± 7.55 77.00 ± 2.65 map (mmhg) xyl 92.36 ± 2.29 77.05 ±3.45* 74.44 ± 5.59* 74.57 ± 4.14* 80.84 ± 4.57* 83.33 ± 7.02 88.09 ± 1.28 85.00 ± 5.00 88.47 ± 1.44 87.36 ± 2.04 xyl/nal 94.35 ± 2.44 81.85 ±2.39* 79.68 ± 4.64* 80.63 ± 2.29* 85.96 ± 5.16 92.93 ± 6.95 90.07 ± 1.04 91.47 ± .82 91.98 ± 2.25 92.68 ± 1.25 rr (breaths/minute) xyl 15.67 ± 1.53 9.00 ±1.00* 9.33 ± 1.53* 10.33 ± 0.58* 11.00 ± 1.73* 13.67 ± 2.08 14.00 ± 1.73 14.67 ± 1.15 15.00 ± 1.00 15.00 ± 1.00 xyl/nal 16.00 ± 1.73 11.67 ±1.15* 12.33 ± 0.58* 13.00 ± 1.00 15.00 ± 1.00 16.00 ± 1.73 16.00 ± 1.73 16.00 ± 1.73 16.00 ± 1.73 16.00 ± 1.73 rt (°c) xyl 38.07 ± 0.15 36.83 ±0.15*‡ 36.57 ± 0.55* 37.30 ± 0.30 37.33 ± 0.45 37.20 ± 0.66 37.50 ± 0.40 37.53 ± 0.45 37.63 ± 0.46 37.70 ± 0.52 xyl/nal 37.93 ± 0.15 37.60 ±0.26 37.50 ± 0.40 37.67 ± 0.25 37.80 ± 0.10 37.90 ± 0.10 37.83 ± 0.25 37.80 ± 0.30 37.83 ± 0.23 37.93 ± 0.06 *significant difference (p < 0.05) from the respective baseline value; ‡significant difference (p < 0.05) from the value of xyl treatment. 271veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 hammad et al. effects of nalbuphine in xylazine-sedated horses dexmedetomidine administration to horses. the addition of nal to xyl produced a greater depression of horses’ response to some stimuli, which agrees with the findings by clarke and colleagues (clarke et al. 1991) when butorphanol was given along with romifidine to horses. according to the designed scoring system, xyl (0.55 mg/kg) provided sedation for 15 minutes. in agreement, hubbell (hubbell 2009) reported sedation for only 30 minutes after administration of 1 mg/kg of xyl to horses. therefore, the shorter sedative effect observed in our study could be parallel to a lower tested dose. in a previous study (taylor et  al.1990), nal was efficient in enhancing detomidine-associated sedation in horses, which agrees with our findings. in our study, xyl/ nal administration achieved longer sedation and analgesia than xyl. similarly, prolonged sedation and analgesia was described in horses treated with romifidine/butorphanol combination compared with those treated with romifidine alone (derossi et al. 2009). administration xyl/nal was associated with only slightly better analgesia than xyl. however, we could hypothesize that the analgesic effect of this combination would be better expressed in horses subjected to a true clinical pain, which responds better to opioid analgesics, compared with the implemented experimental pain (clutton 2010). in this study, xyl was evaluated at a dose of 0.55 mg/ kg. this was based on the publication by fernandes de souza and colleagues (fernandes de souza et al. 2012) in which the same dose followed by a constant rate infusion of xyl (1.1 mg/kg/hour) was associated with mild ataxia in most studied horses. likewise, mild ataxia was the most frequently exhibited ataxia by our horses. this ataxia could be attributed to the muscle relaxant effect of xyl mediated by its binding to α 2 -adrenoreceptors in the spinal cord (sinclair study (30  minutes versus 75 minutes) despite of approximately similar doses used in both. a longer and greater head drop was obtained when nal was given in combination with xyl (xyl/nal treatment) to horses, which might have resulted from the ability of nal to potentiate the sedative effect of xyl or a synergistic action in between. a synergistic sedative effect of opioids and α 2 -agonists combinations was similarly reported by other authors (corletto et al. 2005). for a comprehensive estimation of the sedative effect of the studied treatments, mfss used in this study included the assessment of both head drop and commonly used variables for sedation quality, that is, the horses’ response to auditory, visual, and tactile stimuli (ringer et  al. 2013, schauvliege et al. 2019). the horses’ response to auditory, visual, and tactile stimuli was greatly reduced after xyl administration. similar findings were reported after romifidine administration to horses (clarke et  al. 1991). in this study, xyl maintained reduced reaction to the inflicted stimuli for a shorter duration than head drop. consistent findings were described by rezende and colleagues (rezende et  al. 2015) after 80 90 70 60 50 40 30 baseline 1 3 8 15 23 38 53 68 83 98 113 240 360 480 600 20 10 0 n al b u p h in e p la sm a co n ce n tr at io n (n g /m l) time (minutes) figure 2. the mean plasma concentrations of nalbuphine in xylazine premedicated horses. table vii. pharmacokinetic variables following intravenous administration of nalbuphine (0.3 mg/kg) in xylazine premedicated horses. horse b (ng/h) a (ng/h) β h-1 α h-1 auc (0-inf ) (ng/ml/h) variable mrt h t 0.5β h t 0.5α h k 12 h-1 k 21 h-1 vc (l/kg) v dβ (l/kg) v dss (l/kg) v d area (l/kg) k el h-1 cl tot (l/kg/h) 1 14.8 100.79 0.16 3.5 109.76 4.66 4.22 0.19 3.13 0.58 2.605 20.27 16.66 17.08 0.97 2.73 2 23.8 123.96 0.26 5.01 120.63 3.08 2.67 0.14 4.25 1.03 2.03 12.61 10.41 9.57 1.26 2.49 3 11.01 84.87 0.15 3.02 103.17 4.78 4.52 0.23 2.69 0.48 3.13 27.25 20.67 19.39 0.94 2.91 4 17.42 108.03 0.15 3.94 137.91 5.31 4.53 0.18 3.42 0.68 2.39 17.22 14.41 14.50 0.87 2.18 5 21 90.71 0.49 4.04 73.13 1.41 1.40 0.17 3.37 1.16 2.69 14.29 10.50 8.37 1.71 4.10 mean ± sd 17.61 ± 5.03 101.67 ± 15.33 0.24 ± 0.15 3.90 ± 0.74 108.92 ± 23.94 3.85 ± 1.60 3.47 ± 1.39 0.18 ± 0.03 3.37 ± 0.57 0.79 ± 0.29 2.57 ± 0.40 18.33 ± 0.40 14.53 ± 4.34 13.78 ± 4.74 1.15 ± 0.35 2.88 ± 0.73 b = extrapolated zero-time intercept associated with the elimination phase; a = extrapolated zero-time intercept associated with the distribution phase; β = hybrid rate constant of the elimination phase; α = hybrid rate constant of the distribution phase; auc0 -∞ = area under the curve from zero to infinite time; mrt = mean residence time; t 0.5β  = elimination half-life; t 0.5α = distribution half-life; k 12 = distribution rate constant from central to peripheral compartment; k 21 = distribution rate constant from peripheral to central compartment; v c = the volume of the central compartment; v dβ = volume of distribution in terminal elimination phase; v dss = the volume of distribution at steady state; v d area = apparent volume of distribution; k el = elimination rate constant; cl tot = total body clearance. 272 veterinaria italiana 2022, 58 (3), 265-274. doi: 10.12834/vetit.2408.16506.1 effects of nalbuphine in xylazine-sedated horses hammad et al. administration of 2 mg/kg in horses (dhanjal et  al. 2009). based on this, we used a similar model to conduct the pharmacokinetic analysis of nal. according to the used pharmacokinetic model, the elimination half-life for nal was 3.47  ±  1.39  hours whereas the total body clearance was 2.88  ±  0.73 l/kg/hour. this might indicate higher elimination half-life and lower clearance rate for nal in horses than those (elimination half-life, 0.68 hour; clearance rate, 6.86 l/kg/hour) demonstrated in calves after the administration of a slightly higher nal dose by the same route (coetzee et  al. 2014). in consistent with our data, nal presented a comparable elimination half-life (3.7 hour) after iv administration to humans (aitkenhead et al. 1988). conclusions comparing xyl and xyl/nal treatments elucidated some of the pharmacodynamic effects of nal in xyl-sedated horses. from this, the addition of nal to xyl seemed to have remarkable advantages on the measured pharmacodynamic parameters (improved duration of sedation and analgesia, superior degree of sedation, and better effect on physiological parameters). in addition, ataxia was still acceptable and no considerable adverse events were encountered when nal was combined with xyl. the pharmacokinetics of nal also provided information about the most effective concentration of nal and the clearance rate; both could be used to calculate the effective nal infusion rate. such rate could be further evaluated as an adjunctive agent to xyl for prolonged sedation in horses that could extensively express the role of nal as a sedative adjuvant in horses. acknowledgments the authors would like to thank dr. hanem elgendy (lecturer of pharmacology, faculty of veterinary medicine, university of sadat city) for performing the required pharmacokinetic analysis. authors also thanks dr. mohamed elsunsafty (assistant lecturer of surgery, anesthesiology & radiology, faculty of veterinary medicine, university of sadat city) for his contributions in the study. 2003). ataxia was aggravated and prolonged when nal was combined with xyl. similar data were described by derossi and colleagues (derossi et  al. 2009) and ringer and colleagues (ringer et  al. 2012) after the administration of α 2 -agonists in combination with butorphanol versus α 2 -agonists alone. however, the resultant moderate ataxia was acceptable during various procedures in standing horses (solano et al. 2009, ruiz et al. 2015). for intestinal motility, nal seemed to have a significant effect on this parameter, which was elucidated by non-significant difference between xyl/nal and xyl. conversely, significantly reduced motility was recorded after the administration of a butorphanol bolus to horses (0.1 mg/kg iv) (sellon et  al. 2001). these findings might elucidate the milder effect of nal on intestinal motility than the commonly used agonist-antagonist opioid, butorphanol. the addition of nal to xyl did not exaggerate its depressant effects on sap, dap, map, and rr and even reduced the duration of these changes. this might be caused by a higher incidence of muscle tremors associated with nal administration that might result in a slight degree of sympathetic stimulation. administration of xyl resulted in significant reduction in rt. on the contrary, a significant increase in rt was reported by other authors (seo et al. 2011) after xyl administration. this discrepancy could be attributed to a higher xyl dose used in their study (seo et  al. 2011) than ours, which induced slightly increased peripheral vasoconstriction that might offset body heat loss (dugdale 2010). determining the plasma concentration of nal revealed that a peak concentration of 79.64 ng/ml was attained at 8 minutes after its administration, which coincided with the 15-minute 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10.12834/vetit.2607.16055.1 accepted: 31.08.2021 | available on line: 31.12.2021 1istituto zooprofilattico sperimentale del piemonte, ligura e valle d’aosta, via bologna 148, 10154 turin, italy. 2llp ‘kazakh research institute for livestock and fodder production’, st. zhandosova 51, almaty, kazakhstan. 3llp ‘scientific production center of microbiology and virology’, bogenbai batyr str., 105, almaty, kazakhstan. *corresponding author at: llp ‘scientific production center of microbiology and virology’, bogenbai batyr str. 105, almaty, kazakhstan. tel.: +7 870 58732835, e-mail: saule.daugalieva@mail.ru. chiara beltramo1, talgat karymsakov2, alessandro dondo1, berik aryngaziyev2, aida daugaliyeva2, katia varello1, pier luigi acutis1, saule daugaliyeva3* and simone peletto1 keywords cattle, normalization, real-time qpcr, reference genes, small intestine. summary the use of reference genes is commonly accepted as the most reliable approach to normalize qrt-pcr and to reduce possible errors in the quantification of gene expression. the aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. these sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by mycobacterium avium subsp. paratuberculosis, agent of paratuberculosis. we employed ten pcr assays for commonly used reference genes belonging to various functional classes and then determined their expression stability. the most stable genes were rpl13a and hmbs, followed by tfrc and b-act. normfinder analysis provided similar results with b-act as the best reference gene, followed by rpl13a and tfrc. this validated gene panel may be useful for studies on paratuberculosis aiming to identify genes differentially expressed by qrt-pcr. validation of suitable reference genes for quantitative expression analysis by qpcr in bovine terminal ileum and ileocecal valve reference genes varies in different tissues (lisowski et  al. 2008) and should be evaluated a priori to avoid biased findings (bas et al. 2004). accordingly, a proper evaluation of several reference genes should be performed to validate which and how many genes are needed before any gene expression study (bustin et al. 2009, huggett et al. 2005). the use of a single reference gene is strongly discourage and the use of genes traditionally considered stable has to be carefully considered. vandesompele and colleagues (vandesompele et  al. 2002) demonstrated that the use of a single reference gene can imply an error of up to 20-fold in expression data. additionally, the expression of genes assumed to be stable (i.e., gapdh and b-act) can vary considerably (bustin 2000). the aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. these sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by mycobacterium avium subsp. paratuberculosis (map), the agent of paratuberculosis, a chronic and debilitating introduction quantitative pcr (qpcr) is the election technique for accurate expression profiles determination of selected gene of interest being characterized by high sensitivity, specificity and reproducibility (bustin et  al. 2009). nevertheless, several variables can concur to misleading conclusions, which include biological and technical variance in the gene expression analysis: the amount and quality of the starting material, the rna integrity, the efficiency of retrotranscription and pcr reaction, the differences in biological samples. moreover, the presence of pseudogenes, alternative splicing, health status, storage conditions of the samples may affect the level of gene expression and the pcr efficiency (rekawiecki et  al. 2012). the selection of suitable reference genes is crucial to mitigate any variations arising during the experiment, because they are assumed to be characterized by constitutive and uniform expression level in all the analyzed samples, regardless of tissue differentiation, treatments or experimental design (mcneill et  al. 2007). however, several studies indicate that the expression of 210 xxxxxxxxxxxxxxxx beltramo et al. veterinaria italiana 2021, 57 (3), 209-214. doi: 10.12834/vetit.2607.16055.1 using the bioanalyzer 2100 (agilent technologies). cdna synthesis was carried out starting from 1 µg of rna using the quantitect reverse transcription kit (qiagen). reference genes, primer design and qpcr ten genes usually used as references in qpcr experiments were selected as candidate normalizers (table i). they were tested on a serial 10-fold-dilutions of pooled cdna to determine primer efficiency, slope value and correlation coefficient. cdnas of the eight samples were pooled and 10-fold diluted until 1:100,000 then a five-point calibration curve was constructed for each gene. the qpcr reactions were performed in 25 µl-total volume containing: 1x itaq universal sybr green supermix (biorad), 11 µl of nuclease free water, 200 nm of each primer and 1 µl of cdna. the reactions were set up in triplicates and loaded on a mx3005p qpcr system (agilent technologies) with the following thermal cycle: 95 °c for 5 min; 44 cycles of 95 °c for 30 sec, 56 °c for 45 sec and 72 °c for 30 sec; a melting curve analysis of 95  °c for 1 min and 56  °c for 30  sec. amplification directly from genomic dna and no-template control were included to recognize / granulomatous enteritis of ruminants, characterized by persistent diarrhea, progressive wasting, and eventual death (coussens 2001, dorshorst et  al. 2006, fanelli et al. 2020). materials and methods samples collection, rna extraction and cdna synthesis bovine tissue samples were collected during routine slaughtering procedures from eight holstein friesian cows, at least 2 years old. the distal tract of small intestine and the ileocecal valve were immediately sampled from each animal, rinsed in sterile pbs and preserved in rnalater (qiagen). all samples were stored at 80 °c before rna extraction. total rna was extracted from 50 mg of bovine intestine tissue in rnalater with the rneasy lipid tissue mini kit (qiagen), following the manufacturer’s instructions and performing the optional on-column dnase digestion with the rnase-free dnase set (qiagen). concentration and purity of the rna were determined by spectrophotometer and fluorescent measurements, while rna integrity was evaluated table i. details of candidate reference genes assays. gene symbol gene name function accession no. reference primer sequence amplicon size b-act beta-actine cytoskeletal structural protein ay141970.1 liu et al. 2016 f:gcacaatgaagatcaagatcatc 173 r:ctaacagtccgcctagaagca gapdh glyceraldehyde 3-phosphate dehydrogenase glycolytic enzyme, oxidoreductase in glycolysis and gluconeogenesis u85042.1 reist et al. 2003 f: gtcttcactaccatggagaagg 197 r: tcatggatgaccttggccag hmbs hydroxymethyl-bilane synthase heme biosynthesis nm_001046207.1 lecchia et al. 2012 f: gagaggaatgaagtggacctag 110 r: gcatcataggggctctccc pgk1 phosphoglycerate kinase 1 glycolytic enzyme, polymerase α cofactor protein nm_001034299.1 modesto et al. 2013 f: ggaagggaagggaaaagatgc 92 r: tcccctagcttggaaagtga ppia peptidylprolyl isomarase a (cyclophilin a) accelerate the folding of proteins nm_178320.2 de maria et al. 2010 f: gccccaacacaaatggttcc 95 r: ccctctttcaccttgccaaag rpl13a ribosomial protein l13a member of ribosome proteins nm_001076998.2 modesto et al. 2013 f: ccctggaggagaagagaaagg 104 r: aattttcttctcgatgttcttttcg rps9 ribosomial protein s9 member of ribosome proteins dt860044.1 janovick-guretzky et al. 2007 f: cctcgaccaagagctgaag 62 r: cctccagacctcacgtttgttc sf3a1 splicing factor 3 subunit 1 structural component of the splicing system nm_001081510.1 lecchia et al. 2012 f: ccttaccatgcctactaccgg 144 r: cacttgggcttgaaccttctg tfrc transferrin receptor transferrin receptor nm_001206577.1 modesto et al. 2013 f: tggaaaaatcagttttgctgaa 124 r: gtccaaaaactggaagatttgc ywhaz tyrosine 3-monooxygenase signal transduction by binding to phosphorylated serine residues on a variety of signaling molecules nm_174814.2 modesto et al. 2013 f: ctgaactcccctgagaaagc 165 r: ctgcttcagcttcgtctcct 211 beltramo et al. xxxxxxxxxxxxxxxx veterinaria italiana 2021, 57 (3), 209-214. doi: 10.12834/vetit.2607.16055.1 90%, respectively. cq values for the reference genes varied from 18 to 33. data analysis with genorm ranked the reference genes according to their m-values in decreasing order (figure 1, table ii). all the genes reached m-values less than 1.5 except for ywhaz and gapdh. most of the genes showed stable expression with m-values less than 1. for adequate data analysis, reference genes with m-values less than 1 should be use for a comparison of minor differences in gene expression (hellemans et  al. 2007). the most stable genes were rpl13a and hmbs, followed by tfrc and b-act, while the less stable were gapdh and ywhaz. normfinder analysis provided similar results with b-act as the best reference gene, followed by rpl13a and tfrc; ywhaz and gapdh showed the highest sd values (figure 2). normfinder showed that the lowest number of reference genes for the best evaluation of gene expression is three: in fact, the accumulated sd from b-act, rpl13a and tfrc was 0.4446 and its increment with the addition of the fourth gene pgk1 was negligible (figure 3). discussion rna yields and quality were adequate for qpcr and similar to results obtained by other studies on bovine intestinal rna (weber et  al. 2016, lecchi et  al. 2012). a good quality rna is fundamental for gene expression analysis to avoid wrong conclusion, but it could be a problem for matrices such as intestine. it is very important the preservation of the tissue immediately after the sampling: for this reason, samples are often immediately frozen in liquid nitrogen (hempel et  al. 2016, de luca et  al. 2014, weber et  al. 2016). the samples for this study could not be snap-frozen, but rna was preserved by immediately adding rnalater solution that avoids rna degradation with acceptable quality and yield for qpcr, as it was done also by lecchi and colleagues (lecchi et al. 2016). to exclude genomic dna contamination. stability of the ten candidate reference genes was evaluated by algorithms genorm and normfinder available in the genex software (biomcc). genorm sequentially eliminates the gene that shows the highest variation relative to all other genes, generating an m-value that should be lower than 1.5 (vandesompele et al. 2002). specifically, the lower is the m-value, the more stable is the gene. normfinder compares the individual genes to a global average expression of all the genes in all samples, estimating a standard deviation (sd) for each reference gene. it also calculates the accumulated sd by using multiple reference genes: in this case the random variation among their expression is partially cancelled by sd reduction. plotting the sd from different number of reference genes according to their stability, allows to identify a minimum in the accumulated sd, indicating the number of reference genes giving the lowest sd. results the concentration of the rna extracted from the eight samples by fluorimeter analysis ranged from 550 ng/µl to 1,000 ng/µl. rna purity was assessed as a260/280 ratio (range: 1.5-2.0) and a260/230 ratio (range: 1.0-1.7). rna integrity was checked by determining rin values, which were between 6.6 and 7.9. qpcr experiments showed that all the reference geneassays were expressed in the eight samples and provided a single sharp peak in the melting curve profile corresponding to an amplicon of expected size on agarose gel. only the melting curve for sf3a1 gene assay showed a secondary peak for the last three dilution points, due to primer dimer formation. correlation coefficients and efficiencies for all the standard curves were higher than 0.97 and above table ii. gene expression stability measures determined by genorm for each candidate reference gene. gene name m-value ywhaz 1.959 gapdh 1.712 ppia (cyclophilin a) 1.364 rps9 1.153 pgk1 0.799 b-act 0.714 tfrc 0.643 hmbs 0.552 rpl13a 0.552 genes 2.2 m -v al u e (a n ti l o g 2) 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 rpl13a hmbs tfrc b-act pgk1 rps gapdh ppia ywhaz figure 1. ranking of candidate reference genes according to their expression stability by genorm after stepwise exclusion of the worst scoring genes. 212 xxxxxxxxxxxxxxxx beltramo et al. veterinaria italiana 2021, 57 (3), 209-214. doi: 10.12834/vetit.2607.16055.1 genes in a collection of 33 different bovine tissues comprising small intestine, cecum and colon. they analyzed the expression of seven genes, and sf3a1, hmbs, b-act were the three most stable. this study differed from the previous because it focused on a specific portion of the small intestine, near the ileocecal valve, and on the ileocecal valve itself. our results confirmed b-act and hmbs as optimal reference genes; sf3a1 was also included in our gene panel, but it was discarded for the presence of an unspecific peak in the melt-curve profile of the higher dilution points, probably due to primer dimer formation. this result is apparently in contrast with the results by lecchi and colleagues (lecchi et  al. 2016), but in that study the standard curve for the qpcr analysis was performed with a 4-fold serial dilution, while, in our study, 10-fold serial dilutions were used. this technical difference in the experiment setup results in cqs  >  32 at the last dilution points, and the sf3a1 expression is probably low in the studied intestinal sites, thus resulting in unspecific amplification products at the highest dilutions. so sf3a1 is not helpful as reference gene for qpcr analysis of genes with a low level of expression in bovine terminal ileum tract and ileocecal valve. our analysis demonstrated that ywhaz and gapdh are not suitable for optimal normalization, but they were instead acceptable for lecchi and colleagues (lecchi et  al. 2012). it is important to take into consideration that these conclusions derived by the analysis of the stability of reference genes on 33  different bovine tissues together and are not focused on a particular one, as in this work. considering the gastrointestinal tract, gapdh resulted stable in abomasums, duodenum, jejunum and cecum of lactating and not lactating cows (connor et  al. 2010): it could be considered a good reference gene for gene expression analysis in intestinal tissues, but this conclusion is not true for for the set up of optimal qpcr conditions, the ten reference genes were tested on pooled cdna samples and genomic dna. the selected primer pairs spanned two exons, in order to generate specific melt-curve profile for cdna and genomic dna amplifications and allow the identification of possible dna contamination in the samples. the presence of a single peak on the melt-curve profile and of a single band of the expected size on agarose gel electrophoresis confirmed the gene specific amplifications (peletto et al. 2011). the identification of a list of appropriate reference genes is basilar for a proper determination of gene expression, to reduce the variability introduced during the different steps of the experiment (sahu et  al. 2018). the ideal reference geneshould show a stable expression in the tissue under different experimental conditions, so it is necessary to identify and validate reference genes for each type of sample and for each condition (bustin et al. 2009). genes such as gapdh, b-act and 18s  rrna have been used as reference genes in a great number of studies in the last decades without validation, but their expression can vary considerably, influencing the validity of the results (thelling et  al. 1999, vandesompele et  al. 2002). normfinder identified b-act, rpl13a and tfrc as the best reference genes for qpcr analyses in the terminal tract and ileocecal valve of the bovine small intestine, while for genorm rpl13a and hmbs, followed by tfrc and b-act, were the most stable (figures 2 and 3). both the software gave similar results, with small differences in the ranking of the reference genes, due to the different algorithms used to calculate variability. therefore, the comparison of reference gene rankings from different software/algorithms is advisable for robust results (modesto et al. 2012). the results of this study partially confirmed the conclusions by lecchi and colleagues (lecchi et  al. 2012), regarding the evaluation of suitable reference genes 2.8 sd 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 rpl13a hmbs tfrc b-act pgk1 rps gapdh ppia ywhaz figure 2. ranking of candidate reference genes according to their expression stability by normfinder. no. of genes 0.60 a cc . s d 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 1 2 3 4 5 6 7 8 9 figure 3. normfinder: accumulated standard deviation (sd) for the determination of the optimal number of reference genes. 213 beltramo et al. xxxxxxxxxxxxxxxx veterinaria italiana 2021, 57 (3), 209-214. doi: 10.12834/vetit.2607.16055.1 independently from the infection by histomonas meleagridis (mitra et al. 2016). as reported by several studies, the expression stability of genes to be used as reference genes needs to be carefully validated to ensure reliable data, and the use of software such as genorm and normfinder could help in the selection of suitable genes for each specific sample type/experimental condition. an universal reference gene did not exist and changing sampling site or experimental condition could lead to different conclusions: actually, not all the reference genes previously identified in the bovine small intestine were confirmed as stable in the sampling sites of our study. on the basis of our results, b-act, rpl13a, tfrc and hmbs are stable reference genes for normalization of gene expression data in the terminal tract of the bovine small intestine and ileocecal valve. this validated gene panel may be useful for studies on paratuberculosis aiming to identify differentially expressed genes by quantitative pcr. acknowledgements this research was funded by the italian ministry of health (ricerca corrente 2014 grant izs plv 10/14 rc) and by the science committee of the ministry of education and science of the republic of kazakhstan (grant no. ap09259133). the ileocecal tract. so, these evidences highlighted the importance of stability analysis of the reference genes, in order to take in account all the possible biological variation related to the expression level of genes for normalization. in this study, the analysis on the selected samples identified other two stable genes, tfrc and rpl13a. these genes are not frequently used as reference genes for gene expression analysis in bovine samples and there is few information in literature. both of them were chosen for stability analysis in bovine neutrophils: rpl13a showed a stable expression in these cells (crookenden et  al. 2017), while tfrc was discarded from subsequent analyses because of a high standard deviation (vorachek et al. 2013). in a recent gene expression study aiming to identify putative biomarkers in map-infected cattle, tfrc resulted up-regulated in bovine whole-blood, suggesting caution in the use of this gene as reference gene. also, the use of rpl13a had to be carefully considered, because rrna gene expression may not be a good estimation of the total mrna (vandesompele et  al. 2002), so the exclusive use of rrna genes as reference genes should be avoided. analyses of expression stability on somatic cells from goat milk (modesto et  al. 2013) and ovine whole-blood (peletto et  al. 2011) defined tfrc and rpl13a as the least reliable controls. in contrast, the high stability of tfrc and rpl13a was 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mcneill r.e., miller n. & kerin m.j. 2007. evaluation and validation of candidate endogenous control genes for real-time quantitative pcr studies of breast cancer. bmc mol biol, 8, 107. mitra t., bilic i., hess m. & liebhart d. 2016. the 60s ribosomal protein l13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models. vet res, 47, 105. modesto p., peletto s., pisoni g., cremonesi p., castiglioni b., colussi s., caramelli m., bronzo v., moroni p. & acutis p.l. 2013. evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-pcr in somatic cells from goat milk. j dairy sci, 96, 7932-7944. peletto s., bertuzzi s., campanella c., modesto p., maniaci 281 veterinaria italiana 2022, 58 (3), 281-285. doi: 10.12834/vetit.2342.16499.1 accepted: 10.11.2021 | available on line: 31.12.2022 1department of animal biotechnology, college of veterinary science and a.h., sardarkrushinagar dantiwada agricultural university, sardarkrushinagar, gujarat, india. 2department of veterinary microbiology, college of veterinary science and a.h., sardarkrushinagar dantiwada agricultural university, sardarkrushinagar, gujarat, india. 3department of veterinary parasitology, college of veterinary science and a.h., sardarkrushinagar dantiwada agricultural university, sardarkrushinagar, gujarat, india. *corresponding author at: department of animal biotechnology, college of veterinary science and a.h., sardarkrushinagar dantiwada agricultural university, sardarkrushinagar, gujarat, india. e‑mail: drsushilmohapatra@gmail.com. sushil kumar mohapatra1*, bharat singh chandel1, mehul d. shrimali1, husen r. parsani3, sandipkumar sureshbhai patel2, harsad c. chauhan2, kishan kumar sharma2 and arunkumar chaturbhai patel2 keywords bovine ephemeral fever, buffaloes, cattle, seroprevalence, vero cells. summary bovine ephemeral fever (bef) virus (befv) is an arthropod borne virus that causes bovine ephemeral fever or three-day sickness in cattle and buffaloes. this is the first report on seroprevalence of bef in cattle and buffaloes in gujarat, india. total of 92 animals, 78 cattle and 14 buffaloes from three regions (districts) of gujarat state of india, were screened for the presence of anti-bef antibodies. a total of 27 out of 92 animals were found positive and overall seroprevalence detected was 29.34% (95% ci 20.0-38.6%). a total of 19  out of 78  cattle and 8  out of 14 buffalo’s samples were found positive befv antibodies. species-wise seroprevalence in cattle and buffaloes was 24.35% (95% ci 14.8-33.8%) and 57.1% (95% ci 31.2-83.0%), respectively. there was a statistically significant (p < 0.05) species effect based on the seroprevalence. in cattle, location-wise seroprevalence was observed to be 26.82% (95% ci 13.2-40.3%) and 21.62% (95% ci 8.3-34.8%) in navsari and banaskantha districts, respectively. the effect of location is not statistically significant (p < 0.05). cytopathic effect of vero cells was characterized by rounding, granulation of the cytoplasm within 48-72  hrs of post infection. this was the first report demonstrating the presence of befv in gujarat state. seroprevalence of bovine ephemeral fever virus in gujarat state of india onset and rapid recovery, lasting only 1-3 days, but there are reports of prolonged paralysis and ataxia in some animals following the acute phase of infection (walker and klement 2015). in indian conditions, this disease mostly gets unnoticed due to the short course of viremia in affected animals and thus it is usually treated on symptomatic basis, without attempting a confirmatory diagnosis. the disease has been recorded in gujarat state of india (patel et al. 1993, lunagariya et al. 2015) but neither etiological agent or anti-befv antibodies have been demonstrated. though there has been enough documentation of the presence of competent vector in this region as the vector for bluetongue virus (dadawala et  al. 2012). keeping this view, need of initial work on this disease had been felt. in the present study, an assessment of befv and introduction bovine ephemeral fever (bef), or three-day sickness, is a disease of cattle and buffaloes caused by ephemerovirus of family rhabdoviridae. virus is transmitted by arthropod vectors culicoides spp. and falls under the arbovirus category. though having low mortality rate, bef virus (befv) infections cause significant morbidity. bef associated economic losses are related to poor milk yield, infertility and abortion. sudden onset of fever, as high as 41 °c with severe drop in milk production, anorexia, lethargy, salivation, serous nasal discharge, recumbence, muscle stiffness, lameness and dyspnoea are some noticeable clinical signs associated with bef virus (befv) infection (walker and klement 2015, lee 2019). usually, the disease is characterized by rapid 282 veterinaria italiana 2022, 58 (3), 281-285. doi: 10.12834/vetit.2342.16499.1 seroprevalence of bovine ephemeral fever virus mohapatra et al. elisa for detection of bovine ephemeral fever antibodies elisa was performed by using qualitative antibodies elisa kit (catalogue no.: mbs109097; mybiosource.com, san diego, ca 92195-3308, usa) according to manufacturer’s instructions (https:// www.mybiosource.com/bovine-ovine-elisa-kits/ ephemeral-fever-antibodies-anti-ef/109097). the kit was based on sandwich enzyme immunoassay principle to qualitatively analyze antibodies in bovine serum, plasma or other biological fluids. all the serum samples including the positive and negative standards were loaded as 50 µl volume in the elisa plate. after that, 100 µl hrp conjugate were added and incubated for 1 hour at 37 °c. then, the chromogen solution was added followed by stop solution, strictly as per protocol. the plate was read at 450 nm using elisa reader and absorbance values were used to calculate the positive and negative results. culture of vero cell line and cytopathic effect study vero (african green monkey kidney cells) cell lines, kindly provided by indian veterinary research institute (ivri), izatnagar, up, india and propagated in dulbecco’s modified eagle’s medium (hyclone, italy) containing 10% fetal bovine serum (australian origin, gibco, usa) 50 µg/ml streptomycin and 50  u/ml penicillin (hyclone, italy) in a humidified co 2 incubator at 5% co 2 and 37 °c. centrifugation of blood was carried out for harvesting buffy coat. the buffy coats were frozen and thawed 3 times. harvested buffy coat (300 µl) was added to 25 cm2 cell culture flask containing vero cell monolayer. cells were incubated at 37 °c for virus adsorption. after 1 hour, the buffy coat was removed and fresh cell culture media added. cell culture flask with monolayer was incubated in co 2 incubator at 5% co 2 and 37 °c for 5 days and observed every 24 h intervals for the presence of cytopathic effect (cpe). statistical analysis seroprevalence rates found in each group were compared and statistical differences were analysed using chi-square test or fisher exact probability test. odds ratios (or) with 95% ci were calculated using binary logistic regression to determine the relationship of exposure variables including the species and region on the seroprevalence of bef virus. statistical analysis was carried out using online platform http://vassarstats.net/. the differences were considered to be statistically significant at p < 0.05. befv antibodies in cattle and buffalo in different places of gujarat state of india was carried out, including identification of anti befv antibodies, and investigating the adaptation of virus on the cell culture and cytopathic effect. materials and methods study location and sample collection the present study was conducted in banaskantha (24.085560° n, 72.144234° e), navsari (20.946701° n, 72.952034° e) and junagadh (21.522184° n, 70.457878° e) districts of gujarat, india during 2018 (figure 1). average annual rainfall is 632, 1,793 and 923 mm in banaskantha, navsari and junagadh districts, respectively. a total of 92 cattle and buffalo blood samples of whole blood in edta and serum were collected. the blood was collected from suspected animals. sampled animals which included 78 cattle and 14 buffaloes, had history of bef like symptoms (fever, anorexia, lameness, salivary discharge) and were not vaccinated with bef vaccine. sera were separated using routine protocol and stored at - 20 °c until tested. figure 1. different locations of sample collection for seroprevalence study of bovine ephemeral fever virus. locations were (a) banaskantha (24.085560° n, 72.144234° e), (b) junagadh (21.522184° n, 70.457876° e) and (c) navsari (20.946701° n, 72.952034° e) districts of gujarat, india. a b c 283veterinaria italiana 2022, 58 (3), 281-285. doi: 10.12834/vetit.2342.16499.1 mohapatra et al. seroprevalence of bovine ephemeral fever virus to cattle in banskantha district (or = 1.33; 95% ci 0.46-3.77), but the difference was not statistically significant (p < 0.05). as all buffalo samples were collected from one district (junagadh), hence statistics could not be applied for buffaloes. the infected vero cells showed specific cytopathic effect within 48 hrs of post infection (figure 2). cytopathic effect was characterized using an inverted optical microscope (olympus ix70, olympus, japan), by observing rounding, granulation of the cytoplasm, and finally detachment of vero cells from cell culture flask. detachment of cells was visible on the 3rd day of post infection. discussion in india, bef is an old disease recorded as early as 1919 in parts of punjab of un-partitioned india by meadows and thereafter in tamilnadu (iyer 1924). in recent past, the disease was reported in uttar pradesh (malviya and prasad 1977), himachal pradesh (prasad et  al. 1997) and also in the gujarat area (patel et  al. 1993, lunagariya et  al. 2015), but all these studies encompassed clinical or epidemiological picture of disease. the first direct documentation of the disease in india was claimed by pyasi and colleagues (pyasi et  al. 2020 ) where bef detection has been done by rt-pcr method. therefore, to best of our knowledge, this is the first report on seroprevalence in cattle and buffaloes in gujarat state of india. seroprevalence of befv antibodies in cattle was reported 15.7% in south korea (lim et  al. 2007), 13.6% in taiwan (liao et  al. 1998) and 7-10 % in uganda (nabukenya et al. 2014). in our study, seropositivity in cattle was found as 24.35%. this result was similar to the previous elisa based report by zhagawa and colleagues (zhagawa et al. 2016) where seropositivity was found as 23.2% for dairy and 13.7% for non-dairy breeds and ranging results a total of 78 cattle and 14 buffaloes sera were collected and screened for the presence of anti-befv antibodies (table i). twenty-seven out of 92 sampled animals were found positive in elisa, with an overall seroprevalence was detected of 29.34% (95% ci 20.0-38.6%). nineteen out of 78 cattle, and 8 out of 14 buffaloes samples, were found positive for bovine ephemeral fever antibodies. species-wise seroprevalence in cattle and buffaloes was 24.4% (95% ci 14.8-33.8%) and 57.1% (95% ci 31.2-83.0), respectively. we found that the likelihood of bovine ephemeral fever virus seropositivity was significant higher (p < 0.05) for buffaloes compared to cattle (or = 4.1; 95 % ci = 1.2-13.4). location-wise seroprevalence in cattle was investigated in two districts of gujarat, namely navsari and banaskantha districts. location-wise seroprevalence in navsari district and banaskantha district was found to be 26.8% (95% ci 13.2-40.3%) and 21.6% (95% ci 8.3-34.8%), respectively. cattle samples from navsari district were 1.33 times more likely to have antibodies against bovine ephemeral fever virus compared table i. sero-prevalence of bovine ephemeral fever among cattle and buffaloes in 3 districts of gujarat, india. districts banaskantha navsari junagadh total species bos indicus bos indicus bubalus bubalis number of samples 37 41 14 92 elisa positive 8 11 8 27 elisa negative 29 30 6 65 prevalence (%) 21.6 26.8 57.1 29.3 95% ci 8.3-34.8 13.2-40.3 31.2-83.0 20.0-38.6 figure 2. cytopathic effect of bovine ephemeral fever virus on vero cell lines. visible cytopathic effect in vero cells (b); control vero cells (a). a b 284 veterinaria italiana 2022, 58 (3), 281-285. doi: 10.12834/vetit.2342.16499.1 seroprevalence of bovine ephemeral fever virus mohapatra et al. to infected cells by virus leading to different morphological changes of cell. cytopathic effect of befv was observed in vero cell line (chen et  al. 2010, bakhshesh and abdollahi 2015, zhagawa et al. 2017) and in madin-darby bovine kidney (mdbk) cell line (chen et  al. 2010). higher efficiency of replication and a greater apoptosis induction ability of befv in vero cells compared to mdbk cell line was observed (chen et  al. 2010). in bakhshesh and abdollahi study, vero cells showed cytopathic effect including rounding of cells, chromatin condensation followed by detachment from flasks on the fifth day (bakhshesh and abdollahi 2015). similar kind of morphological changes were observed in our study in vero cells with detachment of cells on the 3rd day of post infection. these results will be useful to increase the awareness among veterinary professionals about the presence of bef in the studied areas, to take up proper therapeutic and preventive measures. the results will be helpful to increase the awareness among veterinary professionals about the presence of bef in the areas concerned, to take up proper therapeutic and preventive measures. from 12 to 26% depending on different provinces of saudi arabia. though a wide range of seropositivity was observed in different regions of china, where 0-81% animals were found positive in large scale four years study (li et al. 2015). in israel, aziz-boaron and colleagues (aziz-boaron et al. 2015) recorded in buffaloes a seroprevalence of 13.79% using serum neutralization test, and lower than that found in our study. we found that the likelihood of bovine ephemeral fever virus seropositivity was significantly higher (p < 0.05) for buffaloes, compared to cattle. higher seroprevalence in buffaloes was probably due to low number of samples screened in junagadh district (average annual rainfall is 912  mm). in our study, we have screened cattle samples from two districts namely, banaskantha (district located in northern gujarat; average annual rainfall is 632 mm) and navsari (district located in southern gujarat; average annual rainfall is 1,793 mm) of gujarat state during the year 2018. cattle samples from navsari region were 1.33 times more likely to have antibodies against bovine ephemeral fever virus compared to cattle in banskantha region. no significant (p < 0.05) region effect was observed. the cytopathic effect (cpe) is induced by apoptosis causing damage 285veterinaria italiana 2022, 58 (3), 281-285. doi: 10.12834/vetit.2342.16499.1 mohapatra et al. seroprevalence of bovine ephemeral fever virus aziz-boaron o., brettschneider s., king r., gelman b. & klement e. 2015. seroprevalence of bovine ephemeral fever virus in domesticated and wildlife species during epidemic and inter-epidemic periods (2000-2009) in israel. transbound emerg dis, 62 (2), 183-187. bakhshesh m. & abdollahi d. 2015. bovine ephemeral fever in iran: diagnosis, isolation and molecular characterization. j arthropod borne dis, 9 (2),195-203. chen c.y., chang c.y., liu h.j., liao m.h., chang c.i., hsu j.l. & shih w.l. 2010. apoptosis induction in befv-infected vero and mdbk cells through src-dependent jnk activation regulates caspase-3 and mitochondria pathways. vet res, 41, 15. dadawala a.i., biswas s.k., rehman w., chand k., de a., mathapati b.s., kumar p., chauhan h.c., chandel b.s. & mondal b. 2012. isolation of bluetongue virus serotype 1 from culicoides vector captured in livestock farms and sequence analysis of the viral genome segment-2. transbound emerg dis, 59, 361-368. iyer v. 1924. three days’ fever of cattle-‘bovine influenza’. vet rec, 4, 277-279. lee f. 2019. bovine ephemeral fever in asia: recent status and research gaps. viruses, 11, 412. li z., zheng f., gao s., wang s., wang j., liu z., du j. & yin h. 2015. large-scale serological survey of bovine ephemeral fever in china. vet microbiol, 176 (1-2), 155-160. liao, y.k., inaba y., li n.j., chain c.y., lee s.l. & liou p.p. 1998. epidemiology of bovine ephemeral fever virus infection in taiwan. microbiol res, 153, 289-295. lim s.i., kweon c.h., tark d.s., kim s.h. & yang d.k. 2007. serosurvey on aino, akabane, chuzan, bovine ephemeral fever and japanese encephalitis virus of cattle and swine in korea. j vet sci, 8, 45-49. malviya h.k. & prasad j. 1977. ephemeral fever a clinical references and epidemiological study in cross bred cows and buffaloes. indian vet j, 54, 440-444. meadows d. 1919. notes on an ephemeral fever in indian cattle resembling south african ‘three days sickness’. vet j, 75, 138-140. patel p.r., suthar b.h., soni v.k., dangaria a.m. & prajapati c.b. 1993. epidemiology, clinical findings and treatment of ephemeral fever in buffalo (bubalus bubalis). in proceedings of the 1st international symposium on bovine ephemeral fever and related rhabdoviruses, beijing, august 1992. australian centre for international agricultural research proceedings, 44, 57-58. prasad b., manuja s., khishtwaria r.s., rao v.n. & singh r.j. 1997. clinical report of ephemeral fever in cattle. indian vet j, 74, 685-686. pyasi s., sahu b.p., sahoo p., dubey p.k., sahoo n., byrareddy s.n. & nayak d. 2020. identification and phylogenetic characterization of bovine ephemeral fever virus (befv) of middle eastern lineage associated with 2018-2019 outbreaks in india. transbound emerg dis, doi: 10.1111/ tbed.13531. nabukenya i., rubaire-akiiki c., olila d., ikwap k. & höglund j. 2014. ethnopharmacological practices by livestock farmers in uganda: survey experiences from mpigi and gulu districts. j ethnobiol ethnomed, 10, 1-14. walker p.j. & klement e. 2015. epidemiology and control of bovine ephemeral fever. vet res, 46, 124. zaghawa a., housawi f.m., al-naeem a., al-nakhly h., kamr a. & toribio r. 2016. risk analysis and seroprevalence of bovine ephemeral fever virus in cattle in the kingdom of saudi arabia. trop anim health prod, 48 (3), 487-92. zaghawa a.a., housawi f., al-naeem a., elsify a. & hegazy y.m. 2017. bovine ephemeral fever epidemics in kingdom saudi arabia: clinical, epidemiological and molecular investigation. j infect dev ctries, 11 (11), 854-860. 183 short communication obed salaan ladron de guevara1, juan jose perez-rivero2*, mario perez-martinez3, fernando ivan flores-perez4 and evangelina romero-callejas5 1 programa de maestria en ciencias de la produccion y de la salud animal, universidad nacional autónoma de méxico, méxico. 2 departamento de producción agrícola y animal, universidad autónoma metropolitana unidad xochimilco, méxico. 3 departamento de morfologia, universidad nacional autónoma de méxico, méxico. 4 facultad de ciencias agropecuarias, universidad autónoma del estado de morelos, méxico. 5 departamento de parasitologia, universidad nacional autónoma de méxico, méxico. *corresponding author at: departamento de producción agrícola y animal, universidad autónoma metropolitana unidad xochimilco, calzada del hueso 1100, colonia villa quietud, delegación coyoacán, ciudad de méxico, c.p. 04960, méxico. tel.: +52 55 54837000 ext. 3658, fax +52 55 54837238, e-mail: jjperez1_1999@yahoo.com, perivet.idea@gmail.com. veterinaria italiana 2019, 55 (2), 183-187. doi: 10.12834/vetit.443.2154.3 accepted: 30.01.2016 | available on line: 30.06.2019 parole chiave prevalenza, oocisti, medicina preventiva, coccidiosi del coniglio. riassunto la coccidiosi è una malattia parassitaria molto comune nei conigli causata da undici specie di eimeria considerevolmente variabili in morfologia e patogenicità. negli allevamenti di conigli da carne la coccidiosi è in grado di determinare gravi perdite economiche. questo studio si è posto come obiettivo quello di valutare la prevalenza stagionale di eimeria  spp. in aziende agricole a conduzione famigliare dello stato del messico. lo studio basato su un campionamento trasversale è stato condotto su giovani conigli (da 20 a 60 giorni) con storie cliniche di diarrea, provenienti da 3 comuni situati nella regione sud-orientale dello stato del messico; per identificare le oocisti sono state utilizzate le tecniche di flottazione e di mc master. nella regione sud-orientale dello stato del messico la prevalenza di eimeria è stata complessivamente del 48,3%. il valore massimo (88%) è stato registrato in amecameca in inverno, mentre quello minimo (5%) in primavera in temamatla. la prevalenza annuale di eimeria intestinalis, la specie più patogena, è stata dell'11,3%. la minima quantità di oocisti per grammo di feci (407) si è avuta in autunno a temamatla, mentre la più alta in estate a cocotitlan (18.330). la più alta prevalenza di eimeria spp. rilevata in autunno e in inverno rende possibile avviare in questa regione programmi di prevenzione su base stagionale. prevalenza stagionale dell’eimeria spp. in conigli di aziende agricole a conduzione famigliare nello stato del messico keywords prevalence, oocyst, preventive medicine, rabbit coccidiosis. summary coccidiosis is the most common parasitic disease in rabbits and it can be responsible for severe economic losses in broiler rabbit farms. eleven species of the eimeria genus, which vary considerably in terms of their morphology and pathogenicity, cause rabbit coccidiosis. the aim of this study was to evaluate the prevalence of eimeria spp. in backyard farms of state of mexico during the different seasons. cross-sectional sampling was performed in young rabbits (20 to 60 day old), with clinical histories of diarrhoea, from 3 municipalities located in the southeastern region of the state of mexico. flotation and mc master techniques were used to identify oocysts. the overall prevalence of eimeria in the southeastern region of the state of mexico was 48.3%. the highest prevalence was recorded during winter (88%) in amecameca, while the lowest prevalence during spring (5%) in temamatla. the annual prevalence of the eimeria intestinalis, the most pathogenic species, was 11.3%. the least amount of oocysts per gram of faeces (407) was obtained in autumn in temamatla, while the highest quantity was observed in summer in cocotitlan (18,330). the highest prevalence of eimeria spp. was detected in autumn and winter, making it possible to establish prevention programs in this region according to the season. eimeria spp. in broiler rabbit: seasonal prevalence in the backyard farms of the state of mexico 184 eimeria spp. in broiled rabbit backyard farms de guevara et al. veterinaria italiana 2019, 55 (2), 183-187. doi: 10.12834/vetit.443.2154.3 the highest rainfall recorded during summer (640.2 mm) and the lowest recorded rainfall during winter, with 20.3 mm4. this study took place in backyard farms, in which rabbits presented diarrhoea and where there had been no parasitological diagnosis for a minimum of 3 months. rabbits in the cocotitlan and temamatla farms were fed with alfalfa (medicago sativa) from the region and vegetable leftovers. in amecameca, rabbits received alfalfa and commercial balanced food. water was provided ad libitum in all farms. coccidiosis is one of the main parasitic diseases in domestic rabbits (yan et  al. 2013). the rabbits in this study were 20 to 60 day old. according to papeschi and colleagues (papeschi et  al. 2013), it is during this age when the disease is more commonly observed, with eimeria spp. infection appearing more frequently from 46 up to 109 days of life. rabbits were kept in collective cages in the farms. for this study, rabbits were placed in individual cages to allow the collection of faeces. after 24  hours they were placed back to the collective cages. during each of the 4 seasons, faecal samples were obtained from 1,399 rabbits in the fattening stage. six-hundred-fifty-one samples were collected in amecameca; 467 in cocotitlán, and 281 in temamatla. the samples were obtained from the individual cage floor (papeschi et  al. 2013), immediately stored in plastic bags, and then delivered to the laboratory. samples were kept at 4 °c until they were analysed for eimeria spp. oocysts by flotation and mc master techniques, using a 10× microscope objective (permin and hansen 1998, coudert et  al. 1995). moreover, oocysts were sporulated, placed in petri dishes with 2.5% potassium dichromate, at room temperature. the length and width of sporulated oocysts were measured using a light microscope (carl zeiss, oberkochen germany) equipped with an eyepiece micrometer with 20× objective to identify the species of eimeria in each of the different seasons, according to the morphometric criteria of kvicerova (kvicerova et al. 2008). prevalence was calculated with a confidence interval of 95% (ci 95). differences in prevalence between seasons and localities were compared using the kruskal wallis test. the mann whitney test was used to compare the number of oocysts per gram of faeces for each season. the statistical in the past few years, broiler rabbit production has grown considerably and has become an important source of animal protein in many developing countries, such as mexico. the success of the rabbit industry depends on many factors, especially good health conditions (varga 1982). coccidiosis is the most common parasitic disease in rabbits. it can cause serious health problems due to diarrhoea, reduction of food intake, and as a result, loss of body weight, affecting animal metabolism in general (freitas et  al. 2010). as such, coccidiosis is responsible for severe economic losses to the industry each year. rabbit coccidiosis is caused by at least 11  species of the genus eimeria (e): e.  coecicola, e.  exigua, e.  flavescens, e.  intestinalis, e.  irresidua, e.  magna, e.  media, e.  perforans, e.  piriformis, e.  stiedai and e.  vejdovskyi. these species vary considerably in terms of their morphology and pathogenicity (kvicerova et al. 2008). eimeria spp. transmission is faecal-oral, through contaminated food, water, caging, as well as other fomites, which serve as common means of spreading intestinal coccidian (kvicerova et  al. 2008). the morbidity and mortality of this disease, in growing rabbits, are between 60% to 90% (jing et  al. 2011, boag et al. 2001, cere et al. 1996). rabbit breeding is a livestock activity in full development, in the southeastern area of the state of mexico. the aim of this study was to evaluate the prevalence of eimeria spp. in broiler rabbit of backyard farms in this area and during the different seasons. in 2013, cross-sectional sampling was performed in an area of 21.67 km2, in the municipalities of amecameca (19° 06' 42.0" n 98° 45' 29.5" w), cocotitlán (19° 13' 58.3" n 98° 51' 23.7" w), and temamatla (19° 12' 04.6" n 98° 51' 50.3" w). all 3 municipalities are located in the southeastern part of the state of mexico. in amecameca, the annual average temperature is 14.1 ºc, ranging from 2.4 ºc during winter to 24  °c in spring1. in cocotitlan, the average was 13.6 ºc, going from 19 ºc during spring to 7  °c in winter2. in temamatla, the average was 12.5 ºc, with temperature as low as 24 ºc in autumn and as high as to 24 ºc in spring and summer3. in addition, in 2013 the annual accumulated rainfall in the state of mexico was 842.9 mm, with 1 instituto nacional para el federalismo y el desarrollo municipal/ secretaria de gobernacion de mexico. 2015. enciclopedia de los municipios y delegaciones de mexico. estado de méxico. amecameca. http://www.inafed.gob.mx/work/enciclopedia/emm15mexico/municipios/15009a.html. 2 instituto nacional para el federalismo y el desarrollo municipal/ secretaria de gobernacion de mexico. 2015. enciclopedia de los municipios y delegaciones de mexico. estado de méxico. cocotitlán. http://www.inafed.gob.mx/work/enciclopedia/emm15mexico/municipios/15022a.html. 3 instituto nacional para el federalismo y el desarrollo municipal/ secretaria de gobernacion de mexico. 2015. enciclopedia de los municipios y delegaciones de mexico. estado de méxico. temamatla. http://www.inafed.gob.mx/work/enciclopedia/emm15mexico/municipios/15083a.html. 4 comision nacional del agua/servicio meteorológico nacional (conagua) 2013. precipitación a nivel nacional y por entidad federativa. https://www. gob.mx/conagua. 185 de guevara et al. eimeria spp. in broiled rabbit backyard farms veterinaria italiana 2019, 55 (2), 183-187. doi: 10.12834/vetit.443.2154.3 analysis was conducted using the palaeontological statistics (past ), ver. 1.81 program, with a significant value of p < 0.05. the annual prevalence in the study area was 48.3% (ci95: 45.7-50.9%). in amecameca annual prevalence was 48.2% (ci95: 44.4-52.1%); in cocotitlan it was 42.8% (ci95: 38.3-47.3%), and in temamatla it was 57.6% (ci95: 51.9-63.4%). there was no significant (p  >  0.05) difference among these results, as shown in table i. prevalence varied significantly (p  <  0.05) during the different seasons: in spring it was 17.1% (ci95: 13.3-21.5%), in summer 48.1% (ci95: 43.0-53.8%), and 56.2% (ci95: 51.1-61.0%) in autumn and in winter 71.3% (ci95: 66.3-75.8%) with significant differences (p < 0.05) (table i and figure 1). regarding the concentration of oocysts per gram of faeces (opg), we observed that there were no table i. sampling scheme and annual prevalence by municipalities and season. sampling (n) positive (n) prevalence (%) ci 95 (%) amecameca spring 162 20 12 6-18 summer 136 59 43 35-51 autumn 213 111 52 49-58 winter 140 124 88 82-94 cocotitlan spring 128 37 29 22-36 summer 108 49 45 35-55 autumn 104 55 53 43-63 winter 127 59 47 39-55 temamatla spring 60 3 5 0-11 summer 82 50 61 51-71 autumn 64 48 75 65-85 winter 75 61 81 71-91 ci 95 = prevalence with a confidence interval of 95%. 4200 15900 750 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 0 10 20 30 40 50 60 70 80 90 100 amecameca cocotitlan temamatla p re va le n ce % 1053 18330 788 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 0 10 20 30 40 50 60 70 80 90 100 amecameca cocotitlan temamatla p re va le n ce % 1049 1121 407 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 0 10 20 30 40 50 60 70 80 90 100 amecameca cocotitlan temamatla p re va le n ce % 1822 1432 2883 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 0 10 20 30 40 50 60 70 80 90 100 amecameca cocotitlan temamatla p re va le n ce % a b c d prevalence opg autumn prevalence opg winter prevalence opg spring prevalence opg summer figure 1. prevalence of eimeria spp. identified in rabbit backyard farms in the state of mexico, méxico, by number of oocysts per gram of feces (opg), municipality and season of the year [spring (a), summer (b), autumn (c) and winter (d)]. significant differences were detected between prevalence during autumn and winter seasons (p < 0.05). kruskal wallis test. no significant differences (p > 0.05) were observed in opg between seasons. 186 eimeria spp. in broiled rabbit backyard farms de guevara et al. veterinaria italiana 2019, 55 (2), 183-187. doi: 10.12834/vetit.443.2154.3 but lower than those recorded during summer in the same municipality (18,330 opg). different eimeria species have been reported as the most prevalent ones: e. perforans in china (35.2%), followed by e. media (31%), and e. magna (28%) (jing et al. 2012). in saudi arabia, the most frequent were e.  coaecicola (70%), e. magna (60%), e.  perforans (60%), and e. media (55%) (abdel-baki and al-quraishy 2013). in the present study, the highest prevalence was found for e.  intestinalis  (11.3%) and e.  magna (11.0%). these species have been recognised as pathogenic. they are associated with the inflammatory lesions in intestines and diarrhoea in rabbits. e. perforans (8.0%), that causes decreased feed conversion, was also present (oncel et al. 2011). a previous study about domestic and wild rabbits indicated that parasite intensity and prevalence could change during the year due to the age of the host and the amount of ingested infective stages (pakandl et al. 2009). in this study, the highest prevalence (71.3%) was reported in winter, which is the coldest and driest season; during autumn, a warm and slightly damp season, prevalence was 56.2%. the lowest prevalence (17.1%) was found during spring, which was the warmest season. similar results have been reported in europe, where the number of cases increases during spring, autumn, and winter, especially in young individuals. susceptibility to infection is determined by geographic location, hygienic conditions, and population density. since a higher number of individuals in a given area increases stress, it therefore, decreases the effectiveness of the immune system (perez-martinez and betancourt 2010, grez et al. 2003). this survey consistently found presence of more than 2 eimeria spp. throughout the samplings. only significant differences (p  >  0.05) when comparing data pertaining to different seasons (figure 1). species of eimeria were found in different municipalities, and were identified on the basis of their morphology. seven species of eimeria were found in all municipalities, 3 of them in 2  municipalities, and e. flavescens in amecamenca only. eimeria intestinalis, the most pathogenic species (oncel et  al. 2011, coudert et  al. 1995), was found in 14.1% of faecal samples (ci95: 11.5-16.8%) from amecamenca, in 4.0% (ci95:  2.3-5.9%) from cocotitlan, and in 16.7% (ci95: 12.4-21.1%) from temamatla (table ii). in a survey conducted in china, coccidian oocyst prevalence was 61.4% in farms with less than 1,000 rabbits (jing et  al. 2012). likewise, in saudi arabia, prevalence of eimeria spp. was reported to range between 73% to 90% (el-shahawi et al. 2012). in both studies, the annual prevalence was greater than the one reported in the present study, although, in terms of seasonal prevalence/year, the results are similar. in this work the amount of opg was also very variable: the maximum amount of opg ranged between 407 and 18,330 with the smaller amount being lower than the one reported in china (jing et  al. 2012) in farms with less than 1,000 rabbits (23,514 opg), where coccidiosis occurs as subclinical in the animals. it has been reported that there is a need for an exposure equivalent to 100,000 opg to produce 80% of mortality. at the same time, when exposed in an experimental way to 50,000 e. stiedai sporulated oocysts for 32 days, rabbits showed a decrease in weight gain (freitas et al. 2010, pakandl et al. 2009). likewise, the amount of opg found by jing et al. (2012) in china (14,252 opg) was similar to those reported in cocotitlan during spring (15,900 opg), table ii. number of rabbits infected with one or more eimeria spp., according to morphometric characteristics, by municipality and season. eimeria spp. (pathogenicity)* oocyst average length × width (µm) amecameca (n = 651) cocotitlan (n = 467) temamatla (n = 281) e. intestinalis (high) 25×17.6 (22.5-27.5×12.5-22.7) 92 sp,su,a,w 19 a,w 47 su,a,w e. vejdovskyi (slight) 32.5×21.2 (32.5×17.5-25) 2 sp 11 sp 16 su e. coecicola (none) 35×19.7 (35×15-22.5) 77 w 44 sp,a 18 su e. stiedai (mild-high) 37.5×18.7 (37.5×17.5-20) 6 w 6 sp 29 su e. media (mild) 30×17.5 (30×17.5) 17 su,a 7 su e. perforans (slight) 22.5×16.2 (20-25×12.5-20) 32 sp,w 73 su,a,w 7 su e. magna (mild) 37.5×22.5 (37.5×20-25) 45 su 34 sp,su 72 w e. exigua (slight) 15×15 (12.5-17.5×12.5-17) 67 su,a,w 44 a e. irresidua (mild) 38.7×22.5 (37.5-40×20-25) 45 a 2 sp 61 sp,a e. piriformis (mild) 30×17.5 (30×17.5) 17 w 19 w e. flavescens (high) 30×25 (30×25) 6 w numbers in parentheses show the length × width range of different eimeria spp. oocyst. *pathogenicity, according to the criteria described by kvicerova and colleagues (kvicerova et al. 2008). sp = spring; su = summer; a = autumn; w = winter. 187 de guevara et al. eimeria spp. in broiled rabbit backyard farms veterinaria italiana 2019, 55 (2), 183-187. doi: 10.12834/vetit.443.2154.3 this study revealed a significant prevalence throughout the year of different types of eimeria  spp., in backyard farms located in the southeastern portion of the state of mexico. further studies are necessary to quantify the economic losses due to the presence of this parasite in the farms. in this study, results showed highest prevalence during autumn and winter, making it possible to establish a prevention program envisaging improvements in hygiene and use of preventive treatment administrated on a seasonal basis. the farm in the temamatla municipality presented a single type of coccidian, during spring. this information is consistent with the one reported by el-shahawi and colleagues (el-shahawi et  al. 2012), jing and colleagues (jing et al. 2012), and oncel and colleagues (oncel et  al. 2011). in all these studies, mixed infections with 3 or more different eimeria spp. were very common. poor hygienic conditions, which may occur when rabbits are in collective cages on the floor most of the time, with the presence of alfalfa and vegetable waste, facilitate contamination and the emergence of eimeria infection (jing et  al. 2012, pakandl et al. 2009). abdel-baki a.s. & al-quraishy s. 2013. prevalence of coccidia (eimeria spp.) infection in domestic rabbits, oryctolagus cuniculus, in riyadh, saudi arabia. pakistan j zool, 45, 1329-1333. abu-akkada s.s., oda s.s. & ashmawy k.i. 2010. garlic and hepatic coccidiosis: prophylaxis or treatment? trop anim health prod, 42, 1337-1343. boag b., lello j., fenton a., tompkins d.m. & hudson p.j. 2001. patterns of parasite aggregation in the wild european rabbit (oryctolagus cuniculus). int j parasitol, 31, 1421-1428. cere n., humbert j.f., licois d., corvione m., afanassieff m. & chanteloup n. 1996. a new approach for the identification and the diagnosis of eimeria spp. media parasite of the rabbit. exp parasitol, 82, 132-138. coudert p., licois d. & drouet-viard f. 1995. eimeria spp. and isospora. eimeria spp. species and strains of rabbits. in biotechnology. guidelines on techniques in coccidiosis research (eckert j., braun r., shirley m.w. & coudert p. eds). office for official publications of the european communities. luxembourg, 52-73. el-shahawi g.a., el-fayomi h.m. & abdel-haleem h.m. 2012. coccidiosis of domestic rabbit (oryctolagus cuniculus) in egypt: light microscopic study. parasitol res, 110, 251-258. freitas f.l.c., yamamoto b.l., freitas w.l.c., almeida k.s., machado r.z. & machado c.r. 2010. eimeria stiedai: metabolism of lipids, proteins and glucose in experimentally infected rabbits, oryctolagus cuniculus. braz j vet pathol, 3, 37-40. grez v., voza t., chabaud a. & landau i. 2003. coccidiosis of the wild rabbit (oryctolagus cuniculus) in france. parasite, 10, 51-57. references jing f., yin g., liu x., suo x. & qin y. 2012. large-scale survey of the prevalence of eimeria spp. infections in domestic rabbits in china. parasitol res, 110, 1495-1500. kvicerova j., pakandl m. & hypsa v. 2008. phylogenetic relationships among eimeria spp. (apicomplexa, eimeriidae) infecting rabbits: evolutionary significance of biological and morphological features. parasitology, 135, 443-452. oncel t., gulegen e., senlik b. & bakirci s. 2011. intestinal coccidiosis in angora rabbits (rryctolagus cuniculus) caused by eimeria intestinalis, eimeria perforans and eimeria coecicola. yyu vet fak derg, 22, 27-29. pakandl m. 2009. coccidia of rabbit: a review. folia parasitol, 56, 153-166. papeschi c., fichi g. & perrucci s. 2013. oocyst excretion pattern of three intestinal eimeria species in female rabbits. world rabbit sci, 21, 77-83. pérez-martínez m. & betancourt-alonso m.a. 2010. coccidiosis hepática en el conejo: aspectos ambientales y clínico-patológicos. ciencia ergo sum, 17, 269-276. permin a. & hansen j. 1998. the epidemiology, diagnosis and control of poultry parasites. in fao animal health manual. rome, fao. varga i. 1982. large-scale management systems and parasite polulations: coccidia in rabbits. vet parasitol, 11, 69-84. yan w., wang w., wang t., suo x.,qian w., wang s. & fan d. 2013. simultaneous identification of three highly pathogenic eimeria species in rabbits using a multiplex pcr diagnostic assay based on its1-5.8srrna-its2 fragments. parasitology, 193, 284-288. 169 veterinaria italiana 2019, 55 (2), 169-172. doi: 10.12834/vetit.887.4400.3 accepted: 29.03.2016 | available on line: 30.06.2019 summary the purpose of this study was to examine the prevalence of antimicrobial resistance among 180 escherichia coli strains isolated from 200 wild pheasants caught in rural areas of the czech republic (eastern moravia) and slovakia (western region). the isolates were also classified into phylogenetic groups by the multiplex pcr method. our findings demonstrated that 130 strains were resistant to ampicillin (72%), 160 strains to cephalothin (89%), and 40 strains to tetracycline (22%). ten strains were found to be resistant to chloramphenicol and sulfamethoxazole/trimethoprim (5.6%). in turn, all strains were sensitive to cefoperazone/ sulbactam, ciprofloxacin, colistin, gentamicin, and piperacillin/tazobactam. ten of the 180 isolates (5.6%) exhibited multi-resistant phenotypes, including resistances against betalactams, aminoglycosides, tetracyclines, sulphonamides, and chloramphenicol. as far as we know, this is the first report describing antimicrobial resistance in e. coli from pheasants. riassunto scopo di questo studio è stato esaminare la prevalenza della resistenza antibiotica nei confronti di 180 ceppi di escherichia coli isolati da 200 fagiani selvatici catturati nelle aree rurali della repubblica ceca (moravia orientale) e slovacchia (regione occidentale). con pcr multiplex gli isolati sono stati classificati anche in gruppi filogenetici. i risultati hanno dimostrato che 130 ceppi erano resistenti all'ampicillina (72%), 160 ceppi alla cefalotina (89%), 40 ceppi alla tetraciclina (22%), dieci ceppi al cloramfenicolo e al sulfametossazolo/ trimetoprim (5,6%). tutti i ceppi erano sensibili a cefoperazone/sulbactam, ciprofloxacina, colistina, gentamicina e piperacillina/tazobactam. dieci dei 180 isolati (5,6%) mostravano fenotipi multi-resistenti, incluse resistenze contro i beta-lattamici, gli aminoglicosidi, le tetracicline, sulfamididolfonammidiche e il cloramfenicolo. per quanto se ne sappia, questo è il primo rapporto sulla resistenza agli antibiotici nei fagiani. caratterizzazione di ceppi di escherichia coli isolati da fagiani (phasianus colchius) nella repubblica ceca e slovacca parole chiave antibiotico, resistenza, fagiano, fauna selvatica. keywords antibiotic, resistance, pheasant, wildlife. an earlier version of this paper was presented at "animal physiology 2014, 21-22 may 2014, prušánky, czech republic" 1vetservis ltd., kalvaria 3, sk-949 01 nitra, slovakia. 2tomas bata university in zlin, faculty of technology, institute of environmental engineering, tgm 275, 760 01 zlin, czech republic. 3tomas bata university in zlin, faculty of technology, institute of food technology, tgm 275, 760 01 zlin, czech republic. 4institute for state control of veterinary biologicals and medicaments, biovetska 5, 949 01 nitra, slovakia. * corresponding author at: vetservis ltd., kalvaria 3, sk-949 01 nitra, slovakia e-mail: ivan.holko@gmail.com. ivan holko1*, magda doležalová2, silvie pavlíčková2, robert gal2, tomas valenta3 and tatiana holkova4 antimicrobial-resistance in escherichia coli isolated from wild pheasants (phasianus colchicus) short communication 170 veterinaria italiana 2019, 55 (2), 169-172. doi: 10.12834/vetit.887.4400.3 antibiotic resistance to escherichia coli in wild pheasants holko et al. obtained data were evaluated using tnw lite 6.5 software (erbalachema brno, czech republic). the in  vitro susceptibility of the isolates against antimicrobial agents was determined using the standard disk diffusion procedure (clsi 2012). the susceptibility, intermediate susceptibility, or resistance of individual isolates was determined according to the minimal inhibitory concentration breakpoints for e.  coli established in the clsi document m100-s22 (2012). the following antibiotic discs (oxoid ltd., besingstoke, hampshire, gb) were used: aminoglycosides: gentamycin (10 µg), streptomycin (10 µg), neomycin (30  µg); penicillins: ampicilin (10  µg); β-lactamase inhibitor combinations: amoxicilin/clavulanic acid 2:1 (30  µg), piperacillin/tazobactam 10:1 (110  µg), sulbactam/cephoperazon (105  µg); cephems: cephalothin (30  µg); quinolones: ciprofloxacin (5µg); polymyxines: colistin sulphate (10  µg); phenicols: chloramphenicol (30  µg); folate pathways  inhibitors: s u l p h a m e t h o x a z o l e / t r i m e t h o p r i m (25 µg); tetracyclines: tetracycline (10 µg). based on the presence/absence of these three fragments, an e. coli isolate could be assigned to one of the main phylo-groups, a, b1, b2 or d. the isolates were assigned to the phylogenetic groups according to presence of the genes chua and yjaa and the dna fragment tspe4.c2.: a(chua-, tspe4. c2-), b1 (chua-, tspe4.c2+), b2 (chua+, yjaa+), or d (chua+, yjaa-). (clermont et al. 2000). a few colonies of each strain were solved in 100 μl of 1x pcr buffer (thermopol buffer, neb, usa), heat-treated to 95 °c for 20 minutes. supernatant after centrifugation (10,000  g/l min) was taken as dna template for all pcr tests. amplicons were visualised by 1% agarose gel electrophoresis and strains were assigned to phylogenetic groups or subgroups (escobar-paramo et al. 2006, carlos et al. 2010). more than half of the strains belonged to a  phylogroup (42% a0, 11% a1). strains belonging to the b1 phylogroup were the 27% whereas the 20% of the strains fell within the combined phylogroups b2 and d (table  i). out of 180 examined strains, 130 strains were ampicillin-resistant (72%) and the remaining 50  strains were ampicillin-intermediate; 160 strains (89%) showed resistance to cephalothin, the other 20  strains were ampicillin-intermediate. ten e. coli strains were resistant to chloramphenicol and sulfamethoxazole/trimethoprim (5.6%) whereas the rest of strains were sensitive to these antibiotics. intermediate sensitivity to amoxicillin/clavulanic acid was determined in 10 strains; all other strains were, in turn, showed to be sensitive. forty strains were identified as intermediately sensitive to neomycin and streptomycin; all other strains were sensitive. all strains were sensitive to cefoperazone/sulbactam, ciprofloxacin, colistin, gentamicin, and piperacillin/ anthropogenic changes in an environment have the potential to affect different aspects of the world, globally. these types of changes may also enrich the population of resistant bacteria and facilitate the dissemination of antibiotic-resistant bacteria, and/ or resistance genes to human pathogens (martinez 2009). several studies have indeed shown the presence of antimicrobial residues in human and animal waste and sewage (kummerer 2009). wildlife is not directly exposed to clinical antimicrobial agents, but can get infected with antimicrobial-resistant bacteria through contact with humans, animals, and the environment. water polluted with faeces appears to be the most vector of contamination (kummerer and henninger 2003). by monitoring and characterizing the prevalence of antimicrobial-resistance in bacteria such as escherichia coli in different populations, including animal, wildlife and humans, it is possible to have a better understanding upon the spread and ecology of antimicrobial-resistance in a one-health approach. the aim of this research was to determine the antibiotic resistance features in 180 strains of escherichia coli isolated from rectal swabs of wild pheasants in eastern moravia and western slovakia and compare results with those publicly available. a total of 180 strains of escherichia coli were collected between 2010 and 2013 from 200 wild pheasants (phasianus colchicus, l. 1758) that were hunted in the regions of eastern moravia czech republic, (100 strains) and western slovakia (80  strains). the rectangular sample area for this study was defined by the following geographical points as shown in figure 1: 1. brno (49° 12´ n 16° 37´ e), 2. nitra (48° 19´ n 18° 05´ e), 3. prievidza (48° 46´ n 18° 37´ e), and 4. olomouc (49° 36´ n 17° 15´ e). the strains were cultured from rectal transport swabs on macconkey agar. suspected colonies were isolated and identified to the species level by using the enterotest24 (erbalachema brno, czech republic) commercial identification microsystem. the microbial species identification success rate was over 98%. 1 4 3 2 poland austria hungary slovakia czech republic figure 1. the map showing the origin of hunted pheasants. 171veterinaria italiana 2019, 55 (2), 169-172. doi: 10.12834/vetit.887.4400.3 holko et al. antibiotic resistance to escherichia coli in wild pheasants in the czech republic, antibiotic resistance is a serious concern. between 2001 and 2005, the antibiotic resistance of e.  coli to fluoroquinolones doubled; ciprofloxacin resistance increased from 8% to more than 20%, and the upward trend continues (nyc et  al. 2011). our research showed no intermediate or resistant strains to ciprofloxacin antibiotics, nor cefoperazone, colistin, gentamicin, and piperacillin/tazobactam. between 1999 and 2000 the 51% of escherichia coli strains isolated from poultry were found to be resistant to ampicillin, the 31% to piperacillin, and the 97% to tetracycline. in the 10% of the examined strains, an increasing resistance to ciprofloxacin and ofloxacin was observed (kolář et al. 2002). a study carried out in slovakia examined the resistance to quinolone antibiotics (nalidixic acid, ciprofloxacin, and enrofloxacin) of commensal escherichia coli isolated from healthy broiler chickens from several different farms. isolates exhibited high resistance to nalidixic acid, ciprofloxacin, and enrofloxacin. the prevalence of resistance was approximately 80% for ciprofloxacin and 50% for enrofloxacin (kmeť et al. 2007). wild birds are important in relation to antibiotic resistance in several different ways: 1) as sentinels, of human activities; 2) as reservoirs and melting pots for antibiotic-resistant bacteria and resistance genes; 3) as disseminators of antibiotic resistance through migration; 4) as sources of resistant bacteria colonising or infecting humans beings (bonnedahl and järhult 2014). the results of our research demonstrate that tazobactam. forty strains (22%) were resistant to tetracycline, while 50  strains were intermediately sensitive and 90  strains were sensitive. results are summarized in table i. the most common multidrug-resistance was observed for ampicillin + cephalothin (140  strains, 78%). of these, 70 strains (39%) were determined to be resistant to another antibiotic, usually tetracycline. a total of 10  strains (5.6%) were multidrug-resistant to more than 3  classes of antibiotics, namely: ampicillin, cephalothin, chloramphenicol, neomycin (intermediate resistance), streptomycin (intermediate resistance), sulfamethoxazole/trimethoprim, and tetracycline. the multidrug-resistant strains belonged to groups a and b1. this finding is consistent with the study by johnson and colleagues (johnson et al. 2006), where isolates from phylogroups a and b1 are normally multi-resistant. our findings demonstrate that escherichia coli isolated from pheasants are highly resistant to ampicillin and cephalothin. the study also demonstrates a considerable resistance to tetracycline. this study shows that a significant antibiotic resistance also occurs in wild animals that have not previously been exposed to antibiotic treatment. our work confirms the high prevalence of antimicrobial resistance to beta-lactam antibiotics in wildlife, which is consistent with other studies. a recent work has, indeed, reported up to 100% resistance to penicillin in e. coli isolated from gulls in ireland (smith et  al. 2014). it can be assumed that the presence of resistant strains is a result of the increasing resistance of bacteria to antimicrobial drugs worldwide. table i. occurence of antibiotic resistence of e. coli in phylogenetic groups a, b1, b2, and d. a n=95 b1 n=48 b2 n=22 d n=15 total n=180 r/i* % r/i* % r/i* % r/i* % r/i* % aamc 0/6 0/6 0/4 0/8 0/0 0/0 0/0 0/0 0/10 0/5.6 amp 78/17 82/18 42/6 87/13 10/12 45/55 0/15 0/100 130/50 72/28 cef 90/5 95/5 48/0 100/0 22/0 100/0 0/15 0/100 160/20 89/11 scf 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 cip 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 cst 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 gen 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 chl 7/0 7/0 3/0 6/0 0/0 0/0 0/0 0/0 10/0 5.6/0 neo 0/21 0/22 0/6 0/12 0/4 0/18 0/0 0/0 0/30 0/17 tzp 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 str 0/12 0/13 0/6 0/12 0/1 0/4 0/1 0/7 0/20 0/11 sxt 8/0 8/0 2/0 4/0 0/0 0/0 0/0 0/0 10/0 5.6/0 tet 21/24 22/25 13/17 27/35 6/9 27/41 0/0 0/0 40/50 22/28 * r/i = number of resistant/intermediate e. coli strains; aamc = amoxicillin/clavulanic acid; amp = ampicillin; cef = cephalothin; scf = cefoperazon/sulbactam; cip =ciprofloxacin; cst = colistin; gen = gentamycin; chl = chloramphenicol; neo = neomycin; tzp = piperacillin/tazobactam; str = streptomycin; sxt = sulphametoxazole/trimethoprim; tet = tetracycline. 172 veterinaria italiana 2019, 55 (2), 169-172. doi: 10.12834/vetit.887.4400.3 antibiotic resistance to escherichia coli in wild pheasants holko et al. in order to elucidate the spread of this phenomenon in other wild animals. acknowledgments this work was supported by internal grant of tomas bata university in zlin (no. iga/ft/2015/012). escherichia coli in wild pheasants follows the trend of increasing antimicrobial resistance to antibiotics. of the 180 examined e.  coli strains, most were resistant to cephalothin and ampicillin (89% and 72% respectively), other strains were intermediately sensitive. no strains were sensitive to these antibiotics. further studies are reasonably warranted allen h.k., donato j., wang h.m., cloud-hansen k.a., davies j. & handelsman j. 2010. call of the wild: antibiotic resistance in natural environments. nat rev microbiol, 8, 251-259. bonnedahl j. & järhult j. 2014. antibiotic resistance in wild birds. upsala j med sci, 119, 113-116. carlos c., pires m.m., stoppe n.c., hachich e.m., sato m.i., gomez t.a., amaral l.a. & ottoboni l.m. 2010. escherichia coli phylogenetic group determination and its application in the identification of the major animal source of faecal contamination. bmc microbiol, 10, 161. clermont o., bonacorsi s. & bingen e. 2000. rapid and simple determination of the escherichia coli phylogenetic group. appl environ microbiol, 66, 4555-4558. clsi. 2012. performance standards for antimicrobial susceptibility testing, 22nd informational supplement, wayne pa: clinical and laboratory standards institute (clsi document m100-s22). escobar-paramo p., le menach a., le gall t., amorin c., gouriou s., picard b., skurnik d. & denamur e. 2006. identification of forces shaping the commensal escherichia coli genetic structure by comparing animal and human isolates. environ microbiol, 8, 1975-1984. johnson t.j., johnson s.j. & nolan l.k. 2006. complete dna sequence of a colbm plasmid from avian pathogenic escherichia coli suggests that it evolved from closely related colv virulence plasmids. j bacteriol, 188, 5975-5983. kmeť v., melicharek i., bobcek r., bidlen l. & szakal t. 2007. dynamics of microbial resistance to antibiotics in comensal escherichia coli in broilers. slov vet cas, 32, 252-253 (in slovak with english abstract). kolář m., pantuček r., bardoň j., vágnerová i., typovská h. & references válka i. 2002. occurrence of antibiotic-resistant bacterial strains isolated in poultry. vet med czech, 47, 52-59. kummerer k. & henninger a. 2003. promoting resistance by the emission of antibiotics from hospitals and households into effluent. clin microbiol infect, 9, 1203-1214. kummerer k. 2009. antibiotics in the aquatic environment a review part i. chemosphere, 75, 417-434. martinez j.l. 2009. the role of natural environments in the evolution of resistance traits in pathogenic bacteria. proc biol sci, 276, 2521-2530. nyc o., urbaskova p., maresova v., prokes m., jindrak v., svihovec j., sechser t., karen i. & hoza j. 2011. consensus use of antibiotics no. iii, the national institute of public health, prague, czech republic. http://www.szu.cz/ konsensy-pouzivani-antibiotik (accessed october. 2014). radhouani h., silva n., poeta p., torres c., correia s. & igrejas g. 2014. potential impact of antimicrobial resistance in wildlife, environment and human health. front microbiol, 5, 23. segura p.a., francois m., gagnon c. & sauve s. 2009. review of the occurrence of anti-infectives in contaminated wastewaters and natural and drinking waters. environ. health perspect, 117, 675-684. skurnik d., ruimy r., andremont a., amorin c., rouquet p. & picard b. 2006. effect of human vicinity on antimicrobial resistance and integrons in animal faecal escherichia coli. j antimicrob chemotherapy, 57, 1215-1219. smith s., wang j., fanning s. & mcmahon b.j. 2014. antimicrobial resistant bacteria in wild mammals and birds: a coincidence or cause for concern? irish vet j, 67, 8. van der bogaard a.e. & stobberingh e.e. 2000. epidemiology of resistance to antibiotics, links between animals and humans. int j antimicrob agents, 14, 327-335. 1 1department of veterinary sciences, university of turin, turin, italy. 2department of veterinary medicine, university of bari, valenzano (ba), italy. *corresponding author at: department of veterinary sciences, university of turin, turin, italy. e-mail: angela.fanelli@unito.it. keywords cattle, buffalo, elisa, q fever, zoonosis. summary a cross‑sectional survey was carried out in dairy cattle and buffalo herds from the southern italy to detect antibodies against coxiella burnetii. from 2014 to 2018, 402 herds were monitored and 50 ml of bulk‑tank milk (btm) per farm was analyzed by indirect elisa. blood samples of animals from positive farms were also taken and analyzed with the same elisa test. the overall seroprevalence was 35% [95% confidence interval (ci):30‑39] at herd level and 13% (95%ci:13‑14) at animal level. herd province seroprevalences ranged from 17% to 75%. the provinces of matera (71%, 95%ci:38‑105) and agrigento (75%, 95%ci:51‑100) showed the highest percentage of infected farms. these results describe the widespread distribution of c. burnetii in livestock from southern italy, highlighting the need to implement a monitoring program for q fever. angela fanelli1*, adriana trotta2, filena bono2, marialaura corrente2 and domenico buonavoglia2 seroprevalence of coxiella burnetii in dairy cattle and buffalo from southern italy veterinaria italiana 2020, 56 (3), xxx‑xxx. doi: 10.12834/vetit.2321.13237.1 accepted: 10.10.2020 | available on line: xx.xx.2020 code. therefore, member countries have the legal obligation to report information on the disease to the oie. national regulations exist also at country level. in italy, q fever is a notifiable disease in humans and it has been listed in the occupational disease list (italian republic, ministry of works decree 14th january 2008). however, the epidemiological situation is not well‑known because of the scarce monitoring in both humans and livestock, as it has been demonstrated for other notifiable animal diseases (fanelli et al. 2020, fanelli and tizzani 2020). indeed, the occurrence of c. burnetii has been investigated in flocks and herds only in limited areas of the country (masala et  al. 2004, rizzo et  al. 2016, guidi et  al. 2017, galluzzo et  al. 2019). the major constraint is represented by the poor knowledge and awareness of q fever in both farmers and veterinarians. considering that monitoring and reporting the infection in livestock is crucial for the prevention of human disease, the objective of this study is to estimate the seroprevalence of c. burnetii in dairy cattle and buffalo herds from southern italy, an area characterized by a closed and interconnected farms network. from 2014 to 2018 402 semi‑intensive farms (herd q fever is a zoonotic disease caused by coxiella burnetii, a bacterium developing spore‑like forms that are highly resistant in the environment. cattle, sheep, and goats are the main reservoirs of the bacteria. they can shed c. burnetii in urine, feces, milk, and birth products, and humans usually acquire the infection through inhalation of contaminated aerosols (arricau‑bouvery and rodolakis 2005). q fever is considered mainly as an occupational zoonosis, being farmers, laboratory workers, veterinarian at high risk of infection (schimmer et al. 2014). consumption of raw/unpasteurized milk and tick bites have also been claimed as possible routes of transmission, but they are probably far less frequent than the airborne one (duron et  al. 2015, gale et al. 2015). q fever is frequently misdiagnosed and underreported. in human, the disease has been associated with a wide clinical spectrum, from asymptomatic to fatal disease. however, in most of the cases, it is characterized by flu‑like symptoms (arricau‑bouvery and rodolakis 2005). in livestock, the infection can cause significant economic losses due to abortion, infertility and subclinical mastitis (van asseldonk et al. 2013). q fever is listed in the oie terrestrial animal health short communication 2 veterinaria italiana 2020, 56 (3), xxx-xxx. doi: 10.12834/vetit.2321.13237.1 c. burnetii in southern italy livestock fanelli et al. positive if the od percent was over 50, doubtful if it was between 40 and 50, and negative if it was under 40. the overall seroprevalence was 35% at herd level (95%ci:30‑39) and 13% (95%ci:13‑14) at animal level, with differences among provinces (table i, figure 2). province seroprevalences ranged from 17% to 75%. when compared to the results from other mediterranean regions, the majority of the provinces present a seroprevalence at the herd level in line with those reported in spain (43%) and portugal (61.1%) (ruiz‑fons et  al. 2010, pimenta et  al. 2015). the high numbers of positive farms in the province of matera (basilicata) and agrigento (sicily) are similar to what described in cattle from central italy (68.5%) (barlozzari et  al. 2020), and it confirms the significant presence of c.  burnetii in the territory of western sicily (galluzzo et al. 2019). some authors have reported a higher risk of being seropositive for animals originating from larger herds (agger et  al. 2013, paul et  al. 2014). in this study, the risk factors of c. burnetii infection on each herd were not assessed, however, the differences detected are unlikely to depend on the herd‑size or management system, considering the similar features of the sampled farms (with the exception of the size of buffalo herds). size ranges from 20 to 50 heads for cattle and 230 to 800 for buffalo) were sampled in 18 italian provinces (figure 1). the sample collection does not represent a random sampling, as it is represented by farms in which abortions due to c. burnetii were detected by rt‑pcr or farms located in the surrounding area (within a radius of 5 km from an infected farm). a multiple sampling strategy was applied to assess the circulation of c. burnetii in the study area. firstly, an elisa test [lsi qfever ruminant serum/milk elisa kit (lsi, france] was performed on 50  ml of bulk tank milk (btm). the use of elisa to test btm has been proved to a be cost‑effective and valuable tool to monitor herds (ryan et  al. 2011). the test was repeated in the herds tested negative after 10 months, in order to consider also animals that were in dry period during the first sampling. ten ml of serum per animal were assessed with the same elisa test in each positive herd. the number of animals to be tested was determined considering the herd size, the expected seroprevalence (20%), the absolute precision desired (5%) and the confidence interval (ci) (95%) (cannon and roe 1982). the assay was performed according to the manufacturer’s instructions. for btm, a sample was considered positive if the od percent was over 40, doubtful if it was between 30 and 40, and negative if it was under 30. for serum, a sample was considered figure 1. map showing the provinces sampled to assess the seroprevalence of c. burnetii. veterinaria italiana 2020, 56 (3), xxx-xxx. doi: 10.12834/vetit.2321.13237.1 3 fanelli et al. c. burnetii in southern italy livestock table i. c. burnetii seroprevalence at both herd and animal level according to provinces. region province herds sampled positive herds prevalence % (95%ci) number of animals tested positive animals prevalence % (95%ci) apulia bari 24 13 54 (34-74) 340 153 45 (40-50) barletta-andria-trani1 1 1 na* 22 9 41 (20-61) brindisi1 1 0 na* foggia 11 5 45 (16-75) 204 91 45 (38-51) lecce1 1 1 na* 16 11 69 (46-91) taranto 46 21 46 (31-60) 560 246 44 (40-48) basilicata matera 7 5 71 (38-105) 60 13 22 (11-32) potenza 11 6 55 (25-84) 62 18 29 (18-40) campania salerno2 104 19 18 (11-26) 247 77 31 (25-37) sicily agrigento 12 9 75 (51-100) 151 22 15 (9-20) caltanissetta 6 2 33 (-4-71) 163 3 2 (0-4) catania 11 2 18 (-5-41) 201 6 3 (1-5) enna 26 11 42 (23-61) 784 27 3 (2-5) messina 41 11 27 (13-40) 531 15 3 (1-4) palermo 46 11 24 (12-36) 1259 24 2 (1-3) ragusa 30 14 47 (29-65) 1092 101 9 (8-11) syracuse 18 7 39 (16-61) 389 21 5 (3-8) trapani 6 1 17 (-13-46) 99 1 1 (-1-3) 1only one herd was sampled for the provinces of barletta-andria-trani, lecce and brindisi, thus it was no possible to compute the seroprevalence at herd level. 2 the buffalo farms included in this study are all located in salerno province. *not applicable. figure 2. choropleth map displaying c. burnetii seroprevalence at herd-level in connection with the first level administrative boundaries. 4 veterinaria italiana 2020, 56 (3), xxx-xxx. doi: 10.12834/vetit.2321.13237.1 c. burnetii in southern italy livestock fanelli et al. precautions (i.e. changing boots and/or clothes) significantly reduce the risk of the infection. data from this study made an important contribution from both public and animal health perspectives. baseline epidemiological data presented herein will be useful for comparative purposes during future studies in areas with circulation of the pathogen. a one health approach, with an integrated surveillance system involving the systematic notification of cases in both humans and animals, is needed to better understand the spread of c. burnetii in the study area. as regards livestock, further investigations should be performed to assess the potential risk factors that could influence the exposure of c. burnetii. this information is crucial to improve the management of biosecurity at farm level, and to prevent the introduction into susceptible populations. financial support this research received no specific grant from any funding agency, commercial or not‑for‑profit sectors. we focused on semi‑intensive farms, where animals are housed in winter and graze during spring and summer. because of that, livestock is at high risk of c. burnetii infection due to both the transmission by infected aerosols derived from contaminated materials left in the environment (such as birth fluids) or by fomites and the exposure to large numbers of infected ticks by grazing pastures during spring and summer. we do believe that the rural reality and the cultural tradition of the livestock farming characterizing our study area have a great influence on the epidemiological framework of the disease. indeed, local farms tend to perform little to no biosecurity prevention practices, and adjacent farms are most likely to share equipment and have human and livestock movements among them. all these factors may contribute to the spread of c. burnetii in the study area. recently, agger and colleagues (agger et  al. 2013) demonstrated that biosecurity is crucial for the prevention of c. burnetii infections. the authors suggested that the veterinarian might bring the bacterium into the farm, and that the hygiene veterinaria italiana 2020, 56 (3), xxx-xxx. doi: 10.12834/vetit.2321.13237.1 5 fanelli et al. c. burnetii in southern italy livestock agger j.f., paul s., christoffersen a.b. & agerholm j.s. 2013. risk factors for coxiella burnetii antibodies in bulk tank milk from danish dairy herds. acta vet scand, 55, 80. https://doi.org/10.1186/1751‑0147‑55‑80. arricau‑bouvery n. & rodolakis a. 2005. is q fever an emerging or re‑emerging zoonosis? vet res, 36, 327‑349. https://doi.org/10.1051/vetres:2005010 327. barlozzari g., sala m., iacoponi f., volpi c., polinori n., rombolà p., vairo f., macrì g. & scarpulla m. 2020. cross‑sectional serosurvey of coxiella burnetii in healthy cattle and sheep from extensive grazing system in central italy. epidemiol infect, 148, 1‑8. https://doi. org/10.1017/s0950268819002115. cannon m. & roe r.t. 1982. livestock disease surveys: a field manual for veterinarians. australian government pub. service, canberra. duron o., sidi‑boumedine k., rousset e., moutailler s. & jourdain e. 2015. the importance of ticks in q fever transmission: what has (and has not) been demonstrated? trends parasitol, 31, 536‑552. https:// doi.org/10.1016/j.pt.2015.06.014. fanelli a., buonavoglia d., martinez carrasco pleite c. & tizzani p. 2020. paratuberculosis at european scale: an overview from 2010 to 2017. vet ital, 56 (1), 13‑21. https://doi.org/10.12834/vetit.1829.9692.3. fanelli a. & tizzani p. 2020. spatial and temporal analysis of varroosis from 2005 to 2018. res vet sci, 131, 215‑221. https://doi.org/10.1016/j.rvsc.2020.04.017. gale p., kelly l., mearns r., duggan j. & snary e.l. 2015. q fever through consumption of unpasteurised milk and milk products ‑ a risk profile and exposure assessment. j appl microbiol, 118, 1083‑1095. https:// doi.org/10.1111/jam.12778. galluzzo p., villari s., geraci f., sciacca c., grippi f., currò v. & chiarenza g. 2019. seroprevalence of coxiella 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da silva g., rabiço â. & simões j. 2015. prevalence of coxiella burnetii antibodies in portuguese dairy cattle herds. trop anim health prod, 47, 227‑230. https://doi. org/10.1007/s11250‑014‑0679‑1. rizzo f., vitale n., ballardini m., borromeo v., luzzago c., chiavacci l. & mandola m.l. 2016. q fever seroprevalence and risk factors in sheep and goats in northwest italy. prev vet med, 130, 10‑17. https://doi. org/10.1016/j.prevetmed.2016.05.014. ruiz‑fons f., astobiza i., barandika j., hurtado a., atxaerandio r., juste r. & garcía‑pérez a. 2010. seroepidemiological study of q fever in domestic ruminants in semi‑extensive grazing systems. bmc vet res, 6, 1‑6. https://doi.org/10.1186/1746‑6148‑6‑3. ryan e.d., kirby m., collins d.m., sayers r., mee j.f. & clegg t. 2011. prevalence of coxiella burnetii (q fever) antibodies in bovine serum and bulk‑milk samples. epidemiol infect, 139, 1413‑1417. https://doi. org/10.1017/s0950268810002530. schimmer b., schotten n., van engelen e., hautvast j.l.a., schneeberger p.m. & van duijnhoven y.t.h.p. 2014. coxiella burnetii seroprevalence and risk for humans on dairy cattle farms, the netherlands, 2010‑2011. emerg infect dis, 20, 417‑425. https://doi.org/10.3201/ eid2003.131111. van asseldonk m.a.p.m., prins j. & bergevoet r.h.m. 2013. economic assessment of q fever in the netherlands. prev vet med, 112, 27‑34. https://doi.org/10.1016/j. prevetmed.2013.06.002. 447 1istituto zooprofilattico sperimentale del piemonte, liguria e valle d'aosta. 2giardino zoologico safari ravenna. *corresponding author at: istituto zooprofilattico del piemonte, liguria e valle d'aosta. e‑mail: lorella.maniscalco@i‑vet.it. lorella maniscalco1*, pier luigi acutis1, cristina biolatti1, daniele r. laguardia2, alessia di blasio1, alessandro dondo1, elena bozzetta1, katia varello1 keywords tiger, zoo, renal cell carcinoma, immunohistochemistry, pax8. veterinaria italiana 2022, 58 (4), 447‑450. doi: 10.12834/vetit.2475.16553.2 accepted: 30.01.2023 | available on line: 31.12.2022 summary a 12‑year‑old intact male panthera tigris presented with pain and weight loss was euthanatized. necroscopical examination revealed a neoplastic mass expanding to the left renal pelvis with metastatic dissemination to local lymph node, adrenal gland, and lung. immunohistochemical characterization was performed revealing co‑expression of both cytokeratin and vimentin and negativity for both pax8 and c‑kit. considering histochemical and immunohistochemical results the tumour was classified as renal cell carcinoma with metastatic spread. this report provides insights into the morphological and immunohistochemical features of renal cell carcinoma in panthera tigris. please refer to the forthcoming article as: maniscalco et al. 2022. renal carcinoma with metastatic spread in a tiger (panthera tigris): morphological and immunohistochemical study vet ital. doi: 10.12834/vetit.2475.16553.2 renal carcinoma with metastatic spread in a tiger (panthera tigris): morphological and immunohistochemical study primary renal tumours are uncommon in domestic feline representing less than 1% of all neoplasms (newkirk, newman et al. 2011, bonsembiante, benali et al. 2016). most tumours diagnosed in domestic feline kidney are lymphomas, representing 78% of the lesions and metastatic lesions (10%) (meuten and meuten 2017, ramos‑vara, edmondson et al. 2017). among primary renal tumours in cats, renal cell carcinoma (rcc) is the most common in domestic animals, including cats (matsumoto, chambers et al. 2018). to the authors’ knowledge, a single case regarding a primary renal tumour in panthera tigris, diagnosed as renal papilloma (kloft, ramsay et al. 2019), was reported, but no reports describing primary renal malignancies are present to date. a 12‑year‑old male tiger (panthera tigris) held in captivity at the safari park, ravenna (ra), italy, developed lethargy, vomiting, constipation and anorexia. the animal was sedated for a complete physical examination and diagnostic work‑up. complete blood cell count and chemistry panel were performed: moderate anaemia, increased urea and creatinine concentration were observed. abdominal ultrasound revealed hydronephrosis of the left kidney. due to the poor condition, the animal was euthanized, and a complete necroscopy was immediately performed. opening the abdominal cavity, a moderate amount (about 150 ml) of sero‑ hematic effusion was observed with moderate and diffuse presence of chronic peritonitis characterized by the presence of chronic fibrinous adherence between intestinal tract and abdominal wall. the gastrointestinal tract was empty. left kidney was discoloured and enlarged by a 4 cm in diameter irregular brown to whitish solid multinodular mass expanding the renal pelvis, involving cortex and medulla causing severe hydronephrosis (fig.1). case report renal cell carcinoma in a tiger maniscalco et al. 448 veterinaria italiana 2022, 58 (4), 447-450. doi: 10.12834/vetit.2475.16553.2 figure 1. kidney with an irregular brown to whitish solid multinodular mass expanding in the renal pelvis and involving cortex and medulla; a severe hydronephrosis.is present. figure 2. kidney. renal cell carcinoma. neoplastic cells are arranged in tubules and papillae surrounded by fibrous stroma. neoplastic cells show moderate anisocytosis and anisocaryosis. hematoxylin and eosin; b: renal cell carcinoma. immunohistochemistry for cytokeratin (ck). neoplastic cells are characterised by diffuse and strong cytoplasmic immunolabelling for ck. streptavidin–biotin–peroxidase method. mayer’s haematoxylin counterstain; c: renal cell carcinoma. immunohistochemistry for c-kit. neoplastic cells are characterised by diffuse lack of immunolabelling for c-kit. streptavidin–biotin– peroxidase method. mayer’s haematoxylin counterstain; d: kidney, immunohistochemistry for pax8. cortical tubules cortical tubules with cytoplasmic immunolabelling for pax8. both left renal lymph node and left adrenal gland appeared irregular and moderately enlarged by a brown to whitish solid mass. splenomegaly and hepatomegaly were also present. in thoracic cavity a moderate amount (about 100 ml) of sero‑hematic pleural effusion was present. multifocal chronic pleuritis was present and examining pulmonary parenchyma, multiple white to brownish 0.5‑8 cm in diameter nodular lesions were observed. representative tissue samples from lungs, liver, kidney, spleen, alimentary tract and renal lymph nodes were fixed in 10% buffered formalin, routinely processed, and paraffin‑embedded. sections of 4 mm were obtained and stained with haematoxylin and eosin and then observed by three pathologists (l.m, k.v, e.b). histopathological examination revealed a renal neoplastic multinodular mass expanding to renal pelvis and medulla end cortex. the neoplastic lesion was composed by polygonal cells arranged in tubules, papillae and solid areas intermingled with abundant desmoplastic stroma. cells were about 20 microns in diameter, polygonal, with well distinct cell borders, moderate amount of cytoplasm and round nucleus with stippled chromatin and one round magenta nucleus sometimes evident. neoplastic cells showed moderate anisocytosis and anisocaryosis, moderate n/c ratio and 0/2 mitoses per high‑power field were present (fig. 2a). maniscalco et al. renal cell carcinoma in a tiger veterinaria italiana 2022, 58 (4), 447-450. doi: 10.12834/vetit.2475.16553.2 449 multifocal areas of the adrenal gland cortex, lymph node and pulmonary parenchyma were effaced by a neoplastic lesion whose histological morphology appeared like the one observed for the renal mass. in order to confirm the diagnosis, immunohistochemistry (ihc) was performed on representative sections of the primary renal neoplasm and metastases applying the following commercial antibodies: anti‑cytokeratin ae1/ae3 (dako, monoclonal, heat‑induced epitope retrieval high ph, 1:50), anti‑vimentin (dako, monoclonal, heat induced epitope retrieval high ph, 1:100), anti pax8 (biocare medical, monoclonal, heat‑induced epitope retrieval high ph, 1:50), anti c‑kit dako (polyclonal, heat induced epitope retrieval high ph, 1:100) ihc was performed using the streptavidin–biotin– peroxidase method. from each block, 4‑mm‑thick sections were cut on polarized slides. all sections were deparaffinized in bio‑clear (bio‑clear, bio‑ optica, 20134 milano, italy), hydrated with graduated ethanol, and finally immersed in distilled water. in order to block endogenous peroxidase activity, slides were treated with 3% hydrogen peroxide in distilled water for 10 min. antigen retrieval was performed with 0.01 mm sodium citrate buffer ph 6.0. the peroxidase reaction was developed using a diaminobenzidine (envision®+ dual link system‑hrp (dab+), dakocytomation, glostrup, denmark) and the sections were counterstained with mayer’s’ haematoxylin. normal skin from a panthera tigris from archive cases was used as positive controls for both cytokeratin and vimentin, while feline normal kidney and feline mast cell tumour were used as positive control for pax8 and c‑kit, respectively. negative controls were obtained with the omission of the primary antibodies. neoplastic cells from both primary and metastatic lesions showed similar immunohistochemical expression. neoplastic cells resulted strongly and diffusely positive for both cytokeratin (fig.2b) and vimentin; interstitial stroma showed no immunoreactivity for cytokeratin while was strongly positive for vimentin. c‑kit was diffusely negative in both neoplastic and stromal compartment (fig 2c). pax8 had strong expression in thick and thin loops of henle in the medulla, renal pelvic urothelium and occasional cortical tubules while neoplastic cells were diffusely negative (fig 2d). negative controls showed no immunostaining to all the antibodies tested. based on these results the tumour was classified as tubulo‑papillary renal carcinoma. these conclusions were first achieved by macroscopic examination, confirmed by histopathology, and followed by immunohistochemical characterization and are in agreement with the immunohistochemical profile of feline renal carcinomas described in literature (ramos‑vara, edmondson et al. 2017). in that paper co‑expression of cytokeratin and vimentin were observed in a large amount of renal cell carcinomas (19 out of 20) together with the 95% of cases expressing pax8. pax8 showed a weak diffuse cytoplasmic positivity in renal tubules similar to what is described in domestic cat kidney but was surprisingly negative in neoplastic cells. since there are no previous reports regarding the expression of this marker in a panthera leo, our results cannot be compared. similarly, c‑kit was not detected in this neoplasia while it is mostly reported as positive in domestic cat rcc (matsumoto, chambers et al. 2018). we can speculate that this neoplasia can represent one of the unusual rcc negative case for these markers or that the immunohistochemical profile of rcc is different in wild felines. this observation suggests that also in panthera tigris, rcc epithelial to mesenchymal transition (emt) transcriptional pathways could be activated, as previously demonstrated for a number of epithelial neoplasms in domestic cat and other animals (matsumoto, chambers et al. 2018). in conclusion, to the best of our knowledge, this is the first report of an rcc with metastatic dissemination in a panthera tigris. our findings provide insights into the morphological and immunohistochemical features of this tumour. renal cell carcinoma in a tiger maniscalco et al. 450 veterinaria italiana 2022, 58 (4), 447-450. doi: 10.12834/vetit.2475.16553.2 bonsembiante, f., et al. (2016). “histological and immunohistochemical characterization of feline renal cell carcinoma: a case series.” j vet med sci 78(6): 1039‑1043. kloft, h. m., et al. (2019). “neoplasia in captive panthera species.” j comp pathol 166: 35‑44. matsumoto, i., et al. (2018). “histopathologic and immunohistochemistry findings in feline renal cell carcinoma.” vet pathol 55(5): 663‑672. references meuten, d. j. and meuten t. l. k. (2017). tumors of the urinary system. tumors in domestic animals. i. john wiley & sons. oxford, uk. newkirk, k. m., et al. (2011). “renal lesions of nondomestic felids.” vet pathol 48(3): 698‑705. ramos‑vara, j. a., et al. (2017). “immunohistochemical profile of 20 feline renal cell carcinomas.” j comp pathol 157(2‑3): 115‑125. 275 veterinaria italiana 2022, 58 (3), 275-280. doi: 10.12834/vetit.2295.15822.2 accepted: 30.07.2021 | available on line: 31.12.2022 1department of veterinary pathology and microbiology, faculty of veterinary medicine, university of nigeria, nsukka, nigeria. 2department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of nigeria, nsukka, nigeria. 3department of veterinary services, ministry of agriculture, anambra state, nigeria. *corresponding author at: department of veterinary pathology and microbiology, faculty of veterinary medicine, university of nigeria, nsukka, nigeria. e‑mail: simeon.okafor@unn.edu.ng. simeon chibuko okafor1*, uju catherine okafor2, donatus lotanna obinwogu3 and john ikechukwu ihedioha1 keywords african swine fever, biochemical analysis, hematological analysis, pigs. summary african swine fever (asf) is a contagious viral disease that affects pigs of all ages, inducing hemorrhagic fever with high mortality and severe threat to pig production. this study investigated the hematological and serum biochemical abnormalities associated with a natural asf virus (asfv) infection in pigs. a total of 100 serum samples of pigs from piggery suspected of asfv infection were screened for antibodies by elisa. thirty-two blood samples from serologically positive pigs and 32 negative pigs were undergo to hematological and serum biochemical analyses following standard procedures. the results showed that the mean values of the red blood cell (rbc) count, total white blood cell (twbc) count, absolute lymphocyte count, absolute monocyte count, serum total protein (tp) and globulin were significantly (p < 0.05) lower while the mean corpuscular volume (mcv), mean corpuscular hemoglobin concentration (mchc), absolute neutrophil count and serum gamma glutamyl transferase (ggt) were significantly (p < 0.05) higher in the infected than the healthy pigs. there were no significant differences (p > 0.05) in the mean values of the packed cell volume (pcv), hemoglobin concentration, absolute eosinophil count, cholesterol, alanine aminotransferase (alt) and aspartate aminotransferase (ast) activities between the infected and healthy pigs. hence, natural asfv infection may have caused alterations in the hematological and serum biochemical parameters in the infected pigs. the generated data could complement the existing laboratory diagnostic techniques such as polymerase chain reaction, direct fluorescence antibody test, indirect fluorescent antibody test and elisa in the diagnosis of asf in pigs. hematological and serum biochemical profiles of a natural african swine fever virus infection in pigs et  al. 2011). contaminated feed as a transmission vehicle for introducing trans-boundary animal diseases onto high-biosecurity swine operations has been recognized as a major risk factor since the introduction of porcine epidemic diarrhea virus into the united states in 2013 (dee et al. 2018). the virus is transmitted oro-nasally in the domestic pigs followed by penetration into the pharyngeal mucosa and through the lymphatics, the virus enters the mandibular or retropharyngeal lymph nodes, where viral replication occurs. the virus then spreads through circulatory system to other organs and tissues of the body (sanders et al. 2012). the disease is characterized by pyrexia (40-42  °c), introduction african swine fever (asf) is a highly contagious hemorrhagic, often fatal viral disease of pigs caused by african swine fever virus (asfv) of the genus asfivirus, in the family asfarviridae. it is a large, enveloped, double-stranded dna virus (alonso et al. 2018) and the only known vector-borne dna virus (dixon et al. 2011). african swine fever is maintained in africa by a natural cycle of transmission between wart hogs and the soft tick vector ornithodorus moubata. a cycle of maintenance between domestic pigs and ornithodoros has been identified in kenya (gallardo 276 veterinaria italiana 2022, 58 (3), 275-280. doi: 10.12834/vetit.2295.15822.2 hematological and serum biochemical abnormalities associated with asf infection in pigs okafor et al. could aid supportive therapy and complement the existing laboratory diagnostic techniques in the diagnosis of asf. materials and methods animals and flock history one hundred pigs from a piggery located in nnewi, anambra state, nigeria, with a natural asf infection, were sampled. the pigs sampled were between 6-7  months of age. the farm with a population of 767 pigs had lost 415 pigs as at the time of sample collection. breed of pigs stocked include large white, landrace, duroc and cambrough. the records at the farm showed the breakdown of the 767 pigs to comprise 346 males and 421 females. drugs used for therapy on the farm include long acting oxytetracycline, penicillin/streptomycin and diminazene aceturate. the study was performed in accordance with the ethical standards and the use of animals complied with local animal laws, guidelines and policies of animal ethics committee of the faculty of veterinary medicine, university of nigeria, nsukka, nigeria. blood sample collection blood samples were aseptically collected via the jugular venipuncture, after the animals were properly restrained using nose snares. blood samples were collected between 9:00 and 11:00 am into two sets of sample bottles (one containing anticoagulant-ethylene diamine tetra-acetic acid-edta for hematology and the other devoid of edta for serum biochemical analysis) and submitted to the laboratory, in cold packs. serological test assay for antibody detection was carried out using asfv-ab rapid eliza test kit (ring biotechnology co ltd, beijing 100176, china) following method described by woah (oie 2016) and the results were interpreted according to the instructions of the manufacturers. hematological analyses the packed cell volume (pcv) was determined by the microhematocrit method, red blood cell (rbc) counts, and total white blood cell counts (twbc) by hemocytometer method while the differential white blood cell counts were determined by leishman technique (thrall and weiser 2002). the hemoglobin concentration was determined by anorexia, severe disseminated hemorrhage, ataxia, and depression in domestic pigs with case-fatality rates approaching 100% (blome et  al. 2013). the clinical forms of the disease include per-acute, acute, sub-acute or chronic and even asymptomatic, with incubation period in natural infections varying from 4 to 19 days and clinical course ranging from less than seven days post-infection in acute form, to several weeks, or even months, in chronic form (beltran-alcrudo et  al. 2017). the major lesions due to asf occur in the circulatory system, lymphoid tissues, and renal system and include hemorrhages under the skin; enlarged, edematous, and completely hemorrhagic lymph nodes with extensive necrosis of the lymphoid follicles; epicardial and endocardial hemorrhages; enlarged, friable, markedly congested and dark-red to black spleen with rounded edges; and petechiae on the capsule of the kidneys with pneumonia, fibrinous pericarditis, and edematous lymph nodes in the chronic form of the disease (sanchez-vizcaino et al. 2015; beltran-alcrudo et al. 2017). the infected animals have marked leucopenia and severe impairment of lymphoid organs, characterized by lymphocytic and significant cellular depletion in spleen, lymph nodes and other lymphoid tissues (ramino-ibanez et al. 1995). pork is the most consumed meat from terrestrial animals, accounting for over 37 percent of global meat intake, followed closely by chicken (35.2%) and beef (21.6%) (fao 2013). however, in a recent report by the food and agricultural organization, pork dropped to second position after poultry as the most consumed meat in the world because of the ravaging effect of african swine fever in china which is the world leading producer of pork (fao 2019). according to beltran-alcrudo and colleagues (beltran-alcrudo et  al. 2017), african swine fever is a severe threat to pig production systems, in that it not only threatens food security and challenges the livelihoods of pig producers and other actors in the supply chain but may also have major consequences on international trade as a result of trade restrictions. the disease, however, is not zoonotic. there are no vaccines available for asf and the supportive therapy usually employed in the management of the disease to reduce morbidity and mortality can be improved with the knowledge of hematological and biochemical profiles of asf infected pigs. in addition, data on the hemato-biochemical profiles of a natural asf infection in pigs are scarce. the present study, therefore, investigated a natural asf infection in a piggery in nnewi, anambra state, nigeria, with special emphasis on the hematological and serum biochemical findings associated with the disease, with the aim of generating data that 277veterinaria italiana 2022, 58 (3), 275-280. doi: 10.12834/vetit.2295.15822.2 okafor et al. hematological and serum biochemical abnormalities associated with asf infection in pigs pigs. there was no significant difference (p > 0.05) in the mean value of the absolute eosinophil count between the infected and healthy pigs (table ii). serum biochemical findings the mean values of the serum total protein and globulin were significantly (p < 0.05) lower while the mean value of serum ggt was significantly (p  <  0.05) higher in the infected than the healthy cyanomethemoglobin method (higgins et  al. 2008) while the mean corpuscular volume (mcv) and the mean corpuscular hemoglobin concentration (mchc) were calculated using standard formulae. serum biochemical analyses serum activities of alanine aminotransferase (alt), aspartate aminotransferase (ast), serum gamma glutamyl transferase (ggt) and cholesterol were determined by colorimetric method using quimica clinica aplicada test kits. total protein was determined by direct biuret method and serum albumin by bromocresol green method using commercial kits. all analyses were read using diatek blood biochemistry analyzer (wuxi hiwell diatek instrument co. ltd, china). serum globulin was calculated by subtracting albumin from total protein. statistical analyses the data generated from the laboratory procedures were statistically analyzed by independent t-test using statistical software program (spss for windows version 20) and the level of significance was considered at p < 0.05. results clinical findings clinical signs such as fever, loss of appetite, depression, recumbency, hyperemia of the skin of the ear, abdomen and legs, respiratory distress, vomiting, emaciation, swollen joints, and lameness, typical of asf were manifested by the pigs sampled in this study. erythrocytic alterations the mean value of the rbc count was significantly (p < 0.05) lower while the mean values of the mcv and mchc were significantly (p < 0.05) higher in the infected than in the healthy pigs. however, there were no significant differences (p > 0.05) in the mean values of the pcv and hemoglobin concentrations between the infected and healthy pigs (table i). leukocytic changes the mean values of twbc count, absolute lymphocyte count and absolute monocyte count in the infected pigs were significantly (p < 0.05) lower than those of the healthy pigs, while the mean value of the absolute neutrophil count in the infected pigs was significantly (p < 0.05) higher than the healthy table i. the erythrocytic profile of pigs infected with african swine fever virus. parameters groups of pigs p-valuesinfected pigs (n = 32) healthy pigs (n = 32) pcv (%) 28.56 ± 1.05 30.00 ± 0.09 0.178 hb conc (g/dl) 10.03 ± 0.34 9.72 ± 0.06 0.364 rbc count (x10⁶) 8.16 ± 0.25a 9.09 ± 0.01b 0.001 mcv (fl) 35.01 ± 0.47a 33.09 ± 0.05b 0.000 mchc (g/dl) 35.23 ± 0.46a 32.38 ± 0.10b 0.000 all data are expressed as mean ± standard error mean (sem); n = number of animals sampled; a, bdifferent alphabetical superscripts on mean in a row indicate significant difference (p < 0.05) between the groups. table ii. the total white blood cell counts (twbc) and differential white blood cell counts of pigs infected with african swine fever virus. parameters groups of pigs p-valuesinfected pigs (n = 32) healthy pigs (n = 32) twbc count (x10³) 17.75 ± 0.67a 29.65 ± 0.13b 0.000 lymphocyte count (x10³) 6.28 ± 0.31a 19.93 ± 0.05b 0.000 neutrophil count (x10³) 9.99 ± 0.29a 6.60 ± 0.05b 0.000 monocyte count (x10³) 0.35 ± 0.03a 2.01 ± 0.05b 0.000 eosinophil count (x10³) 1.11 ± 0.08 1.11 ± 0.02 0.942 all data are expressed as mean ± standard error mean (sem); n = number of animals sampled; a, bdifferent alphabetical superscripts on mean in a row indicate significant difference (p < 0.05) between the groups. table iii. some biochemical changes in pigs infected with african swine fever virus. parameters groups of pigs p-valuesinfected pigs (n = 32) healthy pigs (n = 32) total protein (g/dl) 7.45 ± 0.06a 8.14 ± 0.09b 0.000 albumin (g/dl) 4.92 ± 0.12 5.04 ± 0.06 0.376 globulin (g/dl) 2.53 ± 0.07a 2.99 ± 0.00b 0.000 ggt (iu/l) 8.26 ± 2.23a 0.93 ± 0.03b 0.002 alt (iu/l) 75.23 ± 5.43 64.89 ± 0.66 0.063 ast (iu/l) 120.43 ± 7.73 124.70 ± 1.07 0.586 all data are expressed as mean ± standard error mean (sem); n = number of animals sampled; a, bdifferent alphabetical superscripts on mean in a row indicate significant difference (p < 0.05) between the groups. 278 veterinaria italiana 2022, 58 (3), 275-280. doi: 10.12834/vetit.2295.15822.2 hematological and serum biochemical abnormalities associated with asf infection in pigs okafor et al. the present study may be ascribed to a combination of temporary disruption of bone marrow function and apoptosis of lymphocytes. the significantly higher (p  =  0.000) absolute neutrophil count recorded in the present study may be attributed to a compensatory response of bone marrow stem cells to the depletion of lymphocytes in the lymphoid tissues following asfv induced apoptosis and necrosis. the mean absolute monocyte count was significantly lower (p = 0.000) in the infected pigs than the healthy ones in the current study. afayoa and colleagues (afoyoa et al. 2014) in contrast to the present finding reported a significantly increased monocyte count in the infected pigs in an experimental study. the difference between the two studies could be attributed to the phase of the disease when sample collection was done as the case in the current study may be ascribed to the destruction and depletion of the virus-infected monocytes. the mean value of serum total protein in the infected pigs was significantly lower (p  =  0.000) than the healthy ones. this could be attributed to low dietary protein intake following anorexia (awekew et  al. 2017). protein-losing enteropathy and decreased hepatic biosynthesis of albumin could also be implicated in the hypoproteinemia observed in the present study (nwoha et  al. 2013). in addition, significantly lower globulin (immunoglobulins) level observed in the present study may have contributed to the significantly lower serum protein. the mean value of serum globulin in the infected pigs was significantly lower (p = 0.000). the lower serum globulin correlates with lower absolute lymphocyte count recorded also in this study which was because of asf induced apoptosis of lymphocytes. the mean value of serum ggt in the infected pigs was significantly higher (p = 0.002) than that of the healthy pigs. this finding concurs with the report of afayoa and colleagues (afoyoa et  al. 2014). serum gamma-glutamyl transferase (ggt ) has been shown to be a sensitive marker of cholestasis and has been found to be a valuable tool in the diagnosis of hepatobiliary disorders (kasper et  al. 2018). according to mason and colleagues (mason et  al. 2010), ggt evaluation is an enzymatic liver function test that has been available for several decades and has been initially used as a sensitive indicator of fatty liver disease and hepatitis. the significantly higher serum ggt level recorded in the current study could be attributed to multiple organ damage caused by asf which includes the liver and hepatobiliary system. pigs. however, there were no significant differences (p  >  0.05) in the mean values of serum cholesterol, alt and ast between the infected and healthy pigs (table iii). discussion african swine fever is a highly contagious disease of wild and domestic pigs with high rates of morbidity and case fatality and constitutes a severe threat to pig production. it is listed by the world organization for animal health (woah) as a notifiable disease. however, the disease has no known therapy and vaccines are not readily available. this underscores the need for more research to elucidate the pathophysiology of asf as well as generate data that could complement the existing laboratory techniques in the diagnosis of asf in pigs. the present study, therefore, investigated the hemato-biochemical alterations associated with a natural asf infection in pigs. the mean value of the red blood cell count was significantly lower (p  =  0.001) in the infected pigs. this could be attributed to hemorrhagic blood loss. the asf is a hemorrhagic disease and the significant drop in the rbc count was expected. the significantly higher mcv (p = 0.000) and mchc (p  =  0.000) in the infected pigs recorded in the present study are indicative for the presence of reticulocytes produced by increased erythropoiesis in response to the hemorrhagic blood loss. the mean value of the twbc count was significantly lower (p = 0.000) in the infected pigs than the healthy ones. this finding agrees with the report of afoyoa and colleagues (afoyoa et al. 2014). gomez-villamandos and colleagues (gomez-villamandos et  al. 1995) attributed the leucopenia that occurs in asf to extensive necrosis of lymphocytes in the lymphoid tissues, as lymphocytes represent the largest proportion of leukocytes (territo 2019). in addition, the lower twbc count recorded in the present study was contributed by significantly lower absolute monocyte count in the infected pigs. the mean value of the absolute lymphocyte count was significantly lower (p  =  0.000) in the infected pigs. this finding agrees with the reports of gomez-villamondos and colleagues (gomez-villamondos et  al. 2003) and afayoa and colleagues (afayoa et  al. 2014). this could be attributed to apoptosis of lymphocytes induced by cytokines produced by asfv infected monocytes and macrophages (gomez-villamondos et al. 2003). lymphopenia is mostly associated with conditions that affect the bone marrow including viral infections that temporarily disrupt bone marrow functions (kliegman and st. geme 2019). the lower absolute lymphocyte count in the infected pigs recorded in 279veterinaria italiana 2022, 58 (3), 275-280. doi: 10.12834/vetit.2295.15822.2 okafor et al. hematological and serum biochemical abnormalities associated with asf infection in pigs disease. knowledges of the hemato-biochemical changes may be useful to predict the prognosis of the disease. veterinarians should consider the hematological and biochemical profiles of asf to formulate the effective supportive therapy so as to ameliorate the morbidity and high case fatality caused by the disease. ethical standards the research was carried out in accordance with the ethics and regulations guiding the use of animals, and handling of animals during sample collection complied with institutional animal welfare laws, guidelines, and policies and with ethical standards laid down in the 1964 declaration of helsinki and its later amendments. conclusions the results of the present study support that the disease causes alterations in the hematological and serum biochemical parameters in the infected pigs. the disease is associated with significantly lower rbc count, twbc count, absolute lymphocyte count, absolute monocyte count, serum total protein and globulin levels, and significantly higher mcv, mchc, absolute neutrophil count and serum ggt level. evaluation of hematological and serum biochemical parameters in pigs could therefore be used to complement the existing laboratory diagnostic techniques such as polymerase chain reaction, direct fluorescence antibody test, indirect fluorescent antibody test and elisa in the diagnosis of asf in the case of suspected outbreak of the 280 veterinaria italiana 2022, 58 (3), 275-280. doi: 10.12834/vetit.2295.15822.2 hematological and serum biochemical abnormalities associated with asf infection in pigs okafor et al. afayoa m., atuhaire d.k., ochwo s., okuni j.b., kisekka m., olaho-mukani w. & ojok l. 2014. hematological, biochemical and clinical changes in domestic pigs experimentally infected with african swine fever virus isolates from uganda. bull anim hlth prod afr, 62, 7-22. alonso c., 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on the epidemiology and pathology of african swine fever. j comp pathol, 152 (1), 9-21. sanders e.g., quintas a., perez-nunez d., nogal m. & barroso s. 2012. african swine fever virus uses micropinocytosis to enter host cells. plos pathog, 8 (6), 002754. territo m. 2019. lymphocytopenia. merck manual professional version. kenilworth, nj, merck & co. thrall m.a. & weiser m.g. 2002. hematology. in laboratory procedures for veterinary technicians (hendrix c.m., ed). 4th ed. mosby inc., missouri. 29-74. 215 veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 accepted: 01.09.2021 | available on line: 31.12.2021 1national and oie reference laboratory for brucellosis, istituto zooprofilattico sperimentale dell'abruzzo e del molise “g. caporale”, teramo, italy. 2direzione generale della sanità animale e dei farmaci veterinari, ministero della salute, roma, italy. 3istituto zooprofilattico sperimentale dell'umbria e delle marche “togo rosati”, perugia, italy. *corresponding author at: national and oie reference laboratory for brucellosis, istituto zooprofilattico sperimentale dell'abruzzo e del molise “g. caporale”, campo boario, 64100 teramo, italy. e-mail: d.averaimo@izs.it. fabrizio de massis1, flavio sacchini1, daniela averaimo1*, giuliano garofolo1, pierdavide lecchini2, luigi ruocco2, roberto lomolino2, ugo santucci2, elisa sgariglia3, silvia crotti3, antonio petrini1, giacomo migliorati1, nicola d'alterio1, stefano gavaudan3 and manuela tittarelli1 keywords brucella canis, isolation, dogs, serology, cgmlst. summary brucella canis has been isolated for the first time in italy in a commercial breeding kennel. it was diagnosed after a deep investigation related to the onset of reproductive disorders. animals were tested with direct and indirect techniques. the agent was first detected in two chihuahua aborted foetuses by direct culture. further, it was also isolated from blood samples of dogs hosted in the kennel, which also showed reaction to conventional serological tests (microplate serum agglutination test). the isolates were identified as b.  canis by standard microbiological methods and a bruce-ladder multiplex pcr. to investigate the genomic diversity, whole genome sequencing was used, applying the core genome multilocus sequence typing (cgmlst ). in a first round of serological testing performed on 598 animals, 269 (46.1%) tested positive. in the second round of laboratory testing carried out 4-5 weeks apart, the number of serologically positive dogs was 241 out of 683 tested (35.3%), while the number of dogs positive to isolation was 68 out of 683 tested (10.0%). the pcr showed a lack of sensitivity when compared to direct isolation. the epidemiological investigation did not identify the source of the infection, given the time elapsed from the onset of abortions to the definitive diagnosis of b. canis infection in the kennel. the genomic analyses featured the strains as st21 and, according to the cgmlst, revealed the presence of a tight cluster with a maximum diversity of four allelic differences. the observed limited genomic variation, largely within the known outbreak cut-offs, suggests that the outbreak herein described was likely caused by a single introduction. moreover, in a broader scale comparison using the public available genomes, we found that the closest genome, isolated in china, differed by more than 50 alleles making not possible to find out the likely origin of the outbreak. the lack of updated data on b. canis genome sequences in the public databases, together with the limited information retrieved from the epidemiological investigations on the outbreak, hampered identification of the source of b. canis infection. first isolation of brucella canis from a breeding kennel in italy first identified in 1966 (carmichael 1966), b.  canis is a gram-negative non-motile aerobic intracellular coccobacillus with rough colony morphology when grown on artificial medium. dogs and wild canidae are the only animal species that act as reservoirs of b.  canis under natural conditions (shin and carmichael 1999). canine brucellosis due to b.  canis is a contagious disease characterized by abortions in females and introduction brucellosis is a zoonosis, widespread all over the world, caused by bacteria belonging to the genus brucella. the most clinically relevant brucella species are brucella melitensis, b. abortus, b. suis and b. canis. they tend to be host-adapted, although infections may occur in other animal species, including humans (michaux-charachon et al. 2002). 216 veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 first isolation of brucella canis in italy de massis et al. unlike many bacterial infections, canine brucellosis is not amenable to treatment with antibiotics (nasphv 2012, carmichael and greene 2013). its intracellular persistence makes b.  canis a difficult target for antibiotics, despite its susceptibility in  vitro. treatment failures or relapses are common (carmichael and greene 2013), leading some entities to recommend that infected dogs, regardless of whether they are treated or not, should not be rehomed from infected facilities (usda 2015). canine brucellosis due to b.  canis is considered a zoonosis (blankenship and sanford 1975). the disease is underestimated in man due to general lack of serological testing facilities and misconceptions concerning its prevalence. confirmed cases of human illness due to b.  canis are relatively uncommon, with roughly 50 cases identified in the us since 1973 (daly et  al. 2020). however, because of the vague symptoms and effects in infected people, it is likely that human cases of canine brucellosis are under-diagnosed and underreported (nasphv 2012). those working closely with potentially infected animals, such as in breeding kennels or with stray animals, are considered at higher risk than others (hensel et  al. 2018). culture-positive cases have been reported in laboratory personnel, animal technicians and persons known to have close and frequent contact with infected dogs (carmichael et al. 1980). b.  canis is considered endemic in southern usa, in central and south america and in mexico (flores-castro et al. 1977, brower et al. 2007, küster de paula dreer et al. 2013, krueger et al. 2014, keid et al. 2017). it has been also reported in canada (cosford et  al. 2018). infections with b.  canis have been reported from asian countries such as china (di et al. 2014), japan (hayashi and ysayama 1977), india (yoak et al. 2014) and from african countries such as nigeria (cadmus et  al. 2011) and zimbabwe (chinyoka et  al. 2014). new zealand and australia appear to be free of b.  canis; however, australia has reported some b. suis infections in dogs, mainly in animals used to hunt feral pigs. (mor et al. 2016, rovid spickler 2018, gardner and reichel 1997). cases have been reported also from european countries, such as austria (hofer et  al. 2012), germany (von kruedener 1976, nöckler et al. 2003), hungary (gyuranecz et al. 2011), sweden (holst et  al. 2012, kaden et  al. 2014), switzerland (egloff et  al. 2018), and the united kingdom (taylor 1980, whatmore et al. 2017). the only report recorded in italy so far has been a presumptive b. canis infection in a dog with chronic prostatitis and discospondylitis, detected by pcr (corrente et al. 2010). aims of this paper are to report the first isolation of b. canis from a commercial breeding kennel in italy, to describe the case, and to inform about results of testing. epididymitis, testicular atrophy, prostatitis and infertility in males (wanke 2004). the disease is insidious, and many dogs do not have prominent signs, otherwise some dogs with generalized infection could show uveitis, discospondylitis and other more chronic conditions such as lymphadenomegaly, osteomyelitis, polyarthritis, meningoencephalitis, and pyogranulomatous dermatitis (carmichael and greene 2013). the disease is of particular importance for dog breeders since infection with b.  canis usually ends a dog's reproductive career (carmichael 1990). the most common routes of transmission of b. canis to humans are through contact with infected dogs, which may disseminate the bacteria with their secretions for many months after bacteraemia has ceased, and through direct laboratory exposure (carmichael and shin 1996). dog to dog transmission occurs during breeding, or through oronasal contact with reproductive discharges following abortions. b.  canis may also be shed with urine, feces, and nasal and ocular secretions. pups may be infected in utero or perinatally (carmichael and greene 2013). these bacteria may be transmitted either by the venereal or oral route, more frequently infection occurs following contact with abortive material. in males, urine and seminal fluid represent an important source of infection (george et  al. 1979). prolonged bacteraemia is a typical sign of canine brucellosis that persists from 6 months to 5 years (carmichael et al. 1984). prevalence and spreading of b.  canis infection in canine populations depend upon several individual factors, including age and reproductive status (carmichael and greene 2013, kaufmann and petersen 2019). in endemic countries, infection rates are typically higher in stray animals, as they are more likely to be reproductively intact and active compared to owned pets. breeding kennels can also be sites of higher-than-normal infection rates (carmichael and greene 2013). identifying brucellosis-infected dogs is often challenging. definitive diagnosis requires the culture of b.  canis from the blood of infected dogs, a process that has a relatively low sensitivity, is time-consuming, and somewhat technically impractical to apply to large populations of dogs. some serological methods have been developed as commercial kits and test services are available at diagnostic laboratories. false-positive and false-negative results may occur with these tests (keid et  al. 2009), however, in many situations they are the tests of choice for screening larger populations, such as stray dogs or those entering shelters (carmichael and greene 2013). 217veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 de massis et al. first isolation of brucella canis in italy the nrc, izsam. four-five weeks later, a second round of sampling involved 683 animals and this time both serum and edta blood were collected from each animal. overall, serum samples were collected from 683 dogs and submitted to the nrc, izsam. serological analyses were carried out using a microplate serum agglutination test (msat). the test was carried out modifying the tube agglutination test described by alton and colleagues (alton et  al. 1988), and volumes were adapted to be performed in 96-well u-shaped microplates. briefly, b.  canis strain rm66 was used to prepare msat antigen as previously described (alton et  al. 1988). before testing, serum samples were diluted 1:10 in tris-maleate buffer (tmb) ph 9.0 ± 0.5. the assay was performed by dispensing equal volumes (50 µl) of sera 2-fold serially diluted with tmb and b.  canis antigen in a 96 well u-shaped microplate, to obtain a final dilution ranging from 1:20 to 1:640. plates were incubated at 37 ± 2 °c for 48 h. samples displaying 100% agglutination at a dilution ≥ 1:20 were considered as positive. serology titres were indicated as the highest dilution of serum showing 100% agglutination. furthermore, in order to acquire information on specificity of msat, a panel of 143 samples from owned dogs non-related to the outbreak was also tested with this method. bacteriological investigation a total of 683 dogs of different breed were sampled and a whole blood sample was taken from each of them and submitted to the nrc, izsam. whole blood samples were cultured for detection of brucella spp. according to the literature methods (carmichael and greene 2006, cfsph 2018, gda 2020). briefly, samples were both streaked onto selective farrell’s brucella agar (izsam) and inoculated in enrichment broth supplemented with equine serum (brucella broth, izsam). all media were incubated at 37 ± 1 °c. subcultures in farrell’s brucella agar of all the broth cultured samples were made weekly for a month. plates were examined daily for evidence of growth and were considered negative when no colonies were seen neither from direct culture nor from all subcultures. typical brucella spp. colonies were subjected to specific pcr assay for species identification (oie 2016). pcr assay dna was extracted from all the samples (whole blood and suspected colonies) using the maxwell® 16 blood & cell dna purification kit (promega italia srl, milan, italy), following the manufacturer’s specifications. after extraction, dna was collected in dna/rna free tubes and stored at 4 ± 2 °c until materials and methods background and diagnostic screening in april 2020, two chihuahua aborted foetuses were submitted to the ancona laboratory of the istituto zooprofilattico sperimentale dell'umbria e delle marche (izsum) for the detection of canine reproductive pathogens. these specimens were collected from a kennel where several months of abortion, infertility and reproductive disorders were reported by the private veterinarians caring for the kennel. at time of sampling, the kennel hosted approximately 600 dogs, mostly chihuahua breed, but also some pomeranian, maltese and toy poodle breeds. samples collected from the two foetuses were subjected to standard bacteriological investigations and molecular tests for the following pathogens: canine herpes virus (chv; real-time pcr); brucella spp. (real-time pcr); leptospira spp. (real-time pcr); chlamydia spp. (real-time pcr); mycoplasma spp. (pcr); canine parvovirus (cpv; real-time pcr). brucella spp. were isolated in sheep blood agar, after incubation overnight at 37  °c (± 2 °c). cultures were carried out from brain, spleen, stomach and lung. identification of brucella genus was confirmed by maldi-tof and pcr, both from colonies and pooled viscera. the strain isolated was submitted for identification to the national reference centre for brucellosis (nrc), istituto zooprofilattico sperimentale dell'abruzzo e del molise (izsam), where bruce-ladder assay identified b.  canis (oie 2016). following the confirmation of b. canis isolation in the kennel, a bacteriological and serological survey was carried out, to investigate on the magnitude of the outbreak. samples were collected by local veterinary services according to italian and european regulations for animal welfare. samples were taken from the radial vessel with the vacutainer™ system in anticoagulant tubes, preventing blood from clotting. after collection, blood samples were kept refrigerated until delivered to the laboratory and then stored at 4 ± 2 °c until analysis. population data all dogs were identified with a microchip; data regarding age, gender, and breed were collected from the national and regional databases for the identification and registration of dogs. serological investigation after the confirmation of b.  canis outbreak in the kennel, a first round of serological sampling was carried out on 598 animals and submitted to 218 veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 first isolation of brucella canis in italy de massis et al. were ignored in the calculation of distance between pairs of sample profiles. all sequences were additionally typed using the brucella 9 locus mlst scheme available at https://pubmlst.org/brucella/ (whatmore et  al. 2007) accessible through ridom seqsphere+. statistical analysis data from laboratory information management system were imported in ms access® (microsoft access 2019, redmond, washington, dc, usa), which was used for cleaning and normalizing the dataset. to take into account the uncertainty of the proportion of positive laboratory results over the total tests performed, a beta distribution was used to define the 95% confidence interval of the proportion accuracy. the uncertainty interval was defined as the difference between upper and lower 95% confidence limits. the 95% lower and upper credibility levels (l.c.i. and u.c.l., respectively, composing the credibility interval, ci) of the distribution frequency of positive results were calculated using a bayesian approach (sivia 1996) with a beta distribution (n + 1; n s + 1), where n is the total number of tested samples and s are the tested positive samples. results population data the total number of dogs considered in this study was 683. out of them, 475 (69.5%) were females and 208 (30.5%) were males. the distribution of dogs by age at the time of sampling is shown in figure  1. the dogs in the kennel were 2.8 years old in average, with the oldest dog being 8.5 year old. the most represented breed was chihuahua (605 dogs, 88.6%), followed by spitz (37 dogs, 5.4%), analysis. the reaction mix was prepared using the brucella genus (all species) genesig™ advanced kit (genesig, york house, school lane, chandler's ford, uk) in a final volume of 20 µl consisting of 5 µl of extracted dna and 15 µl of master mix, according to the manufacturer's instructions. the samples were analyzed by real-time pcr. pcr was run in a quantstudio™ 7 pro real-time pcr system (applied biosystems, ca, usa) under the following conditions: enzyme activation step at 95 °c for 2 min, 50 cycles of denaturation at 95 °c for 10 s and data collection at 60 °c for 60 s. a positive control (k+) and a no-template control (ntc) were included in each run. data were analyzed by design and analysis software 2.4.3. whole‑genome sequencing (wgs) a total of 67 b. canis strains each isolated from a single dog were submitted to wgs. the genomic dna extracted from bacterial colonies was quantified with the qubit fluorometer (qubittm dna hs assay; life technologies, thermo fisher scientific inc., waltham, ma, usa). sequencing libraries were prepared using nextera xt library preparation kit (illumina inc., san diego, ca, usa) according to the manufacturer’s instructions. the libraries were sequenced using the illumina nextseq 500 platform, producing 150-bp paired-end reads. after demultiplexing and removal of adapters, reads were trimmed from 50 and 30 ends using trimmomatic tool version 0.36 to discard the nucleotides with quality scores of less than 25. reads shorter than 36 bp were automatically discarded. scaffolds were assembled with spades version 3.11.1 with the careful option selected (bankevich et  al. 2012). read sequences were submitted to sequence read archive of the national center for biotechnology information (ncbi) under the bioproject accession number prjna748851. multilocus sequence typing (mlst) and core genome (cg)mlst analysis genome assemblies produced in our study, along with 67 public genomes available at genbank (accessed on 28 october 2020), were genotyped using cgmlst. the cgmlst profiles were assigned using the task template b. melitensis/suis/canis/ abortus cgmlst with 2076 targets core genes in ridom seqsphere+ software, v4.1.1 (ridom gmbh, münster, germany) as described by janowicz and colleagues (janowicz et al. 2018). multiple spanning tree (mst) was generated by pairwise comparison of cgmlst target genes using default parameters. the neighbour-joining tree was constructed by the distance allele table and circular tree was visualized using itol (letunic and bork 2019). missing values pe rc en ta g e age (years) 30% 25% 20% 15% 10% 5% 0% 17.6% 0-1 17.6% 1-2 14.2% 3-4 7.9% 4-5 7.5% 5-6 5.3% 6-7 1.0% 7-8 0.6% 8-9 28.4% 2-3 figure 1. age distribution of dogs at the time of sampling. 219veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 de massis et al. first isolation of brucella canis in italy identified 138 samples as negative, demonstrating a specificity of 96.5% (ci 92.1%-98.5%). the distribution of seropositivity was different between males and females. overall, 48 males were positive out of 208 (23.1%), while 193 females were positive out of 475 (40.6%). this difference was found as significant (χ2 = 19.52; p < 0.01). when age classes were considered, the distribution of seropositivity was higher in the classes from 2 to 7 years. lower rates of seropositivity were recorded above and under that age interval (table ii). when considering the credibility intervals, the number of seropositives under one year of age was significantly lower than the other age classes. the distribution of seropositivity according to breed is shown in table iii. the seropositivity in poodle breed was significantly higher than the seropositivity recorded for chihuahua breed (χ2 = 9.38; p < 0.01). brucella isolation and identification out of the 683 whole blood samples collected, 68  were positive for microbiological isolation. all strains isolated were confirmed as b.  canis by pcr assay. moreover, 61 samples grew on direct sowing, while seven strains were detected only by subcultures from enrichment broth. furthermore, real-time pcr allowed the identification of 32 positive dogs. results are summarized in table iv. when analysing the performances of the two tests, the cohen’s kappa value obtained was 0.381, poodle (29 dogs, 4.2%), maltese (7 dogs, 1.0%), pug (3 dogs, 0.4%), tibetan mastiff (1 dog, 0.1%), and a cross-breed dog (1 dog, 0.1%). serology in the first round of sampling, 269 tested positive out of 598 animals sampled. given that 15 sera were not analysed because haemolytic, the apparent seroprevalence was 46.1%. in the second round of sampling, out of the 683 serum samples collected, 241 were positive, for an apparent prevalence of 35.3%. among the 241 seropositive animals, 64  were also positive to blood culture (26.6%). in four seronegative animals, b.  canis was isolated from blood cultures (table i). when analysing the performances of the two tests, the cohen’s kappa value obtained was 0.307, considered as fair in the interpretation of strength of agreement suggested by landis and kock (landis and kock 1977). comparing the results of serological testing during the first and second round of sampling, 220 animals remained serologically positive, 45 animals became negative, while 15 animals became seropositive. when considering the same group of animals for the first and second round of sampling, seroprevalence decreased from 46.1% to 40.0%. we also calculated the specificity of msat on 143 sera from owned dogs not-related to the outbreak. msat table i. comparison of positive and negative results obtained by b. canis microplate serum agglutination test (msat) and brucella spp. isolation method. serology (msat) positive negative total isolation positive 64 4 68 negative 177 438 615 total 241 442 683 table ii. distribution of seropositivity by age classes. age class n. tested n. positive % positive l.c.l. u.c.l. 0 1 120 5 4.2% 1.8% 9.4% 1 2 120 40 33.3% 25.5% 42.2% 2 3 194 87 44.8% 38.0% 51.9% 3 4 97 43 44.3% 34.8% 54.3% 4 5 54 26 48.1% 35.4% 61.2% 5 6 51 22 43.1% 30.5% 56.8% 6 7 36 15 41.7% 27.1% 57.9% 7 8 7 2 28.6% 8.5% 65.1% 8 9 4 1 25.0% 5.3% 71.6% total 683 241 35.3% 31.8% 38.9% table iii. distribution of seropositivity by breed. breed n. tested n. positive % positive l.c.l. u.c.l. chihuahua 605 207 34.2% 30.5% 38.1% spitz 37 13 35.1% 21.8% 51.4% poodle 29 18 62.1% 43.9% 77.3% maltese 7 1 14.3% 3.2% 52.7% pug 3 2 66.7% 19.4% 93.2% cross-breed 1 0 0.0% 1.3% 84.2% tibetan mastiff 1 0 0.0% 1.3% 84.2% total 683 241 35.3% 31.8% 38.9% table iv. comparison of positive and negative results obtained on edta blood samples by isolation and real-time pcr for b. canis identification. real-time pcr positive negative total isolation positive 21 47 68 negative 11 604 615 total 32 651 683 220 veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 first isolation of brucella canis in italy de massis et al. strains fell in the st21 group, identifying that closest genome was isolated in china on an unspecified date (figure 3). discussion and conclusions this is the first report of the isolation of b.  canis in dogs in italy, as well as the first report of the occurrence of an outbreak in an italian commercial breeding kennel. moreover, and to the best of our knowledge, this is also the first report worldwide of an outbreak of b. canis infection involving very high number of dogs. until the bacterial isolation described in this study, brucellosis due to b. canis was considered a foreign disease for italy. previous recordings were based on serological evidence only, with the exception of a single report of b.  canis identification by pcr, in a dog with chronic prostatitis and discospondylitis (corrente et  al. 2010). private veterinarians or considered as fair in the interpretation of strength of agreement suggested by landis and kock (landis and kock 1977). the distribution of dogs positive to b. canis isolation was different between males and females. overall, 13 males were positive out of 208 (6.3%), while 55 females were positive out of 475 (11.6%). this difference was found as significant (χ2  =  4.58; p  <  0.05). when age classes were considered, the percentage of dogs positive to bacteriology was higher in the classes from 2 to 3 years, and from 7 to 8 years (table v). however, when considering the credibility intervals, no significant difference was recorded between age classes. the distribution of positivity to isolation according to breed is shown in table vi. the rate of isolation in poodle breed was significantly higher than the rate recorded for chihuahua breed (χ2 = 19.92; p < 0.01) genomic analysis genome assemblies were used to retrieve mlst profiles, which identified for all the strains analysed the sequence type (st) 21. the assignation of the cgmlst profiles comprising 2074 targets retrieved at least 98.7% of them for all the strain analysed. the cluster analysis from the outbreak revealed that all the strains belong to the same cluster (figure 2). the maximum distance observed between strains isolated in the outbreak was four allelic differences, thus confirming the hypothesis that the outbreak has been generated by a single introduction of b. canis in the kennel. the comparison of the cgmlst profiles against the public genomes revealed that the b. canis population was divided in two main groups corresponding to st20, which was previously linked to south, central and north america, and st21 mostly linked to asia and europe (vicente et  al. 2018). the outbreak table v. distribution of dogs positive to b. canis isolation according to age classes. age class n. tested n. positive % positive l.c.l. u.c.l. 0 1 120 6 5.0% 2.4% 10.5% 1 2 120 13 10.8% 6.5% 17.7% 2 3 194 26 13.4% 9.3% 18.9% 3 4 97 11 11.3% 6.5% 19.2% 4 5 54 3 5.6% 2.0% 15.1% 5 6 51 5 9.8% 4.4% 21.0% 6 7 36 3 8.3% 3.0% 21.9% 7 8 7 1 14.3% 3.2% 52.7% 8 9 4 0 0.0% 0.0% 45.1% total 683 68 table vi. distribution of dogs positive to b. canis isolation by breed. breed n. tested n. positive % positive l.c.l. u.c.l. chihuahua 605 54 8.9% 6.9% 11.5% spitz 37 4 10.8% 4.4% 24.8% poodle 29 10 34.5% 19.9% 52.8% maltese 7 0 0.0% 0.3% 36.9% pug 3 0 0.0% 0.6% 60.2% cross-breed 1 0 0.0% 1.3% 84.2% tibetan mastiff 1 0 0.0% 1.3% 84.2% total 683 68 10.0% 7.9% 12.4% 1 1 1 1 1 1 1 1 1 1 1 2 2 gt-9 gt-6 b gt-3 gt-14 gt-8 gt-10 gt-12 gt-1a gt-2 gt-4 gt-5 gt-7 c gt-11d gt-13 1 1 1 1 1 1 1 1 1 1 1 2 2 figure 2. minimum spanning tree generated for 67 dog isolates. each circle represents an allelic profile. the numbers on the connecting lines illustrate the numbers of target genes with differing alleles. 221veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 de massis et al. first isolation of brucella canis in italy brucellosis control and eradication in ruminants would not be acceptable for application in dogs, both for animal welfare and ethical reasons. on the other hand, due to possible health implications, either for dogs and their owners, actions are required to acquire data on distribution of this underhanded zoonosis of pets, as well as for controlling his spread in situations where it occurs. thus, protocols are necessary for proper detection, control, and eradication of the infection in kennels and in owned dogs. laboratory tests are a cornerstone for b.  canis diagnosis and control. serological testing represents a valid screening tool for evaluating the presence of b.  canis in breeding kennels. based on serological results, apparent seroprevalence during the first round of sampling (598 animals) was 46.1% while in the second round of sampling (683 animals) public health authorities have rarely investigated this zoonosis, also due to the lack of regulations on canine brucellosis surveillance or provisions for specific controls in dog international trade for this disease. this contributed to the current lack of data on b.  canis diffusion among the italian dog population, and to the limited knowledge about the disease distribution in the european union (eu) canine population. furthermore, considering its zoonotic potential, uncontrolled spread of b.  canis may have important public health implications. in italy, the veterinary regulations do not provide specific restrictive measures for canine brucellosis caused by b.  canis. actually, brucellosis and agents thereof are included in the list of the zoonosis requiring surveillance on annex i of the so-called “zoonoses directive” (eu 2003). however, in the context of canine brucellosis, current rules for gcf 900497665 1 09 369 776 1 genomic 21 finland gc f 0 00 48 02 75 1 b ruc ca ni 96 72 58 v1 ge no mi c 2 0 err473754 20 err467400 20 er r4 16 54 08 20 gcf 004127195 1 asm412719v1 genomic 21 china gc f 0 00 53 04 95 1 o live ri g en om ic 2 0 err467401 20 gcf 00 029218 5 1 asm 29218v 2 geno mic 21 china 2020-te-128612-2-1 st21 outbreak italy gcf 00 069 158 5 1 as m6 915 8v1 ge nom ic 2 0 s we den gc f 0 01 07 83 35 1 b c an is s cl ge no mi c 2 0 c hil e err485955 20 err473749 20 srr 587 932 1 2 0 er r2 13 65 46 2 0 br az il srr40390 06 20 gcf 0007 4033 5 1 a sm7 4033 v1 g enom ic 20gc f 0 00 36 68 25 1 br uc ca ni uk 10 02 v1 ge no mi c 2 0 er r4 16 54 10 20 err473748 20 er r2 13 65 47 un kn ow n b ra zil gcf 003010715 1 asm301071v1 genomic 21 china gcf 00 259 125 5 1 as m2 591 25v 1 g eno mic 20 err473747 20 gc f 0 01 71 53 65 1 a sm 17 15 36 v1 ge no mi c 2 0 u sa srr 403 900 7 2 0 srr5484406 20 u s gcf 000367305 1 bruc cani f7 05a v1 genomic21 south africa err473755 20 gcf 001715415 1 asm171540v1 21 usa gc f 9 00 49 18 95 1 07 28 59 60 71 ge no m ic un kn ow n b ra sil gcf 001 715 445 1 a sm1 715 44v 1 ge nom ic 20 usa err418017 20 gcf 00029 8575 1 v1 0 for bruce lla canis str 118 genom ic 21 china srr5 4841 36 20 us gcf 000480295 1 bruc cani 04 2330 1 v1 genomic 20 er r2 13 65 49 2 0 br az il err2136545 20 spain err554821 20 er r4 16 54 07 20 gcf 001758265 1 asm175826v1 genomic 21 sweden gc f 0 00 36 72 85 1 br uc ca ni cn gb 13 24 v 1 g en om ic 20 ar ge nt in a err473750 20 sr r6 42 80 9 2 0 a rg en tin a err473752 20err473753 20 srr54 83825 20 us er r4 16 54 09 20 gcf 004127225 1 asm412722v1 genomic 21 china err473746 20 gcf 000 018 525 1 a sm1 852 v1 g eno mic 20 gc f 0 00 37 06 05 1 br uc ca ni cn gb 51 3 v 1 g en om ic 2 0 c hi le err418019 20 err485956 20 err418018 20 gc f 9 00 49 19 15 1 0 7 28 59 6 07 0 ge no m ic 20 b ra si l err473756 20 err473757 20err473751 20 gcf 000238195 1 asm 23819v1 genomic 21 s outh korea err554818 20 err473758 20 err2136548 21 finland err554819 20 lisarb 02 cimoneg 96401 1 529194009 fcg gcf 000370585 1 bruc cani 79 122 v1 genomic 21 japan gcf 900491905 1 96 9626 genomic 20 spain st21 st20 figure 3. neighbour-joining tree of b. canis. the tree was constructed using distance allele table sequences of 68 isolates and mid-point rooted. the label from the outbreak clone is reported in red. sequence type groups are reported in green for st20 and red for st21. 222 veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 first isolation of brucella canis in italy de massis et al. than in males, both for serology and isolation tests. actually, in a free-living dog population and in a situation of uncontrolled spread of the disease, it would be expected to have a similar prevalence in both genders. however, giving that the outbreak occurred in a commercial breeding kennel, this finding could be the result of breeding practices. in fact, one hypothesis is that to maintain a good bloodline not all males were allowed to mate and then only a fraction of them was actually exposed to the infection from the venereal route. the rate of positivity was distributed evenly across age classes, except for dogs aged one year or under. this is suggestive of an infection that remained uncontrolled for a long period of time, favouring the disease spread among dogs of all age classes, both through mating and environmental contamination. similar observations on the low susceptibility of animals under the age of reproduction to brucellosis were also reported in other animal species (nicoletti 1980). however, it is not clear if this resistance or lower susceptibility of young animals is related to immature reproductive system, a reduced time of exposure to infection, or other factors that remain to be investigated. when looking at the breed involved in the b.  canis outbreak, positivity was found significantly higher in poodles, even if this breed was low in numbers compared to chihuahuas. this may suggest that the infection was introduced in the kennel with this breed and then was transmitted to other dogs due to environmental contamination after abortion. the fair agreement recorded between isolation and pcr testing procedures (cohen’s kappa value 0.381), considering that the isolation gave positive results in a higher number of specimens with respect to pcr, suggests that the pcr assay has difficulties in performing on blood matrix. in the literature the sensitivity of pcr for brucella is very variable when performed on blood matrix, ranging from 3.26% (kauffman et  al. 2012) to 97.14% (keid et  al. 2010). this may be due to the presence of pcr-inhibitory molecules like haemoglobin (sidstedt et  al. 2018), which may affect the amplification process through different mechanisms or lead to a failure of amplicons detection. not only the presence of inhibitors, but also the use of antibiotics and heparin blood sampling could alter the sensitivity of the pcr results (mol et al. 2020). to improve the performance of the method, it is possible to proceed with centrifugation of the blood sample and extraction of the dna from buffy coat. however, larger quantities of blood are required for this purpose, and often they are not available from dogs due to the breed size. also because brucella is a facultative intracellular bacterium, the dna yield during extraction may be low. this critical point may be solved by sowing decreased to 35.3%. these data resemble or even exceed the highest values reported in the literature (hensel et al. 2018), giving a clear number of the impact the disease may have in a kennel. the high seroprevalence is probably related and consequent to the high density dog population hosted in the kennel, in addition to inadequate kennel management, both factors contributing to b. canis spreading. we observed a reduced apparent seroprevalence between the first and second sampling. this result is partially explained with the increased number of animal tested during the second sampling. actually, the additional population tested in second sampling was composed by young animals, and most of them resulted seronegative. someone may also argue that the decreased prevalence observed was related to the antibody isotype switching that does occur as the disease progresses, when the most prevalent b. canis specific antibodies found in serum after infection shift from igms to iggs. iggs are characterised by lower agglutination properties compared to igms. in the hypothesis that msat mainly detect igms, msat would have a reduced sensitivity on sera with high concentrations of b. canis specific iggs but low igms. in our opinion, this was not the case and previous studies in human, performed on similar test, showed that even when igms are removed, serum agglutination capacity, despite reduced, was maintained by igg and iga antibodies (marrodan et al. 2001). this suggests that also animals with lower amount of igms compared to iggs should test positive to msat. on the other hand, these observations support the use in parallel of serological tests that evaluate igg antibodies, in addition to igms, this in order to maximize the chance of detecting seropositive animals. finally, the non-optimal specificity observed for msat may have influenced the different prevalence observed between the first and second sampling. in fact, 32  animals that tested positive to msat during the first sampling, with an antibody titer around the cut-off value, turned negative few weeks later. a fair agreement was recorded between isolation and msat testing procedures (cohen’s kappa value 0.307). during the outbreak investigation, we identified four cases of active infection and bacteraemia that tested negative to serological tests. as previously described (carmichael 1990, carmichael and greene 2006) antibody response only appears 5-8 weeks after infection and animals with ongoing bacteraemia may result negative to serological tests for 3-4 weeks post infection. similarly, animals with chronic infection may also result negative to serological screening (carmichael and greene 2006). the study also evaluated the disease prevalence among gender and age. the rate of positivity to laboratory tests was significantly higher in females 223veterinaria italiana 2021, 57 (3), 215-226. doi: 10.12834/vetit.2497.15848.1 de massis et al. first isolation of brucella canis in italy further circumstances prevented the identification of the source of infection in the kennel under study. dogs were kept in a commercial breeding kennel, where epidemiological investigation on movements of breeding dogs were not conclusive. furthermore, from the occurrence of abortions to the diagnosis of canine brucellosis several months elapsed, allowing the infection spreading largely in the kennel, thus hampering the possibility to identify the origin of the infection and, therefore, to trace back its source. public and private veterinarians should be trained in canine brucellosis diagnosis, prevention and control, and should investigate the causes of abortion in dogs considering also b.  canis. more research on the proper use of antimicrobials in affected dogs is advisable, at least until a reliable vaccine will become available. this highlights also the importance of reminding breeders of the clinical signs of canine brucellosis and their responsibility to prevent intra-population and inter-population spread of the disease and possible human infections. surviving puppies can be an important source of b.  canis infection, as they can become permanent carriers and shedders of the pathogen. all cases of canine abortion should be examined for brucellosis by bacterial culture of the foetuses and placentas. commercial breeding kennels should be regularly checked for causes of abortion and international trade rules should foresee testing for b.  canis of imported breeding dogs. finally, more researches are required in order to enhance the performances of the diagnostic tests in terms of sensitivity and specificity, as well as more survey data are needed to determine the current spread of b. canis in italy, in the light to identify the related epidemiological patterns and burden of the disease on italian canine populations, as well as on public health. in enrichment broth and extracting dna from the one-week incubation culture. the genomic analysis of strains isolated from the outbreak revealed that they all belong to the same cluster (figure 1). the maximum distance observed between strains isolated in the outbreak was only of four allelic differences, thus confirming the hypothesis that the outbreak under study has been generated by introduction of a single of b.  canis strain in the kennel. the number of allelic differences was within the published cut-off of genomic cluster found out in a b. melitensis outbreak (janowicz et al. 2018). moreover, these limits were also reasonable if compared with several bovine brucellosis outbreaks. these observations strengthen the idea that a single b.  canis introduction led to unrestrained spread within the kennel, affecting many dogs and puppies. importantly, by surveilling b.  canis sequences from the worldwide database, it was not possible to trace back this strain; however, the availability of the genomes generated by the present study will allow the trace forward and the surveillance of the infection spread from this outbreak. this study highlights the need of further data for an updated genomic surveillance with the aim to avoid public health consequences related to a poor mitigation strategy in fighting the spread of this disease at national and international level. the scarce information about b.  canis contained in the international brucella genomic databases has made not possible to give further details on the genomic differences of the strains isolated, which would have been useful to trace back the infection source. indeed, to better help epidemiological investigations in b.  canis outbreaks, it would be needed to genotype as much b.  canis isolates as possible worldwide, in order to improve the quantity and quality of information stored in the 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106, 102-104. 205 parole chiave agalassia contagiosa, mycoplasma agalactiae, mycoplasma mycoides subsp. capri, mycoplasma capricolum subsp. capricolum, mycoplasma putrefaciens, pecora, capra. riassunto l’agalassia contagiosa (ac) è una malattia seria che colpisce i piccoli ruminanti di molti paesi e si manifesta solitamente con mastite, artrite, keratocongiuntivite, pleuropolmonite e setticemia. il mycoplasma agalactiae (ma) è la principale causa di infezione in pecore e capre; mycoplasma mycoides subsp. capri (mmc, che comprende anche il m. mycoides subsp. mycoides large colony type), mycoplasma capricolum subsp. capricolum (mcc) e mycoplasma putrefaciens (mp) possono dare forme cliniche simili ma sono di solito associati a infezioni nelle capre. per questo studio sono stati prelevati campioni da116 pecore e 16 capre, scelte in seguito a epidemie sospette nella provincia di ardebil in iran, affette dalla forma acuta della malattia. latte, liquido lacrimale e liquido sinoviale sono stati raccolti, esclusivamente dagli animali colpiti, per identificare la causa principale della malattia. dopo la coltura in brodo e agar pplo, 33 dei 132 campioni raccolti (24,2%) sono risultati positivi a specie di mycoplasma. in 18 (12 pecore e 6 capre) il metodo pcr / dgge ha rilevato infezioni miste con specie di mycoplasma responsabili dei casi di agalassia contagiosa dei piccoli ruminanti nel nord-ovest dell’iran keywords contagious agalactia, mycoplasma agalactiae, mycoplasma mycoides subsp. capri, mycoplasma capricolum subsp. capricolum, mycoplasma putrefaciens, sheep, goat. summary contagious agalactia (ca) is a serious disease of small ruminants that occurs in many countries, and is usually characterized by mastitis, arthritis, keratoconjunctivitis, pleuropneumonia, and septicemia. mycoplasma agalactiae (ma) is the main causative agent in sheep and goats but other pathogens including mycoplasma mycoides subsp. capri (mmc, which incorporates the former m. mycoides subsp. mycoides large colony type), mycoplasma capricolum subsp. capricolum (mcc), and mycoplasma putrefaciens (mp) might be involved. they are all usually associated with infections in goats and may cause similar clinical signs. a total of 116 sheep and 16 goats suffering from the acute form of the disease were included in this study. they were recruited following a number of outbreaks suspected to be ca in the ardebil province of iran. milk, lachrymal or synovial fluid were collected exclusively from the affected animals in order to identify the pathogen involved. of the 132 collected samples, 33 (25%) were positive for mycoplasma species by culture in pplo broth and agar. the polymerase chain reaction followed by denaturing gradient gel electrophoresis (pcr/dgge) method identified 18 (12 sheep and 6 goats) of the 33 mycoplasma positive samples with mixed mycoplasma population. in particular, 25 ma (47.2%), 23 mp (43.4%), 4 mcc (7.5%), and 1 mmc (1.9%) were identified. this confirms that the several mycoplasma species rather than the ma only are in circulation, and are able to cause ca in sheep and goats in iran. this is the first report on the isolation and identification of mp, mmc and mcc in infected small ruminant flocks in iran. veterinaria italiana 2018, 54 (3), 205-210. doi: 10.12834/vetit.831.4072.2 accepted: 01.09.2016 | available on line: 30.09.2018 1veterinary aerobic bacterial vaccine quality control laboratory, razi vaccine & serum research institute, agricultural research, education and extension organization (areeo), karaj, iran. 2veterinary aerobic bacterial vaccine department, razi vaccine & serum research institute, agricultural research, education and extension organization (areeo), karaj, iran 3animal and plant health agency (apha), weybridge, woodham lane, addlestone, surrey kt15 3nb, united kingdom. *corresponding author at: veterinary aerobic bacterial vaccine department, razi vaccine & serum research institute, agricultural research, education and extension organization (areeo), karaj, iran. tel.: +98 26 34502892, e-mail: r.ghaderi@rvsri.ac.ir. afshin hajizadeh1, rainak ghaderi2* and roger d. ayling3 species of mycoplasma causing contagious agalactia in small ruminants in northwest iran 206 mycoplasma in small ruminants in iran hajizadeh et al. veterinaria italiana 2018, 54 (3), 205-210. doi: 10.12834/vetit.831.4072.2 organization (fao), iran has 72.5 million small ruminants (68% sheep and 32% goats) of several breeds that are mostly kept together1. this is similar to many other countries in the middle east (awan et  al. 2010). bory and entessar first described ma as a causative agent of ca in iran (bory and entessar 1959). further studies revealed that ma is endemic all over the country (kheirabadi and ebrahimi 2007, sotoodehnia and aerabi 1986). although an inactivated monovalent vaccine adjuvanted with saponin against ma is used to try to control the disease (sotoodehnia et al. 2007), ca persists in iran. despite the current vaccination programme in iran, other ca causing agents have been identified, including ma, mcc, mmc and mp. this study therefore aimed to determine if other mycoplasma species are involved in the occurrence of the disease. materials and methods standard strains four standard isolates were obtained from animal and plant health agency (apha), uk and used as controls: mycoplasma agalactiae nctc 10123, mycoplasma mycoides subsp. mycoides large clony (mmmlc) type nctc 10137, mycoplasma capricolum subsp. capricolum nctc 10154, mycoplasma putrefaciens nctc 10155. sample size and sampling method despite frequent reports of ca in northwest iran and the high population of small ruminants, no previous studies into the presence of ca have been carried out. six cities in the ardebil province located in the northwest of iran, which has a moderately cold mountain climate, were selected as the region for investigation. ca is frequently present in this region, and neighbouring countries may present a risk of disease incursion. a total of 18 flocks with clinical signs of ca were identified, and samples were collected from 132 animals (116 sheep, 16  goats) introduction contagious agalactia (ca) has been known for nearly 200 years (anonymous 2008). the clinical disease was first described by metaxa in italy in 1816 and was given the name contagious agalactia by brusasco in 1871 (madanat et  al. 2001). many countries in the world report ca, but countries in the middle east and mediterranean are probably most affected (kumar et  al. 2014). the main cause of the disease is mycoplasma agalactiae (ma), but mycoplasma mycoides subsp. capri (mmc), which now incorporates m. mycoides subsp. mycoides large colony type (shahram et  al. 2010, vilei et  al. 2006), mycoplasma capricolum subsp. capricolum (mcc), and mycoplasma putrefaciens (mp) are considered as other infectious agents of this syndrome (anonymous 2008, hotzel et al. 2003). contagious agalactia has previously been included in the list b of diseases by the office international des epizooties (oie) (anonymous 2008). ca is a severe infectious disease of small ruminants, with ma being found in sheep and goats, and mmc, mcc, and mp usually detected in goats. some reports state that goats are more susceptible to this disease than sheep (maré 2014, smith and sherman 2009). ca is most commonly transmitted through oral, respiratory, and mammary routes. the incubation period varies between 1 week and up to 2 months. clinical signs in infected animals can be acute or in more severe cases can lead to bacteremia and fever with sub-acute or chronic disease. ca originating agents are carried by circulating blood to susceptible organs such as eyes, mammary gland, and joints (madanat et  al. 2001). the infection of susceptible flocks can cause high morbidity (30-60%) and mortality. mortality rates can be as high as 40-70% in lambs and kids, but is generally lower in adults, although it may still be as high as 20% depending on the mycoplasma species. the clinical signs of ca include: fever, mastitis, arthritis, keratoconjunctivitis, pleuropneumonia, and septicemia (makeps syndrome) with lameness and keratoconjunctivitis affecting between 5-10% of infected animals (anonymous 2008). the organisms can persist for more than 1 year and up to 8 years after clinical recovery in infected animals, which may then serve as a source of infection (bergonier et al. 1997). according to the latest food & agriculture il coinvolgimento di più specie di mycoplasma. in totale sono stati identificati 25 ma (47,2%), 23 mp (43,4%), 4 mcc (7,5%) e 1 mmc (1,9%). questo è il primo rapporto sull'isolamento e l'identificazione di mp in pecore infette e di mmc e mcc nelle capre in iran. i dati confermano inoltre che oltre al mycoplasma agalactiae anche altre specie di mycoplasma sono responsabili dell’agalassia contagiosa nelle pecore e capre dell’iran. 1 http://www.fao.org/faostat/en/#country/102. 207veterinaria italiana 2018, 54 (3), 205-210. doi: 10.12834/vetit.831.4072.2 hajizadeh et al. mycoplasma in small ruminants in iran 5% co 2 for up to 3 days. cultures were observed daily and examined for any indications of mycoplasma growth, including turbidity and changes in colour to the culture medium. those showing signs of growth were used to inoculate fresh pplo agar plates for up to 2 weeks at 37  °c (anonymous 2008) and any cultures obtained were subjected to further testing and also freeze-dried for later examination and identification. the dna of the cultures was extracted using the dna extraction kit (cinnagen, iran) and stored in micro-tubes at -20°c until they were required as template dna in the polymerase chain reaction (pcr) tests. six different pcr’s were performed, generic pcr to detect the mycoplasma species. components for 12-μl reactions contain 6  μl pcr master mix (ampliquor, denmark), 0.7 μl of working that hadn’t received either ca vaccination in the previous year or recent antimicrobial treatment. samples were collected between october 2010 and november 2012. according to clinical conditions, milk samples were aseptically collected from 90  mastitis cases, lachrymal fluid from 12 animals with weeping eyes, and synovial fluid from 30 cases with arthritis (table  i). samples were transported to the laboratory for examination (tola et  al. 1997) within 24 hours at 2-8°c, following the required safety and biosecurity regulations (who 2008). isolation and identification procedures all of the samples were cultured in pleuropneumonia-like organism (pplo) broth and incubated at 37  °c in a humidified incubator with table i. sample size and location. city animal species total samples mycoplasma identified positive samples milk lachrymal fluid joint fluid milk lachrymal fluid joint fluid bileh savar sheep 10 3 3 3 (ma 2, mp 1) 0 0 parsabad sheep 12 3 0 2* (ma 1, mp 2) 0 0 goat 3 0 0 2* (mcc 2, mp 2) 0 0 germi sheep 10 6 4 5* (ma 5, mp 5) 2 (ma 2, mp 2) 1 (ma 1, mp 1) goat 4 0 0 3* (ma 2, mp 2, mcc 1) 0 0 ardebil sheep 16 4 1 6* (ma 5, mp 6) 0 0 goat 5 0 0 2* (mmc 1, mcc 1, ma 1, mp 1) 0 0 meshgin shahr sheep 12 4 1 3 (ma 3) 0 0 goat 4 0 0 1 (ma) 0 0 khalkhal sheep 14 10 3 3 (ma 2, mp 1) 0 0 total 90 30 12 30 2 1 *samples contained more than one mycoplasma species. ma = mycoplasma agalactiae; mp = mycoplasma puterfaciens; mcc = mycoplasma capricolum subsp. capricolum; mmc = mycoplasma mycoides subsp. capri includes the former m. mycoides subsp. mycoides large colony trype. table ii. sequences of primers. targeted species denomination sequence (5'-3') bp size reference general myc23f1729 ctaaggtdagcgagwdaactatag* 102-110 hotzel et al. 2003 myc23r1837 ccccycwtsyttyactgmggc mycoides cluster p1 tatatggagtaaaaagac 253-265 hotzel et al. 1996 p2 aatgcatcataaataattg ma fs1 aaaggtgcttgagaaatggc 375 tola et al. 1996 fs2 gttgcagaagaaagtccaatca mp mput 1 aaattgttgaaaaattagcgcgac 316 peyraud et al. 2003 mput 2 catatcatcaactagattaatagtagcacc mmmlc** mmmlc2-l caatccagatcataaaaaacct 1049 le grand et al. 2004, hotzel et al. 2003, maigre et al. 2008 mmmlc1-r ctcctcatattcccctagaa mcc mccpl1-l agacccaaataagccatcca 1356 le grand et al. 2004, maigre et al. 2008 mccpl1-r ctttcaccgcttgttgaatg * degenerate nucleotides: d = a, g, t; w = a, t; y = c, t; s = g, c; m = a, c. 208 veterinaria italiana 2018, 54 (3), 205-210. doi: 10.12834/vetit.831.4072.2 mycoplasma in small ruminants in iran hajizadeh et al. discussion this study was conducted to determine if mycoplasma species other than ma can be involved in ca in iran. iran is a large country that exceeds 1,648,195 km2 and has a diverse climate. traditional food regimes have led iranian farmers to rear sheep and goats together in common units. considering the close phylogenetic relationships of these 2  ruminant species and the potential for shared mycoplasma flora that produce the clinical signs of ca, knowledge of the organisms causing this disease is required to improve control measures. the ardebil province (17,800 km2) was selected for this study as it has a large population of 2.5 million sheep and goats. a high morbidity rate caused by ca is common in this region. annual vaccinations against ma including an indigenous strain in an inactivated monovalent vaccine adjuvanted with saponin, is in progress. however, ca continues to be reported as an issue across this province and more broadly across iran. there are many reports and articles about the isolation and identification of causative agents of ca from around the world. culture and pcr methods are most commonly used in these studies (anonymous 2008), although more recently real-time pcr methods for ma have been described (oravcová et  al. 2009). we used conventional pcr to detect mycoplasma species, although this method may be less sensitive than the newer developed real-time pcr methods. in this study, out of 33 positive cultures, a total of 53  different isolates were identified. we isolated 4 mcc and 1 mmc from goats, which is similar to that which has been reported in other studies (al-momani et  al. 2006), and 18 mp from sheep. al-momani and colleagues (al-momani et al. 2006) also found mixed populations of mycoplasma species in sheep and goats in jordan. in this study, we targeted animal sites where clinical signs were being observed in order to maximize our chances of detecting and recovering relevant organisms. however, if a more comprehensive sampling regime with multiple sites was used, more isolates may have been obtained. our findings indicated that the milk was the best and most solution (5 pm/μl) from each flanking primer (table ii), and 2.25 μl of dna template plus 2.35 μl of molecular-grade pcr water. pcr reactions were run on an eppendorf pcr system (eppendorf, germany) where an initial 2-minute denaturation at 95  °c was followed by 35 cycles of denaturation (93  °c for 45 seconds), annealing (48  °c for 60 seconds), and extension (72  °c for 60 seconds), with a final elongation step of 72 °c for 10 minutes. in addition, more specific pcrs were used to detect ma and the m.  mycoides cluster: mp, mmc, and mcc. details of the pcr methods and primers are provided in table  ii. all primers were synthesised by mwg biotech (germany). a total of 33 lyophilised samples were sent to the office international des epizooties (oie) contagious agalactia reference laboratory, animal and plant health agency (apha) weybridge, uk for confirmatory testing by pcr and analysis using denaturing gradient gel electrophoresis (pcr/ dgge). the mcauliffe method (mcauliffe et al. 2005) was followed for this process. results the most frequent clinical signs observed among flocks were mastitis, arthritis, and keratoconjunctivitis in descending order. of the 132 collected samples, 33  (25%) were positive for mycoplasma species by culture in pplo broth and agar. twenty six (22.4%) were from sheep samples and 7 (43.8%) from goat samples, the most successful isolation rate (33.3%) was obtained from milk samples. using the pcr/dgge method, 18 of the 33 samples were mixed mycoplasma cultures comprised of isolates from 12 sheep and 6 goats. of the 53 cultures considered, 25 were identified as ma (21 from sheep, 4  from goats) (47.2%), 23 as mp (18 from sheep, 5  from goats) (43.4%), 4 as mcc from goats (7.5%), and 1 mmc from a goat (1.9%). these results were also confirmed in iran by using species-specific pcrs (tables iii and iv, figures 1 and 2). table iii. specific pcrs to detect the m. mycoides cluster. mmc ma mcc mp pcr stages lid temp. c °110 c °110 c ° 110 c ° 110 initial denaturation c/ 300 sec °94 c/300 sec °94 c/300 sec °95 c/120 sec °94 denaturation c/30 sec °94 c/30 sec °94 c/60 sec °94 c/30 sec °94 annealing c/30 sec °64 c/30 sec °49 c/60 sec °55 c/15 sec °48 extension c/30 sec °72 c/60 sec °72 c/60 sec °65 c/15 sec °72 cycles 25 35 35 33 final extension c/420 sec °72 c/600 sec °72 c/600 sec °67 c/600 sec °72 final product (bp) 360 1049 375 1356 table iv. results of pcr tests with specific primers. genus and species of isolates sheep goat total percent mycoplasma agalactiae 21 4 25 47.2% mycoplasma putrefaciens 18 5 23 43.4% mycoplasma capricolum subsp. capricolum 0 4 4 7.5% mycoplasma mycoides subsp. capri 0 1 1 1.9% 209veterinaria italiana 2018, 54 (3), 205-210. doi: 10.12834/vetit.831.4072.2 hajizadeh et al. mycoplasma in small ruminants in iran more positive results may have been obtained. these findings suggest that new approaches toward controlling ca are of great importance. currently, vaccination with a monovalent inactivated vaccine (ma) is performed in iran. considering the multiple mycoplasma species isolated here, a polyvalent vaccine could be developed as it potentially may offer a better protection. acknowledgment we would like to thank all of our colleagues who cooperated and helped us by providing field samples successful sample, which accounted for 90% (30 out of 33) of the positive results. future studies may therefore consider to limit the sampling to milk samples. bacteriological culturing of mycoplasma species requires specialist media and laboratory skills, and is complicated by the potential overgrowth by other bacterial species, it can also select for the more rapid growing mycoplasma species. the use of molecular tests directly on samples, rather than on the culture selection may also increase the detection rate and show a higher prevalence of ca than detected in this study. the pcr/dgge method is useful as it is sensitive and detects mixed infections and identifies the mycoplasma species within a single test, but the equipment and controls that are required with this method mean that the test is not available in many laboratories. in contrast, standard pcr tests can now be performed in many laboratories, but the tests are usually species-specific and multiple tests are required to detect the organisms that are being targeted. the isolation of mp from both goats and sheep demonstrated that mp was the second most commonly isolated organism for ca in iran, and is therefore a major contributor to ca in both sheep and goats. in this study no mcc or mmc was detected in sheep. this is the first report on the isolation and identification of mp, mmc and mcc in small ruminant infected flocks in the ardebil province of iran. working from the samples collected for this study, the identification of all the causative agents of contagious agalactia (ma, mp, mcc, and mmc) by pcr and pcr/dgge indicates and confirms the presence of these agents in small ruminants. this study relied on the culture and identification of mycoplasma species, which gave 33 positive results out of a total of 132 samples collected. had molecular methods been used instead of, or in addition to, culture methods, figure 2. pcr products with specific primers, amplified from mycoplasma mycoides sub. capri. lane1 = positive sample; lane 2, 3 = mmc control; lane 4 = mmmsc control; lane 5 = negative control. 1 2 3 4 5 figure 1. dgge method for identifying the mycoplasmas among the samples. lane1 = negative control; lane 2 = ma control; lane 3 = mmc; lane 4 = mmmsc control; lane 5 = mcc control; lane 6 = m. capripneomonia control; lane7 = m. ovipneomonia control; lanes 8-28 = our samples. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 210 veterinaria italiana 2018, 54 (3), 205-210. doi: 10.12834/vetit.831.4072.2 mycoplasma in small ruminants in iran hajizadeh et al. mycoplasma team at apha (weybridge) especially faye gosney for her skillful technical guidance. this work was fully financed with state funds from razi vaccine and serum research institute under grant no. 2-18-18-87026. and standard strains: dr. g. moazeni, dr. m. lotfi, a. naserirad, dr. s. ataei, dr. a. khafri, dr. khaki, dr. s. moradi, r. banihashemi, m. sekhavati, m. asadpour, s.r. ahmadiniya, a. es. hagi. the author also would like to express his sincere gratitude to members of the al-momani w., halablab m.a., abo-shehada m.n., miles k., mcauliffe l. & nicholas raj. 2006. isolation and molecular identification of small ruminant mycoplasmas in jordan. small ruminant res, 65, 106-112. awan m.a., abbas f., yasinzai m., nicholas r.a., babar s., ayling r.d., attique m.a., ahmed z., wadood a. & khan fa. 2010. first report on the molecular prevalence of mycoplasma capricolum subspecies capripneumoniae (mccp) in goats the cause of contagious caprine pleuropneumonia (ccpp) in balochistan province of pakistan. mol biol reports, 37, 3401-3406. bergonier d., berthelot x. & poumarat f. 1997. contagious agalactia of small ruminants: current knowledge concerning epidemiology, diagnosis and control. rev sci tech off epiz, 16, 848-873. bory g. & entessar f. 1959. etude sur l, agalaxie contagieuse des chevres et des moutons. archiv razi inst, 14, 109-127. hotzel h., sachse k. & pfützner h. 1996. a pcr scheme for differentiation of organisms belonging to the mycoplasma mycoides cluster. vet microbiol, 49, 31-43. hotzel h., frey j., bashiruddin j. & sachse k. 2003. detection and differentiation of ruminant mycoplasmas. methods molecular biology (clifton, nj), 216, 231-245. kheirabadi k.h. & ebrahimi a. 2007. investigation of mycoplasma agalactiae in milk and conjunctival swab samples from sheep flocks in west central, iran. pakistan j biol sc, 10, 1346-1348. kumar a., rahal a., chakraborty s., verma a.k. & dhama k. 2014. mycoplasma agalactiae, an etiological agent of contagious agalactia in small ruminants: a review. vet med int, id 286752. le grand d., saras e., blond d., solsona m. & poumarat f. 2004. assessment of pcr for routine identification of species of the mycoplasma mycoides cluster in ruminants. vet res, 35, 635-649. madanat a., zendulkova d. & pospíšil z. 2001. contagious agalactia of sheep and goats. a review. acta vet brno, 70, 403. maigre l., citti c., marenda m., poumarat f. & tardy f. 2008. suppression-subtractive hybridization as a strategy to identify taxon-specific sequences within the mycoplasma mycoides cluster: design and validation of an m. capricolum subsp. capricolum-specific pcr assay. j clin microbiol, 46, 1307-1316. maré c.j. 2014. contagious agalactia of sheep and goats. georgia uo, ed.), usa. mcauliffe l., ellis r.j., lawes j.r., ayling r.d. & nicholas references ra. 2005. 16s rdna pcr and denaturing gradient gel electrophoresis; a single generic test for detecting and differentiating mycoplasma species. j medical microbiol, 54 (pt8), 731-739. oravcová k., lópez-enríquez l., rodríguez-lázaro d. & hernández m. 2009. mycoplasma agalactiae p40 gene, a novel marker for diagnosis of contagious agalactia in sheep by real-time pcr: assessment of analytical performance and in-house validation using naturally contaminated milk samples. j clin microbiol, 47, 445-450. peyraud a., woubit s., poveda j.b., de la fe c., mercier p. & thiaucourt f. 2003. a specific pcr for the detection of mycoplasma putrefaciens, one of the agents of the contagious agalactia syndrome of goats. mol cell probes, 17, 289-294. shahram m., nicholas r.a.j., wood a.p. & kelly d.p. 2010. further evidence to justify reassignment of mycoplasma mycoides subspecies mycoides large colony type to mycoplasma mycoides subspecies capri. syst appl microbiol, 33, 20-24. smith mc. & sherman dm. 2009. bacterial diseases mycoplasma arthritis. in goat medicine. john wiley & sons, usa, 85-163. sotoodehnia a. & aerabi i. 1986. agalactia disease and its geographical distribution in sheep and goats in iran. archiv razi inst, 36, 75-78. sotoodehnia a., moazenijula a., rad a. & jabbari a. 2007. preparation of agalactia vaccine in fermentor. archiv razi inst, 62, 45-48. tola s., angioi a., rocchigiani a., idini g., manunta d., galleri g. & leori g. 1997. detection of mycoplasma agalactiae in sheep milk samples by polymerase chain reaction. vet microbiol, 54, 17-22. tola s., idini g., manunta d., galleri g., angioi a., rocchigiani a. & leori g. 1996. rapid and specific detection of mycoplasma agalactiae by polymerase chain reaction. vet microbiol, 51, 77-84. vilei e.m., korczak b.m. & frey j. 2006. mycoplasma mycoides subsp. capri and mycoplasma mycoides subsp. mycoides lc can be grouped into a single subspecies. vet res, 37, 779-790. world health organization (who). 2008. guidance on regulations for the transport of infectious substances. who, switzerland, geneva. world organisation for animal health (oie). 2008. contagious agalactia. in oie terrestrial manual. paris. oie, 992-999. 197 1 bihar veterinary college, bihar animal sciences university, patna, bihar 800014, india. 2 regional centre for eastern region-indian council of agricultural research, patna, bihar 800014, india. * corresponding author at: department of veterinary public health & epidemiology, bihar veterinary college, bihar animal sciences university, patna, bihar 800014, india. tel.: 91 9431260790, e-mail: drkaushikvet@gmail.com. parole chiave pollame, e. coli, resistenza antimicrobica, caratterizzazione molecolare, stx 1 gene, stx 2 gene. riassunto in questo studio, condotto su 228 campioni di carne e 24 di uova, sono stati rilevati dalla carne 62 isolati di escherichia coli appartenenti a differenti sierogruppi basati sul sierotipo o, ovvero o29 (10.8%), o8 (7.7%), o40 (6.15%), o2 (4.61%), o60 (3.08%), o106 (3.08%), o42 (1.54%), o87 (1.54%); per ciascun sierotipo o1, o7, o30, o45, o59, o66, o105, o1116, o136, o141, o147, o148, o166, o172, ne è stato individuato uno; 16 (24,62%) sono risultati non dimostrabili e 6 (9,23%) erano di tipi approssimativi. la caratterizzazione molecolare è stata eseguita mirando ai frammenti dei geni di virulenza stx 1 e stx 2 ; 10 (16,12%) trasportavano il gene stx 2 mentre nessuno lo stx 1 . gli isolati di e. coli hanno mostrato un'ampia variazione nel pattern di resistenza contro gli agenti antimicrobici usati (9-90%). la massima resistenza è stata osservata contro cefuroxima (89,1%) e penicillina (89,4%), seguita da ampicillina (80,43%), vancomicina (74,1%), cotrimossazolo (73,1%), cefalotina (60,8%) , ceftriaxone (28,2%), tetraciclina (17,4%), gentamicina (13,0%), amikacina (13,04%), ofloxacina (13%) e ciprofloxacina (6,5%). è stato invece notato un alto grado di suscettibilità nei confronti di amikacina (84,7%) e ciprofloxacina (76%), seguita da gentamicina (71,73) e ofloxacina (60,86%). sono state riscontrate elevate resistenze antibiotiche multiple e identificato un totale di 34 pattern di resistenza. resistenza antimicrobica e caratterizzazione molecolare di escherichia coli nel pollame in india orientale keywords poultry, e. coli, anti-microbial resistance, molecular characterisation, stx 1 , stx 2 . summary in this study 252 poultry samples comprised of poultry meat (n  =  228) and poultry eggs (n = 24) were screened for the isolation of escherichia coli (e. coli). a total of 62 e. coli isolates were recovered from poultry meat. the e. coli isolates belonged to different serogroups based on ‘o’ serotyping of the isolates viz o29 (10.8%), o8 (7.7%), o40 (6.15%), o2 (4.61%), o60 (3.08%), o106 (3.08%), 42 (1.54%), o 87 (1.54%), and 01 serotypes of o1, o7, o30, o45, o59, o66, o105, o1116, o136, o141, o147, o148, o166, and o172. sixteen (24.62%) of the isolates were ut (untypable) and 6 (9.23 %) were rough types. molecular characterisation of the isolates was performed, targeting stx 1 and stx 2 virulence gene fragment. out of 62 e. coli isolates, 10 (16.12%) were carrying virulence gene stx 2 , whereas none of the isolate was carrying stx 1 gene. the e.  coli isolates showed wide variation in resistance pattern against the antimicrobial agents that we used (9-90%). among e. coli isolates, maximum resistance was observed against cefuroxime (89.1%) and penicillin (89.4%), followed by ampicillin (80.43%), vancomycin (74.1%), co-trimoxazole (73.1%), cephalothin (60.8%), ceftriaxone (28.2%), tetracycline (17.4%), gentamicin (13%), amikacin (13.04%), ofloxacin (13%), and ciprofloxacin (6.5%). a high degree of susceptibility was observed against amikacin (84.7%) and ciprofloxacin (76%) followed by gentamicin (71.73) and ofloxacin (60.86%). high multiple antibiotic resistances were observed and a total of 34 resistance patterns were identified. purushottam kaushik1*, anjay1, savita kumari1, shanker dayal2 and sunil kumar1 antimicrobial resistance and molecular characterisation of e. coli from poultry in eastern india veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 accepted: 09.05.2014 | available on line: 30.09.2018 198 veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 and molecular characterisation of stx 1 and stx 2 genes in e. coli isolated from poultry meat. materials and methods sample collection a total of 252 samples constituting of fresh poultry meat (n  =  228) and eggs (n  =  24) were collected randomly from different shops and market of patna, india during september 2010 to march 2013. the samples were transported under cold conditions to the laboratory in the department of veterinary public health, bihar veterinary college, patna, india, and processed within 1 hour of collection. isolation and biochemical characterization ten grams of meat sample and egg white were inoculated in macconkey broth (himedia, mumbai, india) and incubated at 37 °c for 12 hours. samples showing acid and gas production were further inoculated on macconkey agar plates (himedia, mumbai, india) and incubated at 37  °c for 18-24  hours. pure and characteristic pink coloured single colonies selected from macconkey agar were subcultured on eosin methylene blue (emb) agar (himedia, mumbai, india) in order to observe the metallic sheen that is characteristic of e.  coli. the pure colonies were picked up on nutrient agar slants and subjected to standard morphological and biochemical tests (edwards and ewing 1972). serotyping e. coli all the biochemically confirmed isolates of e. coli were submitted to the national salmonella and escherichia centre, central research institute, kasauli, india for serotyping based on somatic (o) antigens. extracting e. coli dna for the detection of virulence genes the bacterial dna was prepared as per dutta and colleagues (dutta et  al. 2011), with slight modification. the isolates were inoculated into 5 ml luria bertani (l-b) broth and incubated at 37  °c for 18 hours. after incubation, 1 ml of the broth culture was centrifuged in a 1.5 ml microcentrifuge tube at 8,000  rpm for 10 minutes. the pellet was washed twice in sterile normal saline solution (nss) (0.85% nacl) then re-suspended in 400  μl of nuclease-free sterile distilled water and boiled for 10  minutes followed by immediate chilling. cell debris was removed by centrifugation at 5,000  rpm for introduction escherichia coli (e.  coli) is a commensal bacterium in humans and animals; however some strains are capable of causing intestinal diseases. this organism is commonly present in the environment and is considered an indicator of faecal contamination of food and water. on the basis of virulence factors, e.  coli has been classified into different pathotypes: enterotoxigenic e.  coli (etec), enteroinvasive e.  coli (eiec), enteropathogenic e.  coli (epec), diffusely adherent e.  coli (daec), enteroaggregative e.  coli (eaec), and enterohemorrhagic or shiga toxin-producing e.  coli (ehec/stec). the latter is most commonly associated with outbreaks of foodborne diseases (bandyopadhyay et  al. 2011). the pathogenicity of ehec/stec is mediated mainly through shiga toxins 1 and 2 encoded by stx 1 and stx 2 genes, respectively. although bovines are considered to be the major carrier for ehec/stec, poultry may also harbour the organism and play an important role in transmission (ferens and hovde 2011). the stx 2 gene is associated with an increased risk of developing hemolytic-uremic syndrome (boerlin et  al. 1999). although reports on the isolation, identification, and characterisation of ehec/stec in humans and animals are available in india, very few reports on the association of ehec/stec in poultry are available in the country (chattopadhayay et  al. 2001, khan et al. 2002, wani et al. 2004). during the past decade, drug resistance to enterobacteriaceae has increased worldwide. since e.  coli is present as gut commensal in humans and animals, it has become one of the microorganisms that are commonly resistant to antimicrobials due to the selective pressure imposed by the antimicrobial drugs used in the treatment of food animals and humans (zhao et  al. 2012). moreover, in animals antimicrobial agents are not only used for therapy and the prevention of bacterial infection, but also for the promotion of growth (van den bogaard and stobberingh 2000). in the poultry sector, maximum numbers of antimicrobial agents are continuously fed to animals in order to promote growth, which further imposes selective pressure on the organism leading to development of antimicrobial resistance. the emergence of antimicrobial resistance among e.  coli of animal origin has important public health implications. various authors report that animals are the source of drug-resistant e.  coli in humans (johnson and nolan 2009, hammerum and heuer 2009, overdevest et  al. 2011). szmolka and colleagues (szmolka et  al. 2012) report common multi-resistance patterns of e. coli from food animals and clinical cases, which suggests that the circulation of multi-drug resistant e. coli circulating in the food chain is significant. the purpose of this study is to determine the prevalence, antimicrobial resistance, e. coli in eastern indian poultry kaushik et al. 199veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 kaushik et al. e. coli in eastern indian poultry was produced by different isolates. we referenced the manufacturer (hi media) for the breakpoint diameter of the zone of inhibition for different antibiotics. multiple-antibiotic resistance was defined as resistance to 2 or more antibiotic classes (shekhar and singh 2014). results bacterial isolation and identification a total of 62 e.  coli out of 228 meat samples was isolated, whereas all egg samples were found negative for e.  coli. the e.  coli isolates belonged to 24 different serogroups: o29 (10.8%), o8 (7.7%), o40 (6.15%), o2 (4.61%), o60 (3.08%), o106 (3.08%), o42 (1.54%), o87 (1.54%), and 1 serotypes each of o1, o7, o30, o45, o59, o66, o105, o116, o136, o141, o147, o148, o166, and o172. other results indicated 16 (24.62%) isolates as ut (untypable), and 6 (9.23 %) as rough types. the molecular characterisation of e. coli by multiplex pcr multiplex pcr targeting stx 1 and stx 2 gene fragment of e.  coli resulted in the amplification of 349  bp amplicon for stx 1 and 110 bp amplicon for stx 2 , as we expected (figure 1). out of 62 e.  coli isolates, 13 (21%) isolates that carried only the stx 2 gene were screened. these belonged to the serogroups o29, o8, o42, and o1. antimicrobial resistance the e.  coli isolates showed a wide variation in resistance patterns (9-90%) against the antimicrobial 5 minutes. the supernatant was used as a template dna for polymerase chain reaction (pcr). detecting virulence genes by multiplex pcr a multiplex pcr protocol was standardized using 2 sets of oligonucleotide primers targeting stx 1 and stx 2 genes (table i). amplification was carried out in a total volume of 25  µl containing 10  pmol each of primer, 50 µm each of dntp, 1.5 mm mgcl 2 , 1u taq dna polymerase, 1x pcr of buffer, and 5 µl template dna. a negative control containing the same reaction mixture except the dna template was included in every experiment. the reaction condition was optimised with initial denaturation at 94  °c for 5 minutes, followed by 35 cycles of denaturation at 94 °c for 1 minute, annealing at 55 °c for 1 minute, and extension at 72  °c for 1 minute. finally, an additional extension was achieved for 5  minutes at 72 °c. amplified products were separated by agarose gel (2% agarose in 1x tris-borate-edta buffer) electrophoresis at 5v/cm for 2 hours and stained with ethidium bromide (0.5 μg/ml) (sambrook et  al. 1989). a standard molecular-size marker (100 bp dna ladder) was included in each gel. we observed and photographed dna fragments in a gel documentation system (biorad, usa). antimicrobial resistance the antimicrobial susceptibility test of isolates was determined using the agar disc diffusion method (wayne 2002). the antibiotic discs included ampicillin (10  µg), amikacin (30  µg), cefuroxime (30  µg), ceftriaxone (30  µg), cephalothin (30  µg), ciprofloxacin (5  µg), co-trimoxazole (25  µg), gentamicin (10  µg), ofloxacin (5  µg), penicillin-g (10  units), tetracycline (30  µg), and vancomycin (30  µg). the isolates were grown on autoclaved mueller hinton broth (himedia, india) for 18 hours at 37  °c. about 100 µl of the inoculum was spread on mueller hinton agar using a sterile disposable l-shaped spreader. antibiotic discs were placed onto the plate using sterile forceps. the plates were incubated at 37  °c for 24 hours and observed for zone of inhibition. the results were interpreted as susceptible, resistant, or moderately susceptible based on the diameter of the zone of inhibition that table i. oligonucleotide primers used in multiplex pcr reaction. primer sequence product size stx 1 f 5'-caa cac tgg atg atc tca g-3' 349 bp stx 1 r 5'-ccc cct caa ctg cta ata-3' stx 2 f 5'-atc agt cgt cac tca ctg gt-3' 110 bp stx 2 r 5'-ctg ctg tca cag tga caa a-3' figure 1. agarose gel electrophoresis showing amplified pcr product of stx 1 and stx 2 gene. lane m = 100 bp dna marker, lane1 = positive control for stx 1 (349 bp amplicon) and stx 2 (110 bp amplicon) gene, lane 2-11 = e. coli isolates from meat samples, lane12 = negative control. 200 veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 e. coli in eastern indian poultry kaushik et al. (13%), and ciprofloxacin (6.5%). a high degree of susceptibility was observed against amikacin (84.7%) and ciprofloxacin (76%) followed by gentamicin (71.73%) and ofloxacin (60.86%) (tables  ii  and  iii). the number of multiple antibiotic resistances pattern of isolates we observed was high and a total of 34 resistance patterns were identified (table iv). agents that have been used. the maximum resistances among e.  coli isolates were observed against cefuroxime (89.1%) and penicillin (89.4%), followed by ampicillin (80.43%), vancomycin (74.1%), co-trimoxazole (73.1%), cephalothin (60.8%), ceftriaxone (28.2%), tetracycline (17.4%), gentamicin (13%), amikacin (13.04%), ofloxacin table ii. antimicrobial susceptibility of e. coli isolates. e. coli serotypes cu g a ci ak ch cf co p va of t ut r r r r r r r s r r r s ut r r r r r r i s r i s i ut r i r i s r s s r i s s o106 i i r i i r s r r r s i o59 i i r r s r s s r i s s ut r r r i i i r s r i s s o106 r s r i s r s r r r s r ut r s r s s r s r r i s r rough i r r r r r s r r i s r rough r r r r s r s r r r r i ut r s r i r r s r r r s s o136 r s r s s r s r r s s s o45 r r r r r r s r r i s s rough r r r s s s s r r r i i ut r r r r s s s r r r r i o40 s s r s s i s r r r i i o40 r s r s s r i s r r r i o40 r s r s s r i r r r i i ut s s r s s s s r r r s i rough r s r s s r i r r r r i ut r i r s s r s s r r r i o147 r s r s s r s r r i i s ut r s r s s r s r r r i s ut r r r s i r s s i r s i ut r i r s s r s r i r s s o141 r s r i s s s r r s s s o7 r s r i s r s r r i s s o40 r s r s s s s r r r i i o87 r s r i r r s r r r s r o29 r s r i s r i r i r s i o8 r s s r r r r s r r s s o29 r i s r s r i r r r r i o8 r s r s s s r r r r s i o60 r s r s s i i r r r r s o172 r i r s s i s r r r s i o42 r i r i r r s r r i s s o2 r s s i s s r i r r r i rough r s r s s r s s r s s s o148 r s r r s i s r r r i i 042 r s r r s i s r r r i r rough r s r s s r s r r r s s o1 r s r i s i s s r r i i cu = cefuroxime; g = gentamicin; a = ampicillin; ci = ceftriaxone; ak = amikacin; ch = cephalothin; cf = ciprofloxacin; co = co-trimoxazole; p = penicilling; va = vancomycin; of = ofloxacin; t, tetracycline; s = susceptible; i = intermediate; r = resistant; ut = untypeable. 201veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 kaushik et al. e. coli in eastern indian poultry (guth et al. 1989, blanco et al. 2004, fratamico et al. 2010, bandyopadhyay et  al. 2011). in the present study however 24 serogroups were detected. they were widely distributed in the eastern part of india. on the contrary to this finding many studies from india reported the isolation of a few numbers of serogroups from poultry (malik et  al. 2002, wani et al. 2004, dutta et al. 2011). the e. coli of serogroups o2, o87, and o172 reported in this study, are associated with shiga toxin producing e. coli (stec), as documented by fratamico and other authors (fratamico et al. 2010, meng et al. 2014), whereas the discussion foodborne diseases are caused by a number of pathogens. of these, e. coli is considered one of the most important. it is responsible for intestinal and extraintestinal diseases in humans (vincent et  al. 2010, scallan et  al. 2011). food plays an important role in the dissemination of e.  coli, causing community-acquired urinary tract infections (vincent et  al. 2010, ferens and hovde 2011, jana and mondal 2013). in the present study, poultry meat samples collected from different retail shops were contaminated with different serogroups of e.  coli. out of 228 poultry meat samples, 62 (27.19%) were positive for e. coli. this finding is both consistent with previous reports from different parts of india (farooq et al. 2009, bonyadian et al. 2011, sahoo et al. 2012, jana and mondal 2013) but higher than the finding of zende and colleagues (zende et  al. 2013), who reported the isolation of e. coli in 16% poultry meat from a small sample size of meat from mumbai, india. our results indicate the contamination of poultry meat with e.  coli. this may be due to a lack of proper sanitation during slaughtering in the local markets or because of contaminated water is used to wash carcasses. in the present study, e.  coli isolates belonged to 24 different serogroups, namely o29, o8, o40, o2, o60, o106, o42, o87, o1, o7, o30, o45, o59, o66, o105, o116, o136, o141, o147, o148, o166, o172; ut (untypable) and r (rough types) were also recovered. most of the serogroups identified have also been reported in other similar studies table iii. oligonucleotide primers used in multiplex pcr reaction. antibiotic number of resistant strain (n = 46) percent β lactums antibiotic ampicillin 37 80.4 penicillin 41 89.13 cephalosporins cephalothin (first generation) 28 60.86 cefuroxime (second generation) 41 89.13 ceftriaxone (third generation) 13 28.26 fluroquinolones ciprofloxacin 3 6.52 ofloxacin 6 13.04 aminoglysides amikacin 6 13.04 gentamicin 6 13.14 sulfonamides co-trimoxazole 34 73.9 tetracyclines tetracycline 8 17.39 glycopeptides vancomycin 34 74.1 table iv. resistance pattern of e. coli serogroups isolated from poultry meat. resistance patterns e. coli serotypes cu-co-va 2 a-cu-ch 1 g-a-p-ch-va 1 cu-co 1 g-p-cu-ch-co-va 1 p-cu-ch 1 a-p-cu-va 2 p-ci-cu-co-va 1 a-p-ci-cu-ch-va 1 a-p-ci-cu-ch-co-va-t 1 a-p-cu-co-va-t 1 a-p-cu-va-t 1 a-p-ci-cu-co-va-t 1 a-p-ch-co-va 1 a-p-ci-ch 1 a-p-cu-ch-co-va-t 2 g-ak-a-p-ci-ch-co-t 1 g-a-p-ci-cu-ch-co-va 2 a-p-cu-ch-co 3 g-a-p-cu-co-va 2 g-a-p-co-va 1 a-p-cu-ch-of-va 1 a-p-cu-ch-co-va 3 a-p-cu-ch-of-co-va 1 a-p-cu-ch-co 4 a-p-co-va 1 a-cu-ch-co-va-t 1 a-cu-ch-co-va 1 p-cu-ch-cf-va 1 p-cu-ch-of-co-va 1 a-p-cu-cf-co-va 1 a-p-cu-of-co-va 1 p-cu-ch-cf-of-va 1 a-p-cu-ch 1 total 46 antibiotics tested for multiple resistance were: cu = cefuroxime; g = gentamicin; a = ampicillin; ci = ceftriaxone; ak = amikacin; ch = cephalothin; cf = ciprofloxacin; co = co-trimoxazole; p = penicillin g; va = vancomycin; of = ofloxacin; t = tetracycline. 202 veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 e. coli in eastern indian poultry kaushik et al. of resistance in e.  coli. the coexistence of virulent and resistant genes in a species may result in the emergence of hybrid plasmids (both resistance and virulence) among e.  coli. this, in turn, poses an increased public health risk (szmolka and nagy 2013). in the present study, all isolates of e.  coli exhibited resistance against 2 or more antimicrobial agents, suggesting the presence of multi-drug resistant (mdr) e.  coli (joshi et  al. 2012, szmolka and nagy 2013). the prevalence of such a large number of mdr e. coli indicates the widespread and extensive use of antibiotics in the area. prevalence of such mdr e. coli has also been observed by shakya and colleagues (2013) in human stool samples from india. the high rate of multiple antibiotic resistance against e.  coli also indicates the injudicious use of several antimicrobial agents for preventive and therapeutic purposes. these all pose serious threats to animal and human health. the present investigation highlights the prevalence of pathogenic e. coli (etec and epec) in poultry meat, which is of importance in the field of zoonosis. the occurrence of high levels of resistance against most of the antibiotics and prevalence of virulent genes call for further attention, and the maintenance of strict hygienic measures in retail meat shops. the present findings also provide information about the occurrence of stec in poultry in eastern india. acknowledgments the authors are thankful to the honorable vice chancellor, bihar agricultural university, sabour, for providing necessary funds and principal, bihar veterinary college, patna for providing infrastructure and facilities to carry out the investigation. we are also thankful to the director cri, kasauli, hp, india, for serotyping the isolates. serogroups o8, o29, o111, and o60 are associated to enteropathogenic e. coli (guth et al. 1989, blanco et  al. 2004, wani et  al. 2004, bandyopadhyay et  al. 2011). thus our findings indicate that meat handlers and consumers are at risk of infection from stec or enteropathogenic e. coli. in this study, molecular characterisation for the presence of stx 1 and stx 2 genes showed the presence of stx 2 gene in 13 (21%) isolates of e. coli. this finding is consistent with other findings from farooq and colleagues (farooq et  al. 2009) and grossmann and colleagues (grossmann et  al. 2005), who reported the stx 2 gene in 11% and 9% of e. coli isolated from pigeons, respectively. various authors reported the occurrence of the stx 2 gene in o45, o18, o75, o64, o89, o91, o17, and o78 serogroups (schmidt et  al. 2000, morabito et al. 2001, farooq et al. 2009, dutta et al. 2011). however, our findings demonstrate the presence of the stx 2 gene in o29, o8, o42, and o1 serogroups. this indicates a variable distribution of virulent genes among different serogroups of e. coli in different geographical area in india. antimicrobial resistance was observed at a range of 9-90%, which shows extreme variation in the susceptibility of poultry meat to e. coli. our findings show maximum resistance against cefuroxime (a cephalosporin) followed by penicillin, ampicillin, and vancomycin. the high level of susceptibility against amikacin and ciprofloxacin that has been observed is consistent with the findings of other studies (mishra et  al. 2002, carraminana et  al. 2004, jana and mondal 2013). these findings might be the result of the indiscriminate use of these antibiotics in poultry or because of sub-therapeutic doses of these antimicrobial agents being administered for the prevention or control of infection. other explanations include transmissible or plasmid mediated drug resistance and mutational changes in the genes that are crucial to the development 203veterinaria italiana 2018, 54 (3), 197-204. doi: 10.12834/vetit.330.1382.2 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accepted: 25.09.2020 | available on line: 31.12.2021 1department of pathobiology, faculty of veterinary medicine, ferdowsi university of mashhad, mashhad, iran. 2department of pathobiology, faculty of veterinary medicine, islamic azad universitygarmsar branch, garmsar, iran. 3department of microbiology and immunology, kashan university of medical sciences, kashan, iran. 4department of pathobiology, faculty of veterinary medicine, shahid bahonar university of kerman, kerman, iran. 5department of avian diseases, faculty of veterinary medicine, university of tehran, tehran, iran. *corresponding author at: department of pathobiology, faculty of veterinary medicine, ferdowsi university of mashhad, mashhad, iran. tel.: +98 51 3880 5642, e-mail: askari.m@um.ac.ir. mahdi askari badouei1*, hossein vaezi2, ali nemati1, ehsan ghorbanyoon2, farzaneh firoozeh3, maziar jajarmi4 and seyed mostafa peighambari5 keywords salmonella infantis, integron, broiler, resistance. summary the poultry industry in iran is the main supplier of protein in the food chain. in the present study, we showed the importance of the possible dissemination of clonally related multiple drug resistant (mdr) salmonella infantis in broiler farms in iran. in total, 156 fecal samples belonging to 23 poultry farms in razavi khorasan province, northeast of iran, were examined for the presence of salmonella serovars. molecular serotypes and serogroups, class 1 and 2 integron types, colistin resistance genes (mcr-1 and mcr-2) and antimicrobial susceptibility patterns were determined on the recovered salmonella isolates. based on pcr analysis, 30 recovered salmonella isolates were identified as s. infantis (23 isolates; 76.6%), s. enteritidis (six isolates; 20%), and one isolate (3.3%) was not serotyped by the applied method. class 1 integrons were detected in 22 isolates (95.6%) and class 2 integrons were not detected in any of the isolates. although colistin resistance was prevalent in disc diffusion test, mcr-1 and mcr-2 genes were not detected. all class 1 integrons carried the cassette aada1 gene. all salmonella isolates were resistant to colistin and amoxicillin/clavulanic acid and mdr patterns were observed in most (96.6%) isolates. this study revealed a high prevalence rate of s. infantis and the presence of class 1 integrons in broiler farms. the presence of the same integron cassettes in the sequenced isolates suggests that strains are clonally related. stringent monitoring programs are required to prevent the spreading of mdr salmonella serovars into food chain via poultry products. high prevalence of clonally related multiple resistant salmonella infantis carrying class 1 integrons in broiler farms in these farms, antibiotics are used therapeutically, prophylactically or for growth promotion purposes (mehdi et  al. 2018). the extensive use of these antibiotics has led to a significant increase in the distribution of multidrug-resistant (mdr) salmonella strains in foods. likewise, these mdr strains can be transmitted to humans through the food chain, or direct contact with poultry and their houses (marshall and levy 2011). salmonellosis is one of the most important bacterial foodborne diseases in both developed and developing countries such as iran (aziz et  al. 2018). human salmonellosis is commonly associated with introduction antimicrobial resistance has become a worldwide public health problem that has a direct impact on food safety (shabana et  al. 2019). the use of antimicrobials has been beneficial for the producers to control and treat salmonella in the food industry (threlfall 2002). however, the overuse of antimicrobial agents, especially in the poultry farms, leads to the emergence and spread of antibiotic-resistant strains (threlfall 2002, irani et al. 2018). in recent years, salmonella species were recognized as a serious and problematic foodborne pathogen in poultry farms on a global level (antunes et al. 2016). 182 veterinaria italiana 2021, 57 (3), 181-188. doi: 10.12834/vetit.2269.13773.1 mdr salmonella infantis in iranian broiler farms badouei et al. co., germany) and incubated at 37  °c for 24-48  h. suspected colonies were cultured into the tsi agar (merck co., germany) and incubated at 37  °c for 24  h. finally, the lactose-negative and h 2 s positive isolates were examined using standard biochemical tests (markey et al. 2013). salmonella serogrouping salmonella serogroups were determined by slide agglutination using antisera against o antigen according to the manufacturer's instructions (bahar afshan, iran). molecular detection of salmonella serovars the dna of salmonella isolates was extracted by using the boiling method as described previously (badouei et  al. 2015). then, the polymerase chain reaction (pcr) was performed to confirm the biochemical identification of the isolates at the genus level using s139 and s141 primers which target inva gene (zahraei salehi et  al. 2013). a multiplex-pcr assay for molecular detection of five important salmonella serovars including s. infantis, s. heidelberg, s. gallinarum, s. enteritidis, and s.  kentucky was performed on all confirmed isolates as described previously (kardos et  al. 2007, zhu et  al. 2015). when salmonella serovars was not identified in the multiplex-pcr, a two-step nested pcr approach was used for molecular identification of s.  infantis (kardos et  al. 2007). twenty-five μl final reaction volume including 3 μl dna extract, 0.3 μm of each primer (table i), 12.5 µl 2x taq dna polymerase master mix red (ampliqon, denmark) and distilled water up to volume of reaction was used in all pcr reactions. the pcr conditions were adjusted on the basis of cited references for each assay (kardos et al. 2007, zhu et al. 2015). for positive controls, s.  infantis (collection isolate, university of tehran), and s. enteritidis (atcc: 13076) were used. detection of class 1 and 2 integrons and colistin resistance genes the presence of gene cassettes containing class 1 and 2 integrons were detected by two pcr assays (table i). all salmonella isolates were screened for the detection of most prevalent plasmid-mediated colistin resistance genes (mcr-1 and mcr-2) using two pcr assays with specific primers (table i). this reaction was conducted in 25 μl volumes based on the protocol described by barbieri and colleagues (barbieri et  al. 2017). all pcr products were electrophoresed on 2% agarose gel at 100  v for 1  h with ethidium bromide and visualized by geldoc 1000 (vilber lourmat, france). the consumption of contaminated poultry and its products. in most cases, the disease is caused by eating raw or undercooked poultry, eggs or egg products (el-prince et al. 2019). nowadays, high rates of mdr salmonella strains are represented as a major threat to public health in iran (nirmala et al. 2018). in many countries, salmonella infantis has been mentioned as a cause of food-borne zoonotic pathogen among serovars of salmonella enterica (merino et  al. 2003, zhao et  al. 2017, borowiak et  al. 2018, wajid et  al. 2019). in iran, many studies show that salmonella infantis serovar is an important public health issue and has become a serious problem for the medical and veterinary communities (firoozeh et  al. 2011, firoozeh et  al. 2014, peighambari et  al. 2018). poultry, especially broilers, are known as one of the main reservoirs for s. infantis in iran (rahmani et al. 2013). today, s.  infantis like other salmonella serovars is becoming resistant to key antimicrobials such as the fluoroquinolones and broad-spectrum β-lactams (gupta et al. 2019). antibiotic resistance genes, play a major role in the evolution of mdr salmonella strains that can be located on chromosomes, plasmids, transposons or integrons (almeida et  al. 2018). the distribution of mdr salmonella strains is mainly related to integrons (kaushik et  al. 2018). integrons are genetic elements that are able to capture antibiotic resistance genes and spread them among sensitive strains, so they have a fundamental role in the emergence of mdr salmonella strains (gillings et al. 2008, kaushik et al. 2018). integrons consist of two major types including chromosomal integrons and mobile integrons (mis). mis are divided into five classes (class 1 to 5) and class 1 integron has been the most commonly reported class in mdr salmonella strains (gillings et al. 2008, kaushik et al. 2018, hossain et al. 2019). the present study was conducted to investigate the prevalence, distribution, antimicrobial resistance patterns and recognition of class 1 and 2 integrons among salmonella serovars from broiler chicken farms in khorasan razavi province, iran. materials and methods isolation and identification of salmonella a total of 156 fecal samples were collected from 23  poultry farms in khorasan razavi province, iran, from september 2013 to october 2013. all samples were transferred to selenite f broth (merck co., germany) and incubated at 37 °c for 16 h. a loopful of the enriched samples were cultured on macconkey agar (merck co., germany) and xld agar (merck 183veterinaria italiana 2021, 57 (3), 181-188. doi: 10.12834/vetit.2269.13773.1 badouei et al. mdr salmonella infantis in iranian broiler farms sequencing of integron class 1 pcr products of class 1 integron of salmonella strains belonging to eight geographically separated farms were sequenced by sanger dideoxy sequencing (seoul, south korea) using the amplification primers. the sequences were compared and analyzed by chromas pro version 1.7.5 technelysium as well as online blast software (http://www.ncbi.nlm.nih. gov/blast/) and integron database integrall (http://integrall.bio.ua.pt/). to confirm the results of detection of class 1 integrons, all positive isolates (possess class 1 integrons) were compared to available whole genome sequencing data. antimicrobial susceptibility test the susceptibility of 30 salmonella isolates to a panel of 27 antimicrobial agents was determined by the agar disc diffusion method and the interpretation of results was carried out according to the clinical and laboratory standards institute (clsi) guidelines (jorgensen et al. 2007, reller et al. 2009, clsi 2018). the antimicrobial agents tested and their concentrations (μg) were: amoxicillin/clavulanic acid (amc; 20/10  µg), amoxicillin (amx; 10  µg), cefixime (cfm; 5 µg), ceftriaxone (cro; 30 µg), cefazolin (cfz; 30 µg), chloramphenicol (chl; 30 µg), chlortetracycline (ctc; 30 µg), ciprofloxacin (cip; 5 µg), colistin (cst; 10 µg), difloxacin (difl; 10  µg), doxycycline (dox; 30 µg), enrofloxacin (enr; 5 µg), florfenicol (flor; 30 µg), flumequine (flu; 30 µg), fosfomycin (fof; 200 µg), furazolidone (fzd; 100 µg), gentamicin (gen; 10 µg), kanamycin (kan; 30 µg), linco-spectin (lp; 15/200 µg), nalidixic acid (nal; 30 µg), neomycin (neo; 30 µg), nitrofurantoin (nit; 300 µg), norfloxacin (nor; 10 µg), oxytetracycline (otc; 30 µg), streptomycin (str; 10  µg), tetracycline (tet; 30 µg), and trimethoprim/ sulfamethoxazole (sxt; 1.25/23.75 µg). each salmonella isolate which were resistant to at least one antibiotic in three or more antimicrobial classes were designated as mdr isolates. results prevalence of salmonella spp. and serovars in total, out of 156 fecal samples tested, 30 (19.2%) salmonella isolates were recovered. in 23 broiler farms in khorasan razavi province salmonella table i. list of primers which were used in this study. target primer sequence (5´ to 3´) size (bp) reference s. enterica s139 gtgaaattatcgccactgtcgggcaa 218 zahraei salehi et al. 2013 s141 tcatcgcaccgtcaaaggaacc s. infantis 558f aacaacgacagcttatgccg variable kardos et al. 2007878f ttgcttcagcagatgctaag 1275r ccacctgcgccaacgct s. heidelberg heli-f acagcccgctgtttaatggtg 782 zhu et al. 2015 heli-r cgcgtaatcgagtagttgcc s. gallinarum biotype gallinarum steb-f tgtcgactgggacccgcccgcccgc 636 zhu et al. 2015 steb-r ccatcttgtagcgcaccat s. gallinarum rhs-f tcgtttacggcattacacaagta 402 zhu et al. 2015 rhs-r caaacccagagccaatcttatct s. enteritidis sdf-f tgtgttttatctgatgcaagag 293 zhu et al. 2015 sdf-r cgttcttctggtacttcagatgac s. kentucky glyf ttccaattgaaacgagtgcgg 170 zhu et al. 2015 gly-r actaaccgcttgggttgttgctgt class 1 integron 5´-cs ggcatccaagcagcaag variable firoozeh et al. 2019 3´cs aagcagacttgacctga class 2 integron hep74 cgggatcccggacggcatgcacgatttgta variable firoozeh et al. 2019 hep51 gatgccatcgcaagtacgag mcr-1 clr5-f cggtcagtccgtttgttc 309 barbieri et al. 2017 clr5-r cttggtcggtctgtaggg mcr-2 mcr2-if tgttgcttgtgccgattgga 567 barbieri et al. 2017 mcr2-ir agatggtattgttggttgctg 184 veterinaria italiana 2021, 57 (3), 181-188. doi: 10.12834/vetit.2269.13773.1 mdr salmonella infantis in iranian broiler farms badouei et al. the main reservoirs for human salmonellosis. our study showed that prevalence of salmonella  spp. were increased dramatically in poultry farms during 2013-2014 in iran; s. infantis had the most significant role in the contamination of broiler farms. similarly, other studies from different regions of iran have serovars were detected in nine farms (39.1%). the kauffman-white group serotyping showed that 23  salmonella isolates (76.6%) belonged to serogroup c (s. infantis), six isolates (20%) belonged to serogroup d (s. enteritidis), and one isolate (3.3%) belonged to serogroups other than a-d. based on pcr analysis, among the 30 salmonella isolates, 23 isolates were identified as s. infantis (76.6%) which belonged to six different farms, and six isolates were identified as s.  enteritidis (20%) which belonged to five different farms, and one isolate (3.3%) was not serotyped by pcr. class 1 and 2 integrons and colistin resistance genes the class 1 integron was detected in 22/23 (95.6%) s.  infantis isolates. among the seven s.  enteritidis isolates, class 1 integrons were identified only in one isolate (14.2%). the class 2 integrons were not detected in any of the obtained salmonella isolates. also, the mcr-1 and mcr-2 genes were not detected in any of the 30 salmonella isolates. sequencing analysis analysis of dna sequencing results revealed that all sequenced isolates harbored an integron class 1 carrying one gene cassette including aada1 gene. phenotypic antimicrobial resistance out of 30 salmonella isolates, all (100%) of s. infantis and s.  enteritidis isolates were susceptible to cefixime, gentamicin, ceftriaxone, norfloxacin, and fosfomycin. also, 96.6% of isolates were susceptible to amoxicillin and ciprofloxacin. all isolates (100%) were resistant to colistin and amoxicillin/clavulanic acid and 93.3% of isolates were resistant to nitrofurantoin and oxytetracycline. mdr patterns were observed in 29 isolates (96.6%). the details of phenotypic resistance to antimicrobials have been presented in table ii and table iii. discussion poultry are one of the most important carriers of salmonella, which carry the bacterium asymptomatically and shed it to the environment through their feces (akbarian et  al. 2010, jajere 2019). this bacterium can survive for a long time in the environment and may be transmitted to human through the consumption of contaminated avian meat and egg products (vt nair et  al. 2018); salmonellosis is one of the most common foodborne diseases in humans worldwide. in many countries, poultry and its products are table ii. resistance (number and percentage) of recovered salmonella isolates from broilers in khorasan razavi province, iran. antimicrobial agent s. infantis (n = 23) s. enteritidis (n = 6) other serovars (n = 1) total (n = 30) βlactams antibiotics: penam penicillins: amoxicillin/ clavulanic acid 23 (100.0) 6 (100.0) 1 (100.0) 30 (100.0) amoxicillin 0 (0.0) 1 (16.6) 0 (0.0) 1 (3.3) cephalosporins: first generation: cefazolin 3 (13.0) 0 (0.0) 1 (100.0) 4 (13.3) third generation: cefixime 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) ceftriaxone 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) polymyxins: colistin 23 (100.0) 6 (100.0) 1 (100.0) 30 (100.0) aminoglysides: gentamicin 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) kanamycin 13 (56.5) 0 (0.0) 1 (100.0) 14 (46.6) neomycin 14 (60.8) 0 (0.0) 1 (100.0) 15 (50.0) streptomycin 13 (56.5) 0 (0.0) 1 (100.0) 14 (46.6) phenicols: chloramphenicol 6 (26.0) 2 (33.3) 0 (0.0) 8 (26.6) florfenicol 7 (30.4) 1 (16.6) 0 (0.0) 8 (26.6) tetracyclines: tetracycline 22 (95.6) 2 (33.3) 0 (0.0) 24 (80.0) chlortetracycline 23 (100.0) 0 (0.0) 1 (100.0) 24 (80.0) doxycycline 23 (100.0) 3 (50.0) 0 (0.0) 26 (86.6) oxytetracycline 23 (100.0) 5 (83.3) 0 (0.0) 28 (93.3) sulfonamides: trimethoprim/ sulfamethoxazole 20 (86.9) 0 (0.0) 1 (100.0) 21 (70.0) quinolones: nalidixic acid 23 (100.0) 3 (50.0) 0 (0.0) 26 (86.6) fluoroquinolones: ciprofloxacin 1 (4.3) 0 (0.0) 0 (0.0) 1 (3.3) difloxacin 0 (0.0) 0 (0.0) 1 (100.0) 1 (3.3) enrofloxacin 0 (0.0) 0 (0.0) 1 (100.0) 1 (3.3) flumequine 15 (65.2) 0 (0.0) 1 (100.0) 16 (53.3) norfloxacin 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) others: fosfomycin 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) furazolidone 22 (95.6) 0 (0.0) 1 (100.0) 23 (76.6) linco-spectin 23 (100.0) 0 (0.0) 1 (100.0) 24 (80.0) nitrofurantoin 23 (100.0) 5 (83.3) 0 (0.0) 28 (93.3) total 23 (51.5) 6 (20.9) 1 (48.1) 30 (45.2) 185veterinaria italiana 2021, 57 (3), 181-188. doi: 10.12834/vetit.2269.13773.1 badouei et al. mdr salmonella infantis in iranian broiler farms integrons are genetic elements that contain a site to integrate a segment of dna that could be disseminated the antimicrobial-resistant genes using a mobile genetic element (mge) such as plasmids and transposons among salmonella spp. the class 1 integron has been the most extensively reported class in the dissemination of resistance genes in salmonella spp. (kaushik et  al. 2018). interestingly, class 1 integrons were detected in most of our s.  infantis isolates and class 2 integrons were not detected in any of the studied isolates. these results are in accordance with a nother study conducted in iran (rahmani et  al. 2013). the class 1 integron seems to be an important player in dissemination of resistant factors among s.  infantis strains in the broiler farms in iran. importantly, the carriage of the same cassette (aada1) within the class 1 integron in eight isolates from different farms strongly suggests the presence of the clonally related s.  infantis in poultry farms in northeast of iran. based on phenotypic antimicrobial susceptibility examination of salmonella isolates in the present study, all s. infantis isolates were resistant to colistin, amoxicillin/clavulanic acid, chlortetracycline, doxycycline, oxytetracycline, nalidixic acid, linco-spectin, and nitrofurantoin. also, all of them were susceptible to amoxicillin, cefixime, reported that s. infantis had the highest frequency of contamination in broiler farms in the same time frame (fallah et  al. 2013, rahmani et  al. 2013). in a study conducted in northern provinces of iran, rahmani and colleagues showed that out of 36  salmonella isolates, 75% (n  =  27) and 25% (n  =  9) were identified as s. infantis and s. enteritidis, respectively (rahmani et  al. 2013). fallah and colleagues reported that out of 44 salmonella isolates, 79.5% (n = 34) were identified as s. infantis; the remaining 18.2% (n  =  8) and 2.3% (one strain) belonged to serogroup d and serogroup  c, respectively (fallah et  al. 2013). however, in other studies conducted by ezzat panah and colleagues and asad poor and colleagues in iran, it was showed that s.  enteritidis had the highest rate (45.3% and 75%, respectively) in broiler farms (ezatpanah et  al. 2013, asadpour et  al. 2014). different results have been obtained in other countries; in colombia, canada, and spain, s.  paratyphi b variant java (76%), salmonella hadar (40.4%), and s.  enteritidis (79.6%) were the most prevalent serovars, respectively (carramiñana et  al. 2004, donado-godoy et al. 2012, mainali et al. 2014). these differences with our study may be related to several factors such as geographical locations, sample selection criteria and hygiene level of broiler farms (firouzabadi et al. 2020). table iii. resistance patterns of salmonella serovars isolated from broilers in khorasan razavi province, iran. resistance patternsa s. infantis s. enteritidis other serovars total lp-c-te-cp-k-na-fr-fm300-sxt-n-cl-d-fm30-s-amc-ff-cte-t 1 -b 1 lp-c-te-k-na-fr-fm300-sxt-n-cl-d-fm30-s-amc-ff-cte-t 2 2 lp-c-te-k-na-fr-fm300-sxt-n-cl-d-s-amc-ff-cte-t 1 1 lp-te-na-fr-fm300-cl-d-fm30-amc-cte-t 3 3 lp-te-na-fr-fm300-cl-d-sxt-amc-cte-t 4 4 cl-amc 1 1 te-fm300-cl-d-amc-t 1 1 lp-c-te-nfx-k-na-fr-fm300-sxt-n-cl-d-fm30-s-amc-df-cte-t 1 1 lp-te-k-na-fr-fm300-sxt-n-cl-d-s-amc-cte-t 2 2 lp,-te-k-na-fr-fm300-sxt-n-cl-d-fm30-s-amc-cte-t 5 5 lp-te-k-na-fr-fm300-sxt-n-cl-d-fm30-s-amc-cte-t 1 1 lp-te-k-na-fr-fm300-sxt-n-cl-d-ff-s-amc-cte-t 1 1 c-na-cl-amc-ff-t-amx 1 1 lp-te-na-fr-fm300-sxt-n-cl-d-fm30-amc-cte-t 1 1 na-fm300-cl-amc 1 1 lp-c-te-na-fr-fm300-sxt-cl-d-fm30-amc-ff-cte-t 1 1 fm300-cl-amc-t 1 1 lp-c-na-fr-fm300-sxt-cl-d-fm30-amc-ff-cte-t 1 1 fm300-cl-d-amc-t 1 1 total 23 6 1 30 aantimicrobial agent tested were amoxicillin/clavulanic acid (amc), amoxicillin (amx), cefazolin (cez), chloramphenicol (c), chlortetracycline (cte), ciprofloxacin (cp), colistin (cl), difloxacin (df), doxycycline (d), enrofloxacin (nfx), florfenicol (ff), flumequine (fm30), furazolidone (fr), kanamycin (k), linco-spectin (lp), nalidixic acid (na), neomycin (n), nitrofurantoin (fm300), oxytetracycline (t), streptomycin (s), tetracycline (te), trimethoprim/sulfamethoxazole (sxt). bno resistance pattern detected. 186 veterinaria italiana 2021, 57 (3), 181-188. doi: 10.12834/vetit.2269.13773.1 mdr salmonella infantis in iranian broiler farms badouei et al. s.  infantis and a strong association between mdr patterns and the presence of class 1 integrons in broiler farms by 2013-2014 in khorasan razavi province, iran. colistin resistance is a major concern because it is the latest treatment of bacterial infection caused by gram-negative bacteria with mdr and carbapenem resistance in humans. all integrons carried the same gene cassette, which indicates that they were clonally related strains which spreaded via a possible common source. the results of the present research highlight the uncontrolled use of antibiotics in broiler farms that may cause the emergence of mdr salmonella strains in broiler products. therefore, there are an emerging need for systematic monitoring and characterizing mdr salmonella serovars in poultry industry in order to prevent the spread to food chain and humans. acknowledgments the authors would like to express their gratitude to dr. adriana cabal (visavet health surveillance center, spain) who kindly provided the positive control strains for colistin resistance genes. ceftriaxone, gentamicin, difloxacin, enrofloxacin, norfloxacin, and fosfomycin. besides, mdr patterns were observed in all of s. infantis isolates (100%). in rahmani and colleagues (rahmani et  al. 2013), and asadpour and colleagues (asadpour et  al. 2014) studies, most of s.  infantis strains were resistant to tetracycline, spectinomycin, streptomycin, sulfamethoxazole, nalidixic acid, and nitrofurantoin; also they observed mdr patterns in 92% of s. infantis isolates which are similar to our findings. in total, our results and also studies of ezatpanah and colleagues (ezatpanah et  al. 2013), asadpour and colleagues (asadpour et  al. 2014), chung and colleagues (chung et al. 2003), and carramiñana and colleagues (carramiñana et  al. 2004), showed that salmonella isolates are highly sensitive to gentamicin and highly resistant to tetracycline. interestingly, in this study, despite observing a high level of phenotypic resistance to colistin, none of the isolates carried the studied resistance genes, mcr-1 and mcr-2. it seems that screening for other types of mcr genes should be considered for future studies on salmonella strains in iran. this study revealed a high prevalence rate of 187veterinaria italiana 2021, 57 (3), 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res, 9, 66. 361 please refer to the forthcoming article as: vilcek et al. 2022. the role of molecular-genetic techniques in bvdv eradication in lower austria. vet ital. 10.12834/vetit.2595.16049.1. specific vaccines (bolin, 1995). however, vaccination cannot change health status of pi animals because their immune response is poor. in principle, vaccination of cattle can reduce the impact of bvdv but does not result in successful eradication in large geographic regions. despite that, it is widely applied in america and asia at least to partially improve health status of cattle herds (van campen, 2010). in contrast, a zoosanitary approach represented by a control program without vaccination has been widely used in many european countries. these control/eradication programmes are based on the identification and elimination of pi animals from the herds (harkness, 1987). this was documented in sweden where the first bvdv eradication programme without vaccination was successfully introduced in 1993 (lindberg and alenius, 1999). later, similar bvdv eradication programmes began in other scandinavian countries (bitsch et al., 2000; nuotio et al., 1999; valle et al., 2005). introduction bovine viral diarrhoea virus (bvdv) is the causative agent of bovine viral diarrhoea and mucosal disease (bvd/md) (baker, 1995; brownlie et al., 1984) which is responsible for significant economic losses in cattle farms (houe, 2003). infected animals develop a spectrum of clinical signs and may suffer from infertility, abortion, malformation and immunosuppression (lanyon et al., 2014). the infection of pregnant animals in the first trimester of gestation (30th – 125th day) is most dangerous because immunotolerant persistently infected (pi) cattle may be born (brownlie et al., 1998). they are constant carriers of virus during their lifetime. pi animals have no or very rare low levels of specific antibodies but they are the main transmitters of bvdv within and between the herds. two different approaches were applied to prevent the spread of bvdv infection in cattle populations. the first approach uses the application of bvdv veterinaria italiana 2022, 58 (4), 361-367. doi: 10.12834/vetit.2595.16049.1 accepted: 31.08.2021 | available on line: 31.12.2022 1university of veterinary medicine and pharmacy in košice. 2 animal health service of lower, austria. *corresponding author at: university of veterinary medicine and pharmacy in košice. e‑mail: stefan.vilcek@uvlf.sk. stefan vilcek1*, wigbert rossmanith2 keywords bvdv, eradication programme, lower austria, molecular-genetic technique, molecular epidemiology, persistently infected animal, reinfection, rt-pcr. summary a voluntary bovine viral diarrhoea virus (bvdv) control programme, which later became a compulsory eradication programme, based on the swedish model was introduced in lower austria in 1997. the persistently infected animals were detected by ag-elisa and all samples were re-tested by the improved single-tube rt-pcr, employing panpestivirus primers targeting the 5’-utr of the virus genome. in 2010, the bvdv eradication programme, which became compulsory from 2004, reached the final stage with only five remaining infected herds in which bvdv was difficult to eradicate. to resolve the problem in those herds, a molecular epidemiology approach was used. no differences in the spectrum of bvdv-1 subgenotypes at the beginning and at the final stage of eradication programme were found. the genetic study revealed the importance of human risk factor when finishing an eradication programme. molecular epidemiology was also used to analyse bvdv isolates associated with re-introductions to bvdv-free herds. the role of molecular-genetic techniques in bvdv eradication in lower austria review molecular-genetic methods in bvdv eradication program in lower austria vilcek et al. veterinaria italiana 2022, 58 (4), 361-367 doi: 10.12834/vetit.2595.16049.1362 at the beginning of the bvdv eradication programme, antibody tests were used to divide cattle farms into infected and noninfected premises. the approaches included the detection of bvdv antibodies in tank milk, spot tests of milk samples from young cows and spot tests of blood samples of young animals aged from 6 to 12 months. specific bvdv antibodies in blood and milk samples were detected by the use of an indirect elisa (svanovirtm, boehringer ingelheim svanova, uppsala, sweden). the non-infected farms were carefully protected with strong biosecurity measures. while cell culture virus isolation was mainly used in sweden to identify pi animals in the group of infected herds, in austria more modern, simple, agelisa and rt-pcr (single-tube reverse transcriptasepolymerase chain reaction) methods were used for this purpose. in infected herds where spot tests of 6-12 months old cattle were bvdv antibody positive or a pi animal had been introduced, a virus clearance was performed. young cattle with maternal derived bvdv antibodies, bvdv antibody negative cattle or cattle with low levels of antibody were tested for bvdv-specific antigen using an agelisa assay (herdcheck bvdv antigen leucocytes, herdchek bvdv antigen/serum, idexx scandinavia, österbybruck, sweden). all samples were retested by rt-pcr employing the panpestivirus 324/326 primer pair, which targeted the 5‘-untranslated region (5’-utr) of the pestivirus genome (vilcek et al., 1994). to facilitate high throughput analysis, virus was detected by rt-pcr in pooled serum samples. in the first two years of the control programme, the results suggested an inadequate sensitivity for the detection of virus antigen by ag-elisa. some samples were negative or doubtful by ag-elisa but positive in rt-pcr assays. to exclude the possibility that this phenomenon was due to an unusual bvdv variant, the genetic typing of incriminated virus isolates was carried out. phylogenetic analysis of bvdv isolates did not confirm this idea because although most of these isolates were found in the well-established bvdv-1f subgenotype, some of them were typed as bvdv-1b and bvdv-1h as well (rossmanith et al., 2001). to improve the results of the commercial ag-elisa kit, the preparation of leukocytes was modified (rossmanith et al., 2001). instead of mixing an equal volume of blood sample and 0.17 m ammonium chloride solution, as proposed in the instruction manual, one part of the blood sample was diluted with four parts of 0.17 m ammonium chloride solution. this was done to increase the efficiency of haemolysis of erythrocytes and to minimise the contamination of the final leucocyte pellet with non-haemolysed erythrocytes. the modified procedure led to a cleaner pellet of leukocytes and increased the ag-elisa test sensitivity. molecular-genetic techniques have benefitted biological research as well as having many practical applications. methods such as rt-pcr, and realtime rt-pcr are widely used for the detection of bvdv in clinical samples. the sequencing of the virus genome fragments coupled with computerassisted phylogenetic analysis are used in molecular epidemiology (cerutti et al., 2016; giammarioli et al., 2008; toplak et al., 2004). the bvdv eradication programme started in lower austria in 1997. after an enormous effort, farmers, veterinarians and other specialists achieved bvdv eradication in this region nearly 10 years ago. as several european countries or regions are introducing bvdv control/eradication programmes, we believe that experience from lower austria can be useful for specialists involved in control of bvdv infection on cattle farms. the aim of this minireview is to show how the molecular-genetic techniques were used in the bvdv eradication programme in lower austria and their contribution to achieve the final stage of eradication and to control of unwanted re-introduction of viral infection. basic principle of bvd eradication programme in lower austria although cattle management in lower austria is to some extent different from that in scandinavia, austrian farmers and veterinarians were inspired by the results of the scandinavian bvdv eradication program. therefore, the control scheme for bvdv infections in cattle herds in lower austria was introduced according to the swedish model in 1997, becoming compulsory in 2004. in principle, the control strategy included the same steps as in scandinavia: (1) dividing the herds into presumed non-infected and infected, (2) protection of noninfected herds and (3) systematic identification of pi animals and virus clearance in the herds by the elimination of infected cattle. more details on the bvd eradication program in lower austria can be found in other papers (rossmanith et al., 2005; rossmanith et al., 2010). importance of diagnostic methods selection as in all bvdv control/eradication programmes based on biosecurity without application of vaccination, the identification of pi animals is the most important step. the selection of good diagnostic methods to identify bvdv infected animals in the herds was also a challenge for specialists working on the eradication programme in lower austria. vilcek et al. molecular-genetic methods in bvdv eradication program in lower austria veterinaria italiana 2022, 58 (4), 361-367. doi: 10.12834/vetit.2595.16049.1 363 circulation of virus in the herds. to resolve this problem, the methods of molecular epidemiology, namely the nucleotide sequencing of pcr products obtained from 5’-utr coupled with the computerassisted phylogenetic analysis were used. to see the relationship between infected herds, the broader collection of 23 bvdv isolates identified in pi animals in the years 2010, 2009, 2008 and 2006 were sequenced and analysed. the genetic typing of bvdv isolates did not reveal the occurrence of any new subgenotype which would be prevalent or specific to the final stage of progress with bvdv eradication programme at the beginning of the voluntary bvdv control program (1997), 5 024 breeding herds took part. from the year 2005 onwards, in the compulsory bvdv eradication programme, nearly all 13 382 herds with animals for breeding have been included. the good progress of bvdv eradication in lower austria is documented in table i. from the introduction of the compulsory programme, the percentage of farms with detected pi animals progressively decreased and the number of bvdvfree herds significantly increased. the bvdv eradication programme finished in 2012 (table i, bolt numbers), when nearly all cattle herds were bvdv-free. the detection of one-tube rt-pcr products was also modified. to avoid the use of carcinogenic ethidium bromide for visualisation of pcr products, a more sensitive and less dangerous silver staining procedure was used (gottlieb and chavko, 1986). for convenience, commercially available polyester films were used to prepare thin layer agarose gels. such gels could be dried and stored at ambient room temperature for future experiments (rossmanith et al., 2001). in 2015, the single tube rt-pcr was replaced by a real-time pcr (viroreal® kit bvd virus, ingenetix ltd, vienna, austria). in addition, sample identification was controlled by a bar code procedure allowing transfer of metadata and diagnostic results directly to the computer. table i. elimination of pi animals from bvdv infected herds. year herds sub-jected to bvd-law in l. austria number of herds with pi animals detected % of herds with pi animals total number of pi animals detected number of bvdv free herds % of bvdv free herds 2005 13 382 248 1,85 511 7 931 59,26 2006 12 857 124 0,96 269 9 982 77,63 2007 12 273 46 0,37 115 11 166 90,98 2008 12 031 22 0,18 45 11 017 91,57 2009 11 733 10 0,09 12 10 951 93,33 2010 10 713 5 0,05 7 10 073 94,02 2011 10 703 5 0,05 14 10 357 96,76 2012 10 369 0 0 0 10 144 97,83 2013 10 105 0 0 0 9 857 97,54 2014 9 530 0 0 0 9 347 98,07 2015 9 262 1 0,01 2 9 048 97,68 2016 8 959 1 0,01 1 8 772 97,91 2017 8 699 1 0,01 1 8 468 97,34 2018 8 478 0 0 0 8 256 97,38 2019 8 076 0 0 0 7 893 97,73 2020 7 766 0 0 0 7 676 98,84 application of molecular epidemiology to resolve problems in last infected herds experience in the field has shown that the most difficult part of the eradication programme was the final stage. in lower austria, 5 farms were the most resistant to finish the eradication programme in 2010. despite enormous effort, the virus was still not eradicated from those farms. at the beginning, it was hypothesized that an unusual bvdv subgenotype might be responsible for continued molecular-genetic methods in bvdv eradication program in lower austria vilcek et al. veterinaria italiana 2022, 58 (4), 361-367 doi: 10.12834/vetit.2595.16049.1364 three cases of unwanted re-introduction of bvdv infections into bvdv-free herds were observed in the period from 2015-2017. the re-introductions were due to purchase of untested animals from infected herds. apart from a young bull for fattening which originated from an infected herd, the import of lambs from hungary and which were housed with pregnant cattle resulted in the birth of a pi calf in 2016. genetic analysis of the viral isolate revealed that the calf was infected with a border disease virus. another pi calf infected with bvdv, born from a purchased untested pregnant heifer, which originated from the neighbouring czech republic, was detected in 2017. since then, no more infections or pi animals have been detected in the bvdv-free cattle livestock of lower austria. to maintain this bvdv-free status, bulk tank milk samples of all herds are tested twice a year for bvdv antibodies with nearly 100 % negative results. in herds without milk production serology is carried out on blood samples from young home-bred cattle. there is a constant challenge to maintain bvdvfree status after eradication has been declared and continuing education remains very important for maintaining a favourable situation (lindberg & alenius, 1999). when unwanted re-introduction will appear, the molecular-genetic methods can significantly contribute to the identification of a pestivirus isolate. bvdv eradication programmes are evolving meanwhile, bvdv eradication programmes in europe have evolved. the austrian bvdv eradication program, similar to that in scandinavia, was based on the application of serological methods to separate infected and non-infected herds and then on the identification of pi animals in infected herds by ag-elisa or rt-pcr (in sweden mostly by virus isolation technique). however, the present control and eradication programmes applied in european countries have changed strategy. pi animals were directly identified by screening the entire cattle population in switzerland (pressi and heim, 2010; pressi et al., 2011) or in newborn calves and their mothers in germany, without application of serological investigation. the serum or blood samples for the identification of pi animals were mostly replaced by tissue samples from newborn calves punched out with special ear tags. virus continues to be mostly detected with sensitive agelisa or one tube real-time rt-pcr methods. this approach with modifications has been applied in many western european countries, such as belgium, uk, ireland (graham et al., 2021; russell et al., 2017) and in other parts of the world (lindberg et al., the eradication programme. while bvdv-1 subgenotypes a, b, d, e, f, g, h and k had been identified in thebeginning of the eradication program (vilček et al., 2001) or in other parts of austria (hornberg et al., 2009; kolesárová et al., 2004; vilček et al., 2003), the subgenotypes b, e, f, g and h were found on the problematic farms at the final stage of the eradication programme. however, the phylogenetic analysis revealed that there were three phylogenetic clusters with the same isolates despite originating from different farms (rossmanith et al., 2014). data in the first cluster of the phylogenetic tree indicated that two farms had the same isolates. their close proximity (around 100 m apart) and employment of common workers explained this observation. the second cluster of farms with the same isolate was served with the same animal carrier and veterinarian. a poor practice on farms falling to this cluster was parking the transport vehicle loaded with animals from herds with unknown bvdv status very close to the animal stable (less than 10 m). such practices may have contributed to the spread of bvdv between animals. the same milk collector or veterinarian regularly visited other farms (the 3rd phylogenetic cluster) with an identical circulating bvdv isolate. their visits could also contribute to the spread of virus. although the exact mode of transmission between herds with identical isolates has not been definitely clarified by genetic study, the results of this molecular analysis significantly contributed to focus on the risk factors for transmission of virus at the final stage of the eradication programme in lower austria. all concerned groups of farmers, animal owners, animal carriers, veterinarians and farm visitors were informed on their critical role to prevent the spread of viral infections in cattle farms. subsequently, the epidemiological situation on the farms investigated has been improved resulting in the final elimination of bvdv from cattle farms and the completion of the bvdv eradication programme in lower austria in 2012 (rossmanith et al., 2014).at the start of the eradication program in lower austria, participants learned that livestock trade, shared grasslands and animal contacts over fences were the greatest risks recognized for the transmission of bvd viruses (rossmanith et al., 2005). at the final stage of the eradication programme the new risk represented by human factor emerged, which was revealed by complex analysis of the cattle management and the application of molecular epidemiology approach (rossmanith et al., 2014). constant danger – re-introduction of bvdv infection it should be mentioned that after finishing the bvdv eradication programme in lower austria in 2012, vilcek et al. molecular-genetic methods in bvdv eradication program in lower austria veterinaria italiana 2022, 58 (4), 361-367. doi: 10.12834/vetit.2595.16049.1 365 and it should be introduced in more, if not all european countries to improve the health status of cattle herds and the welfare of animals. in principle, there are many possibilities to modify programmes to take into account epidemiological specificities of each country. however, in our opinion the omission of serological methods in eradication programmes with direct detection of pi animals by ag-elisa or rt-pcr is not an optimal approach. the serological investigation provides a unique opportunity to obtain epidemiological insights in investigated herds and can significantly contribute to the identification of pi animals with minimal additional economic cost. no doubt, the application of molecular-genetic techniques has become an important part of bvdv control/eradication programmes. the methods are mainly used for the identification of pi animals within herds, in molecular epidemiology studies to find the risk factors for transmission of virus between herds and to identify re-introduced bvdv infection into bvdv-free herds. 2006; moenning et al., 2005). whereas methods of molecular epidemiology were used in the eradication programme in sweden only sporadically (stahl et al., 2005), then more so in lower austria (this paper), and now this approach is widely used for typing of bvdv isolates during control programs (guelbenzu-gonzalo et al., 2016; russell et al., 2017; wenike et al., 2017). a good example is the phylogenetic typing of most bvdv isolates detected in pi animals in scotland (guelbenzu-gonzalo et al., 2016). in switzerland, more than 10 000 isolates were typed so far which revealed epidemiological links between cattle farms related to the history of the swiss federation (stalder et al., 2016; stalder et al., 2018). 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health, faculty of veterinary medicine, university of hama, hama, syrian arab republic. tel.: +963944233966, e‑mail: daremtabbaa@yahoo.com. keywords ihr 2005, coronavirus, global health. summary international health regulations 2005 contributed to the development of public health emergency control programs of international concern. the aim of their application was to mitigate the effects of the spread of such emergencies by proactive measures, stemming from risk assessment and expanding to epidemiological modeling systems that allow a logical extrapolation of what these emergencies can cause, and develop codified international strategies to avoid the occurrence of public health emergencies of international concern as soon as they are early discovered. the covid‑19 pandemic came to be a model in which these regulations were tested in the biggest challenge that human societies have faced since the second world war. the implementation of these ihr 2005 had a great positive role in limiting the spread of the disease, but some gaps, that could have been overcome and mitigate its consequences, appeared during the application of the precautionary economic, social and health quarantine systems. this study examines the importance of ihr 2005 and the main challenges it faced in general, focusing on the results of their application in the area of the covid‑19 pandemic and the gaps that have emerged in the technical, educational and political field and proposals to address them. darem tabbaa* international health regulations and covid-19 pandemic, challenges and gaps veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 accepted: 14.07.2020 | available on line: 31.12.2020 international level, the ihr were developed in 2005 for approval by the health ministers in the member states at the 2007 general assembly meeting of the who and their implementation starts from that date. contributing to this is that globalization has made national and geographical borders increasingly porous from pathogens, and therefore the fight against infectious diseases requires international cooperation and coordination, and this became more evident when the new covid‑19 virus appeared in 2019, providing a strong rationale for global public health management. among the factors that contributed to the review of the ihr are the emergence of the sars epidemic in 2003 and 2 other important challenges that impeded the implementation of the ihr  2005 are the suspension of polio vaccinations in northern nigeria and indonesia's refusal to exchange samples of h5n1 influenza viruses collected in this country with the who. the effective implementation of the 2005 ihr requires overcoming technical, resource, governance, legal, and political challenges. introduction international health regulations (ihr 2005) are a set of legally binding regulations for all world health organization (who) member states. they aim to harmonize the protection of public health while avoiding unnecessary disruption of trade and travel through the development of effective global alert, surveillance and response strategies for all priority public health events. the ihr (1969) have been used by who member states to guide international prevention and control of infectious diseases until june 2007. the ihr (1969) obliged who member states to notify the who of cholera, plague and yellow fever outbreaks in their territories. in addition, the ihr (1969) included requirements for health and vaccination certificates for travelers from infected to non‑infected areas; rerating, disinfecting and disinsecting of ships and aircraft; as well as detailed health measures at airports and seaports in the territories of who member states. in view of the increased risks resulting from various health conditions that cause anxiety at the 238 ihr and covid‑19 pandemic tabbaa veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 support from the who and its member states is required to strengthen national and global capacities to understand the goal of developing and implementing such regulations. the most exciting measure under the ihr  2005 is the announcement of a ‘public health emergency of international concern’ and the consequent adoption of limited‑term ‘interim recommendations‘ for urgent measures to contain the spread of any new pathogen with global concern locally and internationally. the advertising standards for pheic focus on serious, unusual or unexpected events that have public health effects outside the borders of the affected country and may require immediate international action. ihr and the global health system global healthcare spending slowed to 3.2 percent in 2019, from 5.2 percent in 2018. this is the impact of currency shifts and slowing global economic growth caused by geopolitical tensions, including the trade war between the united states and china and the trade war between the uk to plan out of the european union. however, health spending expected to increase during the period 2019‑2023 with a more robust compound annual growth rate (cagr) of 5 percent, compared to 2.7 percent in 2014‑2018. all regions except north america expected to see accelerated average spending growth in the forecast period, with the largest annual increases in the middle east / africa (7.4 percent) and in asia (7.1 percent). likewise, global healthcare spending as a share of gdp is likely to remain at around 10.2 percent until 2023, which is equivalent to 2018. this projected steady state reflects economic improvements and health systems efforts to contain costs. on a per capita basis, spending is likely to continue to spread unevenly, from $ 12,262 in the united states to only $ 45 in pakistan in 2023. high population growth in many developing economies will hamper efforts to bridge this gap. with a world population of 7.7 billion in 2019 to reach 8.5 billion by 2030, meeting health needs will not be easy. however, asian countries are also likely to contribute about half of the global growth in high‑income families (those that earn more than us $ 25,000 annually). population growth, along with increased economic strength and efforts to expand public health systems, are likely to increase spending on health. providing health care for an increasing aging demography is a major concern for governments and health systems. moreover, the life expectancy will rise from 73.7 years in 2018 to 74.7 years by the main challenges facing ihr in the twenty-first century since 2009 there have been six public health emergencies of international concern (pheic) declarations, the 2009 h1n1 (or swine flu) pandemic, the 2014 polio declaration, the 2014 outbreak of ebola in western africa, the 2015‑2016 zika virus epidemic, the ongoing 2018‑2020 kivu ebola epidemic, and the ongoing covid‑19 pandemic, which was declared a pheic on 30 january 2020. emerging infectious diseases often create technical challenges for implementing the international health regulations, even in the most technologically advanced and well‑resourced countries. new emerging pathogens present themselves in unusual or unexpected ways. modern modeling shows that the ability to control the spread of new pathogens is influenced by the transmission rate that occurs before the onset of symptoms or through an asymptomatic infection. this characteristic explains why diseases such as influenza and hiv are more difficult to control than smallpox or sars. improved diagnostic techniques may help public health authorities to identify pathogenic threats and build strategies to enhance reporting processes. resource challenges the requirements of the ihr  2005 challenges will face many countries, especially developing countries, with resource challenges. the ihr 2005 do not include funding mechanisms, which makes each state party bear the financial costs of improving its domestic, intermediate, and national capacities. governance challenges governance challenges include administrative and managerial weaknesses in countries from the local to the national level. only a few countries have assessed their ability to detect and respond to disease threats. legal challenges states parties face legal complications in implementing the ihr  2005 within their national legal and constitutional systems. political challenges the level of political commitments of different countries will demonstrate different challenges in implementing the ihr 2005. although the ihr  2005 contain some provisions that directly address these challenges, active 239 tabbaa ihr and covid‑19 pandemic veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 ihr  2005 identify health‑related events that each country that agrees to bind by the regulations must report to who. in terms of health‑related events that occur in its territory, a state party must notify who of “all events which may constitute a public health emergency of international concern”. these events include any unexpected or unusual public health event regardless of its origin or source. ihr  2005 also require state parties, as far as is practicable, to inform who of public health risks identified outside their territories that may cause international disease spread, as manifested by exported or imported human cases, vectors that may carry infection or contamination, or contaminated goods. ihr 2005 contain a ‘decision instrument’ that helps state parties identify whether a health‑related event may constitute a pheic and therefore requires formal notification to who. the decision instrument focuses on risk assessment criteria of public health importance, including the seriousness of the public health impact and the likelihood of international spread. ihr and covid-19 the covid‑2019 is a persistent global disease caused by novel corona virus (sars‑cov‑2). the outbreak began in wuhan, hubei province, china, in december 2019. the who announced that the outbreak was a public health emergency of international concern on january 30, 2020, and it was recognized as a pandemic on march 11, 2020. until february 9, 2021, there were nearly 107,101,319 cases reported in 213 countries and territories, resulting in approximately 2,338,961 deaths. about 78,980,813 people recovered. in contrast to the sars outbreak in 2003, china demonstrated greater transparency in communication on a daily basis for its epidemiological situation, early participated in the genomic sequencing of the virus in an open‑access database, accepted the presence of the who support team, and took strict control measures including quarantine for millions in wuhan and other cities. the who has established itself as a central institutional center in this case, providing guidance and assistance, coordinating research on vaccines, diagnostics and antiviral drugs, and framing the international response on standard terms under the international health regulations. in order to achieve these regulations, countries must achieve a set of basic capabilities in their national health systems in order to immediately detect report and respond to public health risks and emergencies. this has proven to be one of the major implementation challenges as the ihr lack a dedicated funding mechanism and a formal 2023. the number of people over the age of 65 will exceed 686 million, or 11.8 percent of the total population. this trend will be more pronounced in japan, where the proportion of people over 65 year old is expected to reach nearly 29 percent by 2023; in western europe, the proportion is expected to reach 22%. therefore, spending on the global aged care market (home health, remote patient monitoring, etc.) is likely to exceed $ 1.4 trillion by 2023. dementia currently affects more than 50  million people aged 60 years and over globally. the figure expected to reach 82 million by 2030 and 152 million by 2050. while communicable diseases remain a threat, especially in developing countries, chronic and non‑communicable diseases are also increasing. almost 425 million people were diabetic in 2017. by  2045, this number expected to rise 48 percent to 629 million. china (114.4 million), india (72.9 million), and the united states (30.2 million) topped the list of people with diabetes in 2017 and expected to maintain these proportions until 2045. despite the development of medical technologies, lifestyle factors including smoking, poor nutrition, high blood pressure, obesity, and lack of physical activity remain and contribute to the top ten causes of death. often this human group is more likely affected by the disease pandemics, because most of them suffer from many stubborn and chronic diseases starting from cardiovascular injuries, through chronic respiratory diseases, diabetes, tumors, high blood pressure, etc., and thus their health systems will be unable to meet the needs of treatment and recovery of patients. ihr 2005 represent a major development in the use of international law for public health purposes. one of the most important aspects of ihr  2005 is the establishment of a global surveillance system for public health emergencies of international concern. through these regulations, public health surveillance defined as “the ongoing systematic collection, analysis, and interpretation of outcome‑specific data for use in the planning, implementation, and evaluation of public health practice”. a surveillance system requires structures and processes to support these ongoing functions. the surveillance system includes four main elements: 1) health‑related events under surveillance and their public health importance, 2) purpose and objectives of the system, 3) components and processes of the system, and 4) resources needed to operate it. ihr 2005 remain a valuable but potential framework within which to address infectious diseases across international borders. another challenge to ihr  2005 implementation involves its requirement for significant public health capacity building, particularly with regard to infectious disease surveillance. 240 ihr and covid‑19 pandemic tabbaa veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 covid‑19. these findings must triangulated with the latest risk assessments available for covid‑19 and other assessments such as joint external evaluations, after‑action reviews, simulation exercises, and others to understand the capacity level of countries and to implement priority actions at the national and local levels. the who secretariat is working on the development of a preparedness dashboard to provide real‑time information that based on these capacity assessments. another limitation related to the method based on a deterministic approach; therefore, proportionate or inverse interactions among variables not shown. an effective way of managing airborne infections is applying evidence‑based public health prevention strategies. this method includes scaling up public awareness of behaviors such as hand hygiene and respiratory etiquette, communicating and engaging with local communities about the risks of the outbreak, and putting in place effective public health response measures. national points of entry should also have the capacity to prevent, detect, and respond to potential threats in line with the ihr. many countries have low capacities for preventing the occurrence and spread of outbreaks, such as measles, influenza, ebola virus. therefore, enhancing national preparedness capacities in line with the gaps identified in this study should incorporate action to strengthen points of entry. many countries have made substantial progress in developing effective levels of disease detection, which involves strengthening surveillance and laboratory capacities. the application of lessons learned from previous infectious disease emergencies including the 2002 sars‑cov, 2009 h1n1 influenza pandemic, mers‑cov, ebola virus, and zika virus outbreaks, have helped to strengthen countries’ capacities to effectively detect and verify suspect cases. the early detection of covid‑19 in china and the development of laboratory reagents for testing and genetically sequencing the novel virus are key steps that have supported the early response. seventy six percent of countries have robust detection capacities in place, which enable early detection and verification of potential outbreaks when they occur. mutations accumulating during epidemics increase the replication fitness of the virus in cell culture and increase virulence in an animal model. the phylogenetic tree has a coalescence center with exponential expansion identified by haplotype markers. the color‑mapped phylogenies largely support the 14 identified sub clades. the substantial numbers of samples from the united states show affinity with european lineages rather than those directly derived from east asia. except for the earliest cases, european clades dominate even in samples compliance monitoring mechanism. partially, many of these countries are still far from achieving the required capacities. problems and uncertainties also arise regarding the coordination of the international response to pheic, and the outbreak of covid‑19 underscores these challenges. while the ihr  2005 contain relatively clear obligations regarding due diligence and cooperation in addition to general and routine health measures, national measures in response to the pheic standard are meant to be guided by who's interim recommendations. however, the available practice shows an inconsistent level of compliance, especially with regard to restrictions on travel and trade with the affected countries. the current covid‑19 outbreak tests again the effectiveness and credibility of the ihr 2005 not only as a legal tool but also as a public health tool and framework for guiding narrative political challenges, sovereignty tensions, economic interests and national security considerations. there are serious design and implementation questions that must be addressed urgently to avoid the irreversibility of the ihr  2005 as the only legal framework indispensable to global health security. the covid‑19 pandemic caused an escalation in the number of victims and a collapse in the global economy. it has emerged as a basic test for political leaders. although there are conflicting views about the ability of national political leaders to advance or stand powerless behind a ‘curve’ of rapidly changing dynamics. they are clearly striving to take decisive decisions in controlling health systems in their countries. but they lack data that enable them to actually know the number of people who will be infected, the number of people who will die from this disease, the number of patients who will overwhelm hospitals the more the disease spreads, and how the economic infrastructure of the state could be threatened by such a pandemic. ihr gaps for covid-19 countries differed significantly in their ability to prevent, detect and control outbreaks, with about half of countries reporting operational readiness capabilities to respond to public health emergencies. several factors affect the emergence and spread of infectious disease outbreaks within countries and between regions, including the strength of the capacity of the ihr at the national and local levels, adherence to infection prevention and control measures, climate pressures, and population density. the analyses of the operational readiness index have used to support the development of a draft who strategic preparedness and response plan for 241 tabbaa ihr and covid‑19 pandemic veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 threaten people as a result of changing patterns of life associated with climate change, human‑animal interaction, sustainable life‑threatening problems such as hunger, poverty, pollution, and declining quality of education and others. ihr could not yet been able to apply the concept of ‘one health’ in dealing with emerging and re‑emerging diseases, which has delay the response to such pandemics. in addition, the irresponsible political response in all different aspects caused delayed disease control operations and the emergence of severe economic and social effects on all societies in a way in which the success stories of applying early warning and reassuring response systems to people turned into indicators of horror and severe anxieties. the problems of stopping production, education and movement between countries have catastrophically led to the transformation of health education processes towards diseases into political platforms for the exchange of accusations, and a space for settling scores between countries. public health education (phe), ignoring the concept of ‘one health’ and their impact on the future aspects of the ihr public health competencies, especially with regard to managing epidemic and chronic diseases, are of increasing importance for the global workforce in the healthcare field. it is necessary to strengthen public health education and related disciplines in basic medical education. it also became evident during the covid‑19 pandemic that developing guiding goals that should support the development of university medical education in the areas of public health and considering it a basic requirement has become necessary. the progress of university medical education in the field of public health is also a global challenge to answer a set of questions that include: what we learn in phe? how do we learn phe? when do we learn phe? why do we learn phe? evidence‑based health care has re‑emerged the importance of clinical epidemiology, considering phe a pursuit of postgraduate studies. therefore, the available risk assessments should developed for any pathogen from all animal and environmental sources that can threaten human health and link them with other assessments to understand current capabilities and their implications for people’s lives and the development of societies. the world health organization is working to develop a preparedness dashboard (a data visualization platform that will include a query system) to provide information in real time, and in a virtually based manner on various capacity from western states in the united states. further, european samples tend to associate with lineages that expanded through australia. estimation of mutation rate showed a median of 1.12 × 10‑3 mutations per site‑year (95% confidence interval, ci: 9.86 × 10‑4 to 1.85 × 10‑4). the median tree height was 5.1 months (95% ci: 4.8 to 5.52) mutations in the receptor‑binding domain of the spike protein suggest that these variants are unlikely to reduce binding affinity with ace2. v483a and g476s are primarily observed in samples from the united states, whereas v367f is found in samples from china, hong kong special administrative region, france and the netherlands. the v367f and d364y variants have been reported to enhance the structural stability of the spike protein facilitating more efficient binding to the ace2 receptor. the ability to respond in a country depends on the strength of its preparedness for emergencies, the rapid testing of its causes and risk factors, and the regular updating of national plans and capacities. an effective response to an outbreak depends not only on the availability of adequate human resources and funding, but also on the ability to manage emergency logistics services (including handling supply chains for essential products needed during an emergency). the results that emerged while dealing with the covid‑19 pandemic show that many countries need support to achieve these capabilities, and more support should be provided as a global priority action to enhance health security. it was found that operational readiness capabilities and enabling functionality were low in many low‑resource countries, and this necessitated the need for increased investment in strengthening the capacities of ihr. it also showed the low national preparedness capabilities due to insufficient investment in human capital and weak planning for continuity in support of combating the spread of infectious diseases. perhaps the greatest challenge facing the ihr in addressing new pandemics is the careless to early investigation of potential situations that can figure 1. phylogenetic tree for the sars-cov-2 genomes, 2019-2020. 242 ihr and covid‑19 pandemic tabbaa veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 making control of them start from the beginning of the emergence of disease cases, so that most of the measures taken can stop the spread of the pandemic instead of limiting its occurrence. 4. the inability of ihr to define a well‑informed health reporting and awareness system, and leave plenty of false ideas and information to spread among people and influence the course of control programs. 5. the thoughtlessness of ihr to place control measures for local infection foci's for pathogens that have not yet come into international concern, so that international outbreaks and pandemics can occur. it is necessary to include in these regulations the rules of epidemiological outbreak investigation and methods of implementing quarantine of infection foci's. conclusions ihr 2005 harmonize the protection of public health and avoid unnecessary disruption of trade and travel through the development of effective global alert, surveillance and response strategies for all priority public health events. ihr  2005 contain a ‘decision instrument’ that helps state parties identify whether a health‑related event may constitute a pheic and therefore requires formal notification to who. the decision instrument focuses on risk assessment criteria of public health importance, including the seriousness of the public health impact and the likelihood of international spread. the current covid‑19 outbreak tests again the effectiveness and credibility of the ihr 2005 not only as a legal tool but also as a public health tool and framework for guiding narrative political challenges, sovereignty tensions, economic interests and national security considerations. the covid‑19 pandemic caused an escalation in the number of victims and a collapse in the global economy. it has emerged as a basic test for political leaders. several factors affect the emergence and spread of infectious disease outbreaks within countries and between regions, including the strength of the capacity of the ihr at the national and local levels, adherence to infection prevention and control measures, climate pressures, and population density. during the implementation of ihr procedures in the control of covid‑19 pandemic, some weaknesses that could overcome to mitigate more than the effects of the direct and indirect pandemic has been highlighted in this paper as a challenge for the future development of ihr. assessments, taking into account the commitment to health studies in the field of veterinary medicine, as a basis for assessing the epidemiological situation, and improving national, regional and global surveillance system based on the concept of one health. it has become clear that despite the gains in understanding the pathogen, many countries are unwilling to manage cases within their borders. their health systems were therefore unsuccessful. investments in preparedness should urgently increase to ensure that vulnerable countries are operational, and able to respond to public health events such as the outbreak of covid‑19. future requirements for the development of ihr one of the most important challenges faced by humankind in the twenty‑first century is the experience of dealing with the covid‑19 pandemic. the international community has demonstrated its ability to benefit from international laws in controlling the spread of epidemics and limiting the spread of epidemics in an unprecedented manner. international political differences have not stopped an obstacle for health authorities to impose a global health system that all countries adhere to without exception and implemented popularly at all levels. all countries of the world benefited from the application of ihr, and were able to draw the world's attention to the dynamics of disease development in a real and direct way. they participate effectively in the processes of controlling it. during the implementation of the ihr, some weaknesses that could overcome to mitigate more than the effects of the direct and indirect pandemic highlighted. among these points: 1. lack of strict international rules to ensure the availability of basic prevention elements in all countries, providing the minimum necessary to secure adequate databases for early detection of pathogens that may have international concern (epidemiological surveillance programs, intersectoral cooperation, regional and international coordination, effective programs for public health education in medical higher education). 2. the inability of political systems to hand over the affairs of the integrated health management in the country during crises and disasters to specialists to reduce the problems of issuing irresponsible decisions that can increase the problem rather than get rid of it. 3. failure to apply the concept of “one health” in implementing ihr, delaying early detection of pathogens of international importance and 243 tabbaa ihr and covid‑19 pandemic veterinaria italiana 2020, 56 (4), 237‑244. doi: 10.12834/vetit.2335.13296.1 baker m.g. & fidler d.p. 2006. global public health surveillance under new international health regulations. emerg infect dis, 12 (7), 1058‑1065. doi:10.3201/eid1207.051497. benecke o. & deyoung s.e. 2019. anti‑vaccine decision‑making and measles resurgence in the united states. glob pediatr health, 6. biezen r., grando d., mazza d. & brijnath b. 2019. visibility and transmission: complexities around promoting hand 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organization (who). 2020. who director‑ 295 veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 accepted: 02.12.2021 | available on line: 31.12.2022 1dipartimento di fauna selvatica, istituto superiore per la protezione e la ricerca ambientale (ispra), ozzano dell’emilia (bo), italy. 2direzione generale sanità animale e farmaci veterinari, ufficio 3, sanità animale e gestione operativa del centro nazionale di lotta ed emergenza contro le malattie animali, unità centrale di crisi del ministero della salute, roma (rm), italy. *corresponding author at: dipartimento di fauna selvatica, istituto superiore per la protezione e la ricerca ambientale (ispra), ozzano dell’emilia (bo), italy. e‑mail: giorgia.baiocchi86@gmail.com. giorgia baiocchi1*, andrea marcon1, olivia bessi2, luigi ruocco2 and vittorio guberti1 keywords farms under restriction, kept and wild pigs, simulation. summary african swine fever is a devastating contagious viral disease of kept and wild porcine animals that will challenge the veterinary services involved in its eradication. nowadays, asf represents one of the biggest challenges for the pig sector at a global level. following a number of simulated virus random introductions, the paper estimates the average number of farms (including their type) and animals that will be under restriction, and finally the average distance of infected farms from the nearest rendering plant. the study includes data referring to 101,032 farms with 9,322,819 pigs which are available in the italian national database (bdn). the simulations consider 5 different biogeographic regions with their own domestic pig distribution, breeding systems, and wild boar presence. following an index case in a farm, and in the worst-case scenario, in the 10 km radius of the restriction area, there will be: 2,636 farms in south italy; 470,216 animals in po valley; 147 km in central italy is the longest mean distance from the infected farm to the nearest rendering plant. simulated african swine fever (asf) virus detection in italy: average numbers of farms and pigs under restriction has wild boar as its main epidemiological reservoir, while in the balkans, the involvement of the backyard farms sector plays a key role in the local maintenance of asfv (bellini et al. 2021). in the eu, following an outbreak in domestic pigs, a protection zone (circular, 3 km radius) and a surveillance zone (ring-shaped, 7 km radius) are established, creating a restriction area of 10 km radius around the infected farm/holding [commission delegated regulation (eu) 2020/6871]. during outbreak management, several actions are required by legislation (e.g. prohibition of animal and product movements, stamping-out and disposal of pigs, disinfection of the farm and premises) and the number of farms and pigs involved influences the effort required. in the best-case scenario, only one introduction african swine fever (asf) is a contagious haemorrhagic fever affecting both domesticated and wild pigs belonging to the species sus scrofa. it is one of the most complex and economically devastating diseases, causing a major socio-economic impact in affected countries. asf is an internationally notifiable disease whose presence strongly affects internal and international trades of live pigs and pig products, increasing market price volatility, disrupting usual trade flows and applying selective pressure on small pig farms, changing the structure of the pig farming system in the eu in the last decade (bellini 2021). introduced in georgia in 2007, it spreads north and then west through europe and east through asia, with cases occurring mainly in domestic pigs in asia (vergne et  al. 2020) and wild boars in europe (efsa 2019). in september  2020, the asf virus (asfv) reached the wild boar population in eastern germany on the border with poland (sauter-louis et al. 2021). in central and western europe, the virus 1 european commission 2019. commission delegated regulation of 17 december 2019 supplementing regulation (eu) no. 429/2016 of the european parliament and the council, as regards rules for the prevention and control of certain listed diseases. (eu 687/2020). off j, l  174, 03/06/2020, art.21 par.1 and annex v. 296 veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 asf virus in italy: simulation study baiocchi et al. area, as well as the implementation of additional biosecurity measures and surveillance schemes. in the absence of ornithodorus spp, restrictions will last for 12 months after the last positive result for asfv or specific antibodies in a wild boar (oie terrestrial animal code, 20198), or until the operational expert group recommends lifting the measures based on epidemiological information [regulation (eu) 2016/4299 and commission delegated regulation (eu) 2020/68710]. commission implementing decision 2014/709/eu of 9 october defined a regionalisation approach in 4 different zones, reduced to 3 with the entry into force of eu regulation 2016/429 based on article 71, and implemented by eu regulation 605/202111. these measures relate to the movement of pigs, their products and by-products, including their trade and intra-eu market rules, depending on the presence of the virus and related risks. the presence of asf implies a huge effort for veterinary services, depending on the number of farms and pigs involved, the distribution and abundance of wild boars, and the availability of disposal facilities where pigs or wild boars can be safely disposed of. one of the most pressing arguments to support asf eradication in pigs is the economic loss that the presence of the infection will cause. the greatest economic loss will come from the block on both domestic and international trade in pork and pork products from the infected area; some third-country trading partners may ban the whole country regardless of the geographical distribution of the virus. at a local level, eradication measures impose the block on animal movements and production in the restricted area, with the possibility of derogation for specific cases, which will affect not only pig farms, but all the commercial activities related to pig farming (e.g. holding will be infected and all pigs will be stamped and disposed of safely, usually in a disposal facility [commission delegated regulation (eu) 2020/6872]. however, the census of all pigs and pig holdings within the restriction zone must still be carried out, as well as the implementation of additional biosecurity measures [commission implementing regulation (eu) 605/20213] and a surveillance program to prevent or detect any secondary outbreaks at an early stage. the shortest duration of the restrictions is 15 days for the protection zone (with an additional period of 15 days for surveillance measures in the protection zone), and 30 days for the surveillance zone, to be counted after the mandatory disinfection of holdings and premises. the restriction measures may last longer when the virus is detected in any other location/site of relevance. the competent authority may determine a different duration of the restricted zone on a case by case basis, taking into account factors influencing the risk of disease spread (e.g. category a disease transmitted by vectors, commission implementing regulation 2018/18824) [commission delegated regulation (eu) 2020/6875]. in case of detection of asfv in wild boars, legislation [regulation (eu) 2016/4296 and commission delegated regulation (eu) 2020/6877] requires the establishment of an infected area, possibly including a core area (where the virus was detected) whose size is defined according to the local landscape and distribution of wild boars the average size of the smallest wild boar infected areas in the eu (czech republic, belgium, brandenburg in september 2020) is about 1,000 km2. within the infected area, all wild boars found dead or hunted must be tested and if positive for asf safely disposed of (sante/7113 2015), while movements of domestic pigs are allowed under veterinary supervision. census of pigs and pig farms is mandatory in the infected 2 european commission 2019. commission delegated regulation of 17 december 2019 supplementing regulation (eu) no. 429/2016 of the european parliament and the council, as regards rules for the prevention and control of certain listed diseases. (eu 687/2020). off j, l 174, 03/06/2020, art. 22 par. 3. 3 european commission 2021. commission implementing regulation of 7 april 2021 laying down special control measures for african swine fever. (eu 605/2021). off j, l 129, 15/04/2021, art. 16 and annex ii. 4 european commission 2018. commission implementing regulation of 3 december 2018 on the application of certain disease prevention and control rules to categories of listed diseases and establishing a list of species and groups of species posing a considerable risk for the spread of those listed diseases. (eu 1882/2018). off j, l 308, 04/012/2018, annex table referred to in art. 2. 5 european commission 2019. commission delegated regulation of 17 december 2019 supplementing regulation (eu) no. 429/2016 of the european parliament and the council, as regards rules for the prevention and control of certain listed diseases. (eu 687/2020). off j, l 174, 03/06/2020, art 39 and annex x, art. 55-56 and annex xi, art.58. 6 european parliament and council 2016. regulation of 9 march 2016 on transmissible animal diseases and amending and repealing certain acts in the area of animal health (‘animal health law’). (eu 429/2016). off j, l 84, 31/03/2016, art. 43 par.2 d) iii) and art. 70. 7 european commission 2019. commission delegated regulation of 17 december 2019 supplementing regulation (eu) no. 429/2016 of the european parliament and the council, as regards rules for the prevention and control of certain listed diseases. (eu 687/2020). off j, l 174, 03/06/2020, art.63 and art. 66. 8 https://www.oie.int/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&l=1&htmfile=chapitre_asf.htm. 9 european parliament and council 2016. regulation of 9 march 2016 on transmissible animal diseases and amending and repealing certain acts in the area of animal health (‘animal health law’). (eu 429/2016). off j, l 84, 31/03/2016, art. 31-35 and art. 70. 10 european commission 2019. commission delegated regulation of 17 december 2019 supplementing regulation (eu) no. 429/2016 of the european parliament and the council, as regards rules for the prevention and control of certain listed diseases. (eu 687/2020). off j, l 174, 03/06/2020, art. 67. 11 european commission 2021. commission implementing regulation of 7 april 2021 laying down special control measures for african swine fever. (eu 605/2021). off j, l 129, 15/04/2021. 297veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 baiocchi et al. asf virus in italy: simulation study that perhaps do not receive adequate attention in the preparatory phase of response to epidemics. methods study area our study area is the whole italian territory, except sardinia, which was excluded because asf has been endemic in the island since the 1970s. italy was subdivided into 5 roughly identified macro-areas (figure 1), based on different environmental characteristics (e.g. alps), the density of pig farms and the most common types of farming. the alps macro-area was defined according to the alpine convention (map downloaded from http://webgis.alpconv.org/, accessed 19 october 2019). the po valley macro-area was identified as the area below 150 m a.s.l., bounded by the alps and the apennines. the northern italy macro-area was defined as the area north of 44° n latitude (city of massa, tuscany) that is not included by the alps and po valley macro-areas. the macro-area of central italy was defined as between 44° n latitude (town of massa, tuscany) and 41°.25 n latitude (town of formia, lazio), and the macro-area of southern italy includes the whole territory south of 41°.25  n latitude (town of formia, lazio). wild boar distribution was defined as the area within a 3.5 km buffer around the forested area, while the po valley is wild boar free (monaco et  al. 2003). the forested slaughter, transport, feed production) causing a knock-on effect on the economy. in addition, the trade ban that invariably will follow the epidemic will cause a surplus of pig meat and products on the national territory, leading to a collapse in prices that will affect the whole national pig and pork market. the situation would be further aggravated if the virus were to be detected in wild boars, both because the restriction area is wider thus involving more farms and pigs and because the restriction on farmed animals will last at least one year according to oie rules in order to regain the disease-free status (terrestrial animal health code, art. 15.1.4  2b12). this stricter regulation emerges from the difficulty of monitoring the disease in wildlife, so even if all farms in the area are asf-free, the disease could still spread through the wildlife population and could return to farmed pigs in the area (e.g. oļševskis et  al. 2016, oļševskis et  al. 2020, franzoni et al. 2020). compared to the above, this was found to be applicable in those contexts with free-ranging pigs, as the interaction between these pigs and wild boars increases the risk of asf outbreaks and the efforts needed to eradicate the disease in those areas (franzoni et  al. 2020, tao et al. 2020). the aim of this article is to outline using two sets of simulations the challenges that the local veterinary service will face in case of detection of asf in pigs or wild boars in italy. we quantified the average number and type of farms and reared pigs that will be restricted, as well as the average distance to the nearest rendering plants following the detection of the virus in kept or wild pigs. the simulations consider 5 different areas combining italian biogeographic regions with domestic pig distribution, farming systems and an approximation for wild boars distribution. finally, it must be emphasised that, although this work was theorised at the end of 2021, the subsequent arrival of african swine fever in wild boar in january 2022 in the mountains between piedmont and liguria and in may of the same year first within rome's grande raccordo anulare (gra) and then in the mountains of rieti, made it clear that a quantitative approach albeit in probabilistic terms is indispensable to have an idea of the effort required to manage an outbreak of asf; the number of flocks and animals to be surveyed and planned for slaughter, transport management; rendering capacities including rendering plants location, are all activities that often turn out to be the true criticalities that the veterinary services have to face in the various phases of outbreak management and figure 1. distribution of farms in italy, divided into the five macro‑areas considered. 12 https://www.oie.int/en/what-we-do/standards/codes-and-manuals/ terrestrial-code-online-access/?id=169&l=1&htmfile=chapitre_asf.htm. 298 veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 asf virus in italy: simulation study baiocchi et al. simulations pig farm density and type distribution varied widely among the five macro-areas, so analyses were run independently for each macro-area. we analysed the results of spatial simulations, where a randomly chosen pig farm was infected with asf, which was assumed to be detected immediately, and the number of potentially affected pigs and pig farms was estimated from spatial and capacity data about pig farms (bdn). simulation were iterated for each macro-area as follows. around each randomly-selected virtually-infected farm, following eu regulations [commission delegated regulation (eu) 2020/68713], a circular protection zone was identified with a radius of 3 km centred on the infected farm where the strictest measures of the contingency plan are applied. in addition, a buffer surveillance zone with a radius of 7 km was added, where less stringent measures are applied. within each of these zones, we calculated the number of affected farms and the number of animals involved. as simulations were performed independently for each macro-area, pig farms included in one restriction zone but belonging to another macro-area were excluded from the analysis. this procedure was repeated 1,000 times for each macro-area, the results are reported as area of the country was ex-tracted from corine land cover 2018. data national data on pig farms were extracted from the ‘national database (bdn) of the identification and registration system of italy’, which collects information on all farms and production chains related to animals on the national territory. the database contains accurate and up-to-date information on each establishment, including geographical coordinates, type of production, number of individuals, maximum capacity, movements, etc. these data allow us to depict an accurate scenario of what would happen if asf arrived on a farm anywhere in the country. we selected farms that reported the geographical coordinates, the total number of pigs present or at least the capacity value (i.e. the maximum number of individuals allowed), and belonging to the following types: ‘breeders’, ‘fatteners’ and ‘backyards’. breeders are large farms that house sows and boars for breeding purposes; fatteners are large farms where animals are raised to slaughter size, and backyards are small farms, with the same purpose as fatteners farms, that can have a maximum of 4 pigs for home consumption, and are not allowed to move animals to other farms (sante/7113, 2015). in addition to pig farms, rendering plants (figure 2) were considered to measure their distance from the centre of the outbreak. figure 3. proxy of the distribution area of wild boar divided into the four macro‑areas considered. sardinia was not included in this study. 13 european commission 2019. commission delegated regulation of 17 december 2019 supplementing regulation (eu) no. 429/2016 of the european parliament and the council, as regards rules for the prevention and control of certain listed diseases. (eu 687/2020). off j, l 174, 03/06/2020, art.21 par.1 and annex v. figure 2. locations of rendering plants (red dots). 299veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 baiocchi et al. asf virus in italy: simulation study 2.97 ± 8.72 pigs on backyard farms, 442.19 ± 1,571.36 pigs on breeding farms, and 561.22  ±  1,384.34 pigs on fatteners farms. pig abundance differs among macro-areas (figure  4, table ii) with 347,947 pigs in the alps (3.73%), 6,310,791 pigs in the po valley (67.69%), 1,413,486 pigs in the north italy (15.16%), 880,237 pigs in the central italy (9.44%) and 370,354 pigs in the south italy (3.97%). mean value and range. in addition, we extracted from our simulation results the worst-case scenario, in terms of number of farms and number of pigs, that the whole restriction area (i.e. protection zone and surveillance zone) faced in a single simulation, for each macro-area. a second set of simulations was performed in the scenario where the asf virus is detected in a wild boar population. similar to the previous simulation procedure, a random point was identified within the wild boar territory (figure  3) to represent the index case. two buffer areas were drawn around this point to represent the two areas of different size where eradication measures will be implemented: a 200  km2 circular area (about 8  km radius) and a 1,000 km2 circular area (about 18 km radius) were created, mimicking respectively the guidelines for csf in wild boar (sanco/7032 2010) and the average size of the smallest wild boar-infected eu areas (czech republic, belgium, brandenburg as of september 2020). for both areas (200 km2 and 1,000 km2), we calculated the number of affected pig farms and the number of animals involved, while wild boar population data were not considered. again, simulations are performed for each macro-area independently and are repeated 1,000 times, and the results are reported as mean value and range of the number of farms and individuals located in the wild boar infected area. the distances between the index case and the nearest rendering plant were also calculated, and are reported as mean and standard deviation. results the national database reports that on the national territory excluding sardinia (n  =  14601) there are 128,463 pig farms; of these, 101,032 have the requirements to be included in our study, for a total population of 9,322,819 pigs. observing the types of farms (table i), we can see that the vast majority of farms are of the backyard type (82.65%), followed by the fatteners (10.99%), and the breeders (6.37%). the farms are unevenly distributed among the macro-areas (figure 4, table ii), with 11,348 farms in the alps (11.23%-0.22 farms/km2), 14,070  farms in the po valley (13.93%-0.37 farms/km2), 6,313 farms in the north italy (6.25%-0.21 farms/km2), 41,487 farms in central italy (41.06%-0.48  farms/km2), and 27,812  farms in the south italy (27.53%-0.39 farms/ km2). the average density of farm types for each macro-area is visible in table  iii. moreover, the proportion of the type of farming varies among the macro-areas (figure 5, table  ii). pigs are unevenly distributed among the farms (table i), as the majority of pigs are found within fattening farms (66.83%), followed by breeding farms (30.51%), and backyard farms (2.66%). this, of course, is due to the different number of pigs usually found within each type of farm: table i. number of pig and pig farms in continental italy for each type of farm considered, in brackets as a percentage of the total. the last column reports the mean (and standard deviation) value of the number of pigs per farm for each type of farming. farm type farms pigs mean (sd) backyard 83,498 (82.7%) 248,017 (2.7%) 2.97 (8.72) fatteners 11,102 (11.0%) 6,230,628 (66.8%) 561.22 (1384.34) breeding 6,432 (6.4%) 2,844,174 (30.5%) 442.19 (1571.36) table ii. total number of farms and pigs for each macro‑area, both grouped and divided by farm type. in brackets, the value is expressed as a percentage of the total. macro-area farms pigs by type farms pigs alps 11,348 (11.23%) 347,947 (3.73%) backyard 9,635 26,582 fatteners 1,370 198,033 breeding 343 123,332 po valley 14,070 (13.93%) 6,310,791 (67.69%) backyard 9,445 32,770 fatteners 3,855 4,390,782 breeding 770 1,887,238 north italy 6,313 (6.25%) 1,413,486 (15.16%) backyard 4,174 12,996 fatteners 1,662 990,912 breeding 477 409,578 central italy 41,487 (41.06%) 880,237 (9.44%) backyard 35,943 114,529 fatteners 3,173 499,991 breeding 2,371 265,717 south italy 27,812 (27.53%) 370,354 (3.97%) backyard 24,300 61,138 fatteners 1,042 150,910 breeding 2,470 158,306 table iii. density of holdings for each macro‑area reported both as overall density and as density for each type of farm. densities are expressed as farms per square kilometre. macro-area farm density km2 overall backyard fatteners breeding alps 0.22 0.19 0.03 > 0.01 po valley 0.37 0.25 0.10 0.02 north italy 0.21 0.14 0.06 0.02 central italy 0.48 0.41 0.04 0.03 south italy 0.39 0.34 0.01 0.04 300 veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 asf virus in italy: simulation study baiocchi et al. surveillance zone ‑ 7 km radius in the alps, in the surveillance zone, we found an average of 155.88  ±  118.13 farms (range 0-483), with an average of 1,998.86  ±  4,929.25 pigs (range 0-49,212, figure 7, table iv). in the po valley, we found an average of 170.38  ±  109.13 farms (range  1-449), with an average of 55,089.75 ± 84,960.63 pigs (range 1-412,923). in the north italy, we calculated an average of 91.53 ± 74.40 farms (range 0-308), with an average of 21,003.60  ±  49,753.84 pigs (range  2-289,964). in central italy, we encountered an average of 305.04 ± 233.29 farms (range 3-1,030), with an average of 4,602.49  ±  5,738.38 pigs (range  5-62,018). in the south italy, we found an average of 465.06 ± 468.96 farms (range 1-2,416), with an average of 2,616.07 ± 2,455.85 pigs (range 2-17,654). protection zone ‑ 3 km radius in the alps, in the protection zone, we found an average of 29.96  ±  23.16 farms (range 0-143), involving an average of 373.72 ± 1629.57 pigs (range 0-21,853, figure 6, table iv). in the po valley, we found an average of 21.55±15.60 farms (range 0-75), involving an average of 5,915.87  ±  10,715.67 pigs (range 0-69,712). in the north italy, we found a mean of 14.75  ±  13.56 farms (range 0-65), which involved an average of 3,008.31  ±  8,425.68 pigs (range 0-67,388). in central italy, we found an average of 42.57±37.60 farms (range 0-305), which involved a mean of 592.90  ±  1,427.82 pigs (range 0-12,561). in the south italy, we found an average of 91.60  ±  111.13 farms (range 0-686), which involved an average of 407.32 ± 627.20 pigs (range 0-10,523). figure 4. distribution of farms and pigs among macro‑areas. figure 5. distribution of the type of farms among the macro‑areas. figure 6. boxplots of the number of farms (a) and pigs (b) included in the protection zone, for each macro‑area. the dashed lines show the mean values. y‑axes have been cropped to facilitate reading. figure 7. boxplots of the number of farms (a) and pigs (b) included in the surveillance zone, for each macro‑area. dashed lines show mean values. the y‑axis has been cropped to facilitate reading. 301veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 baiocchi et al. asf virus in italy: simulation study in central italy, we encountered an average of 98.07 ± 119.34 farms (range 0-920), with an average of 1,760.82  ±  3,489.90 pigs (range 0-43,520). in the south italy, we encountered an average of 66.80 ± 130.08 farms (range 0-1,273), with an average of 956.35 ± 1,693.44 pigs (range 0-17,993). wild boar infected area ‑ 1,000 km2 in the alps, in the 1,000 km2 area we encountered an average of 188.15 ± 226.75 farms (range 0-1,290), with an average of 4,527.17  ±  10,294.15 pigs (range  0-94,559, figure 9 and table v). in the north italy, we encountered an average of 104.97  ±  111.88 farms (range 0-594), with an average of 23,491.95  ±  71,065.63 pigs (range 0-581,867). in central italy, we encountered a worst‑case scenario the extraction of the worst-case scenario returned a value of 546 farms and 62,251 pigs for the alps, 503 farms and 470,216 pigs for the po valley, 357 farms and 325,695 pigs for the north italy, 1,150 farms and 62,128 pigs for central italy, and 2,636 farms and 17,775 pigs for the south italy (table iv). wild boar infected area ‑ 200 km2 in the alps, in the 200 km2 area we encountered an average of 41.20 ± 62.34 farms (range 0-455), with an average of 1,014.78 ± 3,707.31 pigs (range 0-48,720, figure 8 and table v). in the north italy, we found a mean of 24.02  ±  33.82 farms (range  0-233), with a mean of 5,114.98 ± 17,436.50 pigs (range 0-177,294). table iv. range and mean value (in brackets) of the number of farms and pigs included in the infected and surveillance areas for each macro‑area. the ‘worst‑case scenario’ shows the highest number of farms or pigs involved in an outbreak in the whole restriction area (10 km radius) in a single simulation. macro-area protection zone surveillance zone worst case scenario farms pigs farms pigs farms pigs alps 29.96 ± 23.16 (0‑143) 373.72 ± 1,629.57 (0‑21,853) 155.88 ± 118.13 (0‑483) 1,998.86 ± 4,929.25 (0‑49,212) 546 62,251 po valley 21.55 ± 15.60 (0‑75) 5,915.87 ± 10,715.67 (0‑69,712) 170.38 ± 109.13 (1‑449) 55,089.75 ± 84,960.63 (1‑412,923) 503 470,216 north italy 14.75 ± 13.56 (0‑65) 3,008.31 ± 8,425.68 (0‑67,388) 91.53 ± 74.40 (1‑308) 21,003.60 ± 49,753.84 (2‑289,964) 357 325,695 central italy 42.57 ± 37.60 (0‑305) 592.90 ± 1,427.82 (0‑12,561) 305.04 ± 233.29 (3‑1,030) 4,602.49 ± 5,738.38 (5‑62,018) 1,150 62,128 south italy 91.60 ± 111.13 (0‑686) 407.32 ± 627.20 (0‑10,523) 465.06 ± 468.96 (1‑2,416) 2,616.07 ± 2,455.85 (2‑17,654) 2,636 17,775 figure 8. boxplots of the number of farms (a) and pigs (b) included in the infected area of 200 km2, for each macro‑area. the dashed lines show the mean values. the y‑axes have been cropped to facilitate reading. figure 8. boxplots of the number of farms (a) and pigs (b) included in the infected area of 200 km2, for each macro‑area. the dashed lines show the mean values. the y‑axes have been cropped to facilitate reading. 302 veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 asf virus in italy: simulation study baiocchi et al. represent about 70% of all farms, while the highest number of pigs is found in the po valley (about 70% of all pigs considered), followed by the north italy (figure 4). this divergence between farms and the number of pigs is due to the proportions of farm types present in each macro-area. the po valley and the north italy have a higher number of pigs because these macro-areas have a higher proportion of fatteners farms (around 25%), about twice as high as the other macro-areas (figure 5). in our simulations, for the scenario with an outbreak of asf, the south italy had the highest average value of farms included in the restriction area, while the po valley had the highest number of pigs involved (table iv). in the second set of simulations, for the scenario with a case of asf in wild boars, central italy had the highest mean value of farms included and the northern italy had the highest number of pigs involved for both the 200 km2 and 1,000 km2 areas (table v). because the proportion of farm types affected by an outbreak likely reflects the distribution of farms present in a macro-area, we expect that a macro-area with a high number of backyard farms faces a different logistical challenge than a macro-area where mostly large farms are present. from our results, we identified three potential scenarios that will follow the detection of asf in italy. south italy ‑ high number of farms our simulations indicate that the south italy will face the highest number of farms involved. the veterinary services will have to manage many backyard farms, often family-owned, for which biosecurity requirements are not as stringent as for commercial farms, and they are often opened and closed on an annual basis, with frequent changes in location and number of pigs. in addition, family farms are geographically dispersed and their census requires a huge logistical effort (regulation 2016/42914). furthermore, a high density of mean of 488.35  ±  473.27 farms (range 0-2,801), with a mean of 9,191.49  ±  13,044.88 pigs (range 0-106,077). in the south italy, we encountered a mean of 305.76 ± 473.11 farms (range 0-3,720), with a mean of 4,677.08 ± 4,687.55 pigs (range 0-30,939). distance from rendering plants the average distance of the outbreak farms from the nearest rendering plant is 78.68  ±  42.76 km (mean ± standard deviation, range 7.12-157.04 km) for the alps, 34.28 ± 25.55 km (range 0.60-163.06 km) for the po valley, 40.34  ±  26.68  km (range 2.06-115.08  km) for the north italy, 147.90  ±  67.59  km (range 23.79-270.43  km) for central italy, and 87.15  ±  72.37  km (range 0.71-262.17 km) for the south italy (figure  10). for the second set of simulations case of asf in a wild boar population the average distance of the case location from the nearest processing plant is 71.59 ± 30.31 km (range 6.00-154,70 km) for the alps, 65.65 ± 43.92 km (range 0.70-223.30  km) for north italy, 158.98  ±  58.19  km (range 29.70-267.90  km) for central italy, and 117.61  ±  66.54  km (range 3.70-261.40 km) for the south italy (figure 10). discussion the number of farms and pigs varies among macro-areas, with the highest number of farms in central italy and in the south italy, which together table v. average value and range (in brackets) of the number of farms and pigs included in the 200 km2 and 1,000 km2 areas for each macro‑area. macro-area 200 km2 area 1,000 km2 area farms pigs farms pigs alps 41.20 ± 62.34 (0‑455) 1,014.78 ± 3,707.31 (0‑48,720) 188.15 ± 226.75 (0‑1,290) 4,527.17 ± 10,294.15 (0‑94,559) north italy 24.02 ± 33.82 (0‑233) 5,114.98 ± 17,436.50 (0‑177,294) 104.97 ± 111.88 (0‑594) 23,491.95 ± 71,065.63 (0‑581,867) central italy 98.07 ± 119.34 (0‑920) 1,760.82 ± 3,489.90 (0‑43,520) 488.35 ± 473.27 (0‑2,801) 9,191.49 ± 13,044.88 (0‑106,077) south italy 66.80 ± 130.08 (0‑1,273) 956.35 ± 1,693.44 (0‑17,993) 305.76 ± 473.11 (0‑3,720) 4,677.08 ± 4,687.55 (0‑30,939) figure 10. boxplots of the distance from the outbreak farm and wild boar case to the nearest rendering plant for each macro area. 14 european parliament and council 2016. regulation of 9 march 2016 on transmissible animal diseases and amending and repealing certain acts in the area of animal health (‘animal health law’). (eu 429/2016). off j, l 84, 31/03/2016, art. 65-68.. 303veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 baiocchi et al. asf virus in italy: simulation study reduce this risk, high levels of biosecurity measures are required to prevent infection from spreading among farms. in fact, large farms apply protocols to prevent disease transmissions, such as disinfection of truck tires, gears, boots and farming tools and are required to record all animal movements, which can help trace the origin of the disease and potentially contaminated products exported from the farm with regard to the average distance from rendering plants, the po valley, considered only for the first scenario, returned the lowest value. however, it should be noted that the distances to the rendering plants were calculated as straight lines, ignoring the orography of the territory which can have a strong impact on the actual distance or the time needed to reach the rendering plants. this is particularly true for the alps, while for the po valley and the north italy, being mostly flat areas, our straight-line distance calculation can be considered as a good approximation for the real distance value. central italy central italy represents the worst-case scenario, as many farms and pigs are involved in both domes-tic pigs and wild boar infection scenarios, and the lack of disposal capacity will challenge any eradi-cation process by increasing costs, time and labour. the geographical distribution and abundance of wild boars in this area are among the highest in europe (pittiglio et al. 2018), creating a potentially explosive situation. in fact, infection reaching the wild boar population would strongly complicate the eradication of the disease, which could even become endemic and spread through the wild boar population to the whole country. furthermore, central italy returned the highest values of the mean distance to rendering plants for both scenarios because rendering plants are simply not present in this macro-area (figure 2). conclusions our simulations outline two possible outcomes for an outbreak of asf in italy, geographically identified. if the outbreak were to occur in the northern part of the country the alps, the north italy and the po valley it would involve a large number of pigs and affect the production system with strong economic consequences. in fact, in this area, we find most of the pig production system (e.g. pig farms, slaughterhouses, charcuterie factories) which in the event of an epidemic would undergo great stress or even a crisis. on the contrary, the veterinary services should be able to manage the outbreak without facing a crisis, since rendering plants are able to dispose of carcasses without being overloaded. backyard farms increases the risk of secondary outbreaks, complicating and prolonging the eradication process. high density of pigs raised in low biosecurity farms is one of the high-risk factors for an outbreak, and further spread of the virus in both domestic and wild pig populations, as reported for the russian federation, eastern eu countries and sardinia (oļševskis et al. 2016, vergne et al. 2016, sanchez-cordon et al. 2018), especially if free-ranging pigs are involved (franzoni et al. 2020, tao et  al. 2020). the wild boar population would therefore contribute to the spread and maintenance of the infection, and represent a possible epi-bridge among backyard farms. iglesias and colleagues (iglesias et  al. 2018) analysing the spread of asf in the russian federation show that, after an initial phase, the disease shows a behaviour as if wild boars and pigs were a single population. regarding the average distance from rendering plants, the south italy returned two different results for the two scenarios, it shows a quite low value for the domestic pig scenario while in the wild boar scenario the value increases strongly (figure 10). this suggests that the spatial distribution of the rendering plants is strongly related to the spatial distribution of the farms, as expected, and that pig farms and the distribution range of wild boars show some type of spatial segregation, which might hinder interspecies spread infection. northern italy ‑ high number of pigs northern italy (which includes both the macro-areas of the north italy and the po valley) will face the highest number of pigs involved, alongside the lowest number of farms for both domestic pigs and wild boars simulation sets. in these macro-areas, there is the highest percentage of large farms (fatteners) representing the majority of the pig population involved. bellini and colleagues (bellini et  al. 2020) conducted a study on the risk of asf introduction in lombardy (the region belongs to both the alpine and the po valley macro-areas): they found that 109 municipalities with 297 pig farms were at very high risk. these farms were selected for targeted surveillance aimed at early detection of asf, in order to prevent its further spread. the main focus of the disease eradication effort will be on pigs culling and carcasses disposal, which should also take into account the possible overloading of rendering plants capacity. developing an application-ready plan for carcasses disposal that takes into account rendering capacity will mitigate the risk of safe disposal system failure. rendering facilities are widely available for these areas, however, the high average number of pigs per farm could cause the disposal system to become overloaded if several large farms are infected at the same time. to 304 veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 asf virus in italy: simulation study baiocchi et al. of backyard farms, and this could cause cascading events leading to a wide and uncontrolled spread of the disease. therefore, although the probability of an outbreak of asf is low, we suggest an updating of the contingency plan to deal with the emergency, taking into consideration the characteristics of the local farming system, together with the availability of rendering plants and their capacities. acknowledgements authors would like to thank the banca dati nazionale of the istituto zooprofilattico dell’abruzzo e del molise ‘g. caporale’ for sharing most of the data included in this study. giorgia baiocchi received a research grant from the project: miglioramento delle strategie e degli strumenti di controllo della peste suina africana in italia (psrc1/2018) funded by ministero della salute and leaded by istituto zooprofilattico dell’umbria e delle marche ‘togo rosati’. if the outbreak were to occur in the macro-areas of central or the south italy, it would involve a large number of farms, probably scattered throughout the territory. this would require a great effort on the part of the veterinary services, which would probably face a crisis trying to keep the restricted area monitored and under control. the pig production system would not be greatly affected by the epidemic, but the pig industry would still be economically affected by the national blockade on the export of the products. a recent study calculated the probability of an asf outbreak for all european countries still free of asf, and for italy, the estimated probability was between  0-0.1 (taylor et  al. 2020, probabilities calculated for the year 2019 based on 2018 data), with the most likely reason for the outbreak being legal trade in infected pigs (or pork meat). although this is a low probability, more than half of the estimated potential asf outbreak locations in that study were in the central or in the south italy, the two macro-areas with the highest density 305veterinaria italiana 2022, 58 (3), 295-305. doi: 10.12834/vetit.2651.16696.2 baiocchi et al. asf virus in italy: simulation study bellini s., scaburri a., tironi m. & calò s. 2020. analysis of risk factors for african swine fever in lombardy to identify pig holdings and areas most at risk of 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the risk of infection by african swine fever virus in european swine through boar movement and legal trade of pigs and pig meat. frontiers vet sci, 6, 486. tao d., sun d., liu y., wei s., yang z., an t., shan f., chen z. & liu j. 2020. one year of african swine fever outbreak in china. acta tropica, 211, 105602. vergne t., korennoy f., combelles l., gogin a. & pfeiffer d.u. 2016. modelling african swine fever presence and reported abundance in the russian federation using national surveillance data from 2007 to 2014. spatial and spatio‑temporal epidemiology, 19, 70-77. vergne t., guinat c. & pfeiffer d.u. 2020. undetected circulation of african swine fever in wild boar, asia. emerg infec dis, 26 (10), 2480. 391 1instituto federal de educação ciência e tecnologia baiano. 2universidade federal do recôncavo da bahia. 3universidade federal de viçosa. 4universidade federal da bahia. *corresponding author at: instituto federal de educação ciência e tecnologia baiano. e‑mail: lilianportovet@gmail.com. please refer to the forthcoming article as: de oliveira et al. 2022. kinetics of antibodies and acute-phase post-vaccinal goats proteins immunized with vaccine against corynebacterium pseudotuberculosis. vet ital. 10.12834/vetit.2440.14841.1. lilian porto de oliveira1*, robson bahia cerqueira2, ângela cristina de oliveira lima2, ana karina da silva cavalcante2, kayck amaral barreto2, vinicius pereira vieira, leandro abreu da fonseca3, maria consuêlo caribé ayres4 treating the cla in sheep and goat herds is practically unfeasible because the bacterium is facultative intracellular surviving and multiplying inside the macrophages. after the cell death, there is the of a necrotic lesion with thick fibrous capsule which hinders the penetration of the majority of antibiotics. vaccination is still the best way to control the disease (belchior et al. 2006; dorella et al. 2006). introduction the caseous lymphadenitis (cla), is one of the most important diseases related to small ruminants’ health. caused by corynebacterium pseudotuberculosis (cp), it results in reduction of weight gain, interferes with the reproductive efficiency, causes early disposal of animals, decrease in production of wool and milk, as well as condemnation of carcasses, leather and whool depreciation (izgur et al. 210; ilhan,2016). keywords acute phase proteins, caseous lymphadenitis, corynebacterium pseudotuberculosis, goats, igg, vaccine. veterinaria italiana 2022, 58 (4), 391-398. doi: 10.12834/vetit.2440.14841.1 accepted: 14.06.2021 | available on line: 31.12.2022 summary the objective of this study was to evaluate the humoral immune response and the innate response of goats immunized with attenuated vaccine against corynebacterium pseudotuberculosis prepared from the strain 1002. one hundred thus, goats were divided into 5 groups (n=20 animals/group). each group was vaccinated as follows: g control: saline solution; g1 107 cfu/ml; g2 -107 cfu/ml re-vaccinated within 21 days; g3 – 106 cfu/ml; g4 – 106 cfu/ml revaccinated within 21 days. blood samples were collected monthly over 12 months and serology was performed through indirect elisa. in order to verify the innate response through of the dosages of acute phase proteins (ceruloplasmin and haptoglobin), samples of five animals from each group were evaluated on days0, 7, 14, 21, 28 days for the groups g1 and g3, and on days 0, 21, 28, 56 days for the groups g2 and g4. the results showed humoral response activation with the production of immunoglobulins above the cut-off point in all groups. the results showed that strain 1002 vaccine induced the antibodies production by the goats’ humoral immune system and that the increase in serum concentrations of haptoglobin and ceruloplasmin may be related of the innate immune response. humoral and innate response in goats proteins immunized with vaccine against corynebacterium pseudotuberculosis corynebacterium pseudotuberculosis vaccine in goats de oliveira et al. 392 veterinaria italiana 2022, 58 (4), 391-398. doi: 10.12834/vetit.2440.14841.1 the defense mechanisms of the immune system to fight the invasion of cp have been of constant interest for researchers aiming at identifying strategies to control the disease through vaccines and diagnostic methods. initially, it was believed that the cellmediated immunity was the sole immune defense mechanism against the agent, but it is known that the immune reaction to this pathogen involves both participation of cell-mediated immunity and humoral response with the production of specific antibodies, acting together to try to eliminate the bacterium (ruiz et al. 2007). the acute phase proteins are constituents of the non-specific innate response, produced mainly by hepatocytes and are involved in inflammation, homeostasis regulation, bacterial growth restriction, before a more effective response of the immune system. studies on the variation of this innate response through the dosage of acute-phase proteins in the vaccine stimulation are still infrequent in the literature and in the caprine species (rudoler et al. 2015). in this context, the objective of this study was to evaluate the response of humoral immune system and the response of acute-phase proteins in goats immunized against cp, using an attenuated experimental vaccine prepared from strain 1002. materials and methods one hundred healthy goats were enrolled in the study, aged from two to three years, serologically negative and without defined breed. the 100 animals were divided into 5 groups of 20 animals each, which received an experimental vaccine prepared from the 1002 live attenuated strain of cp. the vaccine was injected subcutaneously, at a dose of 0,1 ml for each animal. the groups were prepared as follows: group 1 was vaccinated on the day zero with 107 cfu/ml; group 2 vaccinated on the day zero with 107 cfu/ml and revaccinated within 21 days; group 3 vaccinated on the day zero with 106 cfu; group 4 vaccinated on the day zero with 106 cfu/ml and revaccinated within 21 days and control group g which was injected saline solution. blood samples were carried out over 12 months. the procedures performed with the animals were submitted to and approved by the ethics committee for animal use under the number 23007.016893/2013-73 federal university of recôncavo of bahia. for the humoral immune response evaluation in goats’ sera, elisa immunoassay was used. polystyrene elisa microplates were coated with 100µl of somatic antigens of cp, diluted 1:100 in carbonate-bicarbonate buffer 0.05m, ph 9.6 and incubated at 4 ºc for 18 hours. then, plates were washed twice with pbs containing 0.1% tween-20 (pbs-t20). the plates were blocked with 200µl/well of pbs-t20 containing 5% of skimmed milk, during 2 hours at 37ºc. after this period, the plates were washed twice with pbs t-20. fifty µl/well of test sera diluted 1:100 in pbs-t-20 were added and plates were incubated for 1 hour at 37 ºc. after plates were incubation for 1 hour, 5 washing cycles were performed with pbs-t-20 and 50µl of goat anti-immunoglobulin of rabbit were added to the plates, conjugated to peroxidase (dako, santa clara, ca, usa), diluted to 1:10,000 in pbs-t-20. the plates were incubated at 37 ºc for 45 minutes, and then washed five times in pbs-t-20 and incubated with 50µl well of revealing solution (10 ml of phosphate buffer ph 5.1 citric orthophenylenediamine + 4mg + 4µl h202). the reaction was stopped with 25 µl of sulfuric acid (h 2 so 4 ) 4n. the optical density (od) reading was performed in an elisa reader, using a 490nm wavelength filter. the cutoff point considered in the experiment was 0.310 based on indirect somatic elisa test for goats standardized by zerbinati et al. (2007). five serum samples from each group were used for the determination of the electrophoretic profile of proteins (ceruloplasmin, haptoglobin), by the method of polyacrylamide gel containing sodium dodecyl sulfate (sds-page). the g1 and g3 vaccinated groups were evaluated on days 0, 7, 14, 21 and 28 while the g2 and g4 revaccinated groups were evaluated on days 0, 21, 28 and 56. for the fractioning of different protease serum constituents, electrophoretic running was performed on the respective serum samples in polyacrylamide gel containing sodium dodecyl sulfate (sds-page), according to the technique described by modified laemmli (1970). ten μl of blood serum, diluted in 30 μl of phosphate buffer saline (pbs) and 20 μl gel mix were heated for 10 minutes. an aliquot of 5 μl of each sample of blood serum was deposited in duplicate together with the reference standard samples (a marker of molecular weights) in the gel cavities. after the proteins fractioning, the gel was stained for 10 minutes in a solution of 0.25% coomassie blue. a methanol-based decolorizing solution was used to remove the excess of dye. the molecular weight and the concentrations of the protein fractions were determined by computerized densitometry, from the samples scanning. for the molecular weight calculation, markers of molecular weights of 200, 116, 97, 66, 55, 45, 36, 29, 24 and 20 kda were used, in addition to purified proteins. for the densitometric evaluation of protein bands reference curves were manufactured, from the reading de oliveira et al. corynebacterium pseudotuberculosis vaccine in goats veterinaria italiana 2022, 58 (4), 391-398. doi: 10.12834/vetit.2440.14841.1 393 of the standard marker. in the immunoglobulins evaluation, a descriptive analysis was performed of the data and subjected to the analysis of linear mixed model with repeated measures in time considering the dosage as fixed effect and random effect. for the protein evaluation, data were analysed by means of a factor model considering the following variables: dosage, time and their interactions. for multiple comparisons of means, the tukey test was applied at 5% level of significance. results no significantly different (p=0.06) concentrations of immunoglobulins in all the immunized groups were observed when used the indirect elisa during the 12 months of observation, however, vaccinated and re-vaccinated immunoglobulin concentrations are different (p<0.05) different from the control group. in group 1 (107 cfu/ml), an early production of antibodies above the cut-off point was observed at 28 days, and a peak of production in the values of od at 140 days (0.508). this group had a mean (m) and standard deviation (sd) of 0.399 ± 0.085 over the period. a decline of production was observed at 196 days, with varying levels of od, until the end of the period of observation, as shown in figure 1. in group 2 (107 cfu/ml revaccinated at 21 days), an early production was also observed at 28 days, above the cut-off point set at 0.310 od, with peaks of production of 0.610 in the values of od at 140 days. the mean and standard deviation, observed in this group was 0.384 ± 0.101. it was noted a decrease in values of od below the cutoff point at 280 days, with an elevation of these levels to 0.308, remaining until the end of the observational period, as shown in figure1. in group 3 (106 cfu/ml), it was noted at 28 days a beginning of production, as well as in groups 1 and figure 1. means of dosage of total igg using somatic corynebacterium pseudotuberculosis antigen in indirect elisa: control group (saline solution), g1 (dose 107 cfu/ml) and g2 (dose 107 cfu/ml revaccinated at 21 days). figure 2. means of dosage of igg total using somatic corynebacterium pseudotuberculosis antigen in indirect elisa: control group (saline solution), g3 (dose 106 cfu/ml) and g4 (dose 106 cfu/ml revaccinated at 21 days). 2. at 168 days, a peak of production of 0.516 in the od values was observed. at 140 days, a decrease in serum levels was observed close to the cutoff point in contrast to what was observed in group 1 and 2 in the same period. values close to the cut-off point were also observed at 196 days with following periods of increasing and decreasing values until the end of the experimental period, as shown in figure 2. the mean and standard deviation, observed in this group was 0.375 ± 0.105 over the observaton period. in group 4 (106 cfu/ml revaccinated at 21 days) a beginning of immunoglobulins production was also observed at 28 days, with a peak of production of 0.528 in the od values at 84 days. values close to the cut-off point were recorded in different periods of observation: on days 112, 196 and 336, with periods of following increase as shown in figure 2. the mean and standard deviation obtained in this group were 0.361 ± 0.108. regarding the acute phase proteins, analyzing the serum concentrations of ceruloplasmin, it was possible to observe that at 21 days, the group 1 presented statistically significant higher means than the group 3, but equal to the control group (table 1). during the measurement period for this group, no differences were observed among the experimentation days. whereas at 14 days, group 3 showed concentrations 1.5 times higher than group 1, although these means were not statistically different. considering the time factor, no variations were observed in the means of group 3. table i. means of serum concentrations of ceruloplasmin g/l groups of corynebacterium pseudotuberculosis vaccinated animals, g1(107 cfu/ ml) and g3(106 cfu/ml). days of experiment (time) groups 0 7 14 21 28 cv% control 0.190bb 0.782aba 0.868aba 1.240aa 0.624aba 63.87 g1 0.847aa 0.655aa 0.652aa 0.881ba 0.579aa 38.59 g3 1.540aa 0.778aba 1.020aba 0.713ba 0.561ba 56.28 means followed by upper case letters equal in the same column and lowercase letters on the same line do not differ among themselves by tukey test at 5% probability cv = coefficient of variation. corynebacterium pseudotuberculosis vaccine in goats de oliveira et al. 394 veterinaria italiana 2022, 58 (4), 391-398. doi: 10.12834/vetit.2440.14841.1 regarding the revaccinated animals, group 2 showed, on day 21, serum concentrations of ceruloplasmin 1.5 times, when compared with the period before vaccination, with reduction of these concentrations at the end of the measurement periods (table 2). group 4 showed concentrations very similar to group 2 on day 21, but no change in relation to the moment before vaccination and also with a decrease in the concentrations in the final periods. table ii. means of serum concentrations of ceruloplasmin g/l groups of corynebacterium pseudotuberculosis revaccinated animals at 21 days, g2(107 cfu/ml) and g4(106 cfu/ml). table iv. means of serum concentrations of haptoglobin g/l groups of corynebacterium pseudotuberculosis revaccinated animals at 21 days, g2(107 cfu/ml) and g4(106 cfu/ml). table iii. means of serum concentrations of haptoglobin g/l groups of corynebacterium pseudotuberculosis vaccinated animals, g1(107 cfu/ ml) and g3(106 cfu/ml). days of experiment (time) groups 0 21 28 56 cv% control 0.190bb 1.240aa 0.624aba 0.880aba 74.31 g2 0.664aa 1.014aa 0.842aa 0.695aa 46.9 g4 1.172aa 1.198aa 0.515aa 0.694aa 51.46 days of experiment (time) groups 0 21 28 56 cv% control 0.232aa 0.344ab 0.829aa 0.284aa 86.45 g2 0.332aa 0.835ba 0.963aa 0.376aa 67.94 g4 0.939ba 1.113ba 0.608aba 0.354ba 50.11 means followed by upper case letters equal in the same column and lowercase letters on the same line do not differ among themselves by tukey test at 5% probability. cv = coefficient of variation. means followed by upper case letters equal in the same column and lowercase letters on the same line do not differ among themselves by tukey test at 5% probability. cv = coefficient of variation. means followed by upper case letters equal in the same column and lowercase letters on the same line do not differ among themselves by tukey test at 5% probability. cv = coefficient of variation. observing the serum concentrations of haptoglobin, groups 1 and 3 exhibited at 7 days, concentrations higher and different from those of the control group, although not differing among themselves (table 3). group 1 presented means 3.6 times higher at time 7(seven) in relation to 0 (zero). the statistical analysis (p<0.05) revealed no differences among the groups and throughout the period evaluated. days of experiment (time) groups 0 7 14 21 28 cv% control 0.232ba 0.364ba 0.520aba 0.344aba 0.829aa 74.07 g1 0.216ba 0.783aa 0.426aba 0.614aba 0.692aa 47.91 g3 0.661aa 0.626aa 0.624aa 0.314aa 0.500aa 47.71 in the groups of revaccinated animals, it was observed that at 21 days their means differed from the control group. group g2 had 2.4 times higher concentrations of haptoglobin levels than the control group in this period, while g4 presented an increase of 3.2 times in their concentrations, in relation to the control group, as shown in table 4. the statistical analysis (p<0.05) revealed no differences throughout the evaluated period. discussion all the animals were allocated in the same physical space, endemic for cp, and physical contact occurred among the individuals of the different groups throughout the experimental period. they were subjected to the same water and sanitary regime, the same risk of contamination from various etiologic agents, being managed in their natural climatological and edaphological habitat. in other words, they were in the same conditions to the majority of animals kept in extensive husbandry systems in semi-arid regions (almeida et al. 2010). the immunoglobulins production curve showed a dynamic way in all groups, with peaks of production and decrease of immunoglobulins throughout the experimental period, not following the expected standard described in the literature (castelan et al. 2008) that would be a curve of antibody production with low initial production, followed by a growth and stability, with sharp drop after a period of time. these variations in the immunoglobulins curve may be explained by the influence of environmental factors, the nature of the antigen, its concentration, route of administration, genetics of the immunized individual and the vaccination protocol used. all these factors might have influenced the qualitative and quantitative analysis of the immune response during the observation period reflecting the dynamics of the antibody curve (igietseme et al. 2004). the live attenuated vaccine antigen used in this experiment, may have influenced the peaks and declines in the antibody production observed in similar periods in vaccinated groups, since the live attenuated bacterium can multiply itself constantly in the animal body stimulating in various forms the antibody production (santos et al. 2016). in all the tested doses, an antibody production was observed, which can lead to infer that the administration of a single dose of vaccine with lower concentration of antigen can stimulate the animals’ immune system in the production of specific igg against cp. this is an important de oliveira et al. corynebacterium pseudotuberculosis vaccine in goats veterinaria italiana 2022, 58 (4), 391-398. doi: 10.12834/vetit.2440.14841.1 395 may be verified through the concentrations of acute phase proteins, however, this concentration of proteins was not so intense (dorella et al. 2006; simplicio et al. 2015). in this study, for the vaccinated animals group, the haptoglobin concentrations were higher at 7 days of measurement which may be associated with the innate response to vaccination, similar results to those of eckersall et al. (2007) who observed mean values of haptoglobin levels in sheep experimentally infected with cp, significantly greater at 7 days of infection. in the revaccinated group a higher concentration of haptoglobin was observed at 21 days, being that group 4 presented serum levels 3.2 times higher than the animals in the control group. in this group, the vaccinal stimulus provided a better production of haptoglobin, in a longer period of time. simplicio et al. (2017) reported increases of 621% of concentrations of haptoglobin in goats with naturally acquired staphylococcal mastitis when compared with the control group, indicating that haptoglobin is a good inflammatory process indicator. in goats experimentally infected with cp serum, concentrations of haptoglobin were verified significantly higher when compared to the control group (jeber et al. 2016). sheep infected with pas‑ teurella multocida showed high levels of acute phase proteins especially haptoglobin with mean value of 1.65g/dl, whereas the non-infected presented 0.0048g/dl (el-deeb & elmaslemany, 2016), reinforcing the importance of protein as a response to inflammatory processes. in other animal species, and using vaccination stimulus to verify production of acute phase proteins, riber et al. (2015) worked with pigs vaccinated against lawsonia intracellularis with attenuated strain, and found no significant differences in the haptoglobin concentration between the vaccinated and the control group, and the vaccine did not induce detectable levels of igg and ifn-γ. the natural or experimental infection can stimulate the proteins production much more intense, as in the case of haptoglobin and ceruloplasmin, when compared with the use of vaccine antigen in its attenuated form as it was used in this study (dorella et al. 2006). future investigations become necessary to investigate to what extent the innate system is stimulated with attenuated vaccine antigen and even inactivated antigens that constitute the majority of vaccine formulations. from day 28 of the experiment on, a decrease was observed in serum concentrations of ceruloplasmin, haptoglobin, mainly in vaccinated groups, a trend advantage for the industry as manifactoring might require in a smaller quantity of inputs (barbosa et al. 2017). for producers, it facilitates the management by spending less on manpower and purchase of vaccine for revaccination, given the application of a single dose of the immunogen. in groups revaccinated at 21 days (g2 and g4), a faster production of antibodies, that would be the expected pattern, was not observed. this may have occurred because when an antigen stimulates cells b, the process of somatic hypermutations of these antigen-specific cells in germinal centers begins with the purpose of potentiating a secondary response faster and more specific. however, hypermutations may also lead to reduction or inability of affinity with the antigen retarding the secondary response (castelan et al. 2008, lui et al. 2017). production of immunoglobulins was observed up to twelve months of follow up, however, the g2 group showed an increase of immunoglobulins after 364 days, unlike the other groups that had a drop in serum dosages. this can be explained by the antigens accumulation in follicular dendritic cells in the lymph nodes, that would stimulate periodically in a more extended time the memory cells for antibodies production (binns et al. 2007). lima et al. (2017), using an attenuated strain t1 in the dosage of 2x105 and 2x107 at 60 days with revaccination, observed antibody production in sheep, but did not record a statistical significance among the vaccinated and revaccinated groups of animals, results similar to those found in this study, which also did not observed significant differences between the vaccinated and revaccinated groups of animals at 21 days. lima et al. (2017) also indicated that a vaccine dosage, would stimulate the immune system in the antibodies production for at least 12 months, corroborating the results presented in this study. animals without a defined breed, anglo-nubian and cross-bred alpine breeds also had the same pattern recognition over time, inferring that breed does not influence the antigen recognition, since it is assumed that animals without defined breed are more resistant to diseases (vale et al. 2003). breed, therefore, would not be a factor that has influenced the antibodies production curve in this experiment. the acute phase proteins can be indicators of the innate response activation which is the first sign of cells activation related to the immune system inflammatory process. the activation of these cells prepares the immune system for a subsequent specific and more efficient response. as the vaccine under study is attenuated, it was expected that it would stimulate natural infection, stimulating the innate response, which corynebacterium pseudotuberculosis vaccine in goats de oliveira et al. 396 veterinaria italiana 2022, 58 (4), 391-398. doi: 10.12834/vetit.2440.14841.1 that can be explained by the increase of specific adaptive response with elevation in the antibodies production (silva, 2001), being noted in this period, a beginning of production of igg above the cutoff point, i.e., a replacement of the innate response by the adaptive response (castelan et al. 2010). this may also indicate that revaccination does not induce an amplification of the innate response but a stimulation of the humoral response and immunologic memory (lui et al. 2017). in this study, the haptoglobin and ceruloplasmin were the proteins that may be related to the innate response; however, more studies on the dynamics of variations of these proteins should be developed to elucidate the innate response stimulation to vaccination stimulus (rudoler et al. 2015). conclusions the results showed that the lyophilized vaccine induced humoral immune response in goats vaccinated over twelve months, verified by the dosage of total igg, which implies that a revaccination would be indicated after this period and that in a single dose of vaccine a smaller quantity of antigen (106 cfu/ml) can be used for animal vaccination, once there were no statistically significant differences between the groups. haptoglobin and ceruloplasmin were the proteins that may be related with the innate response to stimulation with lyophilized attenuated vaccine against cp. acknowledgments the authors are grateful to infectious disease laboratory of the federal university of recôncavo da bahia (ufrb), department of postgraduate studies in animal science the tropics of the federal university of bahia for scientific support and labovet veterinary products for providing the strains for the experimental vaccines. grand support the authors are grateful to foundation to support research in the state of bahia (fapesb) for financial 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padronização de um antígeno para teste de elisa indireto no diagnóstico da linfadenite caseosa. revista academia ciências animal, 5, 285-293. vale v., freire s., ribeiro m., regis l., bahia r., carminati r., paule b. j. a., nascimento a. & meyer r. 2003. antigen recognition by antibodies against corynebacterium pseudotuberculosis from naturally infected or immunized goats. revista ciências médicina biologia, 2, 192-200. 317 faculty of veterinary medicine, university of teramo, località piano d’accio snc, 64100 teramo, italy * corresponding author at: faculty of veterinary medicine, university of teramo, località piano d’accio snc, 64100 teramo, italy. tel.: +39 0861 266955, fax: +39 0861 266962, e‑mail: sarahsconza@libero.it. parole chiave cavallo, cyathostomini, esame coprologico, monitoraggio epidemiologico. riassunto i cyathostomini sono i principali parassiti dei cavalli per la loro diffusione cosmopolita, per la loro capacità di resistere ai trattamenti antiparassitari con molti antielmintici e per il loro impatto sanitario sia allo stadio adulto che a quello larvale. per implementare i programmi di controllo delle infestazioni da cyathostomini, sono stati proposti i cosiddetti “trattamenti selettivi”, che intervengono esclusivamente sugli animali che eccedono una determinata conta di uova fecali (fec). il presente lavoro ha valutato l'emissione di uova nelle feci di 475  cavalli di 12 scuderie site in 3 regioni italiane. tutte le scuderie analizzate e 224 cavalli (47,6%), sono risultati positivi per la presenza di cyathostomini. centotrentotto cavalli (28,8%) hanno mostrato valori di fec compresi tra 50 e 2.150 uova per grammo di feci (upg) mentre gli altri 86 animali (18,1%) sono risultati positivi solo alla valutazione qualitativa (i.e. < 50 upg). tra i soggetti con fec > 50 upg, 81 (17%) mostravano valori compresi tra 50 e 200 upg mentre 57  (12%) superavano 200 upg. rispetto ai risultati ottenuti in passato nella stessa area di studio, questo lavoro dimostra una riduzione della presenza di cavalli contaminati, a fronte di dati metereologici sovrapponibili nei cinque anni trascorsi dall’ultimo studio. per pianificare programmi di intervento finalizzati contemporaneamente a conservare la salute dei cavalli e a preservare l’efficacia delle molecole antielmintiche, risulta cruciale il monitoraggio continuo dell’epidemiologia e della biologia delle infestazioni da cyathostomini nei cavalli. uova di cyathostomini nelle feci di cavalli del centro italia keywords cyathostomins, faecal examination, horse, epidemiological monitoring. summary cyathostomins, or ‘small strongyles’, are the most important equine helminths because of their worldwide distribution, spread of anthelmintic‑resistant populations, and pathogenic impact. the so‑called ‘selective treatment’ of those animals exceeding a certain faecal egg count (fec) has recently been proposed to implement cyathostomin control programmes. the present study evaluated the extent of egg shedding in 475 horses living in 12 farms from 3 regions of italy. all examined farms and 224 horses (47.6%) were positive for cyathostomins. 138 horses (28.8%) scored positive for cyathostomin fecs with a range of eggs‑per‑gram of faeces (epg) values of 50‑2,150. further 86 horses (18.1%) were positive only under qualitative microscopy (i.e. < 50 epg). of the animals with a fec > 50 epg, 81 (17%) and 57 (12%) showed values of 50‑200 and >  200 epg, respectively. the findings from this study demonstrated a reduced presence of high‑shedding horses compared to results obtained in previous years in the same study areas, despite overlapping climate features in the previous 5 years. a continuing monitoring of epidemiological and biological features of horse cyathostomin infection is crucial for planning intervention programmes aimed to maintaining animal health and preserving the efficacy of parasiticides. sarah sconza*, angela di cesare, raffaella iorio, roberto bartolini, barbara paoletti and donato traversa cyathostomin faecal egg counts in horse farms from central italy veterinaria italiana 2018, 54 (4), 317‑322. doi: 10.12834/vetit.787.3812.1 accepted: 21.09.2016 | available on line: 31.12.2018 318 veterinaria italiana 2018, 54 (4), 317‑322. doi: 10.12834/vetit.787.3812.1 parasiticides in this area during previous years, the present study aims to evaluate cyathostomin occurrence and the fecs trends in farms located in 3 different region of italy and with a long‑lasting history of cyathostomin infection since 2008. materials and methods farms and animals from april to july 2011 and from april to september 2012, individual faecal samples were collected from 475 horses living in 12 farms located in the abruzzo, lazio, and tuscany regions of central italy (figure 1). the study sites were selected based on the following criteria: i. at least 8 permanently resident horses per farm (we selected a range of 8‑131 equines per farm); ii. farms with a previous history of cyathostomin infection or located in areas where these parasites are endemic (traversa et  al. 2009, traversa et  al. 2010, sconza et  al. 2013). our sample included 10 of the 12 farms that had been sampled during previous studies and, according to the owners, fewer than 25% of the horses had changed; iii. anthelmintic treatments had not been administered for at least 4 months prior to testing; introduction small strongyles or cyathostomins (nematoda, strongylidae) are regarded as the most significant horse parasitic helminths (traversa et  al. 2009, kaplan and nielsen 2010). virtually 100% of horses may harbour small strongyles, that may infect all age classes of animals (pickles et  al. 2010, fritzen et  al. 2010). although high worm burdens do not necessarily lead to clinical disease or to faecal egg excretion, the infection can result in malaise, anorexia, colic, and weight loss (love et  al. 1999, corning 2009). the simultaneous emergence of encysted larvae from the gut wall may induce a syndrome known as ‘larval cyathostominosis’, which is characterised by weight loss, diarrhoea, colics, ventral oedema, and a high mortality rate (lyons et al. 2000, von samson‑himmelstjerna 2012). the control of small strongyles relies on the use of benzimidazoles (bz), tetrahydropyrimidines (thp), and macrocyclic lactones (ml) (matthews 2008, kaplan and nielsen 2010). it is well‑known that cyathostomin are resistant to bz and thp. recent studies have additionally proposed an initial resistance to ml, especially ivermectin (lyons and tolliver 2013, geurden et al. 2014). new proposals for the control of horse cyathostomins have focused on preserving the efficacy of parasiticides, and include minimising unnecessary treatments in animals excreting a low number of eggs to increase the proportion of parasites that remain unexposed to anthelmintic (i.e. refugia) (van wyk 2001). these so‑called ‘selective treatments’ aim at keeping parasite burdens below levels that could cause disease, thus simultaneously preserving the efficacy of anthelmintics. the identification of those horses requiring anthelmintic treatment is based on the evaluation of faecal egg counts (fecs). only animals exceeding a certain threshold should be treated. there is a general consensus for a cut‑off is ≥ 200 eggs per gram (epg) of faeces (matthee and mcgeoch 2004, nielsen et al. 2014). a large‑scale study carried out in italy in 2008 (traversa et al. 2009) demonstrated that the efficacy of bz was reduced in about one‑third of the examined premises, and that the efficacy of thp was reduced in about 20‑30% of the examined yards. the results of the study by traversa and colleagues resulted in the selective treatment strategy being promoted among veterinarians. the same study also demonstrated that the percentage of horses with fecs  ≥  200  epg was relatively high (traversa et  al. 2010). however, there are no further studies demonstrating the application of this strategy and thus no updated data on the extent of egg excretion of strongyle eggs in horse farms from italy. following the proliferation of scientific information about the resistance to cyathostomins in horses of central italy sconza et al. figure 1. farm locations. abruzzo, lazio and tuscany: geographic distribution of the farms included in the study. 319veterinaria italiana 2018, 54 (4), 317‑322. doi: 10.12834/vetit.787.3812.1 breeds, such as italian warmblood (250  horses), belgian warmblood (20 horses), haflinger (9 horses), maremmano (4 horses), andalusian horses (6 horses), and mixed‑breed horses (67). the fecs of these 2 categories were compared using a mann‑whitney test. an independent sample t‑test was performed to evaluate differences in the fecs of horses which mostly lived in boxes and those which grazed. the same analysis was applied to test the effect of horse ages on the fecs using 2  age classes, i.e. younger than 2 years and older than 2 years. all statistical tests were performed with medcalc® (version 12.5), with a significance of p < 0.05. results all farms (100%) were positive for the presence of cyathostomin eggs. overall, a 47.6% infection rate was found in the animal populations, with a range of 7.7%‑100% in the single study sites (table  i). 138  horses (28.8%) showed cyathostomin fecs ranging from 50 to 2,050 epg. further 86  horses (18.1%) were positive for up to 50 epg (i.e. only at the qualitative examination). of the animals with iv. willingness of the owners and managers to participate in the study; v. a previous history of reduced efficacy of parasiticides and evaluation of fecs in horse operations in the same regions (traversa et  al. 2009, traversa et al. 2010); vi. owners and practitioners who were aware of the possibility of selective treatments. all horses present in each site were sampled, i.e. 279  mares, 65 stallions, and 131 geldings with an age range of between 6 months and 27 years (mean  =  7  years). horses were kept for sporting purposes (e.g. jumping, 3‑days events, and western shows), pleasure riding, or were retired from previous activities. the majority of horses lived in a box and were allowed into paddocks no more than 3‑4 hours a day (351 of 475 horses). the other horses (124) grazed more than 4 hour a day. faeces were removed regularly. copromicroscopic examinations individual faecal samples were collected from the rectum of each horse and analysed within 24 hours. analysis took place at the faculty of veterinary medicine of teramo, italy, with a standard flotation technique (nano 3 solution with a specific gravity of 1,350), and with a modified mcmaster method with a sensitivity of 50 epg (taylor et al. 2007). up to ~  100 grams of pooled faeces obtained from samples collected in each farm were mixed with oak sawdust and water and incubated for 10 days at 27 °c and ~ 75% relative humidity. one and 2 pooled cultures were prepared for farms with fewer and more than 15 horses, respectively. after incubation, third‑stage larvae (l3) were harvested using baermanisation. approximately 100 l3s for each sample were examined using a light microscope and identified (maff 1986). statistical analysis a descriptive statistical analysis was carried out using microsoft excel® (2014). percentage values were rounded to the nearest integer and climate variables, such as mean, highest, and lowest temperatures; and humidity, rainfall, and wind speed, were collected for the semester prior to the study. these data were differentiated among the 3  enrolled regions and the 3 different study years, and compared using a 1‑way variance analysis. the effect of gender on the fecs was analysed using a 1‑way anova. to compare the effect of the horse breed on the fecs, 2 categories were created: i) western show breeds, such as quarter horse (84 horses), paint (15 horses), and appaloosa (20 horses); and ii) warmblood sconza et al. cyathostomins in horses of central italy table i. distribution of the faecal egg count (fec) of 475 horses within 12 farms in central italy. farm h/y pos neg epg < 50 50 ≤ epg ≤ 200 epg > 200 fec m (± sd) a 1 42 28 14 4 15 9 225.60 (± 377.79) 3 2 14 10 4 3 3 4 167.86 (± 239.07) 0 3 21 8 13 1 4 3 183.33 (± 469.93) 2 4 35 17 18 9 5 3 47.14 (± 133.34) 0 5 8 5 3 2 3 0 43.75 (± 67.81) 0 6 36 20 16 7 10 3 58.33 (± 121.01) 1 7 13 11 2 1 3 7 580.77 (± 59.08) 2 8 13 1 12 1 0 0 3.85 (± 13.87) 0 9 43 4 39 1 3 0 5.81 (± 19.55) 2 10 12 12 0 0 3 9 308.33 (± 157.87) 0 11 131 72 59 33 22 17 305.13 (± 416.88) 3 12 107 36 71 24 10 2 137.50 (± 158.29) 3 h/y = number of horses per yard, pos= number of horses positive for cyathostomins; neg = number of horses negative for cyathostomins; epg = eggs per gram of faeces; m = arithmetic mean; sd = standard deviation; a = number of horses positive for ascarids (parascaris equorum). 320 veterinaria italiana 2018, 54 (4), 317‑322. doi: 10.12834/vetit.787.3812.1 cyathostomins in horses of central italy sconza et al. where a percentage of ‘high‑egg shedders’ was higher than that of ‘low‑egg shedders’ (traversa et al. 2010), could be due to a different age pattern of the horses enrolled in the respective studies. studies showed that the predictive probability for strongyle fec decreased with age (nielsen et al. 2006) and, accordingly, more than 90% of the horses in this study were ≥ 3 year old. in this study, a small number of horses with a fec  >  200  epg were responsible for 83.3% of the total egg output detected at the copromicroscopic examination. these results are consistent with those of a recent uk survey that showed that only a minority of horses were responsible for excreting the majority of strongyle eggs in 22  premises (relf et  al. 2013). in contrast with relf and colleagues (relf et  al. 2013), who found a higher distribution of ‘high shedders’ among horses younger than 2 years (62.4%) compared to the older animals (37.6%), in this study the majority of horses (87.7%) were older than 3 years. only few adult horses, i.e. 16 (6.4%), scored positive for p. equorum eggs and the majority of them (10 of 16) were positive only to the flotation. this finding was likely influenced by the age of the enrolled population, given that horses younger than 3 years were 17.7% of the overall study population, and suggests that most animals were able to develop adequate immunity against this parasitosis. this study can be considered a basis for further studies investigating the application of selective strategies on a larger scale in italy. this is noteworthy considering that horses have a strong tendency to remain at the same level of egg shedding over time, i.e. ‘low‑shedding horses’ are likely to remain this way regardless of any administered treatment, thus justifying selective treatments only in ‘high shedding’ populations (larsen et  al. 2011, nielsen 2012). the weather parameters did not significantly change during this study, however they should be taken into account in larger‑scale studies because they are reported to influence the development of cyathostomin free‑living stages (corning 2009). a fec  >  50 epg, 81 (17%) and 57 (12%) showed epg values of 50‑200 and >  200 epg, respectively (table i). the microscopic examination of the in vitro cultured l3s showed that they belonged exclusively to the cyathostominae subfamily. other than occasional findings of strongyloids and pinworms, 16 horses (3.4%) from 7 sites scored positive for parascaris equorum in both single and mixed infections with cyathostomins. six horses (2.5%) were positive for p. equorum with a range of epg values of 50‑450, while 10 (2.1%) were positive only at the flotation (i.e. epg < 50). infection prevalence in relation to age, sex, and living habits for cyathostomins are shown in figure 2. climate variables did not change significantly from 2008 to 2012 in all study regions (figure 3). the statistical analysis did not show any difference in the distribution of fecs for this study among gender, age, breed, and living habits categories. discussion the data from this study reflect a typical over‑dispersion of parasites (nielsen 2012, lester and matthews 2014), where a minority of horses shed most of the eggs in a population (traversa et  al. 2010, hinney et  al. 2011). in fact many horses harboured low parasite burdens (47.2% of the population had fecs ≤  200  epg), while others carried more moderate parasite levels, and only few individuals shed the highest levels of faecal eggs (12% of the horses showed fecs >  200 epg). there was no linear correlation between fecs and the intestinal cyathostomin burden – thus high levels of egg shedding are not necessarily equivalent to an increased risk of clinical disease for an individual animal (duncan and love 1991, nielsen et al. 2014). the difference between the results of this study and those obtained in the aforementioned 2008 study, i.e. 450 n pos 42 1 13 1 27 9 32 1 12 4 65 49 .9 % 51 3 72 .5 % 33 .4 % 5 5. 0% 40 .0 % 53 .8 % 54 .1 % 46 .8 % 400 350 300 250 200 150 100 50 0 ≥ 3 y ea rs 12 ye ar s < 1 y ea r g el d in g st al lio n m ar e st al le d pa st u re d figure 2. positivity for cyathostomins in relation to age, sex and living habits of 475 horses from 12 farms from italy. n = total amount of horse within each category; pos = positivity within the category; % = positivity percentage in each category. figure 3. graphical representation of the temperature, humidity, rainfall and wind speed trend in tuscany, lazio and abruzzo during the six months prior the studies sample collection (source: www.ilmeteo.it). 321veterinaria italiana 2018, 54 (4), 317‑322. doi: 10.12834/vetit.787.3812.1 sconza et al. cyathostomins in horses of central italy an egg shedding above the treatment threshold. the application of treatment should moreover be thoroughly evaluated on a case‑by‑case basis, considering the age classes, living habits, and risk for diseases caused by other parasites, e.g. p. equorum or strongylus vulgaris. grant support part of this work was funded by progetto speciale “progetto di eccellenza ricercatori”, university of teramo – fondazione tercas. selective treatments may be effective in reducing pasture contamination and the number of parasiticide administrations, and in preserving cyathostomin refugia. importantly, despite scientific consensus that further studies are necessary to ultimately confirm this possible correlation, previous studies have demonstrated certain reductions of anthelmintic resistance on farms using selective treatments (von samson‑himmelstjerna 2009, matthews 2014). the selective treatment strategy requires frequent monitoring of egg excretion, e.g. twice a year (nielsen et  al. 2006), in order to promptly identify those horses with corning s. 2009. equine cyathostomins: a review of biology, clinical significance and therapy. paras vect, 2 (suppl 2), s1. cribb n.c., cote n.m., bouré l.p. & peregrine a.s. 2006. acute small intestinal obstruction associated with parascaris equorum 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horses. j am vet med assoc, 218, 1957‑1960. taylor m.h., coop r.l. & wall k.l. 2007. veterinary parasitology. 3rd ed, blackwell publishing, uk, 1826‑1832. traversa d., klei t.r., iorio r., paoletti b., lia r.p., otranto d., sparagano o.a. & giangaspero a. 2007. occurrence of anthelmintic resistant equine cyathostome populations in central and southern italy. prev vet med, 82, 314‑320. traversa d., milillo p., barnes h., von samson‑himmelstjerna g., schurmann s., demeler j., otranto d., lia r.p., perrucci 191 parole chiave colibacillosi, coniglio, dna microarray, epec atipico, escherichia coli, repec. riassunto nel presente lavoro è stato determinato il profilo dei geni di virulenza di 26 ceppi enteropatogeni di escherichia coli con la tecnica del dna microarray; i campioni sono stati prelevati da 17 focolai di colibacillosi rilevati in conigli provenienti da due regioni del nord italia. i ceppi sono stati classificati secondo il loro biotipo, il sierogruppo e il gruppo filogenetico. è stata anche precisata la distribuzione dei geni di virulenza che codificano il locus di cancellazione degli enterociti (lee), il quello del sistema di secrezione tipo 3 (t3ss), le proteine non-lee traslocate tramite il t3ss e i fattori di adesione. i ceppi testati, ad eccezione di 1, appartengono ai gruppi filogenetici a e b1. è stata osservata, inoltre, un’associazione predominante tra il sierogruppo o103 e il fenotipo ramnosio-negativo (biotipi 12 o 14). il profilo del lee maggiormente riscontrato è stato ler/cest/espa-1/espb-3/ tir-1/eae(beta)/espd-2/escn/eprj. tutti i ceppi possedevano il fattore di adesione rabbit-2 (afr/2) o il ral (rabbit adherence locus) e 24 di essi un ulteriore set costituito da uno o più tra i fattori di colonizzazione efa1/lifa, lpfa e paa. infine, la presenza singola o combinata di proteine effettrici lee e/o non-lee, che codificano i geni espg, cif, map e nle, ha attestato la potenzialità genetica che hanno i ceppi studiati ad indurre lesioni patologiche all'ospite. le tecnologie basate sul microarray, applicate alla valutazione del profilo genetico dell’escherichia coli del coniglio, si sono dimostrate convenienti e affidabili se finalizzate ad indagini a larga scala all’interno di programmi di sorveglianza per monitorare la circolazione profilo dei geni di virulenza di ceppi enteropatogeni di escherichia coli isolati in conigli del nord italia keywords atypical epec, colibacillosis, dna microarray, escherichia coli, rabbit, repec. summary the virulence gene profile of 26 rabbit enteropathogenic escherichia coli strains, isolated from 17 colibacillosis outbreaks located in two regions of northern italy, was determined using an echerichia coli virulence dna microarray. all strains were classified according to their determined biotype, seroand phylo-group. the distribution of virulence genes encoding for the locus of enterocyte effacement (lee), lee type iii secretion system (t3ss), non-lee t3ss translocated proteins and adherence factors was also determined. all strains but one belonged to phylogroups a and b1. a prevalent association between the o103 serogroup with the rhamnose-negative phenotype (biotype 12 or 14) was found. the most prevalent lee profile found in tested strains was ler/cest/espa-1/espb-3/tir-1/eae(beta)/espd2/escn/eprj. all strains possessed either the adhesive factor rabbit-2 (afr/2) or the plasmid rabbit adherence locus (ral) gene and 24 of them an additional individual or combined set of colonization factors efa1/lifa, lpfa and paa genes. finally, the combined or single presence of a set of lee and/or non-lee effector proteins encoding genes, namely espg, cif, map and nle family genes, attested to the genetic potential of investigated strains to induce pathologic lesions to the host. the application of microarray-based technologies in assessing the genetic profile of rabbit e. coli is a reliable, cost-effective candidate for large scale investigations in monitoring programs aimed to survey the circulation of pathogenic strains within rabbit production units, their zoonotic genetic potential and to select e. coli strains eligible for vaccinal prophylaxis in fattening rabbit production. veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.859.4260.2 accepted: 23.04.2016 | available on line: 30.09.2018 1 istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. 2 istituto zooprofilattico sperimentale delle venezie, vicolo mazzini 4 int 5/6, 31020 fontane di villorba, treviso, italy. 3 national research council of canada, 6100 royalmount, montreal, qc, canada h4p 2r2. * corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861 332407, fax: +39 0861 332251, e-mail: p.badagliacca@izs.it. pietro badagliacca1*, fabrizio agnoletti2, ilenia drigo2, valentina merildi1, federica lopes1, cinzia pompilii1, iolanda mangone1, massimo scacchia1, alfreda tonelli1 and luke masson3 virulence gene profiles of rabbit enteropathogenic escherichia coli strains isolated in northern italy 192 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx (adhesive factor rabbit-2), a chromosomal encoded fimbrial adhesin inducing the phenotype of ‘diffuse adhesion’ (da) on surface of hela cells, is associated with virulent rabbit strains (fiederling et  al. 1997). in addition, a putative chromosomal pathogenicity islet gene porcine a/e-associated (paa), which strongly correlates with the a/e phenotype, a chromosomal long polar fimbriae gene (lpf ), thought to contribute to epithelial cell colonization, and a plasmid-encoded fimbriae repec adherence locus gene (ral), involved in the early steps of adhesion, were also found in diarrheal rabbit e.  coli isolates (batisson et  al. 2003, badea et  al. 2003, newton et al. 2004, krejany et al. 2000). the eae and afr/2 genes are the main genetic markers used to define repec strains (milon et  al. 1999). another way to determine e.  coli enteropathogenicity is to assess both the serogroup and sugar fermentation character (biotype), targeted to establishing a link between biotype/serotype and high mortalities (camguilhem and milon 1989). an oligonucleotide virulence microarray, initially developed and validated as previously described (bekal et  al. 2003, bruant et  al. 2006), allowed the detection of an exhaustive list of e.  coli virulence genes as well as antimicrobial resistance genes in e.  coli strains isolated from coastal water and wastewater (hamelin et al. 2006, frigon et al. 2013), camel (salehi et al. 2012), poultry (bonnet et al. 2009), and cattle (staji et al. 2017). in a previous study this microarray was successful in clustering a group of aepec strains from rabbit experiencing diarrhea from a second cluster that grouped non-pathogenic e. coli strains from healthy rabbit, both coming from the same rabbitry (tonelli et al. 2008). the aim of the present work was to develop a methodology for defining virulent genotypes of repec strains by genotyping a collection of rabbit enteropathogenic e.  coli strains isolated from rabbitries affected by colibacillosis in northern italy. materials and methods strain collection, microbiology twenty six e.  coli extracted dna, tested for eae polymerase chain reaction (pcr) signal carried out as previously described (agnoletti et  al. 2004), introduction based on their virulence gene content and overt clinical symptoms, escherichia coli strains are classified into different pathotypes responsible for intestinal or extra-intestinal syndromes. in particular, the attachement and effacing e.  coli (aeec) pathotype, which includes both enteropathogenic (epec) and enterohemorrhagic (ehec) strains, are characterized by the presence of a chromosomal pathogenicity island called the locus of enterocyte effacement (lee), encoding for proteins responsible for the attaching and effacing (a/e) phenotype. aeec strains that are classified as ehec also possess the shiga toxin encoding-genes stx1 and/or stx2. lee genes are organized in five operons, named lee1 to 5. lee1, lee2 and lee3 transcribe the type iii secretion system (t3ss) e. coli secretion (esc) proteins and lee4 transcribes the e.  coli secreted proteins (esp). lee5 operon contains eae, the gene encoding the bacterial adhesin intimin which is involved in the intimate attachment to host epithelial cells, its receptor tir (translocated intimin receptor) and the chaperone cest, translocated from the bacteria to the host epithelial cells through the t3ss structure. the t3ss is also used to inject lee and non-lee encoded effector proteins (espcgp, map; cif, nle) into the host cell, which are responsible for the effacement of host epithelial cells (zhu et  al. 2001, marchés et al. 2003, schmidt and hensel 2004, bertin et al. 2004, thomas et al. 2005, garrido et al. 2006, luo and donnenberg 2011). epec strains also possess the e.  coli adherence factor (eaf) virulence plasmid, that encodes the adhesin bfp (bundle forming pili), which induces localized adhesion (la) of bacterial microcolonies on epithelial cells. the la phenotype is associated with serotypes/serogroups from human diarrhea outbreaks (giron et al. 1991). epec strains, which lack the eaf plasmid, are called atypical epec (aepec) (nuyen et al. 2006). after the initial study by cantey and blake (cantey and blake 1977) on the rabbit diarrheal e. coli-1 strain (rdec-1), strains involved in rabbit colibacillosis outbreaks in several european countries were confirmed as non-toxin producing aepecs inducing a/e lesions (milon et  al. 1999). the pathogenic specificity of rabbit epec (repec) strains lies primarily with the adherence mechanism that complements attachment to host epithelial cells, mediated by intimin. afr-2 virulence of rabbit enteropathogenic e. coli badagliacca et al. di ceppi patogeni nella filiera di produzione del coniglio, valutare il loro potenziale genetico zoonotico, e selezionare ceppi patogeni di e. coli per creare una profilassi vaccinale mirata nelle unità da ingrasso degli allevamenti cunicoli. veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 193 e.  coli isolates were clustered based on their biotype/serogroup/phylogroup profile and analysed according to the virulence gene or marker patterns related to lee structural genes (ler, eae, tir1-2-3, cest, espabd, escjn, eprj), lee and non-lee effector (espcgp, map/orf19; nleabcdefgh, cif) and adherence/colonization factors (afr/1-2, ralg, bfpab, paa, efa1/lifa, lpfa). finally, lee gene variant combinations were classified according to afset et  al. (2008). the chi-square test was used to assess the associations between serogroup, biotype, and phylogroup. statistical significance was expressed with p-value (α <0.05). e. coli dna labeling and hybridization approximately 300 ng of purified genomic dna was labelled with fluorescent cyanine (cy3) dye using the bioprime dna labelling system (invitrogen life technologies, burlington, on, canada) as described previously (bruant et  al., 2006). labelling efficiency and the percentage of dye incorporation were then determined by scanning the dna sample in a nanodrop spectrophotometer from 200 to 700  nm. cy3 dye incorporation was calculated using a web-based percent incorporation calculator (http:// www.pangloss.com/seidel/protocols/percent_inc. html). pre-hybridizations and hybridizations were performed following a protocol derived from hamelin and colleagues (hamelin et  al. 2006). for the hybridizations, 500 ng of labelled dna was dried under vacuum in a rotary desiccator without heating (savant speedvac®, arrayit, usa). dried labelled dna was resuspended in hybridization buffer (dig easy hyb buffer, roche diagnostics, laval, quebec, canada). microarrays were pre-hybridized for one hour at 50  °c with a pre-heated pre-hybridization buffer containing 5x ssc, 0.1% sds and 1.0% bsa. after pre-hybridization, the microarrays were hybridized with a solution that consisted of 25  μl of hybridization buffer, 20  μl of bakers yeast trna (10  mg/ml) (sigma aldrich, st. louis, mo, usa) and 20  μl of sonicated salmon sperm dna (10  mg/ml) (sigma aldrich, st. louis, mo, usa), mixed together with the labelled dna which had previously been denatured. microarrays were hybridized overnight at 50  °c in a slidebooster (model sb800; advalytix, germany). after hybridization, stringency washes were performed with advawash (advalytix, germany) using 1x ssc, 0.02% sds preheated to 50 °c. microarray data acquisition microarray slides were scanned with a scanarray lite fluorescent microarray analysis system (perkin-elmer, missasauga, ontario, canada) using with scanarray gx software (perkin-elmer, foster city, ca). fluorescent spot intensities were quantified were selected from a more extensive collection coming from strains isolated from rabbits affected by diarrheal syndrome. rabbits were 7to 87-day old coming from 17 rabbitries located in the veneto and piemonte regions (northern italy) with variable pathological degree of lesion ranging from liquid content in the caecum to typhlitis, enterotyphlitis and severe enterotyphlitis. an inoculum from the caecal contents of diseased animals was directly plated onto eosin methylene blue agar (emb, oxoid ltd, england) and e. coli isolates identified using the api 20e® system (biomérieux, france) and biotyped according to the method described by camguilhem and milon (camguilhem and milon 1989). one colony was re-isolated on tryptic soy agar (biolife italiana, milan, italy), suspended in phosphate buffer saline and its dna extracted with a genelute bacterial genomic dna kit® (sigma-aldrich, usa) and stored at -20 °c until use. microarray design and e. coli virulence genes or markers the microarray version used in the present study was composed of 70-mer oligonucleotide probes printed in duplicate on corning ultra gaps slides (corning canada, whitby, ontario), targeting for 348 e. coli virulence factors, covering all known e. coli pathotypes, and 95 antimicrobial resistance genes. the tryptophanase (tnaa), beta-glucuronidase (uida), lactose permease (lacy) and beta-galactosidase (lacz) genes were included as positive controls whilst negative controls included empty buffer spots as well as genes for the green fluorescent protein of aequoria victoria (gfp) and the chlorophyll synthase gene of arabidopsis thaliana (at3g). only data related to virulence or virulence-related gene hybridizations were considered in this study, since the antimicrobial resistance profiles of the same strains were analyzed separately (badagliacca et  al. 2014). confirmatory tests independent of the microarray, were performed by specific molecular probes or gene sequencing, as described below. escherichia coli isolates were assigned to a serogroup based on their wzy gene for o7, o15, o22, o24, o26, o28, o45, o53, o55, o56, o59, o66, o86, o91, o98, o103, o104, o113, o114, o117, o121, o123, o126, o127, o128, o138, o139, o141, o145, o146, o147, o148, o149, o155, o157, o172, o174, and o177. assignment to other serogroups was based on rfc gene for o4, wzx for o6, wb for o8, rfb for o9 and o101 and wbdl for o111. e.  coli phylogenetic groups were identified on the microarray based on the combined presence/ absence of the chua gene, the yjaa gene and the dna fragment tspe4.c2, according to clermont and colleagues (clermont et al. 2000). badagliacca et al. virulence of rabbit enteropathogenic e. coli nick title first author et al. 194 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx (eurofins mwg operon, ebersberg, germany). confirmatory test of tir gene as well additional pcrs for variants 1, 2 and 3 of espd, not hybridized by microarray, were performed as described by garrido and colleagues (garrido et al. 2006). additional pcr for the cest gene was performed using primers from bertin and colleagues (bertin et  al. 2004). all pcrs were carried out using universal master mix (pcr master mix 2xtm, promega italia srl, milan, italy), according the provider’s instructions. strains showing an ambiguous signal for repec fimbrial factors afr or ral were submitted to genomic sequencing. dna samples were quantified by using the qubit® dna hs assay kit (thermo fisher scientific, waltham, ma). one ng of dna was used for library preparation by using the nextera xt library prep kit (illumina inc., san diego, ca) according to the manufacturer’s protocol. deep sequencing was performed on the nextseq 500 (illumina inc., san diego, ca) using the nextseq 500/550 mid output reagent cartridge v2, 300  cycles and standard 151  bp pairs-end reads. with quantarray version 3.0 (packard bioscience, informer technologies inc., usa). the mean value for each set of duplicate spotted oligonucleotides was divided by the correction factor taken from the negative control spots. this value was then divided by the average of the empty spots to create a signal-to-noise ratio. oligonucleotide spots with a signal-to-noise fluorescence ratio greater than the established threshold (2 in this study) were considered positive. these ratios were then converted into binary data where a value of 0 indicates a negative probe and a value of 1 indicates a positive probe. confirmatory and additional tests strains showing lee structural genes demonstrating a signal lower than the threshold were confirmed by pcr. a confirmatory pcr test for espb was designed with primer-blast™, after a gene sequence alignment of 2,267 blast hits present in genbank. two primers, forward 5’-gccgtttttgagagcca-3’ and reverse 5-ttacccagctaagcga-3’, were synthesized table i. strain identification, pathological/epidemiological data, biotype and genetic grouping of enteropathogenic escherichia coli strains collected from rabbitries of northern italy. strain* age (day) unit pathological lesion biotype o-type# phylogroup profile° vr/a-1 65 fattening enterotyphlitis b30 nt b1 ii vr/a-2 65 fattening enterotyphlitis b14 o145 a iii tv/b-1 70 fattening typhlitis b30 o103 a i tv/b-2 70 fattening typhlitis b14 nt a i to/c-1 56 fattening enterotyphlitis b12 o103 b1 i to/c-2 42 weaning liquid content in caecum b14 o103 b1 i to/c-3 43 weaning liquid content in caecum b12 o103 b1 i tv/d-1 60 fattening enterotyphlitis b12 o148 a i tv/e-1 68 fattening enterotyphlitis b12 o103 b1 i pd/f-1 50 weaning enterotyphlitis b12 o157 b1 i ve/g-1 50 weaning severe enterotyphlitis b30 o145 b1 ii tv/h-1 25 nest severe enterotyphlitis b30 o103 b1 i tv/i-1 21 nest enterotyphlitis b12 o103 b1 i tv/i-2 42 weaning enterotyphlitis b12 o103 d i tv/l-1 55 fattening enterotyphlitis b14 nt b1 i pd/m-1 7 nest enterotyphlitis b28 o145 b1 iv pd/m-2 48 weaning enterotyphlitis b30 o15 b1 ii tv/n-1 48 weaning enterotyphlitis b12 o103 b1 i tv/o-1 75 fattening enterotyphlitis b12 o126 b1 i tv/l-2 70 females enterotyphlitis b14 o103 b1 i tv/l-3 42 weaning severe enterotyphlitis b12 nt b1 i tv/l-4 43 weaning severe enterotyphlitis b14 o59 b1 i tv/p-1 87 fattening enterotyphlitis b12 o157 b1 i tv/p-2 87 fattening severe enterotyphlitis b14 o103 b1 i tv/q-1 50 weaning severe enterotyphlitis b14 o103 b1 i tv/r-1 77 fattening liquid content in caecum b28 o177 a i * strain identification was defined by province code/alphabetical code of farm-number of strain; # nt, notypeable; ° see table ii. first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 195 strains possessing the ler/cest/espa-1/espb-3/tir-1/ eae(beta)/espd-2/eprj/escn lee gene variants were classified as the afset group d and clustered into two profile differing for afr/2g or ralg fimbrial encoding gene, respectively. two ralg-positive strains differ from this latter group just for the variant tir-3 or espb-2. the combined presence of adherence factors, porcine a/e-associated islet, lymphostatin and long polar fimbriae encoding genes (paa, efa1/ lifa, lpfa) was found in 13 strains with an additional 11 strains showing a positive hybridization for at least one of the three genes. table iii lists the percentage of isolates possessing virulence genes related to lee t3ss-translocated or non-lee t3ss-secreted proteins. the espg protein encoding gene was diffusely detected in the strain collection as well as effector genes map and cif. all strains possessed the non-lee effector c, (nlec), together with a variable presence of nlea, nleb, nled, nlee, nlef, nleg and nleh. the reads obtained were trimmed using a in-house script that trims the first 15 nucleotides and uses as cutoff a mean quality of 30 and the minimum length of 50 bases. trimmed reads were de novo assembled using spades version 3.0.0 (bankevich et  al. 2012). ambiguous nucleotides were solved by local reads remapping by bowtie  2 (langmead and salzberg 2012), followed by alignment visual inspection. results epidemiological data and strain classification table i summarizes the epidemiological data of colibacillosis outbreaks along with the related biochemical or genetic markers used to group the e. coli isolates. no apparent associations were found between epidemiological data (age, production unit, lesion degree) and either the biotype or phylogroup. all but four nontypable strains possessed the o-serogroup gene wzy. all strains but one belonged to phylogroups a and b1 with one strain belonging to phylogroup d. b1 was the prevalent phylogroup (76.9%) and was found mostly associated with the o103, o157, o126 wzy gene as well as notypable ones. an association between the o103 wzy gene and the tspe4.c2 dna fragment (phylogroup b1) with the rhamnose-negative fermentation character (biotype 12 or 14) was found but wasn’t statistically significant (α >0.05). pathotyping and virulence gene profiles table ii shows the virulence-related gene profiles of e.  coli strains and their clustering into four groups based on the presence of the rabbit-specific adherence factor encoding genes (afr/2g or ralg) and variants of the lee structural genes. twenty-four table ii. profiles of locus of enterocyte and effacement (lee) and adherence factor encoding genes in rabbit enteropathogenic escherichia coli. profile n° of involved strains lee structural or secreted proteins* fimbrial adhesin# adherence factors (n) phylogenetic group (n) afset group i 21 ler, cest, espa-1, espb-3, tir-1, eae(beta), espd-2; eprj, escn afr/2g efa1/lifa(12), paa(17), lpfa(19) a (4); b1 (16); d (1) d ii 3 ler, cest, espa-1, espb-3, tir-1, eae(beta), espd-2; eprj, escn, ralg efa1/lifa(3), paa(1), lpfa(2) b1 d iii 1 ler, cest, espa-1, espb-3, tir-3, eae(beta), espd-2; eprj, escn ralg a this study iv 1 ler, cest, espa-1, espb-2, tir-1, eae(beta), espd-2; eprj, escn ralg efa1/lifa, paa, lpfa b1 this study * strains tv/h1, tv/i2 and tv/n1 possess additional escj gene; espd and cest genes were detected by specific pcrs; signal for espb-3 variant was below the threshold signal in strains tv/b2, to/c3, to/d1, tv/l4, tv/p1, tv/r1, vr/a2, ve/g1 and pd/m2, confirmed by specific espb pcr; signal for the tir-1 variant was below the threshold signal in strains tv/b2 and vr/a2, confirmed by specific tir pcr; # strains tv/l-1 and tv/o-1 showing ambiguous signal for repec adhesins were analysed by genomic sequencing. both strains showed 100% similarity with the described sequence of afr/2g (accession number u77302), 72% similarity with the sequence of ralg (accession number u84144). no sequence similar to bfpa (accession number u27184) was found. table iii. frequence of effector enteropathogenic virulence factors in rabbit escherichia coli. gene n° of isolates tested positive lee t3ss-effector espg 24 map/orf19 24 non-lee t3ss-secreted or structural protein cif 24 nlea 20 nleb 13 nlec 26 nled 7 nlee 22 nlef 3 nleg 21 nleh 21 nick title first author et al. 196 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 19 strains (73.1%), respectively. these gene products increase the colonization capability and severity of epithelial damage (newton et al. 2004, batisson et al. 2003). all strains possessed the eprj and escn genes, responsible for secretion of t3ss basal body rod component and general t3ss translocation activity, respectively, both of which are involved in the activation of the inflammation mechanism (miao et  al. 2010). moreover, the confirmatory pcr test for espb gene and additional test for espd gene, together with general positive hybridizations for espa, attests to the ability of examined strains to carry out pore formation allowing the translocation of effector proteins. a lee pattern, namely espg, map, as well as non-lee (cif, nle family gene) effector genes were individually or in combination detected in all strains, confirming the genetic basis underlying the examined strain’s ability to induce epithelial damage and an irreversible cytopathic effect. although no strain in our study possessed a member of the shiga toxin gene family, finding chua gene (a marker for heme transport in ehec o157:h7) in tv/i-2 o103 strain as well as five other strains being characterized as o157 or o145 serogroup, is a reminder of the known plasticity of e. coli. even in light of the recent haemolytic uraemic syndrome epidemics in germany and france caused by bacteriophage-mediated acquisition of the stx2a gene by the enteroaggregative e.  coli o104:h4 strain (scheutz et  al. 2011), the use of the dna microarray technique in screening studies could be a support decision tool for more specific genetic investigations about the recombination capacity of e. coli pathotypes. conclusion despite the low overall number of strains tested, this study confirms that in northern italy rabbit colibacillosis was caused by agents having a defined genotype that was consistently found within a larger and variable virulence gene background. the rabbit species-specific adherence virulence factors, afr/2 or ral, characterize the repec strains. overall, a prevalent attaching and effacing gene profile consisting of espa-1/espb-3/tir-1/eae(beta) places the examined repec strains into the afset d group of atypical epec. the diffuse detection of additional adherence factors as efa1/lifa support this classification. the link between o145 serogroup and the ral adherence factor suggests that routine repec diagnosis should incorporate ral, together with eae and afr/2, as pcr amplification targets. finally, the absence of shiga toxin encoding genes limits the zoonotic potential of tested strains. the application of microarray-based technologies discussion the e.  coli virulence dna microarray technology has proven to be a powerful tool for large scale genetic characterization of rabbit enteropathogenic e. coli and has allowed us to obtain genetic profiles consistent with its pathogenesis. in particular, it has produced a database for more specific analysis of gene products. the strains examined in this study were classified as aepec, thus confirming that the rabbit colibacillosis agents belong to this pathotype (milon et  al. 1999, licois and marlier 2008). these strains possessed the rabbit specific adherence factors afr/2g or ralg, confirming the species-specificity of rabbit colibacillosis agents (repec). interestingly, the afr/2g and ralg genes weren’t detected together in our strains, suggesting that an alternative genotype is expressed by operons of repec adherence locus. although ral and afr/2 genes were not two-way associated with serogroup or biotype, the o145 (3 strains) and o103 (12 strains) serogroups were ral and afr/2 positive, respectively. the classification of strains according to biotype, phylogroup and o-antigen encoding gene, agreed with previous clinical and epidemiological studies on rabbit enteropathies. indeed, all strains, except strain tv/i-2, belonged to phylogroups b1 and a, which generally encompasses e.  coli strains derived from animal origin (clermont et  al. 2000). the most prevalent genetic pattern related to phylogroup/o-antigen/biotype, found in nine strains (about 35% of examined strains), was b1/ o103/rhamnose negative. this is consistent with previous investigations on rabbit colibacillosis in italy (badagliacca et al. 2010, agnoletti et al. 2004). a lee-encoding set of genes covering the cest chaperon and the five operons related to the lee regulator (ler) gene, the eae/tir domain (eae, tir) and the lee type iii secretion system (esp, esc and epr), was detected in all strains. in particular, the most prevalent combination of lee gene variants espa-1/espb-3/tir-1/eae(beta) corresponds to the afset d group. afset and colleagues (afset et  al. 2008) classified atypical epec associated with diarrhea in children into 11 group (a to k group) on the basis of variant distribution of these four lee genes, compared to the phylogenetic group and the presence of the adherence factor efa1/lifa. the afset d group is associated with prevalent a and b1 phylogroup and efa1/lifa gene. therefore, the presence of the efa1/lifa gene in 16 strains (61.5%) and the above referred phylogrouping results strongly correlated with this classification. conversely, the profiles espa-1/espb-3/tir-3/ eae(beta) and espa-1/espb-2/tir-1/eae(beta), found in the ralg-harbouring vr/a2 and pd/m1 strains were not included in the afset classification. finally, the lpfa and paa genes were detected in 22 (84.6%) and first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 197 investigations in monitoring programs aimed to survey the circulation of pathogenic strains within rabbit production units, their 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& robins-browne r.m. 2006. atypical enteropathogenic escherichia coli infection and prolonged diarrhea in children. emerg infect dis, 12, 597-603. 251 1 study center of animal pain, department of veterinary medicine, university of perugia, via s. costanzo 4, 06126 perugia, italy. 2 merial italia spa, viale luigi bodio 37, 20158 milano, italy. 3 college of medical, veterinary & life sciences, university of glasgow, university avenue, glasgow g128qq, united kingdom. 4 college of science and engineering, university of glasgow, university avenue, glasgow g128qq, united kingdom. * corresponding author at: : study center of animal pain, department of veterinary medicine, university of perugia, via s. costanzo 4, 06126 perugia, italy. tel.: +39 075 5854612, e-mail: giorgia.dellarocca@unipg.it . parole chiave icmps-sf, cane, validazione italiana, scala del dolore, validità di costrutto. riassunto obiettivo di questo studio è stato validare la versione italiana della glasgow composite measure pain scale – short form (icmps-sf), finalizzata alla valutazione del dolore acuto nel cane. la versione originale in lingua inglese della scala (la glasgow composite measure pain scale – short form cmps-sf) è stata tradotta in italiano secondo un protocollo standard per assicurarne la validità linguistica e culturale. utilizzando la icmps-sf a 2, 6 e 24 ore dall'estubazione, nove veterinari italiani hanno poi registrato i punteggi relativi al dolore in cani sottoposti a chirurgia ortopedica o dei tessuti molli. la validità del costrutto è stata dimostrata usando il test delle ipotesi. nello studio sono stati reclutati 95 cani, di cui 37 sottoposti a chirurgia ortopedica e 58 ad interventi sui tessuti molli. degli interventi chirurgici effettuati, 23 sono stati considerati lievi, 45 moderati e 27 severi. sono state riscontrate differenze statisticamente rilevanti nei punteggi medi tra i casi ortopedici e quelli dei tessuti molli e tra i tre livelli di gravità attribuiti alle chirurgie. la icmps-sf ha dimostrato validità di costrutto simile alla scala inglese originale, risultando uno strumento valido e affidabile per la valutazione del dolore acuto nei cani da parte dei veterinari italiani. creazione e validazione della versione italiana della glasgow composit measure pain scale-short form (icmps-sf) keywords icmps-sf, dog, italian validation, pain scale, construct validity. summary objective to validate the italian translation of the glasgow composite measure pain scale – short form (icmps-sf) in order to assess acute pain in dogs. the original english-version of the scale (the glasgow composite measure pain scale – short form cmps-sf) was translated into italian according to a standard protocol to ensure linguistic and cultural validity. nine italian veterinary surgeons then recorded pain scores in dogs undergoing orthopaedic or soft tissue surgery using the icmps-sf at 2, 6, and 24 hours post-extubation. construct validity was demonstrated using hypothesis testing. a total of 95 dogs were recruited into the study. thirty-seven dogs underwent orthopaedic procedures and 58 dogs underwent soft tissue procedures. twenty-three, 45, and 27 procedures were classified as mild, moderate, and severe, respectively. statistically significant differences in the median pain scores were demonstrated between orthopaedic and soft tissue cases as well as among mild, moderate, and severe cases. median pain scores decreased with time and changes were statistically significant. the icmps-sf demonstrated construct validity similar to the original english-language scale, resulting in a valid and reliable instrument for the assessment of acute pain in dogs by italian veterinarians. giorgia della rocca1*, rodolfo colpo2, jacqueline reid3, alessandra di salvo1 and e. marian scott4 creation and validation of the italian version of the glasgow composite measure pain scale-short form (icmps-sf) veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.699.3421.3 accepted: 22.02.2016 | available on line: 30.09.2018 252 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx the glasgow cmps-sf was derived from the original glasgow composite measure pain scale (cmps), a structured questionnaire completed by an observer following a standard protocol, which includes the assessment of spontaneous and evoked behaviours, interactions with the animal, and clinical observations (holton et  al. 2001, morton et  al. 2005). the cmps was designed using psychometric principles, which are well established in human medicine for the measurement of complex and intangible constructs such as intelligence, pain, and quality of life. the psychometric approach to a scale design encompasses an established process of item selection, questionnaire construction, and testing for validity, reliability, and responsiveness, which ensures scientific soundness (streiner and norman 2008). validity (criterion, content, and construct) is the most fundamental attribute of an instrument because it provides evidence of the ability of the instrument to do the work it was built for (morton et al. 2005, cook & beckman 2006, streiner & norman 2008). criterion validity is the agreement of a new instrument with an existing ‘gold standard’. in the case of animal pain, a gold standard does not exist, and other forms of criterion validity must therefore be investigated (souza & silva 2005). content validity focuses on the appropriateness and completeness of the items within the instrument. it is deemed to be present when items cover all the relevant aspects that have to be measured without including any extraneous features (bullock & tenebein 2002, streiner & norman 2008). construct validity is demonstrated when hypotheses regarding the attribute(s) in question are upheld by the use of the instrument (crellin et al. 2007). the usefulness of a clinical instrument is markedly enhanced by having an intervention score as a guideline for analgesic treatment. a scoring system provides the veterinary practitioner with a clinical decision-making tool that can be used as an adjunct to their clinical judgement. this was the purpose behind creating the cmps-sf (reid et  al. 2007). the scale comprises 6 behavioural categories, with associated descriptive expressions (items): vocalisation (4), attention to wound (5), mobility (5), response to touch (6), demeanour (5), and posture/activity (5). items are placed in increasing order of pain intensity and numbered accordingly. the observer chooses the item within each category that best describes the dog’s behaviour and ranked scores are summed; the maximum pain score is 24, or 20 if mobility is impossible to assess. consideration of a clinical decision-point for analgesia gave an intervention level for rescue analgesia of 6/24, and 5/20 when section b (mobility assessment) could not be carried introduction pain is a complex, subjective, and emotional experience that is associated with several medical and surgical conditions. recognising pain and assessing its intensity is an integral part of effective pain management. if pain is not recognised, it is unlikely to be treated. moreover, if the intensity of pain is not appreciated, the selection of an appropriately potent analgesic will be hampered, resulting in a lack of pain relief (national research council 2009). at present, there is no ‘gold standard’ in assessing pain in animals. however, a presumptive diagnosis, a clinical examination (including the evaluation of psychomotor changes and pain behavioural expressions), the use of validated pain scales, and the response to therapy are all tools which, especially when combined, can help veterinary practitioners to recognise a painful subject and identify an appropriate therapy. pain scales are a valuable diagnostic aid, and provide the veterinarian with a ready-to-use tool. indeed, attributing a score to a painful condition enables veterinarians to identify a therapeutic approach that is proportional to the degree of pain. unidimensional pain scales including the visual analog scale (vas), the numerical analogue scale (nas), the numerical verbal scale (nvs), and the simple descriptive scale (sds) have been widely used in the assessment of pain in small animals (anil et al. 2002, wiese 2015). unidimensional scales only measure a single parameter associated with pain, namely its intensity, but the contemporary approach to pain assessment emphasises the need to capture the affective component of the pain experience, or ‘how it makes you feel’, because it is this aspect of pain that causes the associated suffering. unidimensional scales require the observer to make a subjective judgement of the animal’s pain. inter-observer variability is a problem when these scales are used in a busy practice environment where several observers may be assessing a single animal at different time-points (holton et  al. 1998). in order to limit this subjectivity, multidimensional scales have been created. these encourage the observer to evaluate different aspects of the patient’s behaviour at rest and during interaction with the observer. a number of multidimensional scales are now available for scoring acute pain in dogs or cats, including the university of melbourne pain scale, the 4avet scales, the glasgow composite measure pain scale – short form (cmps-sf), and the unesp-botucatu multidimensional composite pain scale (unesp-botucatu mcps) (firth and haldane 1999, laboissière 2006, reid et  al. 2007, brondani et al. 2011, brondani et al. 2012, brondani et al. 2013a, brondani et al. 2013b). cmps-sf: validation of the italian version della rocca et al. veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 253 materials and methods linguistic validation linguistic validation was based on the standard linguistic validation process undertaken by the mapi institute (www.mapi-institute.com) and comprised 3 steps: 1. forward translation: the original english tool was translated into italian by 2 independent professional translators who spoke italian as their mother-tongue. the 2 translated versions were synthesised by a third italian mother-tongue person to produce a consensus version. 2. backward translation: the consensus version was back-translated into the source language by 3 independent translators who spoke english as their mother-tongue. a comparison of the source questionnaire with the back-translation was then performed to check the conceptual content of the forward consensus version, in order to assess and control its quality. 3. pilot testing: the clarity, intelligibility, and appropriateness of the words (items) used in the translated version of the scale and cultural relevance of the target language version of the scale to the target population were evaluated by 15 italian veterinarians who offered to participate in the validation process. the aim of this step was to acquire input from people from the representative end-user demographic and to incorporate their feedback. training day a training day was organised by the developer of the original scale with the 15 aforementioned veterinarians. a formal presentation of the basic principles of scale development and validation, with particular reference to the glasgow cmps-sf, was delivered. a focus group discussion followed (pilot testing). finally, participants took part in a practical session that included 4 canine surgical cases (1 orthopaedic, 3 soft tissue). the surgeries were carried out the same day in the surgical unit of the department of veterinary clinical science (university of milan, italy), and were selected for pain scoring in particular. all surgeries involved the administration of analgesic according to standard hospital protocol. the scale was applied once the dogs had fully recovered from the sedative effects of the anaesthetic drugs. after an initial demonstration of the examination protocol by jr, each case was individually scored by participants using the italian composite measure pain scale – short form (icmps-sf). scores were then compared and discussed, and any problems with the use of scale were addressed. out. because it was derived from the cmps, with no new items added, the cmps-sf scale retained the content validity of the original scale. construct validity of the cmps-sf was initially demonstrated by proving the hypotheses that post-surgical pain decreases with time and that orthopaedic surgery is associated with a greater degree of pain intensity than soft tissue surgery. subsequently, further evidence of construct validity and responsiveness of the scale was demonstrated in a study of dogs suffering from painful non-surgical as well surgical conditions, where the magnitude of the change in scores before and after the administration of analgesic corresponded to clinicians’ interpretations of the change in the pain status (better, unchanged, worse) (tait et  al. 2011). accordingly, the cmps-sf has been shown to be suitable for the measurement of acute pain per se, and its use is not limited to post-operative pain. most healthcare measurement instruments have been developed in english. however, an instrument can be used in the international arena if it addresses the same concepts in all languages (guillemin et al. 1993, souza & rojjanasrirat 2011). accordingly, the original instrument needs to undergo a two-stage process to ensure that the translated version is conceptually equivalent to the original instrument; it is culturally relevant and acceptable to the target population within the target country; and it is psychometrically comparable (guillemin et  al. 1993; beaton et  al. 2000; sperber 2004). the process involves a linguistic validation that aims to produce an appropriate translated version that deals with the linguistic and cultural aspects of the target language, followed by a psychometric validation that comprises a statistical evaluation of the properties of the target language version. given the absence of validated tools in the italian language assessing acute pain in dogs, the aim of this study was to validate the italian version of the cmps-sf (icmps-sf), following the international guidelines proposed for cross-cultural validation (beaton et al 2000, streiner & norman 2008, souza & rojjanasrirat 2011). three hypotheses were tested to demonstrate construct validity of the icmps-sf: 1. following surgery, pain decreases with time; 2. in a veterinary context, orthopaedic surgery is associated with a higher degree of pain than soft tissue surgery; 3. if surgical procedures are classified as mild, moderate, and severe, intensity of pain will be mild, moderate, and severe, respectively. della rocca et al. cmps-sf: validation of the italian version nick title first author et al. 254 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx surgeons who had participated in the training day. no restrictions were placed on the age, breed, or sex of recruited dogs. cases that required local anaesthetic blocks (particularly epidural) were excluded from the study due to the effect on post-recovery mobility and the possibility to score up to the maximum 24. psychometric validation study protocol dogs undergoing either orthopaedic or soft tissue surgery were recruited for the study by 9 veterinary table i. icmps-sf. first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 255 varied at the different time points for dogs that had undergone orthopaedic surgery compared with dogs that had undergone soft tissue surgery and to compare pain scores when the surgical severity was classified as mild, moderate, or severe. a formal analysis was then applied. this involved fitting a series of repeated measures 2-way anova (random effect) models to explore time, group (surgery type or surgery severity), and interaction effects. where statistically significant effects were found, follow-up comparisons were performed (using the wilcoxon mann whitney and wilcoxon signed ranks test). a significance level of 0.05 was selected. all statistical analyses were performed using the software statistical package minitab 15. results linguistic validation details of the translation and final consensus version of the icmps-sf are shown in appendix (supplementary materials1) and table i, respectively. despite a high level of discussion and engagement during the focus group, no changes to the consensus version of the scale were suggested. this confirmed the clarity, intelligibility, and appropriateness of the words used in the translated version of the scale and the cultural relevance of the target language version to the target population. psychometric validation animals & surgical procedures a total of 104 dogs were recruited to the study, 52  from university practices and 52 from private clinics. of these, 9 dogs were excluded from the analysis because of missing information or because the mobility category had not been completed. the mean +/sd age of the remaining 95 dogs was 7.1 ± 4.4 years (range: 3 months-14 years). thirty dog breeds were represented, as shown in table ii. details of the surgical procedures that were carried out are shown in table iii. of the procedures that were performed, 37 were orthopaedic and 58 were soft tissue. twenty-three procedures were classified as mild in terms of surgical severity, 45 were moderate, and 27 severe. anaesthetic and analgesic protocols were very variable and consequently are not reported in detail. in summary, with the exception of a number of dogs in 1 private practice where no pre-medication was otherwise there were no restrictions on surgical procedure. all recruited dogs received anaesthesia and analgesia according to the normal practice of the clinic. all dogs were sufficiently recovered from the effects of anaesthesia to allow full participation in the scale’s standard examination protocol. demographic and surgical procedural details, anaesthetic and analgesic administration, time of endotracheal extubation, and post-operative analgesic administration were recorded for each dog. participants recorded pain scores using the icmps-sf at 2, 6, and 24 hours post-endotracheal extubation. statistical analysis surgical procedures were coded according to the type of procedure – soft tissue or orthopaedic – and to the associated surgical severity – mild, moderate, or severe. box plots and descriptive statistics were used initially to gain an impression of how the pain scores table ii. list of dog breeds. dog breeds number labrador 10 mixed breed 30 bichon frisee 1 german shepherd 7 great dane 1 boxer 5 italian greyhound 1 dogue de bordeaux 1 jack russell 2 beagle 1 schnautzer 2 bergamasco shepherd 2 cocker spaniel 2 golden retriever 4 poodle 3 dachshund 4 basset hound 1 pomeranian 1 english bulldog 1 rottweiler 2 cavalier king charles spaniel 2 doberman 2 pekinese 1 miniature pinscher 1 volpino italiano 1 saluki 1 pitbull 2 springer spaniel 1 pointer 1 corgi 1 1 appendix (as supplementary materials) may be requested from the corresponding author. nick title first author et al. 256 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx administered, most dogs were pre-medicated with an alpha2-adrenoceptor agonist or acepromazine in combination with an opioid. induction of anaesthesia was performed with propofol on most occasions and maintained with isoflurane. combinations of morphine, methadone, fentanyl, sufentanil, lignocaine, and ketamine were commonly administered by continuous rate infusion intra-operatively, especially in those procedures classed as moderate or severe. the use of non steroidal antinflaammatory drugs such as carprofen, firocoxib, and meloxicam was largely restricted to the post-operative period, although on occasion carprofen and meloxicam were administered during an earlier stage. other analgesics administered for the purpose of post-operative pain relief were opioids such as tramadol. experimental study orthopaedic (o) vs soft tissue (st) surgery the descriptive statistics for orthopaedic (o) and soft tissue (st) surgical cases at time points 2, 6, and 24 hours are shown in table iv. these findings, combined with those in figure 1, show how the pain score changed over time within these 2 groups. using a 2-way anova, the following hypotheses were tested: a) the median pain score is different between o and st cases; b) the median pain score changes over time; c) there is a statistically significant interaction between time and surgery type. the modelling strategy first fit the full model, including main effects and interactions, and then removed sequentially any terms that were not statistically significant. after fitting the full model, there was not a statistically significant interaction between time and type of surgery, indicating that the rate of decline in pain score with time was the same for o and st. this term was therefore removed and the model re-fit. table v shows the final model. table iii. list of surgical procedures and their classification. procedure surgery type* surgical severity number pelvic # o severe 2 thoracoscopy st mild 1 pyometra st moderate 2 tplo o severe 12 salivary gland excision st moderate 1 ccl repair o moderate 5 amputation limb o severe 1 mastectomy st moderate 2 # elbow o severe 1 patellar luxation o moderate 2 excision femoral head o moderate 2 ulnar ostectomy o severe 1 herniated t/l disc o severe 1 splenectomy st moderate 3 oral tumour st mild 1 bilateral entropion st mild 1 # maxilla o severe 1 thoracotomy st severe 1 # long bone o severe 5 partial excision pinna st moderate 1 teca + bulla osteotomy st severe 1 perianal adenectomy + castration st moderate 2 tibial crest advancement o severe 1 splenectomy + arytenoid lateralisation st moderate 1 extracapsular tendon repair st moderate 1 liver biopsy + mastectomy st moderate 1 arytenoid lateralisation st moderate 1 perianal gland adenoma st moderate 1 maxillectomy o severe 1 cystotomy st moderate 4 punch biopsy granuloma dorsal foot st mild 1 cryptorchid castration st moderate 1 prolapsed 3rd eyelid st mild 1 castration st mild 5 castration + fna prostate st mild 1 ablation of small skin tumour st mild 5 small mass upper eyelid st mild 1 ovariohysterectomy + colposuspension st moderate 1 ovariohysterectomy st moderate 6 skin biopsy st mild 1 aural haematoma drainage st mild 2 reconstruction of shearing injury foot st moderate 1 gastroscopy + biopsy st mild 1 biopsy pad st mild 1 exploratory laparotomy + gastrotomy st moderate 1 plate removal & ext fixator o moderate 1 perineal mass removal st mild 1 ovariohysterectomy + gastropexy st moderate 1 splenectomy + bm biopsy st moderate 1 elbow arthroscopy o moderate 1 enucleation st moderate 1 * o = orthopaedic surgery; st = soft tissue surgery. table iv. descriptive statistics for the pain scores generated by the icmps-sf for orthopaedic (o) and soft tissue (st) cases at 2, 6 and 24hrs. time point (hrs) surgery type no of dogs n* median range q1 q3 2 o 31 6 4 0 8 3 5 st 57 1 3 0 9 1 4 6 o 37 0 3 0 9 2 5 st 55 3 2 0 7 1 3 24 o 36 1 2 0 10 1 4 st 53 5 1 0 6 0 3 n* denotes number of missing observations. q1 and q3 are the lower and upper quartiles of the distribution of pain scores. o = orthopaedic surgery; st = soft tissue surgery. first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 257 following findings: the median pain score at 6 hours was highly likely to be between 0.5  and 1  points lower than at 2 hours; analogously comparing time 24 versus time 2, the median pain score at 24 hours was highly likely to be between 0.5 and 1.5 points lower than at 2 hours. conversely, there was not a statistically significant difference in the median pain scores between 6 and 24 hours, since the 95 % confidence interval includes the 0 value. comparing time 24 versus time 2 and versus time 6  in o dogs yielded the following results: the median pain score at 24 hours was highly likely to be between 1 and 2.5 points lower than at 2 hours and between 0.5 and 1.5 points lower than at 6 hours. there was not a statistically significant difference in the median pain scores between 2 and 6 hours, since the 95% confidence interval includes the 0 value. overall, for both procedures, a 95  % confidence interval for the difference in the median pain score at 2 time points was obtained. mild vs moderate vs severe surgical severity the descriptive statistics for cases classified as mild, moderate, and severe at time points 2, 6, and 24 hours are shown in table vi. when combined with findings from figure 2, these findings show how the pain score changes over time within the 3 – mild, moderate, and severe – groups. the repeated measures 2-way anova model tested the following hypotheses: a) the median pain score is different between mild, moderate, and severe cases; b) the median pain score changes over time; c) there is a statistically significant interaction between time and surgical severity. the modelling strategy fit first the full model, including main effects and interactions, and then removed sequentially statistically significant differences in the median pain score between orthopaedic and soft tissue cases and in the median pain score changes with time were demonstrated (both p-values < 0.001). a series of non-parametric (wilcoxon mann whitney and wilcoxon signed ranks) follow-up comparisons of the median pain scores (overall 95% confidence intervals) were constructed, in the first case between o and st at 2, 6, and 24 hours and in the second case, for o and st separately, between sets of 2 distinct time points. at 2 hours, the median pain score for st was highly likely to be between 1 and 2 lower than the median pain score for o cases. at 6 hours, the median pain score for st was highly likely to be between 1 and 2 lower than the median pain score for o cases. at 24 hours, there was no evidence of a difference in the median pain score, since the 95% confidence interval included 0. overall, both comparisons at 2 and 6 hours showed that the median pain score for o was higher than for st, but at 24 hours, there was no evidence of a difference. comparing time 6 versus time 2 in st dogs yielded the 0 surgery 2 hr score * ** * 6 hr score 24 hr score o st o st o st 5 10 15 20 it al ia n c m p ssf s co re figure 1. box and whisker plot of pain scores for orthopaedic (o) (n = 37) and soft tissue (st) (n = 58) surgical cases at 2, 6 and 24 hrs. each box is the interquartile range; the horizontal line within each box is the median. whiskers represent the range excluding outliers. the asterisk represents an outlier. table v. two-way repeated measures analysis of variance for pain score, using adj(usted) ss for ftests. source df seq ss adj ss adj ms f p-value surgery type 1 97.062 105.937 105.937 14.87 0.000 time (2, 6, 24) 2 72.839 78.280 39.140 25.82 0.000 dog id 93 678.910 678.910 7.300 4.82 0.000 error 172 260.720 260.720 1.516 total 268 1109.532 df = degrees of freedom; ss = sum of squares; ms = mean square; f = statistic and p-value. table vi. descriptive statistics for the pain scores generated by the icmps-sf for cases classified as mild, moderate and severe surgical severity at 2, 6 and 24hrs. time point (hrs) surgery type no of dogs n* median range q1 q3 2 mild 23 0 1 0 7 1 4 moderate 40 5 3 0 9 1 4 severe 25 2 5 2 8 3 5.5 6 mild 21 2 1 0 4 0 2 moderate 44 1 2 0 8 1 4 severe 27 0 4 1 9 2 5 24 mild 20 3 0 0 3 0 1.75 moderate 43 2 1 0 5 1 3 severe 26 1 3 1 10 1 4 n* denotes number missing. q1 and q3 are the lower and upper quartiles of the distribution of scores nick title first author et al. 258 veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx was highly likely to be between 1 and 3 higher than for mild cases, and between 0.5 and 1 higher than for moderate cases, but the median pain score for moderate cases was not statistically different to mild cases. discussion and conclusions this study was performed in order to validate the italian version of the glasgow composite measure pain scale – short form (cmps-sf) and to evaluate acute pain in dogs. the process of translation, cultural adaptation, and psychometric testing were performed according to the rules commonly reported in relevant literature. the results obtained from this analysis confirm linguistic and cultural validation and construct validity of the italian version of the scale for the evaluation of acute pain in dogs. the experimental study was conducted under clinical conditions with no restrictions other than the exclusion of dogs where the use of local anesthetic blocks might interfere with post-operative mobility. the hypotheses tested to assess the construct validity of the scale were 1) the median pain score will change over time as healing takes place; 2) the median pain score will differ between o and st cases; 3) the median pain score will differ between mild, moderate, and severe cases. these hypotheses have been used in human medicine for the validation of pain scales for pediatric patients (bullock & tenenbein 2002, manworren & hynan 2003). a similar method has been described in other studies where pain scales for dogs were validated (morton et  al. 2005, murrell et  al. 2008), and evaluated not only the content and construct validity, but also the responsiveness of the instrument (baeyer & spagrud, 2007). in this study the sensitivity to change was confirmed by changes in pain scores obtained during the post-operative period. there was large variation in pain scores for all dogs within each group (orthopaedic versus soft tissue; mild versus moderate versus severe) and also considerable overlap in pain scores between each group. this is not surprising given the heterogeneous nature of the dogs (ages, breeds), the surgical procedures, and the anaesthetic and analgesic protocols that were employed. nevertheless, our results confirmed that the icmps-sf did perform in accordance with the expected pain profile following surgery when used as a repeat monitoring tool (thus proving hypothesis 1: following surgery, pain decreases with time). additionally, this study demonstrated a difference between the intensity of pain resulting from orthopaedic and soft tissue procedures, icmps-sf scores for the o group being higher than the st group throughout the evaluation period (thus proving hypothesis 2: orthopaedic surgery is associated with any terms that were not statistically significant. the final model (table vii) shows that there was a statistically significant difference in the median pain scores between mild, moderate, and severe cases, and that the median pain score changed with time (both p-values < 0.001). there was no statistically significant interaction between time and surgical severity. follow-up comparisons using 95% confidence intervals (adjusted for multiple comparisons) for the difference in median pain scores over the severity groups at each time point are reported separately below. at 2 hours, median pain score for severe cases was highly likely to be between 2 and 4 higher than for mild cases, and between and 1 and 2 higher than for moderate cases. the median pain score for moderate cases was not statistically different to mild cases. at 6 hours, the median pain score for severe cases was highly likely to be between 1 and 3 higher than for mild cases, but not statistically different to moderate cases, and median pain score for moderate cases was not statistically different to mild cases. at 24 hours, the median pain score for severe cases 0 severity 2 hr score 6 hr score 24 hr score 5 10 15 20 it al ia n c m p ssf s co re * * mild moderate severe mild moderate severe mild moderate severe figure 2. box and whisker plot of pain scores for cases classified as mild (n = 23), moderate (n = 45) and severe (n = 27) at 2, 6 and 24 hrs. each box is the interquartile range; the horizontal line within each box is the median. whiskers represent the range excluding outliers. the asterisk represents an outlier. table v. two-way repeated measures analysis of variance for pain score, using adj(usted) ss for tests. source df seq ss adj ss adj ms f p-value severity 2 187.812 176.96 88.483 14.05 0.000 time (2, 6, 24) 2 71.339 78.280 39.140 25.82 0.000 dog id 92 589.660 589.660 6.409 4.23 0.000 error 172 260.720 260.720 1.516 total 268 1109.532 df = degrees of freedom; ss = sum of squares; ms = mean square; f = statistic and p-value. first author et al. nick title veterinaria italiana 2018, 54 (3), xxx-xxx. doi: 10.12834/vetit.xxxxx 259 could not be carried out (reid et  al. 2007), can be applied. as a result, italian veterinarians can be confident in using the icmps-sf to assess acute pain of any origin in dogs. acknowledgments the authors acknowledge pr. c.m. mortellaro for organising the training day and managing the surgical cases together with dr. d. stefanello and s. romussi; dr. d. bellucci, l. borghi, a. carotenuto, g.m. de benedictis, g. ravasio, r. regorda, c. sozzé and f. staffieri for participation in the experimental study; dr. s. badavelli, i. pavan, m baiesi and g. bruni for the support collecting data in field. financial support funding for the study was provided by merial italia s.p.a. a higher degree of pain than soft tissue surgery). similarly results demonstrated a difference between the intensity of pain resulting from surgical procedures classified as mild, moderate, or severe (thus proving hypotheses 3: the intensity of pain decreases with the degree of surgical severity), although follow-up comparisons showed that at some individual time points the difference between groups did not achieve statistical significance. this may in part be due to the hangover effect of the cri analgesic infusions, which were a feature of the moderate and severe groups, but not of the mild group. additionally, the allocation of cases into mild, moderate, and severe groups was made on a purely subjective basis based on clinical impression. in conclusion, in the 2 sets of analyses, we have been able to demonstrate construct validity of the icmps-sf similar to that demonstrated initially for the original english-language version of the scale. this is sufficient to ensure that the 2 scales will perform in a similar manner in similar circumstances and that the same intervention level of the original english version of 6/24, or 5/20 when section b (mobility assessment) anil, s.s., anil l. & deen j. 2002. challenges of pain assessment in domestic animals. j am vet med ass, 220, 313-319. baeyer v.c. & spagrud l.j. 2007. systematic review of observational (behavioral) measures of pain for children and adolescents aged 3 to 18 years. pain, 127, 140-150. beaton d.e., bombardier c., guillemin f. & ferraz m.b. 2000. guidelines for the process of 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silva j.a. 2005 a métrica da dor (dormetria): problemas teóricos e metodológicos. rev dor, 6, 469-513. 231 1the central laboratory for evaluation of veterinary biologics (clevb), el‑seka el‑baeda st., abbasia, p.o. box 131, egypt. 2food and agriculture organization of united nations (fao), emergency center of transboundary animal diseases (ectad), p.o. box, 2223, giza, egypt. 3department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, postal bag x04, onderstepoort 0110, south africa. 4national laboratory for veterinary quality control on poultry production (nlqp), animal health research institute, p.o. box, 264, giza, egypt. 5food and agriculture organization of united nations (fao), animal production and health division, viale delleterme di caracalla, 00153 rome, italy. *corresponding author at: department of veterinary tropical diseases, university of pretoria, onderstepoort 0110, south africa. tel.: +27 84 958 1925 (mobile), +27 12 529 8069 (office), fax: +27 12 529 8396, e‑mail: daydupe2003@yahoo.co.uk. parole chiave vaccino, catena del freddo, valutazione del vaccino, egitto, influenza aviaria malattia di newcastle, bronchite infettiva. riassunto l'industria avicola egiziana è fonte di sostentamento e di occupazione per milioni di cittadini a basso reddito. l'avicoltura, tuttavia, è un settore sovente coinvolto in episodi di malattie endemiche ed epidemiche. tra queste, l'influenza aviaria ad alta patogenicità e la pseudopeste sono sicuramente quelle più importanti. sebbene in egitto siano disponibili e utilizzati vaccini in grado di ridurre l’incidenza di tali malattie, resta dubbia la loro efficacia. in questo studio sono stati valutati alcuni vaccini virali utilizzati per gli allevamenti avicoli in tre governatorati egiziani. nel 54% dei prodotti selezionati è stata riscontrata una diminuzione del titolo virale spesso correlata a problemi riscontrati nella catena di distribuzione dei vaccini, come presenza di contaminanti sui flaconi vaccinali o interruzione della catena del freddo. i frigoriferi dove sono conservati i prodotti non sono appropriati per la conservazione e stoccaggio di vaccini né sono presenti procedure per la loro manutenzione e stoccaggio. è rivedere o stilare nuove procedure di gestione, manutenzione e distribuzione dei vaccini che prevedano un maggior controllo e rispetto della catena del freddo. controllo sulla gestione, manutenzione e distribuzione dei vaccini aviari in egitto keywords poultry vaccine, cold chain, vaccine assessment, egypt, avian influenza, newcastle disease, infectious bronchitis. summary egypt has a large traditional and exotic poultry sector which is challenged regularly by poultry diseases in endemic and epidemic proportions. the household poultry in particular is a source of livelihoods and employment for millions of low income citizens. highly pathogenic avian influenza (hpai) h5n1 and newcastle disease are the most important poultry diseases in this sector. whereas poultry vaccines are available to reduce the incidence of disease in egypt, their effectiveness is doubtful. we conducted a biological evaluation of selected viral vaccines of poultry in three governorates in egypt. fifty‑four percent of the vaccines had reduced vaccine titres and the effect of secondary vaccine distributions was associated with the observed vaccine titres. external contamination was observed in some vaccines and break in cold chain was reported. whereas no vaccine distributor used purpose‑built vaccine refrigerator, none also had prescribed protocol for vaccine handling or kept record of vaccine. there is a need to review vaccine handling procedure, monitor of vaccine cold chain more critically and review the whole chain that support vaccine distributions in egypt. abdel hakim m. ali1, mohamed m. samy2, folorunso oludayo fasina3*, mohamed k. hassan4, walid h. kilany2,4, souzan el‑mahdy1, ahmed saad2, juan lubroth5 and yilma jobre2 field evaluation of common poultry viral vaccines in egypt: a need for reassessment of the vaccine value chain veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 accepted: 16.10.2016 | available on line: 30.09.2019 232 field evaluation of poultry viral vaccines ali et al. veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 while the recommended cold chain temperature for vaccine maintenance globally is +  2  ºc to +  8  ºc, the marek’s disease vaccine and many of the recombinant vaccines should be kept in liquid nitrogen (‑ 196 ºc) (techathawat et al. 2007, kumru et  al. 2014). from these vaccine storage facilities, supplies are distributed in smaller cold boxes or vaccine carriers to end users. although the success of vaccination programmes in poultry depends partially on the quality of manufactured vaccines, it also depends on handling, the maintenance of cold chain along the marketing and transport processes and the correct application of vaccines to the recipient birds (collett 2013). in addition, the shelf life, potency and immunogenicity of vaccines will determine the level of immunity that an animal develops following vaccination1. for a veterinary vaccine to maintain its potency and immunogenicity, it must not only be stored at the required temperature of + 2 ºc to + 8 ºc, it must also be maintained in a “cold chain” – a continuum of safe handling and procedures on the vaccine from the time of manufacture through the transport processes until used in animals (subramanyam 1989, cheyne 1989, chojnacky et  al. 2010). previous reports have confirmed that incorrect storage temperature (miller and harris 1994, nelson et  al. 2004), interference with maternally derived antibodies (kim et al. 2010), inadvertent placement of vaccines outside the refrigerator and certain other practices like incorrect dilutions and unprofessional administration have all affected immunogenicity and responses to vaccines (miller and loomis 1985, sockey et  al. 1988, casto and brunell 1991). despite the intense vaccination of poultry particularly against h5n1hpai and nd among others, anecdotal evidences from animal health practitioners and farmers, including the continuing and on‑going outbreaks of avian influenza h5n1 and nd have suggested gross vaccine failures and previous scientific evaluations have confirmed these reports (kim et al. 2010, arafa et al. 2012, kilany et al. 2015). in this study, we randomly selected viral vaccines of poultry in three poultry‑dense governorates in egypt, conducted field and laboratory evaluations in order to determine whether the value chain (from handling, cold chain practices, marketing, delivery and use at the farm levels) affects potency, viability, field efficacy and immunogenicity with consequent effect on efficacy. introduction egypt has a large poultry sector with more than 50  thousands commercial producers and the egyptian poultry farming systems include the exotic broilers and layer chicken, indigenous (baladi) chicken, ducks (peking, muscovy, mule and sudani breeds), turkey, geese, ostriches, and quails. day old birds are supplied by both, the traditional and modern hatcheries. at any one time, the total standing poultry population is approximately one billion birds with the commercial poultry sector contributing approximately 90% while the remaining 10% originate from the small‑scale holders and household poultry. these poultry farms are particularly spread in villages and near cities in egypt (ali et al. 2013). the household poultry production is a source of livelihoods and employment for millions of low income citizens. the sector is marked with complex marketing chains and diverse challenges including diseases of considerable economic and public health significance. in the last decade, the financial losses caused by the major epidemic diseases of poultry such as highly pathogenic avian influenza (hpai) h5n1 and newcastle disease (nd) have been enormous (marangon and busani 2007, fasina et al. 2008). vaccines are widely used in the prevention and reduction of incidence as well as control of endemic poultry diseases. for a vaccine to be useful in the maintenance of animal health, it must be pure, safe, potent, and effective (oie 2014). in egypt, approximately 10.4 billion doses of vaccines were used in 2014, the majority (96.43%) of which were imported while only 3.57% were produced locally (central laboratory for evaluation of veterinary biologics ‑ clevb, unpublished data). of these doses, poultry vaccines accounted for 99.7% with an estimated end user value of $236 million (1.7 billion egyptian pounds, clevb unpublished data). in egypt, the clevb is the authorized governmental laboratory for the evaluation and certification of veterinary vaccines and biologicals prior to their release into the markets. the organisation complies with best standards and it is an iso/eic 17025 accredited laboratory which handles both imported and locally produced vaccines. multi‑level vaccine distributions are done primarily using refrigerated trucks to more than 5,000 district and village stores, and further downstream supplies reach the over 50,000 commercial poultry farms. whereas, cold rooms are available in the large integrated farms and with few big distributors, vaccines are kept in the kitchen‑type and household model refrigerators from where the numerous household producers only collect them in small quantities as ready‑to‑use products. 1 poultry hub. 2015. vaccination by poultry crc, 2015. http://www. poultryhub.org/health/health‑management/vaccination/ accessed 2 august 2015. 233 ali et al. field evaluation of poultry viral vaccines veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 at the same time that the vaccines were collected, cold chain storage facilities at the distribution points were inspected and assessed using a validated questionnaire and a checklist (table i). the checklist was a modified guide from the centers for disease control and prevention, usa (cdc 2003) and the us army medical material agency document (usamma 2005). temperature readings were taken from the refrigerators using calibrated thermometers placed in the middle of the storage area (refrigerator) for at least 20 minutes while the questionnaire was answered (bell et al. 2001). at each sampling location, preference was given to collection of the oldest batch where available, and/or those with apparent physical defacement or abnormality. collected vaccines were labelled and transported directly to clevb in ice boxes with hourly temperature taken during transportation with the aid of computerized transit temperature data loggers (range: ‑ 40 ºc to + 85 ºc). any box with logged temperature data outside the range of + 2 ºc to +  8  ºc were excluded and all accepted vaccine vials were stored at clevb facility until evaluated. the median time for vaccine transport from all distributors to the clevb was 4 hours 18 minutes. questionnaires a twenty‑two item pre‑tested questionnaire was administered to each of the selected vaccine distributors (n = 23, table i) to collect information on the procedures and processes around vaccine handling and management of vaccine refrigerators. the instrument was prepared in english and arabic language and all answers were recorded in a microsoft excel® spreadsheet. checklist was used to assess the consistency of the answers against practices at the vaccine distribution stores and where significant deviations from the answers given were observed, a reconfirmation was obtained or the details from the checklist based on observation was utilised. assay procedures the vaccines retrieved from the distributors and their corresponding reserved batches previously retained at clevb were subjected to parallel evaluations by virus titration (for live vaccines) and serological immune response (for inactivated vaccines) according to standard protocols (oie 2014). virus titration for live (attenuated) poultry vaccine newcastle disease (nd) ten‑fold serial dilutions (10‑1 to 10‑9) of nd, ibd and ib materials and methods study areas and participants’ recruitment three governorates (sharqia, dakahlia and fayoum) were purposively selected based on their relative importance as poultry‑dense locations in egypt. in each governorate, two geographically distinct regions were selected, including a more central location, usually in the capital of each governorate where most of the primary vaccine distributors were based, and secondly, a distant district which had high densities of poultry populations with large numbers of secondary distributors. all district‑level vaccine distributors were identified in close consultation with the governorates’ veterinary services. the vaccine distributors were classified into: primary (main) or secondary (sub‑) distributors based on their level of involvement in vaccine distribution. while the primary distributors get poultry vaccines directly from the manufacturing companies or their agencies in egypt and sell them in bulk, the secondary distributors serve as links between the primary distributors and poultry farmers. all participants in the study willingly participated and gave consent for the work to be published. they were informed that they have the right to discontinue participation at any time in the course of the study. vaccine sample collection and analysis eight primary distributors (2 from sharqia, 3 from dakahlia and 3 from fayoum) and 15 secondary distributors (5, 4, and 6 from sharqia, dakahlia and fayoum, respectively) were selected. a total of 41 viral vaccines of poultry were collected and recruited for the study, including 30 live vaccines and 11 inactivated/killed vaccines. of this total, 33 were monovalent (n  =  33) vaccines and 4 were bivalent (n  =  8) vaccines, and these consist of 24  nd, 6  infectious bursal disease (ibd), 4  infectious bronchitis (ib), 5  avian influenza, 1  pox and 1 avian encephalomyelitis (ae) vaccines. these vaccines originated from the 23  distribution points mentioned above. for a vaccine to qualify for inclusion in the study, it had to fulfil the following criteria: (a) it had to be collected from any of the 23  identified distributors within sharqia, dakahlia or fayoum, (b) it had to have been previously evaluated and had to have reserve batch samples kept at clevb to enable paired comparison, and (c) it had to be collected and transported immediately to the laboratory in a temperature‑monitored transport box whose temperature was maintained consistently between + 2 ºc to + 8 ºc. vaccine from any box with temperatures outside this range was not included in the study. 234 field evaluation of poultry viral vaccines ali et al. veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 negative controls and another set was inoculated with the diluent as negative inoculated control. all eggs inoculated with the same dilution were kept in separated tray, incubated at 37 ºc and candled every 24 hours. dead embryos during the first 48 hours post inoculation were discarded as non‑specific. all embryonated eggs were allowed to hatch and the chicks were placed in a brooder and monitored for clinical manifestation of ae for 3 days. the number of chicks that manifested clinical signs of ae were identified and recorded. the eid 50 was calculated as previously mentioned (reed and muench 1938). fowl pox vaccine (fpv) tenfold serial dilution (10‑1 to 10‑9) of the fpv was prepared. five spf chicken embryos (10‑12 day old) were inoculated with 0.2 ml from each dilution on the chorio‑allantoic membranes (cams). five days post inoculation, the surviving embryos were examined for evidence of pock lesions. cams with pock lesions were enumerated as positive and eid 50 was calculated as described above. vaccines were prepared. five (5) specific pathogen free (spf) chicken embryos (9‑11 days old) were inoculated with 0.2  ml of each suspension via the allantoic cavity using /egg. five un‑inoculated chicken embryos of the same age and source were kept as negative un‑inoculated controls and five others were inoculated with the diluent as negative inoculated control. all embryos were incubated at 37  ºc for 5‑8 days and observed daily by candling. nonspecific deaths during the first 24‑hours post‑inoculation were discarded. dead embryos and those that survived during the period of observation were examined for specific lesions associated with each virus. the median egg infectious dose (eid 50 ) was calculated individually according to the method described by reed and muench (reed and muench 1938). avian encephalomyelitis (ae) tenfold serial dilution (10‑1 to 10‑6) of the ae vaccine was prepared. ten spf chicken embryos (5‑6 days old) were inoculated through the yolk sac with 0.2 ml of each dilution. twenty embryos from the same source and age were kept un‑inoculated as table i. factors affecting vaccine immunogenicity, potency and titre in 23 veterinary vaccines storage points in egypt. variables with effect on vaccine efficacy and immunogenicity yes % no % p-value cold chain system has a purpose-built refrigerator (domestic/household refrigerators are not suitable for storage of vaccines). 0 0 23 100 na the refrigerator is situated away from direct heat source. 13 56 10 44 0.77 refrigerator plug is protected e.g. encased to prevent tampering; security marked “do not switch off ” or is hardwired (‘spurred’). 0 0 23 100 na refrigerator is cleaned and defrosted if necessary and regularly (at least every 6 months) 18 78 5 22 0.0001 only pharmaceutical items and biological are stored in the refrigerator i.e. no food, drink are stored in the refrigerator. 10 44 13 56 0.38 a thermometer is available to monitor temperature routinely. 5 22 18 78 0.0001 the thermometer can give the minimum, current and maximum readings daily. 0 0 23 100 na procedures are in place for at least daily recording of temperatures. 0 0 23 100 na recording form or equivalent is used. 0 0 23 100 na minimum, maximum and actual temperature is checked and recorded. 0 0 23 100 na procedures are in place for action to be taken in the event of abnormal temperatures. 0 0 23 100 na vaccines are stored in the cabinet of the refrigerator and not in the refrigerator doors. 4 17 19 83 < 0.0001 items are stored away from the back and sides of the refrigerator and the freezer compartment if it has one. 3 13 20 87 < 0.0001 vaccines are not being stored in the bottom drawer of the floor of refrigerator. unless it is a pharmaceutical refrigerator with custom made wire baskets. the ‘salad’ boxes of domestic refrigerators are not be used. 3 13 20 87 < 0.0001 no more than 66% of the internal volume of refrigerator is filled. 7 30 16 70 0.04 expiry dates are checked regularly at least once a month and these records are documented and filed away. 20 87 3 13 < 0.0001 stock rotation is carried out to ensure that the shortest expiry dates are used first. 20 87 3 13 < 0.0001 a responsibility is attached to a named person and a deputy to monitoring the refrigerator routinely. 12 52 11 48 0.77 electricity disruptions have been experienced. 22 96 1 4 < 0.0001 these electricity outages do occur periodically. 18 78 5 55 0.0001 the vaccine store has an alternative back-up system for power outages. 11 48 12 52 0.77 optimum refrigerator temperature kept between 2 ºc and 8 ºc 9 39 14 61 0.04 235 ali et al. field evaluation of poultry viral vaccines veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 had designed record form or actually checked daily temperature fluctuations (table i). 30% did not fill the refrigerator to more than 66% of its volume, while 87% checked expiry dates and conducted stock rotations routinely. in 96% of the refrigerators tested, electricity interruptions were experienced and 78% had repeated interruptions periodically. finally, only 48% had an alternative back‑up system for electricity interruptions (table i). forty‑one vaccines in 37 vials from 23 distributors and three governorates were assessed in parallel (pairs) in the study including 30 (73%) live and 11  (27%) inactivated vaccines. thirty‑three (80%) of the vaccines were monovalent vaccines and the remaining 20% were bivalent vaccines. twenty‑six (63%) of the vaccines came from 15 secondary distributors while the remaining 15 (37%) were from eight primary distributors. by numbers of sample tested, nd vaccines were 24 (59%), ibd (15%), avian influenza (12%), ib (10%) and 2% each for pox virus and the ae vaccines. some 15 (37%) of the vaccines came from sharqia, while 31.5% each came from dakahlia and fayoum (table ii and iii). vaccine titres reduced vaccine hi titres was observed in 20 (54%) of the 37 vials compared with their retained batch at clevb representing a total of 21 individual vaccines. of the 24 tested nd vaccine samples, eleven (45.8%) showed slight to marked decrease in vaccine titres while 66.7% of the six ibd vaccines failed to maintain titres compared with the retained batch at clevb (table ii and iii). none of the avian influenza vaccines samples maintained vaccinal or hi titres (n = 5) and none of the ib vaccines (n = 4) had reduced vaccinal titres. the only pox vaccine had failed with reduced titre but the ae vaccines maintained its vaccinal titre (table ii and iii). by volume from distribution sources and titres, no significant difference was observed between the reduced titres from primary distributors (53.3%) and secondary producers (50.0%) (p  =  0.84). seven of the 15 vaccines (46.7%) from sharqia, five of the 13 vaccines from fayoum and 9 of the vaccines from dakahlia had reduced vaccinal or hi titres (table  ii  and  iii). there was no specific pattern in vaccinal titre reduction with regards to vaccine producers since vaccines originating from eighteen out of nineteen individual producers had reduced vaccinal or hi titres. it would appear that the secondary distributors had some positive influence on the vaccinal or hi titres of the vaccines (table iv). external contamination (virus) of vaccine vials two out of the three pools showed no embryo tissue culture vaccine (ibd live vaccines) tenfold serial dilution (10‑1 to 10‑8) of the ibd was prepared in a tissue culture micro‑titer plate containing confluent monolayers of chicken embryo fibroblast cells. five wells were inoculated with each dilution using 100 µl/well. five wells were inoculated with positive controls according to the viral vaccine used and five wells were left un‑inoculated as negative control. the plate was sealed, incubated at 37  ºc in 5% co 2 atmosphere for 5‑6 days and examined daily for evidence of cytopathic effect (cpe). wells with cpe were counted and virus titre (tcid 50 ) was calculated (hierholzer and killing 1996). inactivated vaccine groups of twenty spf chicken (aged 3‑4 weeks) were vaccinated with different inactivated vaccine samples collected from the field and the retained samples at clevb. each group was kept in separated isolators and were monitored for 4 weeks. blood samples were collected on day 28 post‑vaccination, sera were harvested and post‑vaccination antibody responses were measured using haemagglutination‑inhibition (hi) test and homologous antigens as described by thayer and beard (thayer and beard 1998). external contamination (viral) of vaccine vials swabs from the external walls of the thirty live vaccines vials were collected and pooled into 3 pools. each pooled swab was inoculated into 5 spf eggs (9‑11 days old). after 24 hours, all dead eggs were examined for the presence of heamagglutinating agents and specific lesion of ib or ibd. allantoic fluids of survivor eggs were inoculated into spf eggs and subjected to further examination as described above. results descriptive statistics a total of 14 (61%) of the evaluated refrigerators in the primary and secondary distributor outlets did not maintain the optimum temperature (2  ºc‑8  ºc) for vaccine storage and all of the refrigerators were domestic (household or kitchen model) type and not purpose‑built vaccine refrigerators. the majority (56%) of the refrigerators were situated away from direct heat sources but none of these devices was protected from electrical surge. fifty‑six percent of the refrigerators had food and water for human consumption in the vaccine storage cabinets and only 22% had ordinary mercury thermometers (and not minimum‑maximum thermometers) for temperature monitoring. none of the respondents recorded temperature daily, 236 field evaluation of poultry viral vaccines ali et al. veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 in egypt and elsewhere. their use in poultry production is aimed at avoiding or minimizing the emergence of clinical disease at farm level and to increase production. we evaluated forty‑one poultry vaccines from the field in egypt and established inconsistencies in the level of vaccinal or hi titres as a measure of immunogenicity in 54% of these vaccines. while the observation cannot be linked directly to vaccine producers due to lack of more definitive empirical data, the same cannot be said of the distribution points. a number of the interviewed distributors confirmed that although they did not assess their vaccines for quality and lesions or produced haemagglutination when compared with the controls. the third pool showed slight haemagglutination activities with chicken red blood cells. the haemagglutinating agent was tested against specific nd h5n1 and h9n2 antisera. the contaminant was positive only against nd antiserum with a mean death time (mdt) of 98 hours. discussions vaccine remains an important component of poultry disease prevention and control measures table ii. comparison of live poultry field vaccine samples with batch samples retained at clevb. s/no. vaccine source governorate tested virus log titer of field sample* (eid 50 /dose) log titer of clevb sample (eid 50 /dose) storage temperature of field samples (ºc) 1 nd lasota sd sharqia ndv ≥ 7.5 ≥ 7.5 15.0 2 nd la sota pd sharqia ndv ≥ 7.5 ≥ 7.5 7.5 3 ibd sd sharqia ibd 5.3 5.3 10.4 4 nd lasota pd sharqia ndv 6.5 7.1 6.5 5 ib pd fayoum ibv ≥ 5.5 ≥ 5.5 4.5 6 nd lasota, pd fayoum ndv ≥ 7.5 ≥ 7.5 10.0 7 nd lasota sd fayoum ndv ≥ 7.5 ≥ 7.5 3.8 8 nd clone pd fayoum ndv 6.9 ≥ 7.5 10.5 9 nd hitchnerb1 sd sharqia ndv ≥ 7.5 ≥ 7.5 11.5 10 nd lasota sd sharqia ndv ≥ 7.5 ≥ 7.5 11.5 11 ibd sd sharqia ibd 5.1 5.3 11.5 12 ibd sd sharqia ibd 4.4 ≥ 4.8 5.3 13 nd lasota sd sharqia ndv ≥ 7.5 ≥ 7.5 8.2 14 ibd sd fayoum ibd ≥ 4.9 ≥ 4.9 9.5 15 nd lasota sd fayoum ndv ≥ 7.5 ≥ 7.5 8.9 16 ib sd fayoum ibv ≥ 5.5 ≥ 5.5 7.0 17 nd hitchnerb1 sd fayoum ndv 7.1 ≥ 7.5 5.5 18 nd lasota pd dakahlia ndv 7.1 ≥ 7.5 12.1 19 nd lasota sd dakahlia ndv 7.1 ≥ 7.5 11.5 20 ibd sd dakahlia ibd 4.6** 4.8** 8.0 21 ibd sd dakahlia ibd 4.6 ** 4.8** 18.0 22 nd hitchnerb1 sd dakahlia ndv 6.9 ≥ 7.5 18.0 23 nd lasota sd dakahlia ndv ≥ 7.5 ≥ 7.5 7.6 24 nd lasota sd dakahlia ndv 7.1 ≥ 7.5 8.5 25 ib + nd clone sd fayoum ndv 7.1 7.1 16.0 sd fayoum ibv ≥ 5.5 ≥ 5.5 16.0 26 pox + ae pd dakahlia pox 4 4.2 17.0 pd dakahlia aev 3.7 3.7 17.0 27 ib + hitchnerb1 pd dakahlia ndv ≥ 7.5 ≥ 7.5 15.0 pd dakahlia ibv ≥ 5.5 ≥ 5.5 15.0 nd = newcastle disease; ibd = infectious bursal disease; ib = infectious bronchitis; ae = avian encephalomyelitis; pd = primary distributor; sd = secondary distributor; ae = avian encephalomyelitis virus; ndv = newcastle disease virus; ibv = infectious bronchitis virus; pox = pox virus. *egg infectious dose (eid) or tissue culture infectious dose (tcid) was used for evaluation; **tcid 50 /dose. note: clebv samples are paired vaccines from the same batch as those collected from the field. they were previously stored as back-up following the evaluation of the samples to be released for farmer use. storage temperature was taken at the same time the questionnaire was administered and does not reflect all the temperature ranges the vaccine have been subjected to previously. 237 ali et al. field evaluation of poultry viral vaccines veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 keep optimum range, negative correlation existed with the measured titres. we concluded that a one‑time evaluation of vaccine storage refrigerators may not be a good indicator to determine continuous maintenance of cold chain and assess the effectiveness of stored vaccines. it is highly likely that the stored vaccines were subjected to freeze‑thawing during the process of cleaning and defrosting, and during electricity interruptions, and the study participants confirmed this hypothesis. previous reports from egypt have confirmed that vaccine failed to protect poultry effectively against challenge pathogens in the field (kim et  al. 2010, arafa et al. 2012, kilany et al. 2015). furthermore, because domestic refrigerators were designed and built for food and drink storage, they may have not met the special temperature requirements for vaccines. purpose built vaccine refrigerators come with temperature regulation mechanisms that ensure narrow variations in internal temperatures, with an on‑going air circulation system that ensures even distribution of temperature which should prevent vaccine from freezing2. in the event of multiple opening and closing of refrigerators for purposes of sale, taking out of food and water, and during the process of cleaning and defrosting, there may be a shift in optimum temperature with implications for stored vaccines. in addition, because no defined procedure or record was established for cleaning and defrosting, and no record of where vaccines effectiveness routinely, there was a likely failure due to intermittent break in cold chain as experienced during electricity interruptions and during vaccine evacuations to clean out the fridge. between 2006 and 2014, a total of 832 billion doses of avian influenza vaccine were released and used to control avian influenza outbreaks, yet the virus continues to circulate in the egyptian poultry (clevb unpublished data). the failure to control outbreaks despite these efforts were associated, among others, with partial immunisation due to low quality vaccine, suboptimal dosages, improper vaccination, continuous shedding and silent transmission, possible mutation of the viruses, and field virus variants which escaped vaccine‑induced immunity (lee et  al. 2004, smith et  al. 2006, taha et  al. 2007, escoria et  al. 2008, domenech et  al. 2009, rudolf et  al. 2010, kilany et  al. 2015). we have provided more revelations on the role of distribution chain. while our ‘on‑the‑study’ survey of refrigerators’ temperatures indicated that 61% of them failed to table iii. comparison of hemagglutination-inhibition arithmetic mean titers of field samples and retained stock samples. s/no. vaccine source governorate virus log titer of the tested field sample (hi*) log titer of clevb sample (hi*) storage temperature of field samples (ºc) 28 locally produced ih5n1 pd dakahlia ai_h5 5 8 17.0 29 imported h5n1 pd sharqia ai-h5 7 8.3 6.5 30 imported h5n1 sd sharqia ai-h5 8.25 8.5 5.3 31 imported h5n1 sd sharqia ai-h5 10 11 4.5 32 imported nd sd sharqia ndv 6.5 6.5 15.0 33 imported nd broiler sd fayoum ndv 7 7.5 8.9 34 imported ndv pd dakahlia ndv 6.4 6.5 15.0 35 imported nd sd fayoum ndv 6.25 6.75 7.0 36 imported nd sd fayoum ndv 7.1 7.1 16.0 37 locally produced h9+nd pd sharqia ai-h9 8.75 9.25 7.5 pd sharqia ndv 8.5 8.5 7.5 *hi = arithmetic mean hemagglutintion inhibition titerlog 2 ; pd=primary distributor; sd= secondary distributor. table iv. odds ratio of effective titres of the field vaccines against some variables using binary logistic regression. variable odds ratio standard error p-value 95% confidence interval optimum storage temperature 0.49 0.32 0.28 0.13; 1.78 primary distributors 0.75 0.49 0.66 0.21; 2.68 secondary distributors 1.33 0.87 0.66 0.37; 4.77 pairwise correlation coefficients between effective titres of the field vaccines and optimum storage temperature, primary distributors and secondary distributors were - 0.1705, - 0.0692 and 0.0692, respectively. 2 ontario ministry of health and long term care. vaccine storage and handling guidelines. gsin: 7540‑19600e may 2013. queen’s printer for ontario, 2013. http://www.health.gov.on.ca/en/pro/programs/ publichealth/oph_standards/docs/reference/vaccine%20_storage_ handling_guidelines_en.pdf. 238 field evaluation of poultry viral vaccines ali et al. veterinaria italiana 2019, 55 (3), 231‑239. doi: 10.12834/vetit.999.5287.1 repeated break in cold chain and previous workers demonstrated this association (thakker and woods 1992). finally, we did not answer whether exposure to adverse high temperature or freezing, and the length of time of exposure actually damaged vaccine and affected its potency in egypt. we advocate for a broader study to assess these limitations and possibly conduct experimental vaccination and challenge in a controlled environment. conclusions while we acknowledged that vaccines were affected by a variety of factors, in egypt, it would appear that viral vaccines of poultry were less effective and efficacious due to poor handling, break in cold chain and poor distribution networks. an overhaul of the vaccine value chain is necessary to improve poultry vaccine efficacy in egypt. statement of animal rights ethics approval was obtained as part of the fao‑ectad‑govs‑nlqp programme number osro/ egy/101/usa. grant support this work was supported by the united states agency for international development (usaid) (grant number aid‑263‑io‑11‑00001, mod.#3) in the framework of osro/ egy/101/usa, through projects jointly implemented by the fao, general organization for veterinary services and nlqp. acknowledgements we thank the staff of clevb for their support and technical assistance. the authors express their sincere gratitude to usaid for its continued support in addressing the endemic hpai situation in egypt. fof was supported by a support for smallholder poultry development personal development grant (associate poultry adviser) facilitated by international network for family poultry development (infpd), the food and agriculture organization of the united nations (fao) and the international fund for agricultural development (ifad) grant number: gcp/int/197/ifa. were kept while these processes were on‑going existed, vaccine immunogenicity, shelf life, potency and titres may have been affected. we have also provided evidence of contaminant on the vial of vaccine sold in egypt. specifically, a lentogenic strain of nd virus was detected. whether this is a vaccine virus (lasota virus, mdt  =  103 hours) or field virus was not established in this study, however, this contamination posed potential risk to vaccinated poultry. because most of live poultry vaccines are used via drinking water, and the vials are immersed in the tanks before opening, it may become necessary to decontaminate the exterior of vaccine vials before use. such decontamination must be followed by adequate rinsing to eliminate potential residual effect of decontaminants on vaccines. distributors, particularly the secondary ones, rarely give attention to personal hygiene and often place extraneous materials in the vaccine fridges. these activities increase the burden of contamination of vaccine vials. approximately 74% of poultry production in egypt remains with the small‑scale producers who rely heavily on these small‑scale secondary distributors for medicine and poultry vaccines (ali et al. 2013). because many of the poultry producers operate with low to moderate biosecurity in farms and most of their animal products end up in the live bird markets, we advocate for pre‑slaughter screening and inspection for zoonotic viruses in poultry. the contribution of low quality vaccines, poor biosecurity measures and the consequent protection failure, disease outbreaks, economic losses, and potential silent spreading of pathogens continue to pose threat to human and animal health. small‑scale distributors must be trained to know their potential roles in poultry diseases transmission and how to conduct responsible storage and vaccine distribution chain. such education may be facilitated by vaccine companies and commercial poultry associations. this study was subjected to certain limitations: we assessed vaccinal and hi titres as a measure of vaccine efficacy and conducted live bird experimental vaccination where necessary but did not follow up with complete challenge studies. in addition, we established the loss in vaccinal titres and associated it with distribution chain but couldn't draw conclusion on the role of cold‑chain because no correlations existed between the two. however, evidence from the distributors confirmed 239 ali et al. field evaluation of poultry viral vaccines veterinaria 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subramanyam k. 1989. vaccine distribution: an operations research study. rev infect dis, 11 (suppl 3), s623‑s628. taha m., ali a., nassif s., khafagy a., elham a., el‑ebiary e., el‑nagar a. & el‑sanousi a. 2008. evolution of new escape mutant highly pathogenic avian influenza h5n1 viruses with multiple nucleotide polymorphisms in egypt, december 2007. in the second international conference of virology (emerging and exotic viral infection: challenging threats of human, animal and plant health), giza, egypt, april 5‑6. issn 0505-401x 28 il canile rifugio, procedure e protocolli filomena iannino, elisabetta finocchi mahne, paolo dalla villa, enzo ruggieri, stefania salucci, greta berteselli, cristina rapagnà, maria luisa danzetta, fabio bellucci, collana di monografie il canile rifugio, procedure e protocolli filomena iannino, elisabetta finocchi mahne, paolo dalla villa, enzo ruggieri, stefania salucci, greta berteselli, cristina rapagnà, maria luisa danzetta, fabio bellucci jan havicksz. steen (leida, 1626 c. leida, febbraio1679) the merry family, l’allegra famiglia, 1668 olio su tela, cm 141 × 110,5 rijksmuseum, amsterdam questa famiglia turbolenta sta facendo molto chiasso: il padre canta a squarciagola mentre alza un bicchiere; la madre e la nonna chiacchierano tra loro; i bambini suonano uno strumento a fiato e fingono di fumare una pipa. il biglietto appeso alla mensola del caminetto recita la morale della storia: “come canta il vecchio, così canterà il giovane”. come si comporteranno i bambini se i loro genitori danno un cattivo esempio? si ringrazia il rijksmuseum di amsterdam per l’immagine di copertina. www.rijksmuseum.nl collana di monografie 28 comitato direttivo silvio borrello, nicola d’alterio, antonia ricci direttore responsabile giovanni savini segreteria di redazione monica bucciarelli, laura ambrogi amministrazione istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale” campo boario, 64100 teramo, italia progetto grafico e impaginazione paola di giuseppe www.veterinariaitaliana.izs.it/index.php/vetit il canile rifugio, procedure e protocolli/ filomena iannino*, elisabetta finocchi mahne, paolo dalla villa, enzo ruggieri, stefania salucci, greta berteselli, cristina rapagnà, maria luisa danzetta, fabio bellucci [teramo] : istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale”, ©2020. 56 pp. (collana di monografie; 28). * f.iannino@izs.it isbn 9788893650038 questa rivista è nata nel 1950 con il nome di croce azzurra. dal 1954 si chiamerà veterinaria italiana. istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale” campo boario, 64100 teramo, italia telefono +39 0861 3321, fax +39 0861 332251 www.izs.it prefazione il presente testo, redatto in collaborazione con medici veterinari liberi professionisti e con il ministero della salute, introduce talune procedure per la corretta gestione dei cani all’interno di canili rifugio. la stesura di appositi protocolli rappresenta un sistema efficace per valutare eventuali criticità e porre in essere azioni correttive da parte di chi si occupa direttamente degli animali, oltre che essere di ausilio nel corso dei controlli operati dalle autorità competenti. questo lavoro fa seguito al manuale di gestione dei canili rifugio pubblicato su “veterinaria italiana” nel 2014 e ne ripropone l’impostazione. i contenuti, tuttavia, sono stati rivisitati tenendo conto dell’apporto di altre istituzioni, quali il ministero della salute e dei cambiamenti sopraggiunti in questi anni a livello normativo. in tale ottica, rivestono primaria importanza i protocolli relativi al benessere animale il cui approfondimento è rimandato al protocollo shelter quality. introduzione ............................................................................................................................................................................................ 7 scopo del manuale ...................................................................................................................................................................... 9 parte i gestione della struttura requisiti strutturali ................................................................................................................................................................................................................. 12 gestione documenti e archiviazione ............................................................................................................................................. 14 piano di emergenza e di evacuazione ......................................................................................................................................... 15 gestione rifiuti .................................................................................................................................................................................................................................. 16 controllo animali infestanti ................................................................................................................................................................................. 18 sicurezza ........................................................................................................................................................................................................................................................ 20 formazione del personale ...................................................................................................................................................................................... 22 controllo accessi .......................................................................................................................................................................................................................... 23 parte ii aspetti sanitari gestione dei nuovi ingressi ................................................................................................................................................................................. 26 gestione sanitaria ..................................................................................................................................................................................................................... 27 benessere: attività psicofisiche e valutazione ............................................................................................................ 31 alimentazione ................................................................................................................................................................................................................................... 33 adozioni .......................................................................................................................................................................................................................................................... 35 eutanasia ....................................................................................................................................................................................................................................................... 37 cessione del cane ...................................................................................................................................................................................................................... 38 parte iii normativa normativa regionale ........................................................................................................................................................................................................... 41 normativa nazionale .......................................................................................................................................................................................................... 50 parte iv bibliografia bibliografia ................................................................................................................................................................................................................................................ 55 collana di monografie 28 veterinaria italiana, collana di monografie, monografia 28, 2020 7 il canile rifugio, procedure e protocolli 30 anni dalla promulgazione della l.q. 281/91, le iniziative fino ad oggi attuate non sono state sempre sufficienti per la prevenzione e per il contrasto del randagismo. è necessario premettere che l’obiettivo della lotta al randagismo è quello di non avere cani vaganti sul territorio e utilizzare i canili solo per ospitare cani smarriti in attesa di rintracciarne i proprietari e cani i cui proprietari hanno rinunciato alla proprietà per qualsiasi motivo (es.malattia del proprietario, indigenza, morte del proprietario, ricovero del proprietario presso strutture sanitarie, ecc.). per ottenere ciò è essenziale • diffondere la cultura del possesso responsabile degli animali attraverso la corretta formazione dei proprietari; • aumentare l’identificazione e registrazione in anagrafe degli animali d’affezione; • sterilizzare i cani vaganti; • promuovere la sterilizzazione dei cani di proprietà. si ricorda che tra le cause più importanti del sovraffollamento dei canili vi è la mancata identificazione dei cani che non permette di restituirli ai padroni e l’abbandono di cucciolate indesiderate. in ogni caso, solo quando le autorità territorialmente competenti applicheranno tutte le disposizioni previste dalla normativa vigente, il randagismo comincerà a diminuire. fino ad allora i canili rifugio dovranno assolvere alla difficile funzione di garantire il benessere dei cani ospitati per lunghi periodi. la sfida di questi ultimi anni è stata quella di migliorare i servizi offerti sia agli animali che ai cittadini, definendo percorsi che possano portare all’ottimizzazione dei livelli gestionali fino alla certificazione da parte di un ente terzo. è essenziale che sia effettuata una costante verifica del mantenimento delle competenze e dei requisiti e la puntuale risoluzione delle non conformità. il soddisfacimento dei requisiti deve essere misurabile e quindi dimostrain italia la lotta al randagismo e la tutela degli animali sono principi cardine definiti dalla legge quadro 14 agosto 1991, n. 281 che ha rappresentato un importante passo in avanti dal punto di vista etico-culturale, riconoscendo agli animali d’affezione il diritto alla vita. la legge 281/91 prevede l’esplicito divieto di soppressione dei cani randagi vaganti quale misura di controllo del randagismo e il loro impiego nella sperimentazione. la diretta conseguenza di ciò è che i cani rinvenuti vaganti, catturati e non restituiti ai proprietari sono ospitati presso canili (per periodi troppo spesso lunghi) in attesa di un’eventuale adozione o fino alla morte. nelle leggi regionali di attuazione della l.q. 281/91 si distinguono principalmente due tipologie di canili, denominate canile sanitario e canile rifugio. il canile sanitario è una struttura di ricovero di prima accoglienza gestita, dal punto di vista sanitario, dalla asl territorialmente competente. nel canile sanitario vengono ricoverati i cani immediatamente dopo la cattura e sono effettuati l’identificazione, la visita clinica, i trattamenti profilattici e, ove previsto dalla norma regionale, la sterilizzazione. il canile rifugio è una struttura destinata al ricovero dei cani fino all’adozione (quindi spesso per lunghi periodi) realizzata e gestita da comuni, singoli o associati, o da privati. nel rifugio l’assistenza veterinaria è assicurata da un medico veterinario libero professionista convenzionato con la struttura e il servizio veterinario della asl territorialmente competente effettua la vigilanza ai sensi del dpr 320/54. nel presente manuale ci occuperemo del canile rifugio, struttura che deve promuovere le adozioni e garantire il benessere degli animali ospitati. in talune regioni d’italia, la presenza di canili rifugio che ospitano animali anche oltre il limite della capienza massima, unitamente a un consistente numero di cani vaganti sul territorio, lascia intuire che, a distanza di quasi introduzione il canile rifugio, procedure e protocolli 8 veterinaria italiana, collana di monografie, monografia 28, 2020 sità delle norme regionali, il gestore di una struttura che ospita 250 cani (destinando loro 3.5 mq/soggetto) potrebbe essere accusato di maltrattamento o considerato un’eccellenza, a seconda del territorio in cui sorge il proprio canile rifugio. la valutazione degli animali (indicatore diretto), anche in relazione al rispetto dei requisiti obbligatori e di quelli facoltativi, è un fattore determinante per un giudizio globale, fermo restando che alla mancanza di un requisito previsto dalla legge dovrebbe conseguire una sanzione amministrativa. è auspicabile che i canili rifugio di vecchia concezione vengano sostituiti da parchi-canile, piccole strutture (50/75 cani) che permettono una gestione ottimale degli animali da parte degli operatori, oltre a presentarsi più fruibili per le visite finalizzate a eventuali affidi. il parco canile potrebbe ospitare, in base anche alle norme regionali, attività autofinanzianti quali corsi di formazione, ambulatori veterinari, pensioni temporanee, cimiteri per cani, ecc. bile alle parti interessate. la normativa vigente stabilisce i requisiti minimi “obbligatori” che ogni struttura che ospita animali deve possedere ma per una gestione idonea è opportuno anche soddisfare taluni “requisiti facoltativi”. il ciclo pdca (plan-do-check-act) rappresenta un sistema efficace per il miglioramento continuo della qualità nel lungo periodo. nel canile, in quanto struttura residenziale, gli ospiti rappresentano la componente di maggior rilievo, quella oggetto di tutela in tutte le attività e nell’intera organizzazione, e quindi deve essere assicurata la corretta relazione intra e interspecifica; per tali motivi, l’applicazione della normativa deve essere considerata solo il primo passo di un percorso di qualità. solitamente l’attività di vigilanza si basa sulla verifica degli aspetti strutturali, della corretta identificazione degli animali (indicatori indiretti di benessere) e la relativa rilevazione di non conformità che possono sfociare in contestazioni anche di carattere penale. tale approccio tuttavia può determinare situazioni paradossali, in quanto data la diverveterinaria italiana, collana di monografie, monografia 28, 2020 9 il canile rifugio, procedure e protocolli mero titolo conoscitivo, uno stralcio della nota della prot. n. 5909 del 31/3/2010 emanata dal ministero della salute direzione generale della sanità animale e dei farmaci veterinari: […] la legge quadro in materia di animali d’affezione e prevenzione del randagismo – legge 281/91 – riguardo alle condizioni di vita degli animali ospitati nei canili, stabilisce che la suddette strutture debbono garantire buone condizioni di vita per i cani e il rispetto delle norme igienico sanitarie; • l’art. 13 del trattato di lisbona, ratificato con legge 2008, riconosce le esigenze in materia di benessere degli animali in quanto esseri senzienti; • la tutela del benessere animale comporta dei costi finanziari, che devono essere sempre determinati secondo il principio costituzionale della buona amministrazione (art. 97 cost.), ossia attenendosi ai criteri di efficienza (rapporto tra risultati raggiunti e risorse impiegate) ed efficacia (capacità di raggiungere gli obiettivi prefissati). premesso quanto sopra, ad avviso di questa direzione, le amministrazioni competenti debbono perseguire un razionale equilibrio tra l’esigenza economica di contenimento dei costi di gestione, al fine di evitare sprechi ed illecite speculazioni, e la tutela del benessere degli animali, in ottemperanza al vincolo giuridico di garantire buone condizioni di vita degli stessi e il rispetto delle norme igienico sanitarie. si ricorda, per inciso, che tale principio è stato affermato anche in diverse sentenze, come, ad esempio, quella n. 1389/2003 del tar puglia, sede di lecce. in ordine alla determinazione di un congruo costo di gestione, precisando che questa direzione nella fattispecie non ha competenza, si ritiene tuttavia opportuno riportare quanto emerso da un’indagine conoscitiva effettuata in ambito nazionale e cioè che, tenendo conto dei costi medi per personale, alimentazione, la finalità di questo manuale è quella di essere una guida facilmente consultabile per gli operatori del settore a qualsiasi livello di responsabilità e per le amministrazioni che si accingono alla costruzione o al risanamento di canili con la prerogativa di ottimizzare la gestione delle strutture attraverso un percorso di qualità. poiché ogni canile ha peculiarità diverse che riguardano collocazione, grandezza, situazione epidemiologica del luogo, ecc., devono essere elaborate specifiche procedure per ogni struttura, pertanto le indicazioni fornite nel presente manuale dovranno essere adattate e integrate alle singole realtà. tutti i protocolli devono essere sviluppati e dettagliati con chiarezza al fine di sviluppare e mantenere standard elevati di gestione del benessere animale e condizioni ottimali di lavoro. nell’elaborazione dei suddetti protocolli si devono considerare i seguenti punti: • creare un ambiente di vita per gli animali ospitati che rispetti le loro esigenze etologiche tale da evitare lo sviluppo di turbe comportamentali, risolverle o almeno non aggravare quelle già esistenti al momento del ricovero; • innalzare il livello di adozioni consapevoli del canile, creando un ambiente consono per le visite del pubblico e aumentando l’adottabilità; • creare un ambiente salubre nel quale si eserciti una adeguata prevenzione e gestione sanitaria; • creare un ambiente sicuro per gli operatori; • stabilire il percorso che porti il canile alla certificazione da parte di un ente terzo indipendente. nella prima e seconda parte del testo sono riportati i fattori indiretti e diretti che concorrono alla caratterizzazione generale di una struttura e soprattutto del benessere degli animali. si coglie infine l’occasione per riportare, a scopo del manuale il canile rifugio, procedure e protocolli 10 veterinaria italiana, collana di monografie, monografia 28, 2020 rimenti garantito un adeguato mantenimento degli animali con importi giornalieri più bassi, a condizione che vi sia capacità gestionale e presenza di personale, dipendente e/o volontario, adeguatamente formato. […] cure e profilassi sanitarie, beni di consumo ed utenze varie, ai fini di una buona gestione risulta appropriato un costo oscillante approssimativamente fra 3,50 e 4,50 euro giornalieri per cane, anche se in taluni casi può essere paparte i gestione della struttura il canile rifugio, procedure e protocolli 12 veterinaria italiana, collana di monografie, monografia 28, 2020 portante è l’orientamento della struttura che dovrebbe essere rivolto a sud e nelle regioni più calde a sud-est. in considerazione della necessità di permettere l’accesso al pubblico, è utile predisporre viali di percorrenza per i visitatori, rivestiti di materiale drenante o ricoperti di ghiaia e parcheggi esterni. i canili rifugio dovrebbero prevedere almeno le seguenti strutture: • un locale adibito a ufficio e a ricevimento del pubblico • un’area per le operazioni di pulizia, lavaggio e disinfezione dei materiali e delle attrezzature e relativo deposito • un locale adibito a cucina o a preparazione dei pasti con annesso deposito per alimenti • un locale refrigerato per lo stoccaggio temporaneo degli animali morti • un locale di deposito rifiuti • spogliatoio e servizi igienici • infermeria/ambulatorio veterinario con possibilità di degenza • locale di deposito farmaci o attrezzatura sanitaria non accessibile a personale non autorizzato • box per il ricovero di singoli soggetti o di più soggetti preferibilmente serviti da aree di sgambamento • reparto isolamento • reparto cuccioli oltre a quanto già previsto dalle singole leggi regionali, al cui capitolo si rimanda per eventuali approfondimenti, è assolutamente necessario tenere in opportuna considerazione una razionale disposizione delle singole strutture, dei locali e delle relative attrezzature come indicato di seguito: • gli spazi siano adeguati alle esigenze fisiche ed etologiche dei cani. le esigenze di spazio variano secondo la mole e l’indole i requisiti previsti dalle norme costituiscono la base per la realizzazione di un canile rifugio che rispetti la nuova visione del rapporto uomo animale e che rappresenti un luogo di lavoro sicuro per tutti gli operatori. il canile rifugio deve essere un luogo aperto ai cittadini, deve comunicare l’impegno a rispettare gli animali nelle loro esigenze fisiche ed etologiche e deve essere il luogo di riferimento per l’educazione della popolazione sul possesso responsabile dei cani. è auspicabile che i canili di nuova costruzione prevedano aree e attrezzature per l’organizzazione di eventi formativi ed educativi e, se costruiti da privati, anche ambulatori o cliniche veterinarie aperti al pubblico. inoltre i canili dovrebbero sempre avere dei locali dedicati agli adempimenti amministrativi e alla conservazione della documentazione relativa alle attività della struttura (uffici). i canili sono classificati dal d.m 5 settembre 1994 “industrie insalubri di i classe” in quanto produttori di cattivi odori, rumori e rifiuti solidi e liquidi, pertanto le strutture di nuova costruzione devono essere collocate lontano dalle abitazioni e dai corsi d’acqua superficiali. molti regolamenti comunali (piani regolatori, regolamenti di tutela igienico sanitaria ecc.) prevedono che queste strutture siano circondate da fasce di verde. è auspicabile comunque che i canili siano circondati da alberi ad alto fusto e siepi, in modo da creare ampi spazi di ombra e integrarli visivamente all’ambiente circostante, creando contemporaneamente un valido isolamento acustico. nella progettazione dei canili di nuova costruzione deve essere considerato con attenzione il raggiungimento di una buona ventilazione ottenuta con accorgimenti strutturali o grazie ad una ben studiata collocazione dell’intera struttura. ciò migliora la situazione igienicosanitaria, allontanando i cattivi odori, mitigando le alte temperature e contribuendo a rendere più gradevole l’ambiente per gli animali, gli operatori e i visitatori. altrettanto imrequisiti strutturali veterinaria italiana, collana di monografie, monografia 28, 2020 13 il canile rifugio, procedure e protocolli • le aree pavimentate siano costruite in materiale lavabile, disinfettabile, non sdrucciolevole e lievemente pendenti (la pendenza non deve superare il 3%) in modo da permettere il rapido allontanamento delle urine e delle acque di lavaggio presso i canali di scolo. è da sconsigliare l’uso di piastrelle in quanto possono rompersi per urti meccanici o possono scollarsi con la pressione dell’idropulitrice. una buona soluzione può essere rappresentata da cemento trattato con resine speciali anche colorate (gradevoli alla vista), resistenti in ambienti esterni e che proteggono il cemento evitando la formazione di buche. • gli spigoli e gli angoli siano arrotondati in modo da evitare il ferimento dei cani e degli operatori nonché gli accumuli di sporco. • tutti i locali di servizio (cucine, ambulatori, ecc.) e i box chiusi siano protetti da zanzariere a maglie fitte che impediscano l’ingresso di zanzare e flebotomi. • tutte le parti erbose siano mantenute costantemente rasate al fine di evitare che diventino ricettacolo di parassiti quali zecche. • le porte di accesso ai box abbiano requisiti di robustezza e facilità di apertura e chiusura da parte degli operatori ma non dei cani. • i cancelli di accesso ai box abbiano la parte inferiore in materiale resistente agli urti e quella superiore in rete elettrosaldata con maglie che impediscano ai cani di infilarci il muso o le zampe (ad esempio con maglie di 4 × 4 cm). • le reti di recinzione sovrastino un muretto di cemento o laterizi. tale muretto deve essere adeguatamente interrato per impedire che gli animali scavino gallerie. • siano sempre presenti aree di sgambamento esterne per consentire a tutti i cani l’esposizione all’aria aperta e un adeguato svolgimento dell’attività fisica. • i box siano forniti di cucce in materiale lavabile e disinfettabile. è auspicabile che le cucce abbiano una parete (o il tetto) smontabile in modo da rendere agevole la pulizia. in aggiunta a ciò, l’esperienza di dei cani e, pertanto, non è possibile riferire misure ottimali valide in ogni situazione. e’ stato osservato che i box suddivisi in tre aree (interna, esterna coperta e aperta scoperta) offrono un migliore riparo dalle intemperie e dal caldo estivo. inoltre, al solo scopo di dare un’indicazione, si ricorda che la deliberazione n. 353 del 2 aprile 2013 della regione emilia-romagna dispone che box individuali, con area di sgambamento aggiuntiva, abbiano dimensione minima di 9 m2, misura che si avvicina maggiormente alle esigenze di spazio degli animali rispetto a quelle previste da numerose leggi regionali. • le strutture di nuova costruzione siano progettate in modo da privilegiare box multipli (2-5 cani). in linea generale nei box multipli i cani dovrebbero disporre unitariamente almeno dello stesso spazio che avrebbero nei box singoli. • siano presenti aree o locali che mantengano temperature adeguate per cani con esigenze particolari (per. es. non al di sotto dei 15° gradi per cuccioli e cani di taglia piccola e a pelo raso). • siano previste delle aree destinate ai cuccioli, adeguatamente arricchite con attrezzature ludiche per il corretto sviluppo comportamentale anche al fine di ottenere un indice di adottabilità ottimale. • siano presenti delle aree di prima accoglienza nelle quali far sostare i cani appena arrivati dal canile sanitario in attesa di essere inseriti in un gruppo o nelle quali formare nuovi gruppi • il reparto destinato ai soggetti problematici sia collocato in un’area distante dall’ingresso, lontano da fonti di stress e non accessibile ai visitatori. cespugli e piante basse possono essere utili al cane timoroso o ansioso per sentirsi più al sicuro. • i box destinati ai cani pericolosi siano contigui, separati da doppie porte (a ghigliottina) azionabili dall’esterno in modo che gli operatori possano trasferire i cani da un box all’altro durante le pulizie, lavorando in sicurezza. il canile rifugio, procedure e protocolli 14 veterinaria italiana, collana di monografie, monografia 28, 2020 procedure per garantire il costante rifornimento di acqua pulita. • siano presenti uffici predisposti per l’accoglienza del pubblico. campo indica che sono da preferirsi quelle a tettuccio piano, quindi fruibile dal cane, rispetto a quelle a tettuccio spiovente. • siano previsti dei sistemi di abbeverata automatica o, in alternativa, ci siano delle • piano annuale di prevenzione (controlli periodici e protocolli profilattici); • piano alimentare; • protocollo di igiene ambientale e disinfezione; • procedura per lo sgambamento (che permetta di verificare l’attività fisica di tutti i cani e la relativa frequenza); • procedura per le adozioni; • procedura per la gestione rifiuti; • procedura di controllo degli animali infestanti; • procedure di biosicurezza per gli operatori; • piano di formazione per l’anno in corso e archivio degli anni precedenti; • procedura di gestione dell’ingresso visitatori; • piano di emergenza ed evacuazione; • procedura per la corretta tenuta dei registri. negli stessi uffici devono inoltre essere conservati i documenti e gli atti che registrino il regolare svolgimento dei processi sopra menzionati al fine di monitorare le attività, individuare le eventuali azioni correttive e rendere l’intero processo trasparente. è auspicabile che piani, procedure e tutti gli atti relativi siano gestiti in maniera informatizzata per permettere una gestione più precisa e all’occorrenza una immediata disponibilità dei dati. presso il canile devono essere conservati i seguenti documenti o le relative copie: • autorizzazione sanitaria rilasciata dall’autorità competente; • organigramma con l’identificazione del personale organico e dei volontari; • convenzione di gestione; • atto di incarico del direttore sanitario; • registro di carico/scarico dei cani (anche informatizzato); • registro farmaci (quando previsto); • registro rifiuti speciali; • documento informativo sull’orario e la modalità di apertura al pubblico; • registro dei visitatori e percorso; • documento informativo sulla procedura di affidamento; • documento di valutazione del rischio per gli operatori; • certificazioni di conformità degli impianti; • schede sanitarie cartacee o informatizzate • regolamento della struttura con annessi protocolli (sanitario, mansionario, gestionale); • autorizzazione ministeriale ad ospitare cani a seguito di sequestro per maltrattamento ai sensi del dm 2/11/2006 (se prevista). presso i locali adibiti ad uffici devono essere inoltre conservati tutti i documenti di programmazione e organizzazione dei seguenti processi: gestione documenti e archiviazione veterinaria italiana, collana di monografie, monografia 28, 2020 15 il canile rifugio, procedure e protocolli il canile può essere coinvolto in disastri ambientali di varia natura (dissesti idrogeologici, esondazioni, terremoti, avvelenamento atmosferico, ecc.) che richiedono una immediata evacuazione. in questo contesto due aspetti appaiono rilevanti: • le modalità di evacuazione; • la sistemazione dei cani evacuati. la modalità di evacuazione deve essere descritta da una apposita procedura che differisce a seconda dell’organizzazione del canile. per la sistemazione dei cani evacuati è auspicabile avere delle apposite convenzioni con le strutture ricettive più vicine. strumenti indispensabili per una pronta ed efficiente evacuazione sono: • gli elenchi degli operatori del canile aggiornati e prontamente accessibili che includano i loro indirizzi e recapiti telefonici, • la planimetria del canile (vedi scheda tecnica) affissa all’ingresso della struttura; • le attrezzature per il trasferimento degli animali (guinzagli, gabbie e automezzi) in numero adeguato al numero di cani; • elenco dei canili, allevamenti e pensioni più vicini in cui venga anche indicata la capacità di accoglienza delle strutture è necessario fare particolare attenzione che i percorsi di fuga siano sempre mantenuti liberi e che gli idranti e gli estintori siano sottoposti a controllo periodico, secondo quanto previsto dalla normativa in vigore e dalla casa produttrice. la procedura di evacuazione e i singoli allegati devono essere aggiornati ogni volta che si verifichino le seguenti condizioni: • variazioni negli edifici per quanto attiene sia alle strutture sia agli impianti; • variazioni organizzative; • nuove norme che richiedono modifiche; • mutazioni nelle esigenze di sicurezza; • significative variazioni numeriche degli animali nei box; • variazioni nei recapiti degli operatori; • variazioni nei percorsi di fuga. piano di emergenza e di evacuazione la planimetria deve riportare: i box con l’indicazione della capienza; gli altri locali e le relative destinazioni di uso; le uscite di emergenza; i percorsi di fuga (colorati); le attrezzature antincendio (estintori, idranti, ecc.); la segnaletica di sicurezza; i punti di erogazione dell’acqua; la posizione del quadro con sganciatore elettrico; la posizione del rubinetto per la chiusura del gas; le cassette di medicazione. scheda tecnica i. planimetria. il canile rifugio, procedure e protocolli 16 veterinaria italiana, collana di monografie, monografia 28, 2020 i rifiuti prodotti dal canile sono riconducibili alle seguenti categorie: • rifiuti urbani o rifiuti assimilati o assimilabili ai rifiuti solidi urbani; • rifiuti sanitari pericolosi; • rifiuti speciali. rifiuti assimilati o assimilabili ai rifiuti solidi urbani rientrano in questa tipologia i rifiuti che derivano da attività di routine del canile e non presentando rischio tossico o infettivo e sono assoggettati alle modalità di gestione dei rifiuti urbani. la raccolta ed il deposito devono essere organizzati in modo tale da evitare cattivi odori, disordine e inquinamento ambientale. per quanto riguarda la raccolta differenziata di vetro, plastica, metallo, carta e cartone devono essere rispettate le specifiche disposizioni comunali. gli scarti di giardinaggio possono essere smaltiti in appositi contenitori da compost ed essere utilizzati sui terreni della stessa struttura oppure, se di modica entità, dopo adeguata riduzione di volume, possono essere depositati presso un contenitore riportante la dicitura “rifiuto organico”. gli indumenti monouso, quando non utilizzati per attività medica o infermieristica, possono essere smaltiti presso gli appositi contenitori recanti la dicitura “raccolta indifferenziata”. rifiuti sanitari pericolosi sono rifiuti derivanti dalle attività ambulatoriali, chirurgiche, mediche ed infermieristiche condotte presso gli ambulatori dei canili sanitari e rifugio o comunque praticate sugli animali del canile. questi rifiuti devono essere identificati tramite il codice cer (catalogo europeo dei rifiuti). le quantità prodotte e la tipologia devono essere riportate sui formulari di identificazione che devono essere conservati in ordine cronologico per 5 anni. i rifiuti sanitari pericolosi sono suddivisi in: 1. rifiuti sanitari pericolosi a rischio infettivo; 2. rifiuti sanitari pericolosi a rischio non infettivo. rifiuti sanitari pericolosi a rischio infettivo sono rifiuti provenienti da attività veterinaria che rispondono ad almeno una delle seguenti caratteristiche: • siano contaminati da agenti patogeni per l’uomo o per gli animali; • siano venuti a contatto con un qualsiasi liquido biologico, secreto od escreto, per il quale sia ravvisato, dal medico veterinario, un rischio di patologia trasmissibile attraverso tali liquidi. tutti i rifiuti pungenti e taglienti usati devono essere considerati rifiuti sanitari pericolosi a rischio infettivo per la possibilità di trasmettere al personale agenti patogeni. questi rifiuti devono essere conferiti in appositi contenitori rigidi a tenuta, recanti la scritta “rifiuti sanitari pericolosi a rischio infettivo” e il simbolo del rischio biologico. i contenitori, una volta riempiti, devono essere chiusi ed inseriti in un altro apposito contenitore rigido. rifiuti sanitari pericolosi a rischio non infettivo appartengono a questa categoria i farmaci citotossici e citostatici. rifiuti speciali acque reflue le acque reflue di lavaggio di un canile (sanitario, rifugio, pubblico o privato), non possono essere destinate a spandimento sul suolo, gestione rifiuti veterinaria italiana, collana di monografie, monografia 28, 2020 17 il canile rifugio, procedure e protocolli almeno 2 anni. i canili possono essere dotati di impianti di termodistruzione autonomi, altrimenti il responsabile del canile si può avvalere di una ditta di trasporto autorizzata dalla asl per l’avvio alla termodistruzione. deve essere presente un frigorifero dedicato allo stoccaggio temporaneo delle carcasse in attesa del prelievo da parte della ditta. la carcassa deve essere trasportata in un sacco monouso sufficientemente resistente e a tenuta. la sepoltura, quando prevista dalla normativa, può rappresentare una valida soluzione per parchi canile di piccole dimensioni. smaltimento feci le feci rimosse con le acque di lavaggio possono essere smaltite come acque reflue (vedi paragrafo acque reflue). le feci raccolte a secco possono essere immesse in contenitori a tenuta e rimosse periodicamente tramite ditte autorizzate. devono invece essere depurate in loco o avviate ad un depuratore. la depurazione in loco può essere effettuata tramite sistemi di depurazione biologica (impianto a fanghi attivi, fitodepurazione, percolatore, ecc.). le acque reflue depurate possono essere scaricate in acque superficiali (canali, torrenti o eventualmente fossi poderali). nel caso non sia possibile ricorrere a tali sistemi si possono immettere tutti i reflui (feci e acque di lavaggio) in una vasca di raccolta autorizzata e a tenuta, di capacità adeguata, senza trattamenti, e smaltirli tramite ditte autorizzate. smaltimento carcasse le carcasse dei cani non soggette a provvedimenti di polizia veterinaria devono essere avviate ad un impianto autorizzato di termodistruzione, previa certificazione veterinaria da conservare presso gli uffici del canile per rifiuto caratteristiche gestione rifiuti assimilati o assimilabili ai rifiuti solidi urbani non presentano rischio tossico o infettivo come i rifiuti urbani rifiuti sanitari pericolosi a rischio infettivo contaminati da agenti patogeni per l’uomo o per gli animali o venuti a contatto con qualsiasi liquido biologico per il quale sia ravvisato, dal medico veterinario, un rischio di patologia trasmissibile devono essere identificati tramite il codice cer. le quantità prodotte e la tipologia devono essere riportate sui formulari di identificazione rifiuti sanitari pericolosi a rischio non infettivo farmaci citotossici e citostatici devono essere identificati tramite il codice cer. le quantità prodotte e la tipologia devono essere riportate sui formulari di identificazione rifiuti speciali acque reflue e deiezioni depurate in loco o avviate a depurazione scheda tecnica ii. rifiuti. il canile rifugio, procedure e protocolli 18 veterinaria italiana, collana di monografie, monografia 28, 2020 nei canili il controllo di specie animali infestanti (pest) assurge a particolare importanza in considerazione della gravità dei danni che possono causare tra i quali ricordiamo: • danni meccanici alla struttura; • imbrattamento dell’ambiente; • imbrattamento e distruzione delle scorte alimentari; • diffusione di malattie infettive. gli infestanti più comuni sono rappresentati da mosche, zanzare, flebotomi, blatte, coleotteri, ratti, topi, uccelli. nei canili rifugio sono più frequenti le infestazioni in quanto le strutture sono spesso collocate in aree periurbane circondate da campagna e provviste di aree verdi destinate allo sgambamento. i mezzi di controllo degli infestanti sono molteplici e tra questi negli ultimi anni sono andate affermandosi strategie di lotta integrata (integrated pest management ipm). i programmi di ipm sfruttano diverse informazioni (come ad esempio il ciclo di vita del parassita, l’interazione parassita/ambiente, le modalità di diffusione, la sensibilità ai vari metodi di lotta ecc.) per raggiungere una maggiore efficacia e una riduzione dell’uso degli agenti chimici. le strategie di lotta integrata comprendono: • monitoraggio e identificazione dei pest; • prevenzione; • controllo. monitoraggio il monitoraggio si basa sull’ispezione visiva e sull’uso di trappole. l’ispezione visiva degli ambienti viene effettuata per rilevare i segni della presenza di specie infestanti (materiali rosicchiati, animali o insetti morti, ragnatele, escrementi, impronte, ecc.). l’uso di trappole specifiche consente di rilevare la presenza di infestanti non rilevabili alla ispezione visiva. prevenzione la prevenzione si basa su: 1. pulizia dei locali, delle cucce e degli ambienti esterni che deve essere accurata e a cadenza giornaliera e deve essere preceduta dalla rimozione di eventuali ostacoli fisici (scatole, attrezzi, ecc.); 2. eliminazione di ogni eventuale fessurazione delle strutture murarie, delle porte e degli infissi; 3. installazione di reti metalliche su tutte le finestre; 4. cura costante del verde (le zone a manto erboso devono essere rasate e le zone cespugliose devono essere potate regolarmente). controllo il controllo degli infestanti è solitamente affidato a ditte specializzate, tuttavia conoscere i principali metodi di controllo è importante per verificare il lavoro svolto. i mezzi di controllo possono essere fisici, biologici, chimici e meccanici. mezzi di controllo fisici • ultrasuoni; • vapore; • congelamento con azoto liquido. mezzi di controllo biologici si basano prevalentemente sull’utilizzo di insetti antagonisti ed agenti patogeni (virus, batteri, protozoi, nematodi). nei canili può essere utile l’utilizzo del bacillus thuringiensis per il controllo delle zanzare. mezzi di controllo chimici i principi attivi utilizzabili nella lotta ai pest sono riportati nel regolamento ue sui biocidi n. 528/2012. controllo animali infestanti veterinaria italiana, collana di monografie, monografia 28, 2020 19 il canile rifugio, procedure e protocolli mezzi di controllo meccanici i mezzi di controllo meccanici sono rappresentati dalle trappole. in commercio esistono molti tipi di trappole finalizzate alla cattura sia di roditori che di insetti. formazione e addestramento degli operatori gli operatori devono essere adeguatamente addestrati sulla rilevazione dei segni di presenza di pest (presenza di materiali rosicchiati, feci, carcasse, impronte, ecc.) e sulle misure di prevenzione e controllo. piano di monitoraggio il piano di monitoraggio deve essere redatto da un esperto e prevedere una appropriata procedura documentabile. piano di prevenzione il piano di prevenzione deve essere redatto da un esperto e prevedere una appropriata procedura documentabile. piano di controllo il piano di controllo deve essere redatto da un esperto e prevedere una appropriata procedura documentabile che deve essere adeguata periodicamente in base ai dati di monitoraggio. scheda tecnica iii. piano di controllo pest. il canile rifugio, procedure e protocolli 20 veterinaria italiana, collana di monografie, monografia 28, 2020 il responsabile del canile deve effettuare una corretta valutazione dei rischi per gli operatori e attuare una efficace prevenzione. i rischi più comuni sono rappresentati da: • infortuni; • zoonosi. infortuni possono essere riconducibili a 3 gruppi principali: • infortuni dovuti a caratteristiche strutturali dei locali e degli ambienti del canile; • infortuni dovuti alla conduzione delle attività; • infortuni dovuti ad aggressioni di animali ospiti. gli infortuni dovuti a caratteristiche strutturali si verificano soprattutto quando le costruzioni non sono ben progettate e ben manutenute. tra le cause più frequenti ricordiamo: • pavimenti sdrucciolevoli e soluzioni di continuità che possono causare scivolamenti e cadute; • angoli, spigoli e rifiniture che possono causare tagli, graffi, escoriazioni; • impianti elettrici che possono causare folgorazioni. le attività lavorative che più frequentemente possono essere causa di infortuni sono rappresentate da: • immagazzinamento e prelievo di fusti (alimenti, agenti chimici, ecc.); • spostamenti di animali. gli infortuni dovuti ad aggressioni di animali ospiti possono essere riconducibili a: • eccessivo affollamento dei box; • presenza di animali con disturbi comportamentali; • eccessiva familiarità degli operatori con gli animali e conseguente diminuzione dell’attenzione. prevenzione infortuni gli infortuni legati alle caratteristiche delle strutture possono essere prevenuti attuando le misure riportate nel capitolo “requisiti strutturali”. gli infortuni legati allo svolgimento delle attività possono essere prevenuti predisponendo rigorose procedure per l’esecuzione standardizzata delle attività lavorative. gli infortuni legati a eccessiva familiarità o a errato atteggiamento verso gli animali possono essere ridotti o eliminati con un’adeguata formazione degli operatori. in ogni caso deve essere prevista un’assicurazione per la struttura e/o per gli operatori/ volontari. zoonosi per zoonosi si intende qualsiasi malattia e/o infezione trasmessa direttamente o indirettamente dagli animali all’uomo e viceversa; possono essere causate da batteri, miceti, parassiti, virus e veicolate da vettori. i più comuni agenti di zoonosi sono: • campylobacter; • salmonella; • escherichia coli; • giardia; • dermatofiti • leishmania; • leptospira; • echinococco; • toxocara canis; • ancylostoma; • dirofilaria immitis; • dirofilaria repens. sicurezza veterinaria italiana, collana di monografie, monografia 28, 2020 21 il canile rifugio, procedure e protocolli • controllo clinico dei cani in ingresso; • profilassi vaccinali; • trattamenti antiparassitari per parassiti interni e esterni; • disinfestazione degli ambienti; • pulizia accurata, disinfezioni dei locali; • dieta individuale, sana e bilanciata per favorire un buono stato di salute e una adeguata risposta immunitaria; • uso di dispositivi di protezione individuale per gli operatori; • eventuale separazione fisica/quarantena degli animali (da valutare bene per gli effetti negativi sul benessere a causa dell’isolamento sociale). le modalità di trasmissione possono essere: 1. morsi o graffi; 2. contatto con sangue infetto e altri liquidi biologici; 3. puntura di insetti; 4. contatto con liquami; 5. semplice contatto con l’animale. prevenzione benché presso i rifugi arrivino animali transitati dal canile sanitario e quindi già sottoposti a controlli sanitari, è comunque importante adottare misure preventive delle zoonosi quali: gli operatori prima di entrare nella parte del canile che ospita gli animali devono cambiarsi nei locali destinati a spogliatoi per indossare adeguati indumenti, eventualmente monouso gli addetti alle operazioni di pulizia devono indossare stivali con suole antiscivolo lo spostamento dei cani e la somministrazione di alimenti devono avvenire secondo precisa procedura l’eventuale impiego di sostanze chimiche disinfettanti che provocano irritazione delle vie respiratorie deve essere comunicato per tempo a tutti gli operatori. l’accesso ai locali potrà avvenire solo al termine dell’effetto tossico della sostanza chimica usata a fine lavoro gli operatori devono lasciare gli indumenti in appositi contenitori di raccolta distinti per quelli da avviare al lavaggio e quelli monouso. se sono entrati in contatto con animali o luoghi sospetti di infezione dovranno provvedere alla propria disinfezione prima di passare nella cosiddetta zona pulita scheda tecnica iv. sicurezza operatori. il canile rifugio, procedure e protocolli 22 veterinaria italiana, collana di monografie, monografia 28, 2020 tutti gli operatori del canile devono essere adeguatamente formati, a partire dal responsabile che sarebbe opportuno ottenesse un riconoscimento di idoneità da parte del servizio veterinario della asl territorialmente competente (così come previsto per allevamenti e pensioni in talune regioni). il responsabile deve garantire che tutti gli operatori abbiano una formazione e un addestramento adeguati allo svolgimento delle proprie attività per: • operare in relazione al proprio livello di competenza; • costruire un buon rapporto con gli animali, gli altri operatori e i volontari; • conoscere adeguatamente le esigenze etologiche e sanitarie degli animali; • conoscere le procedure applicate nel canile e la normativa vigente. come già detto in premessa è auspicabile che il canile rifugio eroghi formazione con personale esperto. oltre ai corsi di formazione e di aggiornamento è opportuno prevedere anche periodi di tirocinio per ogni operatore. formazione del personale il responsabile del canile d’intesa con il direttore sanitario deve redigere un piano annuale di formazione e aggiornamento per tutto il personale, definendo: gli obiettivi gli argomenti il target dei partecipanti la durata il periodo di erogazione. scheda tecnica v. piano di formazione. veterinaria italiana, collana di monografie, monografia 28, 2020 23 il canile rifugio, procedure e protocolli al canile accedono quotidianamente sia coloro che vi lavorano (direttore, operatori, veterinari, fornitori, volontari) sia visitatori, pertanto l’ingresso nelle diverse aree deve essere diversificato secondo le attività e il ruolo svolto. alcune strutture quali ambulatorio, infermeria, sala operatoria, locale di deposito della scorta farmaci, box degli animali malati o pericolosi, possono essere frequentati esclusivamente da personale autorizzato dal responsabile del canile o dal medico veterinario. gli elenchi delle persone autorizzate per le strutture ad accesso limitato devono essere conservati presso gli uffici per tutto il periodo di validità. i visitatori esterni hanno accesso al canile in orari prestabiliti e resi noti tramite apposita cartellonistica affissa all’ingresso della struttura. possono essere programmati appuntamenti su richiesta telefonica in orari al di fuori dell’apertura. tutti i locali non direttamente destinati al ricovero degli animali devono essere debitamente identificati. la presenza di apposita cartellonistica segnala i percorsi per il pubblico che visita la struttura durante gli orari di apertura del canile. è opportuna la presenza di piantine in cui si segnala la localizzazione del luogo in cui ci si trova. si ricorda che è auspicabile che i canili siano aperti al pubblico per più giorni alla settimana e preferibilmente durante i giorni prefestivi e festivi. controllo accessi all’interno della struttura è vietato l’ingresso con mezzi motorizzati non autorizzati non è consentito ai visitatori accedere nelle aree dove sono ospitati gli animali senza la presenza e il consenso degli operatori addetti gli operatori non autorizzati non possono accedere ai locali ad accesso limitato (per es. ambulatorio, uffici) durante gli eventi, il pubblico deve percorrere le vie segnalate da apposite frecce o da indicazioni equivalenti che indichino il percorso più breve e diretto alla struttura dove l’evento avrà luogo i volontari possono accedere al canile per collaborare nelle attività di pulizia della struttura e di cura degli animali, previa apposita autorizzazione nella quale siano definiti orario e modalità di frequenza scheda tecnica vi. modalità di accesso al canile e ai locali. la domanda di autorizzazione deve essere presentata al responsabile del canile specificando: nome; cognome; data di nascita; indirizzo; estremi di un documento d’identità; autodichiarazione relativa all’assenza di carichi penali pendenti per maltrattamento di animali; partecipazione a corsi sulla cura degli animali; giornate di disponibilità. per i giovani volontari di età compresa tra16 e 18 anni è necessaria l’autorizzazione di chi esercita la patria potestà. scheda tecnica vii. richiesta di autorizzazione allo svolgimento di attività di volontariato nel canile. parte ii aspetti sanitari il canile rifugio, procedure e protocolli 26 veterinaria italiana, collana di monografie, monografia 28, 2020 tie e per questo motivo è essenziale porre particolare attenzione alle procedure di gestione dei nuovi ingressi. l’ingresso di nuovi cani nella struttura rappresenta un momento di elevato stress per gli animali e un rischio di introduzione di malatgestione dei nuovi ingressi documentazione di scorta all’ingresso di un cane il cane in ingresso nel canile rifugio proveniente dal canile sanitario è scortato dalla seguente documentazione: a) documento di iscrizione all’anagrafe canina attestante: codice identificativo del microchip; razza del cane; sesso; data o periodo di nascita; mantello; taglia; nome; data iscrizione; segni particolari; note. b) documento dell’autorità che dispone il ricovero e la motivazione (ad esempio rinvenimento, cattura, rinuncia alla proprietà, sequestro). nel caso di rinuncia alla proprietà deve esserene specificata la motivazione. c) cartella clinica contenente almeno le seguenti informazioni: visita clinica con l’indicazione della data, esito ed eventuali diagnosi; esami di laboratorio, con l’indicazione della data e dell’esito; terapie effettuate con l’indicazione della data, dei dosaggi e della durata; trattamenti antiparassitari sia esterni che interni con l’indicazione della data, dei dosaggi e della durata del trattamento; vaccinazioni con indicazione della data; indicazione e descrizione di eventuali disturbi comportamentali. nel caso il cane non provenga dal canile sanitario, è importante che il responsabile del rifugio stabilisca una procedura per la raccolta delle informazioni sopra riportate scheda tecnica viii. documentazione nuovi ingressi. nel registro di carico e scarico degli animali ospitati devono essere riportate almeno le seguenti informazioni: microchip dell’animale; data di entrata; provenienza; generalità del proprietario (sindaco per i cani vaganti o privato cittadino in caso di rinuncia); data e causa di morte; data di adozione/affidamento; destinazione del cane in caso di affidamento o adozione. scheda tecnica ix. registro di carico e scarico. veterinaria italiana, collana di monografie, monografia 28, 2020 27 il canile rifugio, procedure e protocolli segna ed a qualsiasi titolo sono tenuti alla denuncia per qualunque nuovo caso di malattia o di morte improvvisa che si verifica entro otto giorni da un caso precedente non riferibile a malattia comune già accertata.” protocolli di prevenzione e cartelle cliniche prevenzione la prevenzione delle malattie infettive nel canile si basa su una conduzione gestionale che limiti l’esposizione ai patogeni e mantenga gli ospiti del canile in uno stato di salute tale da renderli meno inclini ad ammalarsi. in tale ottica ogni canile rifugio deve redigere ed attuare un piano per analisi di laboratorio e per le profilassi antiparassitarie e vaccinali. tale piano deve essere redatto in relazione alla situazione epidemiologica del territorio, del canile ed eventuali obblighi previsti dalle singole norme regionali (es. leishmania in alcune regioni). a tal proposito si sottolinea come l’accertamento delle cause di morte degli animali, anche attraverso esami necroscopici, ha una funzione di prevenzione di nuovi focolai e inoltre contribuisce al monitoraggio delle cause di morte diventando un indice della qualità di gestione del canile. nel piano annuale dei controlli periodici possono essere compresi: visite cliniche, controlli sierologici, esami emocromocitometrici, biochimico-clinici e esami delle feci. oltre alla regolare profilassi per gli ectoparassiti, l’alta incidenza di malattie trasmesse da vettori in alcune zone (per es. leishmaniosi, filariosi, erhlichiosi, borreliosi, ecc.), rende indispensabile oltre al trattamento diretto degli animali anche disinfestazioni ambientali periodiche. i protocolli vaccinali variano secondo l’area geografica, il numero di animali ospitati, l’intensità del turn-over, l’anamnesi collettiva e individuale e la situazione del canile e sono la gestione sanitaria di un canile rifugio ha come obiettivi fondamentali: • tutelare il benessere degli animali; • prevenire le patologie degli animali; • curare le patologie di qualsiasi natura degli animali; • prevenire le malattie trasmissibili all’uomo. la gestione sanitaria del canile vede a capo un medico veterinario come direttore sanitario ma a vari livelli e a diverso titolo coinvolge tutti gli operatori. una buona gestione sanitaria porta al miglioramento delle condizioni generali di benessere degli animali e degli operatori e alla riduzione dei costi delle spese mediche riducendo l’incidenza delle malattie e migliorando l’indice di adottabilità. per ottenere ciò, tutte le attività del canile e in maniera particolare l’alimentazione, la gestione corretta delle nuove introduzioni, l’igiene ambientale, la gestione controllata del personale e del pubblico in entrata e in uscita devono essere oggetto di costante monitoraggio. al fine di una corretta gestione della salute degli animali, oltre alla conoscenza della normativa sull’utilizzo dei farmaci, i canili rifugio dovrebbero essere provvisti di: • box di isolamento; • locale di deposito farmaci e antisettici ad uso topico; • locali adibiti ad ambulatorio o clinica o ospedale veterinario; • sala e box di degenza. è inoltre utile potersi avvalere di un laboratorio di analisi (interno o esterno). è fortemente auspicabile che nei canili rifugio privati dotati di ambulatori o cliniche veterinarie vi siano accessi separati per i cani padronali e quelli del canile. si ricorda che secondo il dpr 320/54 “i veterinari liberi esercenti, i proprietari e i detentori di animali anche in temporanea congestione sanitaria il canile rifugio, procedure e protocolli 28 veterinaria italiana, collana di monografie, monografia 28, 2020 tipo di intervento tempi vista medica con esame obiettivo generale almeno due volte l’anno esame feci e trattamento per parassiti intestinali l’esame almeno due volte l’anno e il trattamento secondo necessità trattamento antiparassitario per parassiti interni su indicazione veterinaria in relazione alla situazione epidemiologica e al tipo di prodotto usato trattamento antiparassitario per parassiti esterni su indicazione veterinaria in relazione a tipo di prodotto usato e a situazione climatica esami sierologici (p.e. leishmania, dirofilaria) almeno una volta l’anno nelle zone endemiche; con frequenza da stabilirsi in corso di epidemia; secondo i piani regionali laddove previsti vaccinazioni secondo protocollo individuale tutti gli interventi possono essere fatti con maggior frequenza su alcuni o su tutti i cani se particolari situazioni cliniche lo richiedano. altri esami e altre azioni preventive possono essere inseriti nel piano di prevenzione in base alla situazione epidemiologica o geografica del canile. scheda tecnica xi. piani di prevenzione. essere separati, pertanto nella cartella clinica vanno annotate anche le eventuali diagnosi di disturbi comportamentali e i percorsi terapeutici messi in atto da personale esperto in medicina comportamentale del cane. è opportuno inserire nella cartella clinica un prospetto che riporti i dati e le informazioni illustrati nella scheda tecnica xii. stabiliti del medico veterinario responsabile della struttura in coerenza anche con quanto già effettuato nel canile sanitario. le linee guida per la vaccinazione del cane e del gatto stilate dal vaccination guidelines group (vgg) della world small animal veterinary association (wsava) pubblicate e aggiornate nel 2015 possono rappresentare un ottimo riferimento per la scelta dei vaccini di base (core) o facoltativi (non core) e dei protocolli vaccinali. nelle stesse linee guida sono reperibili utili indicazioni circa gli esami sierologici necessari per la documentazione e il monitoraggio delle risposte immunitarie e un paragrafo sulle reazioni avverse. nella scheda tecnica xi sono riportati gli interventi di prevenzione di base da attuare su tutti gli animali. cartella clinica nella cartella clinica devono essere conservati tutti i documenti di registrazione attestanti i controlli, le diagnosi, le terapie, gli interventi chirurgici, i trattamenti e qualsiasi altro documento che può fornire informazioni circa la salute del cane. le cartelle cliniche aggiornate consentono di avere sempre a disposizione la storia clinica del cane e rappresentano la base per elaborare un puntuale quadro epidemiologico della struttura. è opportuno ricordare che il benessere fisico e quello psicologico del cane non possono per i trattamenti profilattici obbligatori previsti dalla normativa si rimanda alla legge 281/91, art. 2, comma 5. nei canili italiani sono, di solito, ritenuti “core” le seguenti vaccinazioni: parvovirus-2 canino; virus del cimurro; adenovirus-2 canino. leptospira interrogans sierogruppo canicola leptospira interrogans sierogruppo icterohaemorrhagiae leptospira interrogans sierogruppo australis leptospira kirschneri sierogruppo grippotyphosa solitamente rientrano nelle “non core” le seguenti vaccinazioni: bordetella bronchiseptica; virus parainfluenzale-3; borrelia burgdoferi; herpesvirus-1canino; leishmania infantum; coronavirus canino; microsporum canis; babesia canis. i vaccini devono essere conservati in frigorifero, secondo le indicazioni del produttore e usati entro la data di scadenza. scheda tecnica x. vaccinazioni. veterinaria italiana, collana di monografie, monografia 28, 2020 29 il canile rifugio, procedure e protocolli gestione dell’igiene ambientale e della disinfezione tra i disinfettanti che possono essere utilizzati: • alcool • composti a base di cloro • aldeidi • iodofori • composti di ammonio quaternario. gli alcool sono battericidi ma non sporicidi e agiscono denaturando le proteine. i composti a base di cloro hanno un ampio spettro di attività, sono economici e sono veloci da utilizzare, ma sono anche corrosivi ed instabili. le aldeidi devono essere usate in soluzione acquosa e possono essere utilizzate sia sotto forma gassosa che liquida. possiedono azione battericida, fungicida, virulicida e a determifoto nome; microchip; razza; sesso; mantello; taglia; data di nascita (se conosciuta o indicativa); data di ingresso nel canile; motivo di consegna al canile (per es. rinvenuto vagante, sequestrato, consegnato dal proprietario). visite cliniche (tutte le visite effettuate compresa quella presso il canile sanitario) data di effettuazione, nome del veterinario, esito esami di laboratorio data, richiedente ed esito trattamenti antiparassitari data, prodotto usato, veterinario vaccinazioni data, vaccino usato, veterinario terapie data, protocollo terapeutico, veterinario prescrittore interventi chirurgici data, ambulatorio di esecuzione, equipe chirurgica visite comportamentali data, diagnosi, nome del veterinario, terapia prescritta dieta tipo di dieta, durata, veterinario prescrittore scheda tecnica xii. cartella clinica. far uscire i cani e sistemarli in luogo asciutto. rimuovere tutte le parti mobili. tutti gli oggetti presenti, in maniera particolare ciotole e abbeveratoi, devono essere accuratamente detersi e risciacquati. lavare e disinfettare con prodotti efficaci ed attendere i tempi di azione. la scelta dei prodotti è fatta dal direttore sanitario o da un veterinario da lui delegato. risciacquare abbondantemente con acqua calda in modo da rimuovere i residui di disinfettante. allontanare l’acqua in eccesso. far rientrare i cani solo quando l’ambiente è sufficientemente asciutto. n.b. anche le aree di sgambamento devono essere tenute pulite con la rimozione almeno quotidiana dei rifiuti organici solidi. scheda tecnica xiii. gestione dell’igiene ambientale e della disinfezione. nati valori di ph anche sporicida. l’aldeide più usata è la glutaraldeide. gli iodofori, come soluzioni di iodio, sono utilizzati come disinfettanti della cute e delle mucose. le soluzioni diluite hanno un elevato potere battericida ma la presenza di materiale organico riduce la loro attività. i composti quaternari d’ammonio sono ampiamente utilizzati come disinfettanti e antisettici e normalmente sono inattivati da acqua dura, sapone e residui anionici. possiedono azione fungicida, battericida e virulicida (limitatamente ai virus lipofilici). il loro uso è ottimale per la sanitizzazione ambientale di superfici quali pavimenti, sanitari e muri. ferma restando quindi la possibilità di scegliere i prodotti più confacenti alle esigenze pratiche, si ribadisce che le operazioni di pulizia (con acqua, idropulitrici, ecc.) devono essere eseguite solo dopo aver fatto allontanare il cane dal box. il canile rifugio, procedure e protocolli 30 veterinaria italiana, collana di monografie, monografia 28, 2020 la prescrizione avviene con ricetta elettronica come riportato nel manuale operativo della ricetta elettronica veterinaria di cui al link: https://www.ricettaveterinariaelettronica.it/ manuale.html. i documenti d’acquisto dei farmaci devono essere conservati per tre anni. gli operatori che affiancano il veterinario nelle somministrazioni devono essere adeguatamente formati e rispettare rigorosamente la posologia prescritta. la legislazione vigente prevede che vengano immediatamente segnalate le sospette reazioni avverse ai farmaci al ministero della salute o ai centri regionali di farmacovigilanza (http://www. salute.gov.it/farmacovigilanzavetmodule/ farmacovigvetservlet). gestione dei farmaci la prescrizione del farmaco può essere fatta solo dal direttore sanitario o da un veterinario da lui delegato. le modalità di tenuta delle scorte dei medicinali deve rispettare quanto indicato dagli art. 80; 82 e 84 del dlgs. 193/2006. i nominativi dei medici veterinari responsabili delle scorte devono essere indicati nella domanda di autorizzazione alla scorta presentata ai servizi veterinari della asl competente per territorio con l’indicazione delle ulteriori strutture presso le quali risultano responsabili della tenuta di scorte. i medicinali devono essere custoditi in idonei locali chiusi o in armadi accessibili solo al veterinario responsabile delle scorte e devono essere conservati, in luogo adeguato, pulito, non esposto a umidità, luce o sbalzi termici. veterinaria italiana, collana di monografie, monografia 28, 2020 31 il canile rifugio, procedure e protocolli necessitano dell’accesso alle aree di sgambamento per almeno un’ora, due volte al giorno. per migliorare la capacità di socializzazione e apprendimento e diminuire lo stress dei cani è altresì importante seguire le seguenti indicazioni: • gli operatori devono istaurare un rapporto di scambio comunicativo (visivo, gestuale e vocale) con i cani; • i cuccioli senza madre devono essere posti in una nursery con un adulto di buona indole che li aiuti nelle relazioni sociali intraspecifiche; • devono essere organizzate aree attrezzate con percorsi e oggetti ludici; • devono essere organizzate attività mirate a migliorare le capacità cognitive, sociali e di apprendimento; • devono essere impostati percorsi mirati di riabilitazione per i soggetti che manifestano disturbi comportamentali. esercizi che stimolano l’attività mentale, condotti per almeno 25 minuti al giorno, esercitano un’importante azione sinergica con l’attività motoria. detti esercizi possono riguardare giochi condotti con o senza l’uso di arricchimento ambientale, attività di abituazione (p.e. a pettorine e collari) e attività di educazione. attraverso la stesura di un programma e di procedure che permettano di verificare lo svolgimento delle attività dei cani è possibile la collocazione dei cani in canile può essere causa di stress attribuibili a molte cause. le più frequenti sono rappresentate da: • spazi confinati, spesso ristretti; • dinamiche relazionali alterate o assenti sia con l’uomo che con altri cani; • mancanza di esercizio fisico; • mancanza di stimoli ambientali. i segnali di stress possono essere rappresentati da: alterazioni del comportamento alimentare (es. coprofagia), movimenti in circolo prolungati (circling), paure, fobie, intolleranza verso altri animali, iperattività o leccamento, grattamento e vocalizzazioni eccessive. l’attività fisica può ridurre gli effetti avversi del confinamento, soprattutto se associata al contatto con l’uomo e a esercizi mirati alla stimolazione mentale. la necessità di attività motoria varia secondo l’età, la mole, la razza, il periodo fisiologico, lo stato di salute, tuttavia è possibile individuare riferimenti temporali minimi per ottenere effetti positivi sullo stato di benessere psicofisico dei cani. i cani che hanno accesso ad una zona esterna direttamente dal box, necessitano di attività di movimenti in aree di sgambamento per almeno un’ora al giorno. cani che non hanno accesso diretto ad una zona esterna del box (fermo restando l’obiettivo di dismettere questo tipo di strutture), benessere: attività psicofisiche e valutazione di seguito si elencano le principali attività psicofisiche di base: educazione e training; abituazione all'uso di collari, pettorine, museruola e guinzaglio; abituazione alle manipolazioni; attività di socializzazione con conspecifici e e con persone (vedi capitolo “adozioni); accesso quotidiano alle aree di sgambamento e, dove possibile, passeggiata al guinzaglio per favorire il comportamento di esplorazione dell’ambiente; attività ludico-sportive con inserimento di arricchimenti ambientali che permettono al cane di sviluppare e allenare le proprie capacità. scheda tecnica xiv. attività psicofisiche. il canile rifugio, procedure e protocolli 32 veterinaria italiana, collana di monografie, monografia 28, 2020 tale protocollo prende in considerazione quattro principi di benessere (corretta alimentazione, ricovero adeguato, buono stato di salute, comportamento appropriato) e consente di: • ottenere informazioni in tempo reale sullo stato di benessere dei cani; • identificare eventuali fattori di rischio. il protocollo è stato studiato per poter essere utilizzato non solo da autorità competenti veterinarie ma anche da operatori di canili adeguatamente formati sull’applicazione del protocollo e sulle nozione di base in materia di comportamento, benessere, salute e gestione dei cani in canile (vedi capitolo “formazione del personale”). anche calcolare il numero degli operatori di cui il canile deve disporre. tutte queste attività migliorando lo stato psicofisico e le competenze sociali dei cani, contribuiscono ad aumentare l’indice di adottabilità e a diminuire il numero di restituzioni al canile dei cani adottati (vedi paragrafo “adozioni”). oltre alle ispezioni interne e ai controlli istituzionali, è consigliabile effettuare almeno annualmente una auto-valutazione della situazione del canile sul benessere degli animali ospitati, che può essere fatta attraverso l’applicazione del protocollo shelter quality (http://www.izs.it/izs/engine/raservefile. php/f/pdf_pubblicazioni/protocolloshelterquality_it_maggio2018.pdf ). veterinaria italiana, collana di monografie, monografia 28, 2020 33 il canile rifugio, procedure e protocolli l’alimentazione è fondamentale per un buono stato di salute degli animali e pertanto la dieta deve essere valutata dal direttore sanitario o da un veterinario da lui delegato. la dieta deve rispondere alle esigenze nutritizionali (caloriche, proteiche, vitaminiche, ecc.) degli animali ma anche rispondere a criteri di appetibilità e per la sua formulazione si devono prendere in considerazione: • l’età dell’animale • la mole • la razza • la stagione • l’attività fisica • lo stato fisiologico o parafisiologico (p. es. gravidanza) • la presenza di patologie è importante redigere un programma alimentare nel quale vengano definiti: • la tipologia di alimenti (ad esempio preparati industriali o alimenti base per la preparazione dei pasti); • le modalità di preparazione; • le modalità e i tempi di somministrazione; • le quantità; • le misure igieniche da rispettare nella preparazione e nella somministrazione. eventuali esigenze dietetiche particolari riferibili a stati patologici (per es. insufficienza renale, diabete ecc.) o a specifici momenti fisiologici (per es. allattamento, crescita, ecc.) devono essere annotati nella cartella clinica. alimenti gli alimenti devono essere conservati in ambienti puliti, asciutti e protetti da agenti infestanti (topi, ratti, ecc.). i locali di deposito degli stessi e la cucina devono avere finestre protette da zanzariere. gli spazi degli ambienti e i lavandini devono essere ampi per garantire i movimenti degli operatori e le operazioni di pulizia. i pasti devono essere somministrati a orari fissi e regolari. la dieta può basarsi sull’uso di preparazioni estemporanee di alimenti o di prodotti industriali (crocchette, mangimi umidi inscatolati). nei canili è possibile utilizzare sottoprodotti di origine animale costituiti da rifiuti di cucina e ristorazione come indicato dalla nota congiunta delle direzioni generali della sanità animale e della sicurezza alimentare del 28.12.2015 del ministero della salute. è comunque vietato l’utilizzo per l’alimentazione di olio di cucina esausto e rifiuti di cucina e ristorazione costituiti da residui di alimenti già somministrati al consumatore finale. tali sottoprodotti possono provenire solo da imprese alimentari registrate e riconosciute in base reg.(ce) 852/2004 e reg. (ce) 853/2004, e il loro utilizzo è consentito solo nell’ambito della medesima provincia in cui sono ubicati anche i canili. l’utilizzo di tali sottoprodotti è subordinato a: • registrazione ai sensi dell’art. 23 del reg. ce 1069/2009; • registrazione o riconoscimento ai sensi del reg. (ce) 852/2004 o ai sensi del reg. (ce) 853/2004 dell’industria alimentare produttrice dei sottoprodotti; • trattamento termico; • il responsabile o gestore del canile deve notificare all’autorità competente locale l’utilizzazione dei sottoprodotti; • il proprietario dell’azienda produttrice deve detenere apposito registro in cui annotare la data dell’invio e il peso stimato dei sottoprodotti. inoltre, i canili rifugio possono utilizzare i pet food non idonei per motivi commerciali ai sensi della nota dgsaf prot. n. 8665 del 9 aprile 2020. le preparazioni estemporanee devono essere alimentazione il canile rifugio, procedure e protocolli 34 veterinaria italiana, collana di monografie, monografia 28, 2020 modalità di somministrazione la somministrazione degli alimenti rappresenta un momento di grande importanza nell’interazione cane/uomo e cane/cane. si sconsiglia l’uso di mangiatoie collettive. ogni cane dovrebbe disporre della propria ciotola in modo da rispettate le prescrizioni quantitative per ogni animale. per lo stesso motivo anche le mangiatoie a getto continuo fornite di “serbatoi” sono da sconsigliare. queste ultime inoltre riducono l’interazione con gli operatori privandoli anche del ruolo di gestori delle risorse alimentari. un buon sistema di distribuzione dell’acqua può essere costituito da abbeveratoi a riempimento automatico, che garantiscono la costante fornitura d’acqua anche nella stagione calda. prodotte sulla base delle indicazioni del direttore sanitario del canile, in considerazione delle esigenze nutritive specifiche degli animali. l’alimentazione con prodotti industriali (crocchette o mangime umido) è sicuramente più agevole ed in molte situazioni consigliabile. in commercio esistono mangimi studiati per soddisfare particolari esigenze in condizioni fisiologiche (crescita, allattamento, gravidanza) o patologiche. è auspicabile che anche nei canili dove sono somministrati esclusivamente prodotti industriali sia conservato un registro di carico e scarico su supporto cartaceo o informatizzato che consenta di tenere sotto controllo sia le scorte che le scadenze. somministrazione alimenti gli alimenti devono essere riposti in ciotole singole ben pulite; la quantità e la tipologia di alimento deve rispettare quanto disposto dal veterinario nel piano dietetico; a fine pasto le ciotole devono essere ripulite da eventuali avanzi e lavate accuratamente. somministrazione acqua le ciotole per l’abbeveraggio devono essere lasciate a disposizione dell’animale con acqua fresca e pulita. scheda tecnica xv. alimenti. veterinaria italiana, collana di monografie, monografia 28, 2020 35 il canile rifugio, procedure e protocolli • i cani siano puliti e alloggino in box ben tenuti; • la rumorosità, soprattutto quella dovuta all’abbaiare dei cani, sia contenuta mediante accorgimenti che limitano le fonti di stress per gli animali. il canile deve essere un luogo nel quale le famiglie possono incontrare gli animali e operare una scelta di adozione informata e consapevole. il canile come presidio di lotta al randagismo deve essere il punto di riferimento per lo sviluppo e la diffusione del concetto di possesso responsabile. particolare cura dovrà essere data alla distribuzione di materiale divulgativo che informi su tutti gli aspetti connessi alla presenza di un cane in famiglia. la permanenza dei cani nei canili rifugio dovrebbe essere temporanea e finalizzata all’adozione. ogni struttura dovrebbe individuare formalmente un responsabile delle adozioni con specifica formazione. nel processo di adozione i fattori principali da analizzare sono due: • le aspettative dell’adottante; • la capacità dell’adottante di gestire un determinato soggetto (che dipende dall’esperienza e dalla formazione). conseguenze da evitare sono: • restituzione/abbandono/maltrattamento; • aggressioni (che a volte possono essere molto gravi). l’iter dell’adozione è particolarmente complesso e deve incentrarsi sull’incrementare il numero di cani adottati e limitarne al massimo il loro ritorno in canile. un cane adottato e poi restituito al canile è sottoposto a stress elevato dovuto ai cambiamenti dei riferimenti sociali e ambientali che richiedono sforzi adattativi che vengono frustrati con possibile insorgenza di stati ansiosi dell’animale. per incrementare le adozioni è necessario incentivare le visite al canile, utilizzare i social network per pubblicizzare e far conoscere i cani del canile pronti per l’adozione, attraverso foto e schede con caratteristiche sanitarie e caratteriali. per aumentare il numero di visite al canile è necessario che: • gli orari di apertura al pubblico siano tali da far sì che i visitatori abbiano un’ampia possibilità di accesso soprattutto durante i giorni festivi; • il canile abbia un aspetto gradevole, curato architettonicamente, pulito, ordinato, e ben mantenuto anche per quanto riguarda la distribuzione e la manutenzione del verde; adozioni domande per chi vuole adottare di seguito si riporta uno spunto per lo sviluppo di materiale divulgativo rivolto ad aspiranti adottanti:: 1. tutti i membri della famiglia sono d’accordo a prendere un cane? 2. hai abbastanza tempo da dedicare al cane? 3. hai calcolato l’impegno temporale necessario per le uscite quotidiane (per i cani giovani almeno 4) per l’espletamento delle esigenze fisiologiche e per praticare l’attività motoria di cui ogni cane ha bisogno? 4. hai preventivato i costi che dovrai affrontare (alimentazione, igiene, spese mediche)? 5. sei adeguatamente informato sugli adempimenti da mettere in atto quando il tuo cane frequenta luoghi pubblici (per. es. uso di buste e palette per rimuovere gli escrementi)? 6. hai considerato che il cane, rappresenta un impegno aggiuntivo nel tenere pulita la casa? 7. hai previsto un adeguata sistemazione per il cane durante i periodi di vacanza? 8. hai considerato che il cane è un animale sociale che soffre se costretto alla solitudine per molte ore al giorno? 9. sei pronto ad impegnarti a lungo termine? scheda tecnica xvi. materiale divulgativo per l’adottante (volantini e poster). il canile rifugio, procedure e protocolli 36 veterinaria italiana, collana di monografie, monografia 28, 2020 queste informazioni, possono aiutare a identificare non solo l’indice di adottabilità dei cani ma anche l’idoneità delle persone ad adottare un cane. l’adozione deve essere registrata anche sul registro di carico e scarico. il passaggio di proprietà deve essere riportato sull’anagrafe canina regionale e i documenti aggiornati devono essere forniti all’adottante. è opportuno consegnare all’adottante anche una copia della cartella clinica nella quale devono essere riportate eventuali diagnosi di malattie e trattamenti profilattici, terapeutici, chirurgici e comportamentali effettuati. l’adottante prima dell’adozione deve documentare la maggiore età con carta di identità o patente di guida e l’assenza di condanne penali per maltrattamenti ad animali con autocertificazione. le generalità dell’adottante devono essere indicate su apposita scheda, nella quale dovranno anche essere riportate le dichiarazioni circa la reale disponibilità di risorse adeguate dell’adottante (per es. spazi idonei) e i doveri e le responsabilità che deve sottoscrivere. si coglie l’occasione per precisare che mentre le adozioni dai canili rifugio sono improntate sulle “garanzie di buon trattamento” (281/91) e sul rispetto della normativa vigente (p.e. iscrizione in anagrafe e sterilizzazione) esistono purtroppo altre forme di adozione che non danno le medesime garanzie. infatti in molti casi i cani recuperati dal territorio non vengono fatti passare dai canali ufficiali per cui vengono ceduti, anche tramite trasporti non idonei senza identificazione e sterilizzazione. la scheda tecnica “materiale divulgativo per l’adottante” è un esempio di indicazioni da fornire a coloro che intendono adottare. l’adozione dovrebbe essere preceduta da un periodo di affidamento temporaneo di almeno tre mesi. l’affidamento deve essere registrato in anagrafe, in quanto comporta un cambio di detentore e l’interruzione, da parte del comune, dell’erogazione del mantenimento del cane. l’affidamento temporaneo deve avvenire in seguito a una dichiarazione scritta dell’adottante che accetta controlli da parte del canile che possono avvenire in qualsiasi momento, e senza preavviso. detti controlli devono essere fatti da personale debitamente formato che utilizza check list validate dal veterinario della struttura o da un veterinario comportamentalista. l’affidamento definitivo avviene se il comportamento del nuovo proprietario dimostra una corretta gestione del cane. i dati delle adozioni e degli affidi devono essere registrati e conservati in un apposito registro (anche informatico) e devono riguardare almeno le seguenti informazioni: • nome e cognome del proprietario; • data dell’affido/adozione; • data dell’eventuale restituzione al canile; • breve relazione dell’operatore cinofilo o del veterinario comportamentalista o del direttore sanitario sulle condizioni di salute del cane e sulle motivazioni che ne hanno determinato la restituzione. veterinaria italiana, collana di monografie, monografia 28, 2020 37 il canile rifugio, procedure e protocolli la legge quadro 281/91, in materia di animali d’affezione e prevenzione del randagismo, ha stabilito che la soppressione di cani e gatti può avvenire solo con eutanasia e soltanto se “gravemente malati o incurabili o di comprovata pericolosità”. alla comprovata pericolosità fa riferimento l’art. 672 c.p. “omessa custodia e malgoverno degli animali”, di competenza degli organi preposti all’ordine pubblico, a difesa dell’incolumità fisica delle persone (minacciata da un cane) e il regolamento di polizia veterinaria, che tra le misure restrittive per contenere la propagazione di malattie infettive e zoonosi comprende l’abbattimento forzato degli animali. la legge n. 189/04 vieta qualunque uccisione degli animali per crudeltà o in assenza di necessità (art. 544 bis c.p.); la stessa legge punisce “chiunque, per crudeltà o senza necessità, cagiona una lesione ad un animale ovvero lo sottopone a sevizie o a comportamenti o a fatiche o a lavori insopportabili per le sue caratteristiche etologiche” (art. 544 ter c.p.). per non incorrere nel reato di maltrattamento, l’eutanasia, così come indicato dall’etimologia di questo termine, non deve provocare alcuna sofferenza all’animale. i cani su cui è esercitata l’eutanasia, in genere, sono già in stato di stress dovuto ad una patologia fisica o comportamentale. è compito del medico veterinario condurre l’eutanasia in maniera da non aggravare lo stato di stress o provocare ulteriori sofferenze quali dolore e ansia. per questo motivo, prima di eseguire le procedure di eutanasia vere e proprie, é indispensabile che il veterinario scelga, in base ad aggiornate conoscenze medico-scientifiche, il miglior protocollo anestesiologico a seconda del caso. i farmaci impiegati per l’eutanasia dell’animale devono essere detenuti e somministrati solo dal veterinario che adotta tutte le precauzioni per evitare rischi da contatto accidentale. gli animali soppressi devono essere distrutti o posti in luoghi inaccessibili ad altri animali; la procedura di eutanasia dei cani riconosce le seguenti fasi: eutanasia valutazione verificare se il cane è in una delle condizioni previste per l’abbattimento (stato di comprovata pericolosità, malattia grave o incurabile). trasporto dell’animale in luogo idoneo e contenimento il contenimento deve essere praticato da personale esperto in maniera da non impartire stress, angoscia o aumentare lo stato di sofferenza del cane. anestesia i protocolli anestesiologici sono scelti in base alle buone pratiche veterinarie. indipendentemente dal protocollo utilizzato, l’anestesia indotta nell’animale deve essere sempre profonda. somministrazione dei farmaci per l’eutanasia i farmaci per l’eutanasia devono essere somministrati da un medico veterinario. compilazione dei documenti e dei registri previsti la morte deve essere riportata nel registro di carico e scarico con indicazione della data di decesso e motivo della morte. smaltimento dell’animale soppresso l’animale deceduto deve essere avviato a un impianto inceneritore. se il canile non possiede un proprio impianto, deve essere stoccato in congelatore sino al ritiro da parte della ditta autorizzata che provvederà al trasporto presso un impianto di incenerimento autorizzato. scheda tecnica xvii. eutanasia. il canile rifugio, procedure e protocolli 38 veterinaria italiana, collana di monografie, monografia 28, 2020 • somministrazione del farmaco eutanasico; • compilazione dei documenti e registri previsti (certificato di morte, scarico dell’animale dal registro di carico/scarico e comunicazione all’anagrafe canina) • smaltimento dell’animale soppresso secondo la normativa in vigore. • valutazione; • trasporto dell’animale in luogo idoneo e contenimento (da tener presente che questa fase può essere eliminata qualora le condizioni fisiche del cane rendano difficoltoso o stressante lo spostamento); • anestesia profonda, preceduta da eventuale sedazione; in talune regioni è prevista la possibilità di cedere il proprio cane al canile a titolo definitivo. tale prassi, che trova giustificazione in caso di decesso o impossibilità (fisico/economica) del padrone, deve essere oggetto di controllo da parte del servizio veterinario ufficiale al fine di evitarne un ricorso incontrollato. la richiesta di cessione deve essere presentata al sindaco e deve essere prevista la registrazione in anagrafe per evitare che la stessa persona possa riprendere altri animali nelle medesime condizioni. cessione del cane parte iii normativa veterinaria italiana, collana di monografie, monografia 28, 2020 41 il canile rifugio, procedure e protocolli regione abruzzo legge regionale n. 47 del 18 dicembre 2013 norme sul controllo del randagismo, anagrafe canina e protezione degli animali da affezione bollettino ufficiale telematico della regione abruzzo speciale n. 127 del 27 dicembre 2013. deliberazione n. 213 del 28 marzo 2011 approvazione, ai sensi dell’art. 2 della l.r. 21 settembre 1999, n. 86, del programma di prevenzione del randagismo della regione abruzzo 2011-2013 bollettino ufficiale regione abruzzo n. 28 del 22 aprile 2011 legge regionale n. 9 del 7 maggio 2007 cimiteri per animali d’affezione bollettino ufficiale regione abruzzo n. 27 dell’11 maggio 2007 legge regionale n. 8 del 23 gennaio 2004 modifiche ed integrazioni alla l.r. 21 settembre 1999, n. 86 bollettino ufficiale regione abruzzo n. 1 (straordinario) dell’11 febbraio 2004 legge regionale n. 86 del 21 settembre 1999 norme sul controllo del randagismo, anagrafe canina e protezione degli animali da affezione bollettino ufficiale regione abruzzo n. 39 del 13 ottobre 1999 legge regionale n. 31 del 9 aprile 1997 finanziamento della costruzione delle strutture di ricovero per cani e gatti nonché per la prevenzione del randagismo bollettino ufficiale regione abruzzo n. 9 del 20 maggio 1997 legge regionale n. 27 del 3 aprile 1995 istituzione del servizio volontario di vigilanza ecologica bollettino ufficiale regione abruzzo n. 10 del 28 aprile 1995. legge regionale n. 34 del 31 maggio 1994 finanziamento costruzione canili sanitari bollettino ufficiale regione abruzzo n. 25 del 24 giugno 1994 legge regionale n. 15 dell’11 febbraio 1992 norme sul controllo del randagismo,istituzione dell’anagrafe canina e sulla protezione degli animali da affezione bollettino ufficiale regione abruzzo n. 8 del 5 marzo 1992 legge regionale n. 26 del 6 aprile 1989 modifiche ed integrazioni alla lr 16.6.87, n. 31 concernente: “tutela e valorizzazione del cane da pastore abruzzese” bollettino ufficiale regione abruzzo n. 17 del 26 aprile 1989 regione basilicata legge regionale n. 46 del 30 novembre 2018 disposizioni in materia di randagismo e tutela degli animali da compagnia di affezione legge n. 35 del 6 dicembre 2017 promozione delle terapie, dell’educazione e delle attività assistite con gli animali. succ. mod. con lr 29 giugno 2018, n. 11 delibera regionale n. 20160000022 del 12 gennaio 2016 presa d’atto dell’accordo tra il ministero della salute, la regione basilicata e l’ente nazionale protezione animali onlus (enpa) per l’avvio nella regione basilicata del progetto pilota contro il fenomeno del randagismo legge regionale n. 7 del 4 febbraio 2003 disciplina del bilancio di previsione e norme di contenimento e realizzazione della spesa per l’esercizio 2003 bollettino ufficiale regione basilicata n. 11 del 4 febbraio 2003 legge regionale n. 3 del 24 febbraio 2009 normativa regionale il canile rifugio, procedure e protocolli 42 veterinaria italiana, collana di monografie, monografia 28, 2020 bollettino ufficiale della regione calabria n. 15 dell’11 marzo 2000 legge regionale n. 41 del 5 maggio 1990 istituzione anagrafe canina, prevenzione randagismo e protezione degli animali bollettino ufficiale della regione calabria n. 4 del 12 gennaio 1990 regione campania deliberazione giunta regionale n. 209 del 27 giugno 2014 recepimento dell’accordo tra il governo, le regioni e le province autonome del 24 gennaio 2013 in materia di identificazione e registrazione degli animali d’affezione approvazione del disegno di legge recante “tutela degli animali d’affezione e prevenzione del randagismo” deliberazione n. 2131 del 7 dicembre 2007 priorità, modalità e termini per la concessione dei contributi previsti dalla legge regionale 16/2001 recante: “tutela degli animali d’affezione e prevenzione del randagismo” bollettino ufficiale della regione campania n.1 del 7 gennaio 2008 deliberazione n. 1214 del 23 settembre 2005 modifiche alla delibera di giunta regionale n. 3438 del 12 luglio 2002, concernenti le linee guida interpretative della l.r. 16/01 in materia di tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione campania n. 58 del 9 novembre 2005 deliberazione n. 1276 del 7 ottobre 2005 priorità, modalità e termini per la concessione dei contributi previsti dalla legge regionale 16/2001 recante “tutela degli animali d’affezione e prevenzione del randagismo” bollettino ufficiale della regione campania n. 55 del 31 ottobre 2005 deliberazione n. 3438 del 12 luglio 2002 linee guida interpretative della l.r. 16 del 24 novembre 2001, concernente la tutela degli animali d’affezione e la prevenzione del randagismo cimiteri per animali d’affezione bollettino ufficiale regione basilicata n. 10 del 01 gennaio 2009 legge regionale n. 6 del 25 gennaio1993 norme sulla prevenzione e sul controllo del randagismo. istituzione anagrafica canina e protezione degli animali di affezione bollettino ufficiale regione basilicata n. 3 del 29 gennaio 1993 provincia autonoma di bolzano legge provinciale n. 9 del 15 maggio 2000 interventi per la protezione degli animali e prevenzione del randagismo bollettino ufficiale della regione bolzano n. 23 del 30 maggio 2000, supplemento ordinario. legge provinciale n. 16 dell’8 luglio 1986 interventi per la protezione degli animali bollettino ufficiale della regione bolzano (prov.) n. 31 del 22 luglio 1986 regione calabria decreto del presidente della giunta regionale n. 32 del 11 maggio 2015 decreto del presidente della giunta regionale n. 51 del 19 maggio 2014, modificativo del dpgr-ca n. 197 del 20 dicembre 2012 razionalizzazione degli interventi in materia di randagismo:istituzione di una rete di canili sanitari nel territorio della regione calabria modifiche e integrazioni decreto del presidente della giunta regionale n. 197 del 20 dicembre 2012 razionalizzazione degli interventi in materia di randagismo: istituzione di una rete di canili sanitari nel territorio della regione calabria. obiettivo svet bollettino ufficiale della regione calabria n. 2 del 16 gennaio 2013 legge regionale n. 4 del 3 marzo 2000 modifiche ed integrazioni alla legge regionale 5 maggio 1990, n. 41 recante: istituzione anagrafe canina, prevenzione randagismo e protezione degli animali veterinaria italiana, collana di monografie, monografia 28, 2020 43 il canile rifugio, procedure e protocolli n. 20 (norme per il benessere e la tutela degli animali di affezione) bollettino ufficiale della regione friuli venezia giulia n. 11 del 18 marzo 2015 legge regionale n. 20 dell’11 ottobre 2012 norme per il benessere e la tutela degli animali di affezione bollettino ufficiale della regione friuli venezia giulia n. 42 del 17 ottobre 2012 legge regionale n. 134 del 10 giugno 2011 modifiche al decreto del 6 giugno 2002, riformulandone in particolare gli articoli relativi all’anagrafe canina e alla strutture di ricovero bollettino ufficiale della regione friuli-venezia giulia n. 25 del 22 giugno 2011 decreto del presidente della regione n. 171 del 6 giugno 2002 legge regionale n. 39/1990. regolamento di esecuzione della legge regionale 4 settembre 1990, n. 39, in materia di tutela degli animali domestici per il controllo e la prevenzione del fenomeno del randagismo. istituzione dell’anagrafe canina bollettino ufficiale della regione friuli venezia giulia n. 27 del 3 luglio 2002 legge regionale n. 39 del 4 settembre 1990 norme a tutela degli animali domestici per il controllo e la prevenzione del fenomeno del randagismo. istituzione dell’anagrafe canina bollettino ufficiale della regione friuli-venezia giulia n. 108 del 5 settembre 1990 regione lazio deliberazione della giunta regionale n. 621 del 25 ottobre 2016 deliberazione della giunta regionale n. 43 del 29 gennaio 2010 deliberazione n. 394 del 29 maggio 2009 istituzione dell’osservatorio per i diritti degli animali d’affezione e la prevenzione del randagismo. attività di promozione dell’anagrafe canina regionale bollettino ufficiale della regione lazio n. 27 del 21 luglio 2009 bollettino ufficiale della regione campania n. 42 del 9 settembre 2002 legge regionale n. 16 del 24 novembre 2001 tutela degli animali d’affezione e prevenzione del randagismo bollettino ufficiale della regione campania speciale del 29 novembre 2001 legge regionale n. 36 del 2 novembre 1993 tutela degli animali d’affezione e istituzione dell’anagrafe canina bollettino ufficiale della regione campania n. 48 dell’8 novembre 1993 regione emilia romagna deliberazione n. 353 del 2 aprile 2013 approvazione dei requisiti strutturali e gestionali per le strutture di ricovero di cani e gatti, oasi e colonie feline bollettino ufficiale della regione emilia romagna n. 121 dell’8 maggio 2013 legge regionale n.3 del 29 marzo 2013 modifiche alla legge regionale 17 febbraio 2005, n. 5 (norme a tutela del benessere animale). bollettino ufficiale della regione emilia romagna n. 3 29.03.2013 delibera giunta regionale 647/2007 indicazioni tecniche in attuazione alla legge regionale 5/05 relativa alla tutela del benessere degli animali. parziale modifica alla delibera 394/06 bollettino ufficiale della regione emilia romagna n. 75 del 5 giugno 2007 legge regionale n. 5 del 17 febbraio 2005 norme a tutela del benessere animale bollettino ufficiale della regione emilia romagna n. 30 del 18 febbraio 2005 legge regionale n. 27 del 7 aprile 2000 nuove norme per la tutela ed il controllo della popolazione canina e felina bollettino ufficiale della regione emilia romagna n. 61 del 10 aprile 2000 regione friuli-venezia giulia legge regionale n. 5 del 13 marzo 2015 modifiche alla legge regionale 11 ottobre 2012, il canile rifugio, procedure e protocolli 44 veterinaria italiana, collana di monografie, monografia 28, 2020 bollettino ufficiale della regione liguria n. 9 del 13 aprile 1994 regione lombardia legge regionale n. 15 del 29 giugno 2016 evoluzione del sistema sociosanitario lombardo: modifiche ai titoli v e viii della legge regionale 30 dicembre 2009, n. 33 (testo unico delle leggi regionali in materia di sanità) bollettino ufficiale della regione lombardia n. 27, suppl. del 4 luglio 2016 legge regionale n. 33 del 30 dicembre 2009 testo unico delle leggi regionali in materia di sanità bollettino ufficiale della regione lombardia n. 52, 3° suppl. ord. del 31 dicembre 2009 regolamento regionale n. 2 del 5 maggio 2008 regolamento di attuazione della legge regionale n. 16 del 20 luglio 2006 (lotta al randagismo e tutela degli animali di affezione) bollettino ufficiale della regione lombardia n.19 del 9 maggio 2008 legge regionale n. 16 del 20 luglio 2006 lotta al randagismo e tutela degli animali da affezione bollettino ufficiale della regione lombardia n. 30 del 24 luglio 2006 (supplemento ordinario n. 1 del 25 luglio 2006) legge regionale n. 30 dell’8 settembre 1987 prevenzione del randagismo tutela degli animali e della salute pubblica bollettino ufficiale della regione lombardia n. 36 del 9 settembre 1987 (supplemento ordinario n. 2 del 9 settembre 1987) regione marche legge regionale n. 18 del 20 aprile 2015 modifiche alla legge regionale 20 gennaio 1997, n. 10 “norme in materia di animali da affezione e prevenzione del randagismo” bollettino ufficiale della regione marche n.37 del 30 aprile 2015 delibera della giunta regionale n. 1172/2005 regolamento regionale n. 1 del 27 gennaio 1997 regolamento di attuazione della legge regionale n. 89 del 14 dicembre 1990 norma sulla detenzione, allevamento e commercio di animali esotici bollettino ufficiale della regione lazio n. 4 del 10 febbraio 1997 deliberazione n. 920 del 21 dicembre 2006 revoca della deliberazione di giunta regionale n. 176 del 18 febbraio 2005 e adozione nuove linee guida relative all’applicazione del microchip, quale sistema di identificazione ai fini dell’anagrafe canina ed al rilascio del passaporto europeo per cani, gatti e furetti bollettino ufficiale della regione lazio n. 4 del 10 febbraio 2007 deliberazione n. 487 del 3 luglio 2007 approvazione linee guida per la ripartizione dei fondi regionali per l’attuazione dei piani di controllo delle nascite attraverso la sterilizzazione dei cani randagi catturati e/o a rischio di riproduzione incontrollata e per la costruzione e/o il risanamento dei canili pubblici. revoca della dgr 1370/98. 30-8-2007 bollettino ufficiale della regione lazio n. 24 del 30 agosto 2007 legge regionale n. 34 del 21 ottobre 1997 tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione lazio n. 30 del 30 ottobre 1997 legge regionale n. 89 del 14 dicembre 1990 norme sulla detenzione, l’allevamento ed il commercio di animali esotici bollettino ufficiale della regione lazio n. 36 del 29 dicembre 1990 regione liguria legge regionale n. 23 del 22 marzo 2000 tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione liguria n. 7 del 12 aprile 2000 legge regionale n. 16 del 24 marzo 1994 nuove norme in materia di randagismo veterinaria italiana, collana di monografie, monografia 28, 2020 45 il canile rifugio, procedure e protocolli legge regionale n. 7 del 4 marzo 2005 nuove norme per la protezione dei cani e per l’istituzione dell’anagrafe canina bollettino ufficiale della regione molise n. 6 del 16 marzo 2005 legge regionale n. 11 del 4 marzo 1992 norme per la protezione dei cani e per l’istituzione dell’ anagrafe canina bollettino ufficiale della regione molise n. 5 del 16 marzo 1992 regione piemonte deliberazione della giunta regionale n. 10-7753 del 30 ottobre 2018 variazione al bilancio di previsione finanziario 2018-2020. finanziamenti statali per attività sanitaria in materia di prevenzione del randagismo di animali. deliberazione della giunta regionale n. 32-7387 del 7 aprile 2014 recepimento dell’accordo rep. n. 5/cu del 24/1/2013 in materia di identificazione e registrazione degli animali da affezione bollettino ufficiale della regione piemonte n. 17s1 del 24 aprile 2014 legge regionale n. 27 del 4 novembre 2009 disciplina del rapporto persone-cani per la prevenzione della salute pubblica e del benessere animale bollettino ufficiale della regione piemonte n. 45 del 12 novembre 2009 legge regionale n. 22 del 6 agosto 2009 disposizioni collegate alla manovra finanziaria per l’anno 2009 bollettino ufficiale della regione piemonte n. 31 del 7 agosto 2009 decreto del presidente della giunta regionale n. 10 del 25 giugno 2008 integrazioni al regolamento regionale 11 novembre 1993, n. 2 (regolamento per la tutela e controllo degli animali da affezione) bollettino ufficiale della regione piemonte n. 27 del 3 luglio 2008 deliberazione n. 35-5274 del 12 febbraio 2007 recepimento ed attuazione dell’accordo sancito il 6 febbraio 2003 tra il ministero della salute, le regioni e le provincie autonome di trento e di bolzano in materia di benessere degli animali da compagnia e pet-therapy bollettino ufficiale della regione marche n. 93 del 22 ottobre 2010 regolamento regionale n. 2 del 13 novembre 2001 attuazione della legge regionale 20 gennaio 1997 n.10 norme in materia di animali da affezione e prevenzione del randagismo e succ. modd. bollettino ufficiale della regione marche n. 134 del 22 novembre 2001 legge regionale n. 74 del 29 dicembre 1997 modificazioni alla legge regionale 20 gennaio 1997, n. 10 “norme in materia di animali da affezione e prevenzione del randagismo” bollettino ufficiale della regione marche n. 3 del 9 gennaio 1998 legge regionale n. 25 del 18 marzo 1997 contributo una tantum ad associazioni protezionistiche che gestiscono canili e rifugi per cani. bollettino ufficiale della regione marche n. 22 del 27 marzo 1997 legge regionale n. 10 del 20 gennaio 1997 norme in materia di animali da affezione e prevenzione del randagismo bollettino ufficiale della regione marche n. 8 del 24 gennaio 1997 regione molise delibera di giunta regionale n. 806 del 18 dicembre 2012 programma 2013-2015 per la prevenzione del randagismo e per la gestione dell’anagrafe canina bollettino ufficiale della regione molise n. 4 del 1 febbraio 2013 legge regionale n.12 del 24 giugno 2011 modifiche ed integrazioni alla legge regionale n. 7 del 4 marzo 2005, recante “nuove norme per la protezione dei cani e per l’istituzione dell’anagrafe canina” bollettino ufficiale della regione molise n. 18 del 1 luglio 2011. il canile rifugio, procedure e protocolli 46 veterinaria italiana, collana di monografie, monografia 28, 2020 bollettino ufficiale della regione puglia n.  18 del 10 febbraio 2020 deliberazione della giunta regionale n. 1223 del 4 luglio 2013. linee guida attuative dell’art. 2 della l. 281/91 e degli artt. 6 e 8 della l.r. 12/95 in materia di prevenzione del fenomeno del randagismo bollettino ufficiale della regione puglia n. 102 del 24 luglio 2013 deliberazione n. 2505 del 27 novembre 2012 tutela degli animali di affezione e prevenzione del randagismo. contributi destinati ai comuni e all’unione dei comuni della regione puglia per la campagna di sterilizzazione di cani padronali e per la realizzazione e/o ampliamento di canili sanitari, di proprietà comunale bollettino ufficiale della regione puglia n. 186 del dicembre 2012 legge regionale n. 34 del 12 dicembre 2006 modifiche e integrazioni alle leggi regionali 9 agosto 2006, n. 26 (interventi in materia sanitaria) e 3 aprile 1995, n. 12 (interventi per la tutela degli animali d’affezione e prevenzione del randagismo) bollettino ufficiale della regione puglia n. 166 del 15 dicembre 2006 legge regionale n. 15 del 31 luglio 1996 integrazione della legge regionale 3 aprile 1995, n. 12 concernente gli interventi per la tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione puglia n. 86 del 7 agosto 1996 legge regionale n. 12 del 03 aprile 1995 interventi per la tutela degli animali d’affezione e prevenzione del randagismo bollettino ufficiale della regione puglia n. 39 del 18 aprile 1995 regione sardegna deliberazione n. 34/9 del 3 luglio 2018 direttive in materia di lotta al randagismo e protezione degli animali di affezione approvate con la delib.g.r. n. 17/39 del 27 aprile 2010. modifica art. 4 e allegati n. 9, 10, 11 recepimento del d.p.c.m. 28.02.2003 recante “accordo tra il ministro della salute, le regioni e le province autonome di trento e di bolzano, in materia di benessere degli animali da compagnia e pet-therapy” bollettino ufficiale della regione piemonte n. 10 dell’8 marzo 2007 legge regionale n. 9 del 4 luglio 2005 modifiche alla legge regionale 19 luglio 2004, n. 18 (identificazione elettronica degli animali da affezione e banca dati informatizzata. abrogazione della legge regionale 13 aprile 1992, n. 20) bollettino ufficiale della regione piemonte n. 27 del 7 luglio 2005 legge regionale n. 18 del 19 luglio 2004 identificazione elettronica degli animali da affezione e banca dati informatizzata. abrogazione della legge regionale 13 aprile 1992, n. 20 (istituzione dell’anagrafe canina) bollettino ufficiale della regione piemonte n. 29 del 22 luglio 2004 legge regionale n. 39 del 7 aprile 2000 cimiteri per animali d’affezione bollettino ufficiale della regione piemonte n. 15 del 12 aprile 2000 d.c.r. 697/1993 decreto del presidente della giunta regionale n. 4359 regolamento n. 2 dell’11 novembre 1993 regolamento per la tutela e controllo degli animali da affezione bollettino ufficiale della regione piemonte n 47 del 24 novembre 1993 legge regionale n. 34 del 26 luglio 1993 tutela e controllo degli animali da affezione bollettino ufficiale della regione piemonte n. 31 del 4 agosto 1993 regione puglia legge regionale n. 2 del 7 febbraio 2020 norme sul controllo del randagismo, anagrafe canina e protezione degli animali da affezione. abrogazione della legge regionale 3 aprile 1995, n. 12 (interventi per la tutela degli animali d’affezione e prevenzione del randagismo) veterinaria italiana, collana di monografie, monografia 28, 2020 47 il canile rifugio, procedure e protocolli regione sicilia deliberazione n. 468 del 19 novembre 2018 linee guida per il contrasto e la prevenzione nella regione siciliana del fenomeno del randagismo. decreto dell’assessore della salute n. 2440 del 28 novembre 2011 criteri e modalità per la concessione dei contributi previsti dall’art. 20, commi 1 e 2 della legge regionale 3 luglio 2000 n. 15 legge regionale n. 15 del 3 luglio 2000 istituzione dell’anagrafe canina e norme per la tutela degli animali da affezione e la prevenzione del randagismo direttiva assessorato per la sanità n. 1059 del 12 giugno 2009 controllo del randagismo misure a tutela dell’incolumità pubblica decreto 13 dicembre 2007 linee guida per il controllo del randagismo e bandi per la concessione di contributi da destinare al risanamento dei rifugi esistenti e alla costruzione di rifugi sanitari, all’attuazione di piani di controllo delle nascite e al mantenimento di animali bollettino ufficiale della regione sicilia n. 4 del 25 gennaio 2008 circolare n. 300 del 13 febbraio 2007 benessere animale, randagismo, stato di applicazione della legge regionale 3 luglio 2000, n. 15 decreto del presidente della regione siciliana n. 7 del 12 gennaio 2007 regolamento esecutivo dell’art. 4 della legge regionale 3 luglio 2000, n. 15 “istituzione dell’anagrafe canina e norme per la tutela degli animali d’affezione e prevenzione del randagismo” bollettino ufficiale della regione sicilia n. 15 del 6 aprile 2007 decreto del presidente della regione siciliana n. 15 del 27 giugno 2002 regolamento concernente i requisiti dell’albo delle associazioni per la protezione degli animali bollettino ufficiale della regione sicilia n. 47 dell’11 ottobre 2002 deliberazione n. 17/39 del 27 aprile 2010 delibera della giunta regionale n. 44/35 del 14 dicembre 2010 trasferimenti alle aziende sanitarie locali per l’identificazione elettronica animale e per la gestione anagrafe animale delibera della giunta regionale n. 38/13 del 9 novembre 2010 legge 14 agosto 1991, n. 281 e legge regionale 18 maggio 1994, n. 21. contributi ai comuni per la lotta al randagismo e la gestione dei canili e ripartizione tra le aziende sanitarie locali dei fondi regionali e statali per la prevenzione del randagismo delibera n. 17/39 del 27 aprile 2010 l.r. n. 21/1994 e s.m.i. direttive in materia di lotta al randagismo e protezione degli animali d’affezione ministero della salute circolare 2725/p i.8.d/318 del 27 luglio 2006 revoca dell’obbligo di vaccinazione antirabbica per i cani in ingresso in sardegna decreto del presidente della giunta regionale n. 1 del 4 marzo 1999 regolamento di attuazione della legge 14 agosto 1991, n. 281 e della legge regionale 18 maggio 1994, n. 21 e della legge regionale 1° agosto 1996, n. 35 sulla prevenzione del randagismo bollettino ufficiale della regione sardegna n. 13 del 29 aprile 1999 legge regionale n. 35 del 1 agosto 1996 integrazioni e modifiche alla legge regionale 18 maggio 1994, n. 21, recante: «norme per la protezione degli animali e istituzione dell’ anagrafe canina» bollettino ufficiale della regione sardegna n. 25 dell’8 agosto 1996 legge regionale n. 21 del 18 maggio 1994 norme per la protezione degli animali e istituzione dell’anagrafe canina bollettino ufficiale della regione sardegna n. 17 del 21 maggio 1994 il canile rifugio, procedure e protocolli 48 veterinaria italiana, collana di monografie, monografia 28, 2020 tela degli animali d’affezione e la prevenzione del randagismo) bollettino ufficiale della regione toscana n. 41 del 26 ottobre 2009 legge regionale n. 90 del 4 dicembre 1998 modifiche ed integrazioni della legge regionale 8 aprile 1995, n. 43 “norme per la gestione dell’anagrafe del cane, la tutela degli animali d’affezione e la prevenzione del randagismo” bollettino ufficiale della regione toscana n. 42 del 10 dicembre 1998 legge regionale n. 43 dell’8 aprile 1995 norme per la gestione dell’anagrafe del cane, la tutela degli animali d’ affezione e la prevenzione del randagismo bollettino ufficiale della regione toscana n. 28 del 18 aprile 1995 legge regionale n. 89 del 30 dicembre 1989 lr 4/ 87 istitutiva dell’ anagrafe canina. modifiche ed integrazioni all’art. 14 bollettino ufficiale della regione toscana n. 3 del 10 gennaio 1990 provincia autonoma di trento legge provinciale n.4 del 28 marzo 2012. protezione degli animali d’affezione e prevenzione del randagismo bollettino ufficiale provincia di trento n. 14 del 3 aprile 2012 regione umbria legge regionale n. 11 del 2015 testo unico in materia di sanità titolo xvi, capo iv (norme per il benessere e la tutela degli animali di affezione), capo v (prevenzione e controllo del fenomeno del randagismo) e capo vi (divieto di detenzione e utilizzazione di esche avvelenate) deliberazione n. 255 del 10 giugno 2013 deliberazione della giunta regionale n. 69 del 19 gennaio 2005 accordo tra ministero della salute, regioni e province autonome di trento e di bolzano in materia benessere degli animali da compagnia, cimiteri e pet-therapy. recepimento e linee guida vincolanti legge regionale n. 15 del 3 luglio 2000 istituzione dell’anagrafe canina e norme per la tutela degli animali da affezione e la prevenzione del randagismo bollettino ufficiale della regione sicilia n. 32 del 7 luglio 2000 regione toscana delibera n. 943 del 6 ottobre 2015 linee guida per l’istituzione del soccorso animali delibera n. 1153 del 30 novembre 2015 recepimento dell’accordo stato regioni e province autonome n. 60/csr del 25 marzo 2015, che approva le “linee guida nazionali per gli interventi assistiti con gli animali (iaa)”, in armonizzazione con la l.r. 59/2009. legge regionale n. 9 del 20 gennaio 2015 disciplina dei cimiteri per animali d’affezione bollettino ufficiale della regione toscana n. 4 del 23.1.2015 delibera n. 1233 del 22 dicembre 2014 linee d’indirizzo per l’accesso degli animali d’affezione in visita a degenti presso strutture sanitarie e ospedaliere pubbliche e private accreditate decreto del presidente della giunta regionale n. 53/r del 1 ottobre 2013 modifiche al d.p.g.r. 4 agosto 2011, n. 38/r regolamento di attuazione della legge regionale 20 ottobre 2009, n. 59 decreto del presidente della giunta regionale n. 38 del 4 agosto 2011 regolamento di attuazione della legge regionale 20 ottobre 2009, n. 59 “norme per la tutela degli animali. abrogazione della legge regionale 8 aprile 1995, n. 43” (norme per la gestione dell’anagrafe del cane, la tutela degli animali d’affezione e la prevenzione del randagismo) bollettino ufficiale della regione toscana n. 39 del 5 agosto 2011 legge regionale n. 59 del 20 ottobre 2009 norme per la tutela degli animali. abrogazione della legge regionale 8 aprile 1995, n. 43 (norme per la gestione dell’anagrafe del cane, la tuveterinaria italiana, collana di monografie, monografia 28, 2020 49 il canile rifugio, procedure e protocolli modifica della legge regionale 28 dicembre 1993, n. 60 “tutela degli animali d’affezione e prevenzione del randagismo” e successive modificazioni bollettino ufficiale della regione veneto n. 62 del 24 giugno 2014 deliberazione della giunta regionale n. 272 del 6 febbraio 2007 linee guida per una regolamentazione uniforme dell’igiene urbana veterinaria nel territorio della regione veneto. completamento del recepimento dell’accordo tra il ministero della salute, le regioni e le province autonome di trento e bolzano in materia di benessere degli animali da compagnia e pet-therapy. modifica d.g.r. 243 del 7 febbraio 2006 deliberazione della giunta regionale n. 243 del 7 febbraio 2006 linee guida per una regolamentazione uniforme dell’igiene urbana veterinaria nel territorio della regione veneto. completamento del recepimento dell’accordo tra il ministero della salute, le regioni e le province autonome di trento e bolzano in materia di benessere degli animali da compagnia e pet-therapy. legge regionale n. 60 del 28 dicembre 1993. tutela degli animali d’affezione e prevenzione del randagismo. bollettino ufficiale della regione veneto n. 111 del 31 dicembre 1993. bollettino ufficiale della regione umbria n. 8 del 23 febbraio 2005 regione valle d’aosta dgr n. 1162 del 28 giugno 2013 modifiche ed integrazioni delle linee guida regionali per la tutela degli animali d’affezione linee guida regionali per la tutela degli animali d’affezione approvate con dgr n. 1731 del 24.08.2012 ai sensi art.4 comma 2 della l.r. n. 37/2010 dgr n. 1731 del 24 agosto 2012 linee guida regionali per la tutela degli animali d’affezione linee guida regionali per la tutela degli animali d’affezione ai sensi art. 4 comma 2 della l.r. n. 37/2010. legge regionale n. 37 del 22 novembre 2010 nuove disposizioni per la tutela e per il corretto trattamento degli animali di affezione. abrogazione della legge regionale 28 aprile 1994, n. 14 bollettino ufficiale della regione valle d’aosta n. 51 del 14 dicembre 2010 legge regionale n. 14 del 28 aprile 1994 norme per la tutela e per il corretto trattamento degli animali di affezione bollettino ufficiale della regione valle d’aosta n. 21 del 10 maggio 1994 regione veneto legge regionale n.17 del 19 giugno 2014 il canile rifugio, procedure e protocolli 50 veterinaria italiana, collana di monografie, monografia 28, 2020 decreto del presidente della repubblica 8 febbraio 1954, n. 320 regolamento di polizia veterinaria gazzetta ufficiale n. 142 del 24 giugno 1954 legge 14 agosto 1991 n. 281 legge quadro in materia di animali di affezione e prevenzione del randagismo gazzetta ufficiale n. 203 del 30 agosto 1991 ministero della sanità decreto 14 ottobre 1996 norme in materia di affidamento dei cani randagi gazzetta ufficiale n. 300 del 23 dicembre 1996 (annullato con decreto 19 novembre 1998) ministero della sanità circolare 14 maggio 2001, n. 5 attuazione della legge 14 agosto 1991, n. 281. gazzetta ufficiale n. 144 del 23 giugno 2001 decreto del presidente del consiglio dei ministri 28 febbraio 2003 recepimento dell’accordo del 6 febbraio 2003 recante disposizioni in materia di benessere degli animali da compagnia e pet-therapy gazzetta ufficiale n. 52 del 4 marzo 2003 legge 20 luglio 2004, n.189 disposizioni concernenti il divieto di maltrattamento degli animali, nonché di impiego degli stessi in combattimenti clandestini o competizioni non autorizzate gazzetta ufficiale n.178 del 31 luglio 2004 ministero della salute decreto 13 maggio 2005 determinazione dei criteri per la ripartizione dei fondi per la prevenzione e lotta al randagismo, previsti dalla legge del 29 dicembre 2003, n. 376. gazzetta ufficiale n. 169 del 22 luglio 2005 decreto 2 novembre 2006 individuazione delle associazioni e degli enti affidatari di animali oggetto di provvedimento di sequestro o di confisca, nonché determinazione dei criteri di riparto delle entrate derivanti dall’applicazione di sanzioni pecuniarie gazzetta ufficiale n. 19 del 24 gennaio 2007 ministero della salute ministero dell’economia e delle finanze decreto 06 maggio 2008 determinazione dei criteri per la ripartizione tra le regioni e le province autonome delle disponibilità del fondo per l’attuazione della legge 14 agosto 1991, n. 281, recante: «legge quadro in materia di animali di affezione e prevenzione del randagismo». gazzetta ufficiale n. 185 dell’8 agosto 2008 ministero del lavoro, della salute e delle politiche sociali decreto 28 luglio 2009 disciplina dell’utilizzo e della detenzione di medicinali ad uso esclusivo del medico veterinario gazzetta ufficiale n. 230 del 3 ottobre 2009 ministero del lavoro, della salute e delle politiche sociali decreto 26 novembre 2009 percorsi formativi per i proprietari dei cani gazzetta ufficiale n. 19 del 25 gennaio 2010 ministero della salute ordinanza 14 gennaio 2010 proroga e modifica dell’ordinanza 18 dicembre 2008, come modificata dall’ordinanza 19 marzo 2009, recante: «norme sul divieto di utilizzo e di detenzione di esche o di bocconi avvelenati» gazzetta ufficiale n. 33 del 10 febbraio 2010 legge 4 novembre 2010, n. 201 ratifica ed esecuzione della convenzione europea per la protezione degli animali da compagnia, fatta a strasburgo il 13 novembre 1987, nonché norme di adeguamento dell’ordinamento interno gazzetta ufficiale n. 283 del 3 dicembre 2010 normativa nazionale veterinaria italiana, collana di monografie, monografia 28, 2020 51 il canile rifugio, procedure e protocolli ministero della salute ordinanza 12 luglio 2019 norme sul divieto di utilizzo e di detenzione di esche o di bocconi avvelenati gazzetta ufficiale n.196 del 22 agosto 2019 ministero della salute ordinanza 18 luglio 2019 proroga dell’ordinanza contingibile e urgente 6 agosto 2013, e successive modificazioni, concernente la tutela dell’incolumità pubblica dall’aggressione dei cani gazzetta ufficiale n.196 del 22 agosto 2019 accordo 24 gennaio 2013 accordo, ai sensi dell’articolo 9, comma 2, lettera c), del decreto legislativo 28 agosto 1997, n. 281, tra il governo, le regioni e le province autonome di trento e bolzano, le province, i comuni e le comunità montane in materia di identificazione e registrazione degli animali da affezione. (rep. atti n. 5/cu). 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(ce) 1069 2009. 24. mornement k.m., coleman g.j., toukhsati s. & bennett p.c. 2010. a review of behavioural assessment protocols used to determine the adoption suitability of australian shelter dogs. j appl anim welf sci, 13(4), 314-329. 25. natoli e., totino r., alfieri l., vassallo g., donato s. & fantini c. 2001. determinazione della personalità dei cani ospitati presso il presidio canile sanitario per la formulazione di schede individuali ai fini dell’adozione. il progresso veterinario, lvi, 12. 26. newbury s., blinn m.k., bushby p.a., cox c.b., dinnage j.d., griffin b., hurley k.f., isaza n., jones w., miller l., o’quin j., patronek g.j., blackmore m.s. & spindel m. 2010. guidelines for standards of care in animal shelters association of shelter veterinarians. 27. notari l. 2004. dal canile a casa vostra. edizioni calderini de il sole, bologna (italia). 28. petrantoni g. 2007. problemi di bioetica isbn 9788893650038 35 parole chiave e. coli fecale aviare, gruppi filogenetici, polli, resistenza antimicrobica, virulenza. riassunto l'obiettivo dello studio è stato quello di determinare i caratteri di virulenza e di resistenza antimicrobica di 100 ceppi di e. coli fecali isolati da polli clinicamente sani in algeria. la maggior parte degli isolati apparteneva ai filogruppi a (45%) e b1 (37%) e mostrava una grande diversità nei profili genetici. sono stati rilevati i geni fimh, tsh, entb, iuta, irp2, fyua, iron, sita, etsa, etsb, eita, iss, trat, ompt, hlyf, vat, ibea, cvaa, cvab5’, cvab3’, cvac, cma e cbi. le combinazioni tra i geni di virulenza hanno permesso di definire 67 profili di virulenza. sono stati rilevati alti tassi di resistenza (62‑97%) per amoxicillina, amoxicillina‑acido clavulanico, cefazolina, fluorochinoloni, tetraciclina, trimetoprim, sulfonamidi e sulfametossazolo/ trimetoprim e il 93% dei ceppi presentavano resistenza multipla. sono stati identificati i geni bla tem , bla shv , bla ctx‑m‑1 , teta, tetb, qnrb, qnrs1, sul1, sul2, sul3, dfra1, dfra7, dfra12 e dfra14; gli integroni di classe 1 sono stati rilevati nel 49% degli isolati. una percentuale del 37% dei ceppi era resistente al mercurio, con la presenza del gene mera. lo studio riporta la presenza, nei ceppi aviari isolati da tamponi fecali, di geni di virulenza di origine plasmidica caratteristici dei ceppi expec associata ad un’elevata resistenza agli antibiotici di prima linea e di integroni di classe 1: un rischio per la salute umana e animale. geni di virulenza, resistenza antimicrobica e correlazione filogenetica di ceppi di e. coli isolati da allevamenti di broiler in algeria keywords avian fecal e. coli, broiler chickens, phylogenetic groups, virulence, antimicrobial resistance. summary the objective of the study was to determine the virulence and antimicrobial resistance traits of 100 fecal e. coli strains isolated from clinically healthy chickens in algeria. most of isolates belonged to phylogroups a (45%) and b1 (37%) and showed a great diversity in dna profiles. the genes fimh, tsh, entb, iuta, irp2, fyua, iron, sita, etsa, etsb, eita, iss, trat, ompt, hlyf, vat, ibea, cvaa, cvab5’, cvab3’, cvac, cma and cbi were detected. combinations of virulence genes defined 67 virulence profiles. high resistance rates (62‑97%) were noted for amoxicillin, amoxicillin‑clavulanic acid, cefazolin, fluoroquinolones, tetracycline, trimethoprim, sulfonamides and sulfamethoxazole/ trimethoprim, and 93% of strains were multidrug‑resistant. combinations of resistance phenotypes defined 59 resistance patterns. the genes bla tem , bla shv , bla ctx‑m‑1 , teta, tetb, qnrb, qnrs1, sul1, sul2, sul3, dfra1, dfra7, dfra12 and dfra14 were identified and class 1 integrons were detected in 49% of isolates. a rate of 37% of strains was resistant to mercury, with the presence of mera gene. the study reports the presence in the avian strains isolated from fecal swabs of virulence genes of plasmid origin characteristic of expec strains associated with high resistance to first‑line antibiotics and class 1 integrons, this augurs a risk for human and animal health. veterinaria italiana 2019, 55 (1), 35‑46. doi: 10.12834/vetit.799.3865.2 accepted: 07.07.2016 | available on line: 31.03.2019 laboratory of cellular and molecular biology, faculty of biological sciences, university of sciences and technology houari boumediene, pb 32 el-alia, bab ezzouar, 16111 algiers, algeria *corresponding author at: laboratory of cellular and molecular biology, faculty of biological sciences, university of sciences and technology houari boumediene, pb 32 el-alia, bab ezzouar, 16111 algiers, algeria. tel.: +213 21 24 79 13, fax: +213 21 24 72 17, e-mail: bakourrabah@gmail.com, rbakour@yahoo.fr. chahinez messaili, yamina messai and rabah bakour* virulence gene profiles, antimicrobial resistance and phylogenetic groups of fecal escherichia coli strains isolated from broiler chickens in algeria nick title first author et al. 36 veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx flora in terms of virulence and antimicrobial resistance. in this context, the objective of this study was to assess the prevalence of virulence factors and antimicrobial resistance and to determine the phylogenetic groups in fecal e.  coli strains isolated from clinically healthy broiler chickens. materials and methods bacterial strains one hundred non‑repetitive avian fecal e. coli strains (one isolate, one chicken) were recovered from intestines of 45‑47 day old clinically healthy chickens using sterile cotton swabs. chickens were from five poultry farms in the center of algeria: rouiba, shaoula, lakhdaria, tizi‑ouzou, bejaia. strains were identified using api 20e identification system (biomérieux, france) and by pcr detection of iuda gene (beta‑glucuronidase) (clermont et al. 2013). phylogenetic grouping and genotyping of isolates phylogenetic groups of strains were determined as previously described (clermont et al. 2013). molecular typing of strains was performed by enterobacterial repetitive intergenic consensus‑pcr (eric‑pcr) using primer eric2 (messai et al. 2008). eric profiles are compared visually and those dissimilar by one band or more were considered as different. pcr detection of virulence‑associated gene the dna template for pcr was extracted using the boiling method (feria et  al. 2002). all isolates were screened for the presence of 31 virulence associated genes (vags) by multiplex and simplex pcr (table i). e.  coli ec7372, ecor66 and cft073 were used as control strains. biofilm formation assay biofilm formation (bf) ability was assessed as previously described (naves et  al. 2008). briefly, a 100 fold dilution in lb medium of an overnight bacterial culture was distributed in 96‑well polystyrene microplate. after incubation at 37  °c for 24 h, the optical density (od) of bacterial growth was measured at 630  nm. plate was then emptied, washed with sterile distilled water, air dried for 20 min and stained with 130 µl of 1% crystal violet for 5 min. dye was discarded, and plate was washed five times to remove excess dye and air dried for 1 hour. the biofilm‑bound dye was eluted with 130 µl of 95% ethanol and the absorbance was measured at 570 introduction most escherichia coli are commensal bacteria present in the gut of humans and warm‑blooded animals. however, some strains can harbor virulence genes and might be associated with various intestinal (inpec strains) or extraintestinal (expec strains) infections in humans and animals (bélanger et  al. 2011). different combinations of virulence factors define pathotypes, which are specific of a type of infection. the expec group includes upec (uropathogenic), nmec (newborn meningitic), sepec (septicaemia associated) and apec (avian pathogenic) strains (bélanger et al. 2011). eight phylogenetic groups are now recognized in e. coli: seven (a, b1, b2, c, d, e, f) belong to e.  coli sensu stricto, whereas the eighth is the escherichia cryptic clade i (clermont et al. 2013). a and b1 groups generally include commensal strains and certain intestinal pathogens while b2 and to a lesser extent d groups are characteristic of extraintestinal pathogens (ewers et al. 2007, mellata 2013). avian colibacillosis caused by apec strains is one of the major causes of economic losses in the poultry industry in algeria and throughout the world; it manifests by various injuries as airsacculitis, peritonitis, polyserositis and sepsis. apec strains are characterized by the expression of various virulence factors including adhesins, toxins, iron uptake systems and serum resistance (johnson et  al. 2008, bélanger et  al. 2011, mellata 2013). there are genotypic similarities between apec and human expec, mainly upec and nmec, suggesting zoonotic potential among apec strains (bélanger et al. 2011, mellata 2013). in addition to the virulence factors, antibiotic resistance of bacteria determines their impact in infectious diseases. the increase of antibiotic resistance has worldwide reached alarming proportions; it is inherent in large part to the widespread use of antibiotics in intensive livestock, especially poultry (mellata 2013). healthy poultry is considered the main reservoir of virulence genes and antibiotic resistance (rodriguez‑sieck et al. 2005, ewers et al. 2009). pathogenic strains result generally from commensals by the acquisition of infectious capacity through horizontal transfer of virulence genes (johnson and nolan 2009, mellata et al. 2010). in addition to inter‑animal transfer, virulent and/or resistant bacteria and genes can even reach humans via contaminated environment and food chain (graham et  al. 2008, vincent et  al. 2010). in order to better understand and monitor the emergence of pathogens and antibiotic‑resistant strains from healthy animal reservoir, it is necessary to know the presence and prevalence of virulence factors and antibiotic resistance. these investigations are very important in algeria, because of the lack of data on this issue in this country, especially since veterinary practices, farming conditions and environment have a considerable impact on the evolution of intestinal first author et al. nick title veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx 37 used (µg/ disk): amoxicillin (amx) (25 µg), amoxicillin/ clavulanic acid (amc) (20  µg/10  µg), cefazolin (cz) (30 μg), cefotaxime (ctx) (30 μg), ceftazidime (caz) (30 μg), nalidixic acid (na) (30  µg), ciprofloxacin (cip) (5 µg), pefloxacin (pef) (5  µg), ofloxacin (ofx) (5  µg), tetracycline (te) (30  µg), kanamycin (k) (30  µg), netilmicin (net) (30 µg), gentamicin (gm) (15 µg), sulfonamides (sss) (200  µg), trimethoprim (tmp) (5  µg), sulfamethoxazole/trimethoprim (sxt) (1.25  µg/23.75  µg) and colistin (cs) (50  µg). e.  coli atcc 25922 was used as a control strain. multiple antibiotic resistance index (mar) was used to check the antibiotic resistance. mar is calculated as a ratio a/b, ‘a’ is the number of antibiotics to which the isolate is resistant and ‘b’ is the total number of antibiotics to which it is exposed. nm. the specific biofilm formation index (sbf) was determined as follow: sbf = t ‑ c/g. t is od 570 value of wells containing strains to be tested; c is od 570 of control wells containing bacteria‑free medium and g is od 630 of bacterial growth. strains were classified as weak biofilm producer (sbf ≤ 1) or strong biofilm producer (sbf > 1). antimicrobial susceptibility testing antibiotic susceptibility was tested by the disk diffusion method according to guidelines of antibiogram committee of the french society for microbiology (ca‑sfm 2013) (www. sfm‑microbiologie.org). the following disks of antibiotics (bio‑rad, marnes la coquette, france) were table i. all primer sequences used in pcr for detecting e. coli virulence associate genes. — cont’d gene primer sequence (5’-3’) annealing temp. (c°) expected size (bp) reference adhesion papa atggcagtggtgtcttttggtg cgtcccaccatacgtgctcttc 63° 720 johnson and stell 2000 fimh tgcagaacggataagccgtgg gcagtcacctgccctccggta 63° 508 johnson and stell 2000 afa/drabc ggcagagggccggcaacaggc cccgtaacgcgccagcatctc 63° 559 johnson and stell 2000 bmae atggcgctaacttgccatgctg agggggacatatagcccccttc 63° 507 johnson and stell 2000 sfa/focde ctccggagaactgggtgcatcttac cggaggagtaattacaaacctggca 63° 410 johnson and stell 2000 tsh gggaaatgacctgaatgctgg ccgctcatcagtcagtaccac 60° 420 johnson et al. 2006a protectins kpsmtii gcgcatttgctgatactgttg catccagacgataagcatgagca 63° 272 johnson and stell 2000 kpsmtiii tcctcttgctactattccccct aggcgtatccatccctcctaac 63° 392 johnson and stell 2000 serum resistance iss cagcaacccgaaccacttgatg agcattgccagagcggcagaa 63° 323 johnson et al. 2008 trat ggtgtggtgcgatgagcacag cacggttcagccatccctgag 63° 290 johnson et al. 2008 iron-related entb atttcctcaacttctggggc agcatcggtggcggtggtca 57° 371 el fertas-aissani et al. 2013 iron aagtcaaagcaggggttgcccg gacgccgacattaagacgcag 63° 667 johnson et al. 2006a fyua gcgacgggaagcgatgattta taaatgccaggtcaggtcact 56° 547 el fertas-aissani et al. 2013 irp2 aaggattcgctgttaccggac tcgtcgggcagcgtttcttct 57° 287 el fertas-aissani et al. 2013 iuta ggctggacatcatgggaactgg cgtcgggaacgggtagaatcg 63° 300 johnson and stell 2000 sita agggggcacaactgattctcg taccgggccgttttctgtgc 59° 608 johnson et al. 2006a eita acgccgggttaatagttgggagatag atcgatagcgtcagcccggaagttag 60° 450 johnson et al. 2006a etsa caactgggcgggaacgaaatcagga tcagttccgcgctggcaacaacctac 60° 284 johnson et al. 2006a etsb cagcagcgcttcggacaaaatctcct ttccccaccactctccgttctcaaac 60° 380 johnson et al. 2006a continued nick title first author et al. 38 veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx (šeputienė et  al. 2010); mercuric reductase mera (bass et al. 1999). class 1 integrons were searched by multiplex pcr targeting int1, sul1 and qaceδ1 genes (messai et al. 2008). pcr products of positive reactions for qnrb, qnrs, bla ctx‑m , dfra1, dfra5, dfra7 and dfra12 were sequenced and analyzed with the blast and fasta programs of the national center of biotechnology information (www.ncbi.nlm.nhi.gov). conjugation assay and plasmid analysis conjugation assay was performed for the cefotaxime resistant strain using sodium azide resistant e.  coli bm21 as recipient. exponential culture of donor isolate (1 volume) and recipient (2 volumes) were inoculated as a spot on brain heart infusion agar (bhia). after overnight incubation at 37 ºc, transconjugants were selected on mueller hinton agar supplemented with cefotaxime (4 μg/ml) and sodium azide (300 μg/ml). plasmid dna was extracted by alcalin lysis method (kado and liu 1981) and analyzed by electrophoresis on 0.7% (wt/vol) agarose gels at 5 volts/cm. plasmid size was estimated by using reference plasmids, prk2013 (48 kb) and pip173 (126.8 kb). plasmid incompatibility group was determined by a pcr‑based replicon typing method (carattoli et al. 2005). the screening of isolates for esbl production was performed by the double‑disc synergy test (ddst) (messai et al. 2008). heavy metal susceptibility was assessed by the agar‑dilution method on mueller hinton medium, the following heavy metal concentrations were tested: hgcl 2 : 2.7, 13.57, 27.15 and 54.3 µg/ml; cucl 2 and zncl 2 : 100, 200, 400, 800, 1600 and 3,200 µg/ ml; pb(no3) 2 : 400, 800, 1,600, 2,400 and 3,200 μg/ ml; cd(no 3 ) 2 . 4h 2 o: 7, 12.5, 25, 50, 100, 200, 400 and 800 μg/ml. mic values indicative of metal resistance were: 200 μg/ml for cd2+, 1,600 µg/ml for zn2+, 3,200 µg/ml for cu2+ and pb2+ (calormiris et al. 1984) and 27.15 µg/ml for hg2+ (edlund et al. 1996). detection of antibiotic resistance genes and integrons simplex and multiplex pcr were used for the detection of following resistance genes as previously described: β‑lactamases bla tem , bla shv and bla ctx‑m (messai et  al. 2008); plasmid mediated quinolone resistance (pmqr) genes qnra, qnrb, qnrs, and qepa (figueira et  al. 2011); aminoglycoside‑modifying enzyme aac(6’)-ib (figueira et al. 2011); tetracycline efflux pumps teta and tetb (guardabassi et  al. 2000); dihydropteroate synthases sul1, sul2 and sul3 (frank et  al. 2007, messai et  al. 2008); dihydofolate reductase dfra1, dfra5, dfra7, dfra8 and dfra12 table i. table i. all primer sequences used in pcr for detecting e. coli virulence associate genes. — cont’d gene primer sequence (5’-3’) annealing temp. (c°) expected size (bp) reference toxins vat aacggttggtggcaacaatcc agccctgtagaatggcgagta 58° 420 restieri et al. 2007 hlya aacaaggataagcactgttctggct accatataagcggtcattcccgtca 63° 1177 johnson and stell 2000 hlyf ggccacagtcgtttagggtgcttacc ggcggtttaggcattccgatactcag 63° 450 johnson et al. 2008 cnf1 aagatggagtttcctatgcaggag cattcagagtcctgccctcattatt 63° 498 johnson and stell 2000 colicins cvaa atccgggcgttgtctgacgggaaagttg accagggaacagaggcacccggcgtatt 63° 319 johnson et al. 2006a cvab5’ tggccacccgggctctttcactggagtt atgcgggtctgcagggtttccgactgga 63° 550 johnson et al. 2006a cvab3’ ggcccgtgccgcctcctatttta tcccgcaccggaagcaccagttat 63° 247 johnson et al. 2006a cvac cacacacaaacgggagctgtt cttcccgcagcatagttccat 63° 679 johnson and stell 2000 cbi acaagacagcaccagttatgggtatt gttgttggttttgttggcgtagttat 63° 430 johnson et al. 2006b cma cagcgccattaccccataaatagtga ggttcgttcgccggtgtaagcgttag 63° 498 johnson et al. 2006b miscellaneous ompt tcatcccggaagcctccctcactactat tagcgtttgctgcactggcttctgatac 63° 496 johnson et al. 2008 ibea aggcaggtgtgcgccgcgtac tggtgctccggcaaaccatgc 63° 170 johnson and stell 2000 first author et al. nick title veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx 39 iron acquisition and transport, entb, iuta, irp2, fyua, iron, sita, etsa, etsb and eita were found in 77%, 17%, 14%, 13%, 19%, 35%, 4%, 6% and 16% of strains, respectively. serum resistance‑associated genes, iss and trat, were present at rates of 20% and 58%, respectively. capsular genes kpsmtii and kpsmtiii were not found. a rate of 27% of strains harbored ompt and avian hemolysin hlyf genes. the vat gene was present in 5% of strains and, hemolysin a and cytotoxic necrotizing factor 1 genes, hlya and cnf1, were absent. the invasion gene ibea was detected in a single strain (1 %). the colicin v, b and m operon genes, cvaa, cvab5’, cvab3', cvac, cma and cbi, were present in 12%, 14%, 10%, 2%, 10% and 10% of strains, respectively, revealing a total of 24% of strains harboring a presumptive plasmid colv and/or colbm (colv: 14%, colbm: 6%, colv+colbm: 4%). all strains had a biofilm formation (bf) ability, of which 20% were strong biofilm producers. the combinations of the different virulence factors allowed to distinguish 67 virulence profiles including 0 to 16 virulence genes, and based on the gene combination “iuta, hlyf, iss, iron, ompt”, 5% of strains could be assigned to apec pathotype (johnson et  al. 2008) (table ii). the fisher’s exact test showed significant association between colv operon and phylogenetic group b1, with a rate of 29.7% in b1 strains versus 11.1% in the non‑b1 strains (p = 0.021). antibiotic susceptibility testing showed high level of resistance to amoxicillin (97%), amoxicillin‑clavulanic acid (72%), cefazolin (73%), nalidixic acid (97%), ofloxacin (78%), ciprofloxacin (62%), pefloxacin (68%), tetracycline (90%), trimethoprim (75%), sulfonamides (75%) and sulfamethoxazole/ trimethoprim (69%). resistance rates of 53%, 6% and 5% were observed statistical analysis for comparison of rates, fisher’s exact test was used, p < 0.05 was considered significant. results phylogenetic analysis allowed to assign isolates to phylogroups a (45%), b1 (37%), b2 (3%), c (1%), d (3%), e (4%), f (4%), i (1%) and unknown (2%). molecular typing of strains by eric‑pcr showed 80 different genetic profiles. the phenotypic and genotypic screening for 31 virulence‑associated genes (figure 1) showed the presence of fimh gene in almost all strains (91%), while the other adhesin genes (papa, bmae, sfa/focde, afa/drabc) were absent. the temperature‑sensitive hemagglutinin gene tsh was detected in 2% of strains. genes of 0 10 20 30 40 50 60 70 80 90 100 �m h pa pa ts h bm ae sf a/ fo cd e af a/ dr ab c kp sm t ii kp sm t iii en tb iu ta fy ua irp 2 iro n si ta et sa et sb ei ta tr at is s om pt hl ya hl yf va t cn f1 ib ea cv aa cv ab 5’ cv ab 3’ cv ac cb i cm a p re va le n ce % phylogenetic groups a b1 other: b2, d, c, f, e, i, uk figure 1. prevalence of virulence-associated genes and their distribution according to phylogenetic groups. table ii. virulence and antimicrobial resistance patterns of some representative e. coli strains. — cont’d phylo-group dna profile strain virulence gene profile antimicrobial resistance pattern resistance and integron genes a e40 s67 fimh entb trat hlyf colv amx amc cz na cip pef ofx te sss tmp sxt blatem teta e73 s111 fimh entb trat eit vat bf amx amc cz na cip pef te k sss tmp sxt blatem teta sul2 sul3 e38 s65 fimh entb yers hlyf colv amx na cip pef ofx te k sss teta tetb sul2 e53 s84 fimh entb sita iss ompt hlyf amx amc cz na cip pef ofx te sss tmp sss tmp sxt blatem blashv e80 s118 fimh iron sita iss ompt hlyf amx amc cz na cip pefofx bla tem e22 s37 fimh entb yers sita trat ompt hlyf amx amc cz na cip pef ofx te k sss tmp sxt hg teta sul1 sul2 dfra12 qace∆1 mera e39 s66 fimh entb iron sita iss hlyf colbm amx cz na cip pef bla tem e8 s14 fimh entb iuta sita ets trat iss colbm colv amx amc cz na cip pef ofx te k sss hg blatem blashv tetb sul2 int1 e78 s116 fimh entb iron sita eit trat iss ompt hlyf amx amc cz na cip pef ofx te sss tmp sxt blatem blashv teta sul2 qace∆1 int1 e45 s72 fimh tsh entb iron iuta sita ets eit trat iss ompt hlyf colv amx amc cz na cip pef ofx te k sss tmp sxt bla tem bla shv tetb sul1 sul2 dfra7 int1 qace∆1 continued nick title first author et al. 40 veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx by conjugation in association with an inci1 plasmid of about 118 kb. tetracycline resistance genes teta and tetb were detected in 74% and 12% of the strains. plasmid mediated quinolone resistance determinants qnrb and qnrs1 were present in 12% and 1% of strains. sulfonamide resistance genes sul1, sul2 and sul3 were identified in 14%, 53% and 10% of strains and trimethoprim resistance genes dfra1, dfra7, dfra12 and dfra14 were present in 5%, 7%, 13% and 43% of strains, respectively. the combinations of resistance phenotypes allowed distinguish 59 antibiotic resistance patterns for kanamycin, netilmicin and gentamicin. one strain (1%) was resistant to cefotaxime and positive for ddst. all strains were susceptible to ceftazidime, amikacin and colistin (figure 2). molecular identification by pcr and sequencing of resistance genes (figure  3) revealed the presence of broad‑spectrum beta‑lactamase (bsbl) genes bla tem , bla shv and extended‑beta‑lactamase (esbl) bla ctx‑m‑1 allele in 70%, 50% and 1% of isolates, respectively. esbl allele bla ctx‑m‑1 and, amoxicilline, cefazolin and cefotaxime resistance phenotypes of the cefotaxime‑resistant strain were transferable table ii. virulence and antimicrobial resistance patterns of some representative e. coli strains. — cont’d phylo-group dna profile strain virulence gene profile antimicrobial resistance pattern resistance and integron genes b1 e47 s75 fimh entb iuta sita trat amx amc na cip pef ofx te k sss bla tem tetb sul2 e8 s12 fimh entb iron ompt hlyf bf amx amc cz na cip pef ofx te k sss tmp sxt blatem teta dfra5 int1 e29 s53 fimh sita trat iss ompt colv amx amc na cip pef ofx te k sss tmp sxt hg blatem teta sul2 dfra5 mera int1 e37 s63 fimh entb iron sita trat iss ompt amx amc cz na cip pef ofx te sss tmp sxt hg bla tem bla shv teta sul2 dfra1 dfra5 mera int1 e69 s103 fimh entb iron sita iss ompt hlyf amx cz na cip pef ofx te bla tem bla shv teta e59 s93 fimh entb sita eit iss ompt hlyf colv amx amc cz na cip pef ofx te k sss tmp sxt blatem teta sul2 dfra5 mera int1 e31 s55 fimh entb iron sita trat ompt colbm colv amx amc na te k blatem blashv teta e64 s98 fimh entb iron sita trat iss ompt hlyf colv amx amc cz na cip pef te sss tmp sxt hg blatem blashv teta sul2 dfra5 mera int1 e52 s83 fimh iron iuta sita eit iss ompt hlyf colv amx amc cz na ofx te k blatem teta dfra5 int1 e63 s97 fimh entb iuta sita trat iss ompt hlyf colv amx amc cz na cip pef te k sss tmp sxt blatem blashv teta tetb sul2 dfra7 int1 e19 s31 fimh entb iron sita trat iss ompt hlyf colbm amx amc na cip pef ofx te k sss tmp sxt hg teta sul1 dfra12 qace∆1 e50 s81 fimh entb iron iuta sita trat ompt hlyf colv amx amc cz na cip pef ofx te k sss tmp sxt blatem blashv teta sul2 dfra5 mera int1 e16 s27 fimh tsh entb iron iuta sita trat iss colbm colv amx amc cz na cip pef ofx te k sss tmp sxt blatem tetb sul1 sul2 sul3 dfra7 qace∆1 e51 s82 fimh iron iuta sita eita trat iss ompt hlyf colv amx amc cz na cip pef ofx te sss tmp sxt blatem blashv teta sul2 dfra5 int1 e72 s110 fimh entb iron fyua irp2 iuta sita eit trat iss ompt hlyf colv bf amx amc cz na cip pef ofx te sss tmp sxt hg tetb sul2 dfra5 mera int1 d e12 s19 fimh iuta sita ets ompt hlyf colbm amx na cip pef te sss tmp sxt bla shv teta sul2 b2 e33 s58 fimh entb amx amc ofx qnrb e20 s35 fimh entb trat amx amc cz na cip pef ofx te sss bla tem teta e37 s64 fimh entb ompt amx amc cz na cip pef ofx te k sss tmp sxt blashv teta sul3 dfra5 int1 f e54 s86 iuta ibea sita colv bf amx amc cz na cip pef ofx te k net sss tmp sxt blatem tetb sul1 sul2 dfra7 qace∆ int1 e61 s95 fimh entb iuta sita ets trat ompt hlyf bf amx amc cz na cip pef ofx te k sss tmp sxt blatem teta sul2 dfra5 int1 e60 s94 fimh entb iuta sita ets eit trat ompt hlyf colv amx cz na cip pef ofx te k sss tmp sxt blatem teta sul2 dfra5 int1 e30 s54 fimh entb iuta fyua sita ets trat ompt hlyf vat colbm amx amc cz na cip pef ofx te k sss tmp sxt hg blatem blashv teta sul2 dfra5 mera int1 e e9 s15 fimh iron iuta sita trat iss ompt hlyf colbm colv amx amc cz ctx na cip pef te k sss tmp sxt hg bla tem bla shv bla ctx-m-1 teta sul1 sul2 sul3 dfra12 qace∆1 mera c e5 s6 fimh entb fyua, irp2 iuta trat iss ompt hlyf colbm amx amc na ofx te hg blashv teta first author et al. nick title veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx 41 agar dilution mics of cd2+, zn2+, cu2+, pb2+ and hg2+ were, respectively, from 25 µg/ml to 50 µg/ml, 100 µg/ml to 400 µg/ml, 800 µg/ml to 1,600 µg/ml, 3,200 µg/ml and 2.7 µg/ml to >  54.3 µg/ml. a total of 37 strains were resistant to mercury, of which 26 (70.2%) harbored mera gene, and 69.2% of mera were associated with class 1 integrons. all strains were susceptible to the other heavy metals tested. there were significant associations between antimicrobial resistance and virulence factors. (resistance to 1‑7 antibiotic families); 93% of strains had a multidrug‑resistance (mdr) phenotype, they resisted to at least three antibiotic classes, and 95% had a mar index from 0.27 to 0.72. no statistically significant association was found between antibiotic resistance and phylogenetic groups. class 1 integrons were detected in 49% of strains, fourty five (91.8%) of them lacked the 3’‑conserved sequence (3’‑cs) that contains qaceδ1 and sul1 and one lacked only sul1. in these cases, sulfonamides resistance was conferred by sul2 and/or sul3. furthermore, 10% of strains contain qaceδ1 gene in absence of integrons. p re va le n ce % 0 10 20 30 40 50 60 70 80 90 100 a m x a m c c z c tx c a z n a o fx pe f c ip te k n et g m a n ss s tm p sx t c s h g phylogenetic groups a b1 other: b2, d, c, f, e, i, uk figure 2. prevalence of e. coli antimicrobial resistance phenotypes and their distribution according to phylogenetic groups. p re va le n ce % phylogenetic groups a b1 other: b2, d, c, f, e, i, uk 0 10 20 30 40 50 60 70 80 bl a t em bl a s h v bl a c tx -m -1 te ta te tb qn rb qn rs 1 su l1 su l2 su l3 df ra 1 df ra 14 df ra 7 df ra 12 qa ce ∆ 1 m er a in t1 figure 3. prevalence of e. coli antimicrobial resistance genes and their distribution according to phylogenetic groups. table iii. association between e. coli virulence genes and antimicrobial susceptibility phenotypes (p < 0.05). % of virulence gene among resistance strains % of virulence gene among susceptible strains p virulence gene (%) antimicrobials (% resistance , % susceptibility) amc (72, 28) cz (73, 27) cip (62, 38) pef (68, 32) ofx (78, 22) te (90, 10) k (53, 47) sss (75, 25) tmp (75, 25) sxt (69, 31) hg (37, 63) iron (19) 22.22-10.71 20.53-14.81 25.8-7.89 0.035* 18.60-9.37 19.23-18.18 18.89-20 18.87-19.15 18.67-20 18.67-20 20.29-16.13 18.91-19.05 fyua (13) 12.5-14.28 8.21-25.92 0.039** 11.29-15.79 10.29-18.75 8.97-27.27 0.034** 14.44-0 9.43-17.02 8-28 0.016** 10.66-20 7.25-25.80 24.32-6.35 0.014* irp2 (14) 11.11-21.43 13.51-33.33 0.001** 9.68-21.05 10.29-21.05 10.26-27.27 15.55-0 9.43-19.15 8-32 0.005** 13.33-16 7.24-29.03 27.03-6.35 0.006* iuta (17) 20.83-7.14 19.18-11.11 24.19-5.26 0.014* 22.06-6.25 17.95-13.64 18.89-0 24.53-8.51 20-8 17.33-16 18.84-12.90 13.51-19.05 sita (35) 40.28-21.43 36.99-29.63 48.39-13.15 0.0004* 44.11-15.62 0.006* 37.18-27.27 36.67-20 43.39-25.53 40-20 37.33-28 40.57-22.58 35.13-34.92 trat (58) 58.33-57.14 56.16-62.96 58.06-57.89 52.94-68.75 53.85-72.73 63.33-10 0.001* 67.92-46.81 0.043* 62.66-44 64-40 62.32-48.39 67.57-52.38 iss (20) 23.61-10.71 21.91-14.81 29.03-5.26 0.004* 26.47-6.25 0.017* 20.51-18.18 20-20 18.87-21.28 20-20 18.67-24 20.29-19.35 21.62-19.05 ompt (27) 31.94-14.28 28.77-22.22 38.70-7.89 0.0009* 35.29-9.37 0.007* 26.92-27.27 28.89-10 32.07-21.27 29.33-20 0.001* 29.33-20 0.001* 31.88-16.13 0.001* 27.03-26.98 hlyf (27) 27.78-25 28.77-22.22 37.09-10.53 0.004* 33.82-12.5 0.030* 24.36-36.36 28.89-10 26.41-27.66 20.67-28 26.67-28 27.54-25.81 24.32-28.57 cvaa (12) 15.28-3.57 15.07-3.70 17.74-2.63 0.027* 16.18-3.12 12.82-9.09 13.33-0 16.98-6.38 14.67-4 12-12 13.04-9.68 10.81-12.69 cvab5’(14) 19.44-0 0.004* 16.44-7.40 19.35-5.26 17.65-6.25 14.10-13.63 15.55-0 22.64-4.25 0.009* 16-8 14.67-12 15.94-9.68 13.52-14.28 positive* and negative** significant association between virulence gene and antimicrobial resistance (p < 0.05). nick title first author et al. 42 veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx and bonnet and colleagues (32%) (bonnet et  al. 2009). the gene sita belongs to sitabcd operon encoding an iron and manganese abc transport system, whose role in virulence and resistance to oxidative stress of apec was demonstrated (sabri et  al. 2008). the gene sita was mostly associated with pathogenic strains than fecal ones; however, its rate in our strains (35%) was higher than those reported in fecal strains (27%, 19%) (amabile de campos et al. 2008, kemmett et al. 2013). genes eita and etsa/etsb are located in the eitabcd and etsabc operons encoding putative iron abc transporters identified in high pathogenic apec and induced in vivo during infection in chickens (johnson et al. 2008, tuntufye et al. 2012); their prevalence (16%, 4%, 6%) was relatively lower than that reported by johnson and colleagues in commensal strains (43%, 43%, 44%) (johnson et  al. 2008). serum resistance is one of the pathogenicity mechanisms of apec strains, there is a correlation between serum resistance and the ability of bacteria to persist in body fluids and internal organs (mellata et  al. 2003). genes iss and trat involved at least in part in serum resistance, were detected at rates below (20%, 58%) those reported in fecal e. coli by bonnet and colleagues (trat, 86.3%) (bonnet et al. 2009) and johnson and colleagues (iss, 60%) (johnson et al. 2008), consistent with results of hiki and colleagues (iss, 20.5%) (hiki et al. 2014) and higher than in kemmett and colleagues (iss, 10%) (kemmett et al. 2013). the avian hemolysin gene hlyf, epidemiologically associated to the most virulent apec, was found at prevalence (27%) close to that of hiki and colleagues (28.2%) (hiki et  al. 2014). the serine protease gene ompt, involved in providing defense against cationic antimicrobial peptides secreted by the epithelial cells and macrophages, was present at rate (27%) close to result of hiki and colleagues (29.5%) (hiki et  al. 2014), but under those recorded by rodriguez‑sieck and colleagues (45.2%) (rodriguez‑sieck et  al. 2005), johnson and colleagues (42%, 47%) (johnson et  al. 2008) and bonnet and colleagues (46.7%) (bonnet et  al. 2009). the invasion‑related gene ibea was present in our strains at low rate (1%) compared to that previously reported in fecal strains (7.7%, 16%) (rodriguez‑sieck et  al. 2005, kemmett et  al. 2013). the prevalence of vat gene (5%) matches that reported by kemmett and colleagues (6%) (kemmett et  al. 2013) in fecal strains. biofilm is a form of bacterial resistance to antimicrobials, opsonization and phagocytosis. rate of strong biofilm producers among our strains was lower (20%) than the 30% reported by skyberg and colleagues (skyberg et al. 2007). most of the detected genes (iuta, fyua, irp2, iron, fimh, cvac, trat, iss, sita, ompt, hlyf, cvaa, etsa, etsb, eita, tsh) have been described in pathogenicity islands associated with virulence plasmids in apec, of which colv and colbm plasmids as papec‑o1, resistance to ciprofloxacin was distinguished by its association to 7 virulence genes (iuta, iron, sita, iss, ompt, hlyf, cvaa) (table iii). discussion most of our isolates (82%) belonged to a and b1 phylogenetic groups in accordance with many previous studies (johnson et  al. 2008, bonnet et  al. 2009, hiki et  al. 2014). the clonal relationship investigated by eric‑pcr showed a large genetic diversity among strains. almost all of our isolates (99%) possessed at least one of the examined virulence genes. type 1 fimbriae are ubiquitous among e.  coli strains including apec, they are involved in the initiation of the colonization of respiratory tract epithelium (wooley et  al. 1998). according to our result (2%), tsh was reported at low frequency (10%‑11.2%) in avian fecal e.  coli, while it was described at higher rates (49.7%‑97.7%) in apec strains; it would have a role in the colonization of tracheal mucosa and in the development of lesions in the air sacs, it was proposed as a marker of apec (dozois et  al. 2000, dozois et  al. 2003, amabile de campos et  al. 2005, bonnet et  al. 2009). seven iron‑related genes were detected; enterobactin was reported among pathogens and commensals; however, it was found in our study at a rate higher (77%) than in commensals (3%‑13.2%) and close to those in apec (40%‑75%) (amabile de campos et  al. 2008, bonnet et  al. 2009). the transformation of enterobactin to salmochelin (c‑glucosylation) mediated by irobcden gene cluster can prevent siderocalin binding (dozois et  al. 2003, garénaux et  al. 2013). gene iron representative of this gene cluster was detected at rate (19%) close to that reported in fecal strains by johnson and colleagues (21%) (johnson et al. 2008), but lower than in study of bonnet and colleagues (62.4%) (bonnet et  al. 2009). aerobactin and yersiniabactin were reported at high frequency in pathogenic strains (amabile de campos et al. 2008, bonnet et al. 2009), their significant role in apec virulence was demonstrated (dozois et al. 2003, tuntufye et al. 2012). yersiniabactin allowed evasion of siderocalin and prevents reactive oxygen species production by innate immune cells. the rate of aerobactin was close to those of fecal e. coli in certain studies (12.2%‑15%) (amabile de campos et  al. 2008, bonnet et  al. 2009, kemmett et  al. 2013) but lower than in others (25.9%, 35.5%) (rodriguez‑sieck et  al. 2005, johnson et  al. 2008). the prevalence of yersiniabactin (14%) is consistent with that reported in fecal e. coli by amabile de campos and colleagues (13%) (amabile de campos et al. 2008) and kemmett and colleagues (11%) (kemmett et al. 2013), whereas it was lower than in studies of rodriguez‑sieck and colleagues (30.1%) (rodriguez‑sieck et  al. 2005), johnson and colleagues (30%) (johnson et  al. 2008) first author et al. nick title veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx 43 links between antimicrobial resistance and expec strains in animal food products, specifically chicken meat, and human infections were observed (vincent et al. 2010). bsbl genes bla tem and bla shv were detected in a large number of strains, as already reported (gyles 2008, bonnet et  al. 2009, wang et  al. 2013), they would be the cause of resistance to amoxicillin, amoxicillin‑clavulanic acid and cefazolin in our strains. conversely, esbl bla ctx‑m‑1 , located in transferable inci1plasmid, was found in the single cefotaxime‑resistant strain (1%). the prevalence of broad cephalosporin resistance and esbls varies by country, our results were in accordance with those of studies from europe and china (de jong et  al. 2012, wang et  al. 2013). ctx‑m‑1 esbl and inci1 plasmids are among the most widespread, particularly in animal strains including poultry (caratolli 2009, johnson and nolan 2009); however, it should be noted that ctx‑m‑1 type is reported for the first time in algeria in this study. plasmid mediated quinolone resistance genes qnrb (12%) and qnrs1 (1%) were detected, their presence among avian e.  coli strains was reported with varying frequency by country. their transfer from animal to human was reported, they contribute to the emergence of highly quinolone‑resistant bacteria mainly due to mutations in the dna gyrase and topoisomerase iv genes (szmolka and nagy 2013). a rate of 92% of tetracycline‑resistant strains had teta and/or tetb, they are the most frequently involved among avian strains with a predominance of teta (guerra et  al. 2003, bonnet et  al. 2009, johnson et  al. 2012). the three dihydropteroate synthase genes sul1, sul2 and sul3 were detected in 72.3% of sulfonamides‑resistant strains; they play an important role in sulfonamide resistance and are significantly related to integrons and transposons. consistent with our result, sul2 gene was the most widely distributed in avian e.  coli (guerra et  al. 2003, wang et  al. 2013). genes dfra1, dfra7, dfra12 and dfra14 were detected alone or in combination in 77.3% of trimethoprim‑resistant strains; dfra1, dfra12, dfra14 and dfra17 were the most commonly identified, inside integrons (guerra et  al. 2003, machado et  al. 2008). integrons are important contributors in the emergence and dissemination of antimicrobial resistance, half of our strains carried class 1 integrons, the most frequently detected in avian e. coli. our prevalence is equal to that reported in greece (49.6%) (vasilakopoulou et  al. 2009) and higher than those recorded in portugal (22.5%) (machado et  al. 2008) and germany (36%) (guerra et  al. 2003). the majority of detected integrons lacked sul1 and qaceδ1, this truncated structure was already described, it generally contains sul3 at the 3'‑end and is linked to is26, which probably is the cause of the 3'‑cs deletion (dawes et al. 2010, sáenz papec‑o2‑colv and papec‑o1‑colbm (johnson and nolan 2009, mellata et  al. 2010). the simultaneous presence of several of these plasmid‑borne virulence‑associated genes and, operon colv (cvaa, cvab, cvac) and/or colb/m (cbi, cma) genes in 24% of our strains augurs that the latter harbored colv and/or colbm plasmids. these virulence plasmids have an important role in pathogenicity, evolution from commensal to pathogenic state and zoonotic risk (johnson and nolan 2009, mellata et  al. 2010). in addition to plasmid genes, some of our isolates possessed certain chromosomal genes (fyua, vat, ibea), which characterize apec strains (johnson et al. 2008). the phylogroup b2 strains were characterized by the presence of little virulence genes, mainly chromosomal and ubiquitous (entb, fimh), compared to other groups and the majority of strains carrying presumptive colv plasmids belonged to phylogroup b1 (61.1%), this finding is not in accordance with previous reported data (johnson et  al. 2008). no statistical association with phylogenetic groups was noted for the remaining virulence factors. the combination of virulence factors showed a diversity in virulence profiles (n = 67). five percent of our isolates possessed the gene combination “iuta, hlyf, iss, iron, ompt” defined as the most significantly genes associated with highly pathogenic apec strains (johnson et  al. 2008), and 12% harbored at least 4 genes of this combination. these findings were in agreement with results from hiki and colleagues (hiki et al. 2014). the prevalence of virulence factors found among our strains differ for some of them from those reported in other countries, environmental conditions (feed, production systems, veterinary practices) can modulate the distribution of virulence determinants (amabile de campos et al. 2008, bonnet et al. 2009, kemmet et al. 2013) high resistance rates were observed for amoxicillin, amoxicillin‑clavulanic acid, cefazolin, fluoroquinolones, tetracycline, trimethoprim, sulfonamides and sulfamethoxazole/ trimethoprim. these results reflect the general trend worldwide both for fecal and apec strains; however, resistance rates vary by country. in comparison, our rates are significantly higher than those recorded in the usa (johnson et  al. 2012), europe (de jong et  al. 2012), canada (bonnet et al. 2009), japan (hiki et al. 2014), and lower than those from egypt (mohamed et  al. 2014), china (wang et  al. 2013). the range of antibiotics used in algeria for prophylaxis, therapy and growth promotion covers various families; this can directly affect antimicrobial resistance of endogenous bacteria. furthermore, the environment can also be a source of resistant organisms and resistance genes for animals (bélanger et  al. 2011). nick title first author et al. 44 veterinaria italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/vetit.xxxxx resistance was associated with fewer virulence genes in comparison to ciprofloxacin‑susceptible strains (graziani et  al. 2009). the molecular mechanisms underlying association between resistance and virulence remains to understand. this study, the first in algeria devoted to virulence and antimicrobial resistance of fecal strains from healthy broiler chickens, reported the presence of expec virulence genes typically found in pathogenicity islands located on plasmids, particularly colv and colbm plasmids. high prevalence of mdr phenotype was observed, with resistance to first line antibiotics including amoxicillin‑clavulanate, fluoroquinolones and trimethoprim‑sulfamethoxazole, as well as various plasmidic resistance genes and class1 integrons. intensive chicken farming in the current conditions in algeria really constitutes a source of virulence and antimicrobial resistance genes that may spread and exacerbate virulence and resistance of animal and human pathogenic strains. this situation should incite to take measures at the level of farming conditions and veterinary practices. et al. 2010). the presence of the gene qaceδ1 in the absence of integrons would probably be the result of a selection pressure by quaternary ammonium compounds which are, among disinfectants, the most used in the poultry industry. the mercury resistance (37%) can result from an anthropogenic selection pressure or co‑selection by antibiotics. the detection of mera gene associated in the majority of cases to class 1 integrons is indicative of the presence of the transposon tn21 which carries mercury resistance operon (mer) and an integron (in2). this transposon allows co‑selection by antibiotics and mercury. this finding is in agreement with that already reported among avian strains (bass et al. 1999, johnson et al. 2012). many 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characterizing the apec pathotype. vet res, 36, 241‑256. sabri m., caza m., proulx j., lymberopoulos m.h., brée a., moulin schouleur m., curtiss iii r. & dozois c.m. 2008. contribution of the sitabcd, mnth, and feob metal transporters to the virulence of avian pathogenic escherichia coli o78 strain chi7122. infect immun, 76, 601‑611. 269 department of veterinary sciences, university of turin, turin, italy *corresponding author at: department of veterinary sciences, university of turin, largo p. braccini 2‑5, 10095 grugliasco (to), italy. e‑mail: gessica.giusto@unito.it. parole chiave sutura dentata, pressione di scoppio, cieco, colon, chiusura di enterotomia, cavallo, chirurgia, flessura pelvica, sutura non dentata. riassunto in questo studio sono stati raccolti l’intestino cieco e quello crasso da 24 cavalli macellati; per ogni campione è stata eseguita un’enterotomia di 8 cm suturata con tecnica dentata o usando glycomer‑631. sono stati confrontati la cicatrizzazione, l’aspetto, la lunghezza del materiale, la pressione di rottura e i costi associati a ciascun tipo di materiale. il tempo di cicatrizzazione è stato significativamente più breve (cieco p  =  0,034, flessione pelvica p = 0,039) con le suture dentate (cieco 610,4 secondi, flessione pelvica 699,3 secondi) rispetto a quelle non dentate (cieco 661,0 secondi, flessione pelvica 743,1 secondi). la lunghezza del materiale di sutura si è dimostrato essere significativamente inferiore (cieco p < 0,0001, flessione pelvica p  <  0,0001) nelle prime (cieco 28,1 cm, flessione pelvica 32,0 cm) rispetto alle seconde (cieco 41,6 cm, flessione pelvica 46,6 cm) mentre non ci sono state differenze significative (cieco p  =  0,294, flessione pelvica p  =  0,430) nella pressione di rottura (suture dentate: cieco 172,5 mmhg, flessione pelvica 188,9 mmhg; non dentate: cieco 178,3 mmhg, flessione pelvica 183,3 mmhg). l'uso di suture dentate ha un costo maggiore ma è più veloce, lascia meno materiale di sutura nel tessuto e sostiene una pressione di rottura paragonabile alle suture non dentate; sono dunque una valida alternativa per la chiusura dell'enterotomia dell'intestino crasso nei cavalli. comparazione ex vivo di suture con o senza dentatura per la chiusura delle entorotomie nelle flessure pelviche e dell’intestino cieco nei cavalli keywords barbed suture, bursting pressure, caecum, colon, enterotomy closure, equine, gastrointestinal surgery, horse, pelvic flexure, unbarbed suture. summary in this study the caecum and large colon were harvested from 24 slaughtered horses. on each sample, an 8‑cm long enterotomy was performed. enterotomies were closed using either barbed or unbarbed glycomer‑631. we compared the time to close, appearance, length of suture material, bursting pressure, and costs associated with each type of material. our findings demonstrated that time to close was significantly shorter (caecum, p  =  0.034; pelvic flexure, p  =  0.039) using barbed sutures (caecum 610.4 seconds; pelvic flexure 699.3 seconds) than unbarbed sutures (caecum 661.0 seconds, pelvic flexure 743.1 seconds). the length of suture material used was significantly less (caecum, p  <  0.0001; pelvic flexure, p < 0.0001) with barbed (caecum 28.1 cm, pelvic flexure 32.0 cm,) compared with unbarbed sutures (caecum 41.6 cm; pelvic flexure 46.6 cm). there were no significant differences in bursting pressure (caecum, p  =  0.294; pelvic flexure, p  =  0.430) between barbed (caecum, 172.5 mmhg, pelvic flexure, 188.9 mmhg) and unbarbed sutures (caecum 178.3 mmhg, pelvic flexure 183.3 mmhg). the cost was higher using barbed sutures. however, the use of barbed sutures was faster, left less suture material in the tissue, and sustained comparable bursting pressure to unbarbed sutures. we therefore conclude that barbed sutures are a valid alternative to unbarbed sutures for closing large intestine enterotomy in horses. gessica giusto*, vittorio caramello, francesco comino and marco gandini ex vivo comparison of barbed and unbarbed sutures for the closure of caecal and pelvic flexure enterotomies in horses veterinaria italiana 2019, 55 (3), 269‑274. doi: 10.12834/vetit.691.3383.2 accepted: 09.01.2016 | available on line: 30.09.2019 270 barbed and unbarbed sutures for enterotomy closure in horses giusto et al. veterinaria italiana 2019, 55 (3), 269‑274. doi: 10.12834/vetit.691.3383.2 the pelvic flexure as well as the caecum in its entirety, were harvested at a local abattoir from 24 slaughtered horses. horses were aged 12‑18  months, with no apparent gastrointestinal disease. specimens were stored in lactated ringer’s solution at 4  °c before testing. the enterotomy closures and testing were completed within 6 hours of the death of the horse. samples were randomly assigned into 1 of 2 groups (unbarbed or barbed) by means of a random numbers generator1. the caecum and the pelvic flexure of the 24 horses were randomly assigned to 1 of 2 groups. in the unbarbed group, a hand‑sewn technique with glycomer 631 (glycomer 631, biosyn, covidien italia, segrate milano, italy) was used to close the enterotomy site. in the barbed group, the same technique was applied, but barbed glycomer 631 (barbed glycomer 631, vloc‑90, covidien italia, segrate milano, italy) was used. the same surgeon (mg) and assistant (gg), performed all of the procedures while the intestines were lying on a table (caecum) or a colon tray (pelvic flexure). procedures on all specimens an 8‑centimetre‑long enterotomy was performed on the caecal apex between the lateral and ventral bands and on the pelvic flexure on the antimesenteric side. specimens were closed using a hand‑sewn double‑layer technique (gandini et  al. 2013, rakestraw et  al. 2012) that included a full thickness, simple continuous suture pattern over‑sewn with a cushing pattern. the suture bite size was approximately 3 mm from the incision edge, and 5 mm in length. in the enterotomies for the unbarbed group, the suture was tied and cut after the first layer. the second layer was performed with a new strand of the same suture material. all knots were buried in the second suture layer. in the enterotomies for the barbed group, the suture was started by passing the needle through the welded loop of the suture thread. when the first layer was completed, the pattern was continued for 2 passages, exceeding the end of the length of the enterotomy by approximately 10 mm. the second layer was completed with a second strand of the same suture material. again, after the second layer was completed, the suture was finished by taking 2 additional bites that exceeded the length of the enterotomy. introduction enterotomy procedures are performed in the large intestine during equine surgery, and various closure techniques have been described in vivo and in vitro (johnston et al. 1997, rosser et al. 2012, gandini et al. 2013, rakestraw et al. 2012). successful enterotomy closure techniques involve ensuring watertight tissue approximation, maintaining luminal diameter, withstanding increasing intraluminal pressures, and limiting the amount of exposed suture material. the hand‑sewn, 2‑layer suture technique is favoured for use in the caecum (rakestraw et al. 2012) and large colon (gandini et al. 2013, rakestraw et al. 2012). a new barbed suture material was recently made available to surgeons. barbed suture material is characterised by the presence of a welded loop at one end and barbs cut into the body of the thread distributed circumferentially along the thread. this produces a suture material that does not require the tying of a knot at the start or end of the suture line. this, in turn, reduces the amount of suture material that is exposed, and improves the strength of the suture line by eliminating the need for knots, which are otherwise considered the weakest portion of a suture line. in addition, the shear stress is distributed along the entire length of the suture (zaho et al. 2013). while it was initially intended for human plastic surgery (ruff 2006, ruff 2013), barbed suture has proven effective in other fields such as orthopedics, gynecology (maheshwari et  al. 2015, medina et  al. 2014, manoucheri et  al. 2013, chamsy et  al. 2013, greenberg 2010), and gastrointestinal surgery for performing end‑to‑end anastomosis and closing enterotomies in dogs (hansen et  al. 2012, ehrhart et al. 2013, omotosho et al. 2011, miller et al. 2012), pigs (demyttenaere et al. 2009), and humans (nemecek et  al. 2013, tyner et  al. 2013). to date, 4 reports have been published on the use of barbed sutures in horses for intestinal anastomosis and laparoscopic procedures (nelson et  al. 2014, ragle et  al. 2013, albanese 2013). our hypothesis is that barbed suture could prove to be a valuable option for enterotomy site closure in horses, and could favourably compare with unbarbed suture material used for the same purpose. the aim of this study was to compare barbed suture with conventional (unbarbed) suture materials for enterotomy closure in horses. the following items were compared: construction time, amount of suture material used, macroscopic appearance, bursting pressure, mode of failure, and cost. materials and methods intestinal specimens intestinal samples from the large colon, including 1 www.random.org. 271 giusto et al. barbed and unbarbed sutures for enterotomy closure in horses veterinaria italiana 2019, 55 (3), 269‑274. doi: 10.12834/vetit.691.3383.2 which the construct failed during bursting pressure testing. we identified 3 possible modes of failure relative to the suture line: (1) failure at the knot (knot slippage) for unbarbed suture, (2) failure at the suture (suture breaking), and (3) failure at the tissue (tearing of the tissue). the following were also noted: failure at the tissue and tearing at the enterotomy site, tearing adjacent to the enterotomy, or tearing at a distant location. cost the cost for each enterotomy in euros was calculated according to pricing from a local surgical supply distributor and the number of strands used in each procedure. statistical analysis normality of the data was evaluated using the shapiro‑wilk normality test. for caecal enterotomies, a mann‑whitney test was employed to compare the data that had non‑normal distributions (construction time and bursting pressure) while an unpaired t‑test with a welch correction applied for unequal variances was employed to test the length of the suture material that was used. for pelvic flexure enterotomies, an unpaired t‑test with welch’s correction applied for unequal variances was used to compare all variables. all statistical analyses were performed with commercially available software (graphpad prism 6, graphpad software, san diego, ca), with significance set at p < 0.05. results results are summarised in table i. macroscopic appearance and exposed suture material all enterotomies had a similar appearance macroscopically, with a smooth inverted surface. in many specimens in the unbarbed group, there was some exposed suture material where the last knot was tied (8 of 12 in the caecum and 10 of 12 in the large colon), while no exposed suture material was evident in the barbed group. macroscopic appearance and exposed suture material after completion, all enterotomies were evaluated for differences in serosal appearance and the presence of exposed suture material. construction time time (in seconds) of enterotomy closure was defined as the time from the first suture bite to the cutting of the excess suture thread. length of suture material the initial length of the suture strand was measured with a digital caliper, just after opening the package. the length of suture material used to complete the closure was measured by subtracting the residual length of the suture at the end of the procedure from the initial length. in the barbed group enterotomies, only the material trimmed after completion of the suture was measured, while in the unbarbed group enterotomies, the suture material trimmed from the initial knot was also measured. bursting pressure the bursting strength of each specimen was determined by specimen inflation using a previously described water immersion test (gandini et al. 2006). briefly, a cannula connected to a compressed air tank was inserted into the lumen, and a similar cannula, connected to a calibrated mercury sphygmomanometer, was inserted at the other end (or at the same end in the caecal enterotomy samples). the intestine was submerged in water, and the lumen was inflated with air (1  l/min). luminal pressures were measured continuously, and were video‑recorded to determine the peak pressure at which each specimen failed or leaked. this was detected by the presence of air bubbles in the water and a drop in pressure measured by the sphygmomanometer. mode of failure mode of failure was defined by the location at table i. construction time, length of suture material and bursting pressure of intestinal enterotomies closed with barbed or unbarbed suture material. construction time (sec) 95% ci interval length of suture material (cm) 95% ci interval bursting pressure (mmhg) 95% ci interval caecum unbarbed (mean) 661.0 629.0-693.0 41.6 40.7-42.51 178.3 167.4-189.3 barbed (mean) 610.4* 575.7-643.5 28.1* 27.7-28.56 172.5 163.5-181.5 pelvic flexure unbarbed (mean) 743.1 713.2-755.2 46.6 42.7-44.63 183.3 172.8-193.8 barbed (mean) 699.3* 671.6-727.1 32.0* 31.3-32.69 188.9 177.8-200.0 * indicates a statistically significant difference (p < 0.05) with values of the unbarbed group. 272 barbed and unbarbed sutures for enterotomy closure in horses giusto et al. veterinaria italiana 2019, 55 (3), 269‑274. doi: 10.12834/vetit.691.3383.2 unbarbed sutures were used, some exposed suture material was evident, particularly where the last knot of the second layer was tied. this observation is consistent with what has previously been reported (gandini et al. 2013). despite the care taken to bury all knots, it was impossible to completely avoid this occurrence. in our opinion, this could be a result of any of the following: the large volume of knots, the surgical technique, or the ex vivo setting, where tissue compliance could be impaired, thus making it more difficult to completely bury the knots. barbed sutures have several advantages compared to conventional sutures. as has been previously reported, barbed suture holds the edges in place, and does not slide backward between 2 bites (nemecek et  al. 2013). this allows suturing without the help of an assistant to keep tension on the suture thread. we found this property particularly useful in large intestine enterotomies. in our study, closure with barbed suture significantly reduced the length of suture material required, and significantly reduced surgical time. the actual time difference was minimal thus it is not likely to have an effect in a clinical setting. clinically, the use of barbed sutures could potentially improve healing by leaving a smaller amount of suture material in the tissue (trimbor et al. 1989). as has been noted in a previous study (nelson et  al. 2014), we also found that the difference in construction time could partly be due to the difference in length of the suture material used (45  cm of the barbed suture vs 76 cm of the unbarbed suture) (nelson et  al. 2014). this could result in a different amount of suture that needs to be pulled through the tissue after each bite, with loss of time occurring when handling the long, unbarbed sutures. avoiding knot placement could prevent weakening of the construct, reduce surgical time, and reduce the amount of foreign material left in the patient. while surgical time and suture material are reduced by using barbed suture, our results do not support the finding that knots are the weakest point of a suture line (mulon et al. 2010), at least when applied with glycomer 631 on equine intestines. in fact, none of the constructs with unbarbed suture failed at the knot in our study, because tearing of the tissue always occurred first. while nelson and hassel (nelson and hassel 2014) found a significant difference in bursting strength between anastomoses performed with barbed or unbarbed suture material, we found that there was no difference in bursting pressure between enterotomy closures with barbed or unbarbed sutures. this could be because we used a double layer pattern in the caecum and colon, while they used a single layer inverting suture (nelson et  al. 2014). another difference between the results construction time in all intestinal tracts, enterotomy closure with barbed suture (caecum 610.4 sec, 95% ci 575.7‑643.5  sec; pelvic flexure 699.3 sec, 95% ci 671.6‑727.1 sec) was significantly faster (caecum p = 0.034; pelvic flexure p  =  0.039) than with unbarbed suture (caecum 661.0  sec, 95% ci 629.0‑693.0 sec; pelvic flexure 743.1 sec, 95% ci 713.2‑755.2 sec). length of suture material used the length of the suture material that was used was significantly shorter (caecum p < 0.0001; pelvic flexure p < 0.0001) in the barbed group (caecum 28.1  cm, 95% ci 27.7‑28.56 cm; pelvic flexure 32.0 cm, 95% ci 31.3‑32.69 cm) in all intestinal tracts, compared to the unbarbed group (caecum 41.6 cm, 95% ci 40.7‑42.51 cm; pelvic flexure 46.6 cm, 95% ci 42.7‑44.63 cm). bursting pressure the difference in bursting pressure between the 2 groups was not significantly different (caecum p  =  0.294; pelvic flexur, p  =  0.430) for all intestinal tract specimens with barbed (caecum 172.5  mmhg, 95% ci 163.5‑181.5  mmhg; pelvic flexure 188.9  mmhg, 95% ci 177.8‑200.0  mmhg) or unbarbed sutures (caecum 178.3 mmhg, 95% ci, 167.4‑189.3 mmhg; pelvic flexure 183.3 mmhg, 95% ci 172.8‑193.8 mmhg). mode of failure all constructs failed by tearing tissue on one side of the enterotomy. in most instances, the seromuscular layer started tearing at pressures below bursting pressure without causing leakage. when the submucosal and mucosal layer tore, complete bursting occurred. cost the cost of each caecum and pelvic flexure closure was 15.1 euros for the unbarbed group and 84.12 euros for the barbed group. discussion the results of our study showed that there were significant differences between barbed and unbarbed suture materials in terms of construction time and length of suture material used. bursting pressure and mode of failure were not affected by suture material. the macroscopic appearances were similar, with a smooth inverting surface, but when 273 giusto et al. barbed and unbarbed sutures for enterotomy closure in horses veterinaria italiana 2019, 55 (3), 269‑274. doi: 10.12834/vetit.691.3383.2 described here would increase costs in the clinical setting, but avoids using a contaminated strand for the second layer. the cost‑per‑enterotomy is then 5.5‑fold higher for barbed sutures, and this could influence the surgeon’s choice. evaluating the use of barbed suture material on cadavers could be regarded as a limitation of our study. injured tissue handled during exploratory laparotomy may respond differently to the passage of suture material than healthy tissue, and may exhibit differences in bursting pressure. furthermore, it has been hypothesised that intestinal motility could affect the behaviour of barbed suture lines (giusto et  al. 2019). intestinal circular contractions, especially in pathological conditions, could cause a progression of the intestinal wall onto the barbs of the suture material, thus causing stenosis. however, one study shows that intestinal motility did not affect the use of barbed suture material in healthy pigs (giusto et al. 2019). our results suggest that barbed suture is a viable alternative to unbarbed suture for enterotomy closure in horses. another possible application of our results would be in laparoscopic intestinal biopsies (schambourg et al. 2006, bracamonte et al. 2008), where barbed sutures could allow effective intracorporeal suturing of the enterotomy without knot tying. however, further in  vivo testing is necessary before applying the use of barbed suture material in patients. acknowledgements the authors would like to thank dr dylan gorvy for his help with the english language. reported in our study and nelson’s may be because of the different methods that were used to measure bursting pressure. in our study, we distended the specimens with air and considered air bubbles leaking from the enterotomy to determine the maximal pressure sustained by the construct (gandini 2006, gandini et al. 2013) while nelson and hassel used stained fluid, which could have different behaviour (nelson et al. 2014). as has been previously reported, we noticed that the barbed suture was easier to handle, although there was some dragging through the tissues (nemecek et al. 2013). this may cause damage by creating larger suture tracts when the suture is pulled through tissue, but we were unable to objectively evaluate this issue in our study (nelson et al. 2014). another study showed that the use of barbed suture during intestinal closure in pigs did not cause more damage or inflammation than unbarbed suture (demyttenaere et al. 2009). barbed suture proved effective for enterotomy closure and gastrointestinal anastomoses in pigs and humans, but there are drawbacks to its application because suture patterns with exposed barbs could cause intestinal damage or obstruction (demyttenaere et  al. 2009, giusto et  al. 2019, buchs et  al. 2012, burchett et  al. 2013). to more closely mimic the clinical setting, we chose to use the cushing pattern as either a single layer or as the second layer in a 2‑layer closure. this pattern also prevents problems related to exposed suture barbs, whereas a lembert pattern would achieve the same results in terms of exposed suture barbs, but with more tissue inversion both in 1‑layer or 2‑layer closures. to perform the second layer in closures we preferred to use a new strand 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thoreson a.r., cha s.s., moran s.l., an k.n. & amadio p.c. 2013. beyond the square knot: a novel knotting technique for surgical use. j bone joint surg am, 95 (11), 1020‑1027. 157 parole chiave india, parametri biochimici, parametri ematologici, elisa indiretto, toxoplasmosi, felini selvatici, parco zoologico. riassunto scopo di questo studio è stato quello di indagare sulla presenza di anticorpi anti-toxoplasma gondii in tigri reali del bengala (panthera tigris tigris), leoni asiatici (panthera leo persica), leopardi (panthera pardus) ed elefanti (elephas maximus indicus) del mahendra chaudhury zoological park, chhatbir, punjab (india). su 20 campioni di siero prelevati durante la stagione invernale ed esaminati in elisa, 1 campione di siero prelevato da un leone (5%) è risultato positivo mentre 3 prelevati da tigri e uno da un altro leone (20%) hanno dato esito dubbio o sospetto; nel prelievo effettuato dagli stessi animali nel periodo dei monsoni, 4 campioni di siero, prelevati da 2 tigri e 2 leoni (20%), sono risultati positivi mentre un campione prelevato da una terza tigre (5%) ha dato esito dubbio o sospetto. rispetto ai negativi, gli animali risultati sieropositivi e dubbi o sospetti all’elisa hanno evidenziato valori di globulina, creatinina, azoto ureico, fosforo e creatina chinasi significativamente più elevati. analogamente, i livelli di albumina, glucosio, calcio, sodio e ferro rilevati nel gruppo sieronegativo sono risultati significativamente più bassi. la presenza di anticorpi anti-toxoplasma gondii è stato rilevata anche in un roditore catturato nelle aree adiacenti al parco. questo studio evidenzia per la prima volta l’esposizione di tigri reali del bengala e leoni asiatici al t. gondii. dimostra inoltre un’associazione significativa tra sieropositività e alterazione di alcuni parametri ematici e biochimici. presenza di anticorpi anti-toxoplasma gondii in tigri reali del bengala (panthera tigris tigris) e leoni asiatici (panthera leo persica) in india keywords biochemical parameters, haematological parameters, indirect elisa, india, toxoplasmosis, wild felids, zoological park. summary the purpose of this study was to detect the antibodies against toxoplasma gondii in royal bengal tigers (panthera tigris tigris), asiatic lions (panthera leo persica), leopards (panthera pardus), and elephants (elephas maximus indicus) residing in the mahendra chaudhury zoological park, in chhatbir, punjab (india) during winter and monsoon seasons. using indirect elisa, 20 serum samples were analysed during the winter season. results indicated that 1 lion (5%) tested seropositive, and 3 tigers and 1 lion (20%) were considered suspect. during the monsoon, 4 individuals (2 tigers and 2 lions, 20%) were seropositive, whereas only 1 tiger (5%) gave suspected results. significantly higher globulin, creatinine, blood urea nitrogen, phosphorus, and creatine kinase values were recorded in seropositive and suspected groups. levels of albumin, glucose, calcium, sodium, and iron decreased significantly in the seronegative group. results from sero-testing 40 rodents trapped in and around the park depicted the presence of antibodies against toxoplasma gondii in 1  individual. this study reveals the haemato-biochemical alterations in both seropositive and suspected wild felids for toxoplasmosis. moreover, it provides the first serological evidence of t. gondii exposure in wild felids, notably royal bengal tigers and asiatic lions, in india. veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 accepted: 05.09.2016 | available on line: 30.06.2019 guru angad dev veterinary and animal sciences university, ludhiana, punjab 141004, india * corresponding author at: department of veterinary parasitology, college of veterinary science, guru angad dev veterinary and animal sciences university, ludhiana, punjab 141004, india. e-mail: moudgil.aman@gmail.com. first record of toxoplasma gondii antibodies in royal bengal tigers (panthera tigris tigris) and asiatic lions (panthera leo persica) in india aman d. moudgil*, lachhman das singla, amrita sharma and mandeep singh bal 158 veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 toxoplasma gondii antibodies in wild felids moudgil et al. longitudes at an altitude ranging between 180 to 300  metres above main sea level. the zoological park (mahendra chaudhury zoological park, which falls under sub-mountain undulating zone of the province) considered in this study is comprised of animals and birds belonging to around 61 different species, some of which were rare or endangered. the animals targeted in this study were kept in close confinements with adequate wandering space. the wild felines were fed with buffalo meat (carabeef ) and ad libitum water; elephants were provided with green fodder and sugarcane with ad libitum water. ethical aspects and sampling frame the ethics committee for animal experiments of guru angad dev veterinary and animal sciences university granted the ethical approval (iaec/2013/27-43) to conduct this study. the blood samples from 11 rb tigers, 3 asiatic lions, 2 leopards, and 4 elephants (negative control) were collected twice (in february and august – across winter, monsoon, and summer seasons) in 2013. the wild animals were restrained in squeeze cages and blood samples were taken from the dorsal coccygeal vein. the blood samples were collected in vacutainers with anticoagulant and were stored at - 20°c directly for haematological parameters. sera were separated from the blood samples collected in the vacutainers with coagulation activators by centrifugation and stored at -  20°c for further immunological and biochemical analysis. serological screening a commercial indirect elisa (ielisa) kit from cusabio® (wuhan hi-tech medical devices park, china) was used in order to detect t.  gondii antibodies. the kit was used according to the manufacturer’s instructions. the percent positivity (%p) of samples was obtained as follows: mean od of sample _______________________ mean od of critical control x100percent positivity (%p) = od is the optical density of the sample. this is compared with the optical density of the critical control (provided in the kit), and helps in evaluating the values of percent positivity of the samples. samples with % p value less than 90% were considered seronegative. those with more than 110% were considered seropositive, and the values in-between were considered suspected. thus, the animals were divided into 3 groups, as per the infectivity pattern: seropositive, suspected, and seronegative, and further analyses of haemato-biochemical alterations were carried out among these groups. sero-testing of wild rodents (n  =  40) was also introduction toxoplasma gondii, an obligate intracellular protozoan parasite, is the etiological agent of the ubiquitous zoonotic disease, toxoplasmosis (tedesco 2004). it has the potential to infect almost all homeothermic animals (lopes et al. 2008, liu et al. 2012). approximately, one-third of the world human population is chronically infected with t.  gondii (tenter et  al. 2000). toxoplasmosis can be harmful in immunocompromised humans and pregnant women, especially if a woman is infected during the first trimester of her pregnancy (dubey and beattie 1988). it is also responsible for causing abortions in sheep (dubey and beattie 1988). domestic and wild felids are the only definitive hosts for toxoplasma gondii. they thus play the most important role in the life cycle of the detrimental parasite, as they shed environmentally resistant infective oocysts (miro et  al. 2004). the oocysts shed by infected wild felids in captivity are not only a source of infection to other non-infected captive wild felids, but also to other zoo animals, zoo keepers, zoo veterinarians, as well as to the persons visiting zoos (thiangtum et al. 2006). in india, conservation strategies for the royal bengal (rb) tigers (panthera tigris tigris) were recently implemented following a serious reduction in the rb population (banerjee 2013). though toxoplasmosis is asymptomatic in felids and rarely results in clinical signs, it can result in breeding failure in felids (thiangtum et al. 2006). serological studies are better suited to detect t. gondii infection in comparison to faecal sample examination for oocysts because wild felids shed the oocysts for only a shorter period of time (dubey and thulliez 1989). the significant role of t. gondii in the pathophysiology of wild felids and the epidemiological role of wild felids in the transmission of toxoplasmosis in india has not yet been established. this study was planned to adjudge the seropositivity of t. gondii in rb tigers, asiatic lions (panthera leo persica), leopards (panthera pardus), and elephants (elephas maximas indicus), and thus address the lack of relevant literature regarding the prevalence of toxoplasmosis in wild felids in this region. it also aims to compare the associated alterations in the haemato-biochemical parameters of the seropositive individuals to those of healthy seronegative animals. materials and methods study area and management conditions the punjab state is located between 29”  30’  n to 32”  32’  n latitude and 73”  55’  e to 76”  50’  e 159veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 moudgil et al. toxoplasma gondii antibodies in wild felids results at the beginning of the study period (winter), 1  animal was seropositive (lion) and 4 were suspected (3 tigers and 1 lion) for t. gondii by ielisa. by the end of the monsoon season, 4 animals were seropositive (2 tigers and 2 lions) and 1 (tiger) was suspected. the antibody titres of 2 tigers and 1 lion exceeded the positive threshold value by the end of the study period. the anti-toxoplasma antibody titres showed only a slight increase with the change of seasons, while the animals in seronegative titre range at the beginning of the study remained in the same range. all the elephants and leopards were seronegative in both seasons (figure 1), with percent optical density (od) values ranging between 57.54%-78.82% and 30.53%-57.07% for leopards and elephants, respectively. the serum biochemical studies targeting seropositive, carried out in order to assess their probable role in the transmission of toxoplasmosis to wild felids. the rodents were captured from the fields and jungle area around the zoological park by applying rat-catcher machines, and blood was collected by using the retro-orbital plexus puncture method. haemato‑biochemical analysis the haematological analysis of the blood samples for all of the animals (except leopards and elephants) was carried out on fully automated analyser, advia 2120 haematology system (siemens health care diagnostic inc. deerfield, il, us). analysis included haemoglobin level (hb), total erythrocyte count (tec), total leukocyte count (tlc), packed cell volume (pcv), and differential leukocyte count (dlc). biochemical parameters were analysed by using commercial kits of siemens health care diagnostics inc. il, u.s.a. these included total bilirubin (tbil), aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alkp), total protein (tp), albumin (alb), globulin (glo), blood urea nitrogen (bun), creatinine (cre), uric acid (ua), creatine kinase (ck), blood glucose (glu), sodium (na), potassium (k), chloride (cl), calcium (ca), phosphorus (p), and iron (fe). thus, haemato-biochemical alterations were studied in detail only for rb tigers and asiatic lions, as the levels of all the haematological and biochemical parameters fell within normal range in the cases of leopards and elephants. statistical analysis the analysis of variance (one-way anova) of haemato-biochemical parameters among the different groups was done using spss 16.0 software (marco et al. 2000). infected non-infected suspected 140 120 100 80 60 40 20 0 t1 t2 t3 t4 t5 t6 t7 t8 t9 t1 0 t1 1 l1 l2 l3 e1 e2 e3 e4 le op 1 le op 2 monsoon titres winter titres figure 1. antibody titres against toxoplasma gondii in royal bengal tigers (t), asiatic lions (l), indian elephants (e), leopards (leop) in mahendra chaudhury zoological park, in chhatbir, punjab, india. table i. serum biochemical values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status. biochemical parameters tbil (mg/dl) ast (u/l) alt (u/l) alkp (u/l) tp (g/dl) alb (g/dl) glo (g/dl) bun (mg/dl) cre (mg/dl) glu (mg/dl) groups group i (seropositive) 0.74±0.50 * 41±14.25* 51.2±7.79* 33.4±6.91* 7.56±0.53* 3.26±0.26* 4.3±0.43* 69.6±11.95* 3.26±0.50* 29.8±7.01* group ii (suspected) 0.66±0.34 * 45.4±18.90* 46.4±10.55* 32.8±4.21* 7.38±0.19* 3.26±0.24* 4.12±0.18* 64.2±8.67* 3.06±0.39* 23±3.74* group iii (seronegative animals) 0.96±0.38* 51.5±10.13* 46.11±10.81* 33.28±6.73* 7.36±0.46* 4.03±0.44** 3.33±0.36** 50.61±5.19** 2.08±0.29** 68.72±12.5** normal range (nr) shrivastav et al. 2011 (tigers), jani and sabapara 2010 (lions) 0.4-3.2 14.4-84.0 21.2-109.0 16.28-87.9 3.7-8.7 2.1-4.6 1.6-4.1 6.5-48.2 1.6-4.6 66-124 values indicated as mean± standard deviation; *values differing significantly at p < 0.05; tbil = total bilirubin; ast = aspartate aminotransferase; alt = alanine aminotransferase; alkp = alkaline phosphatase; tp = total protein; alb = albumin; glo = globulin; bun = blood urea nitrogen; cre = creatinine; glu = glucose. 160 veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 toxoplasma gondii antibodies in wild felids moudgil et al. recorded in animals of the seronegative group. individually, the glucose concentration values for all of the suspected and seropositive tigers and lions were below the normal range, whereas the values for seronegative individuals were within the normal range (table i). the values for ua, k, and cl did not vary significantly among any of the 3 groups. the values of ca, na, and fe in the suspected and seropositive groups were significantly (p  <  0.05) lower than the seronegative group, whereas the levels of p and ck were significantly higher in the animals of the seropositive and suspected groups than the values recorded for seronegative animals (figures 2 and 3). all the haematological parameters varied non-significantly except for tlc and neutrophils percentage, where the values recorded in the seropositive and suspected groups were significantly suspected, and seronegative groups including tigers and lions revealed no significant difference in the values of tbil, ast, alt, alkp, and tp and values of all the parameters were in the normal range in both seasons irrespective of the group (table i). the values of alb recorded in the seropositive and suspected group were significantly (p  <  0.05) lower than the values recorded in the seronegative group (table i). the globulin level showed a significant increase in the seropositive and suspected groups as compared to the seronegative group. the bun values in both seasons for both seropositive and suspected groups were significantly (p  <  0.05) higher than those recorded in the seronegative group (table  i). the creatinine levels of the animals in the seropositive and suspected groups were significantly higher than the animals of the seronegative group. the glucose levels of the seropositive and suspected groups were significantly (p < 0.05) lower than those 350 200 250 300 150 100 50 0 ck * ** * ** *** ** * ** na cl fe seropositive suspected seronegative figure 3. creatinine kinase (ck), sodium (na), chloride (cl) and iron (fe) values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status. superscripts '*', '**' and ‘***’indicate values differing significantly (p < 0.05). table ii. haematological values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status haematological parameters hb tlc tec pcv platelets n l e m groups group i (seropositive) 14.02±1.39* 18,840±2,013.34* 8.53±0.69* 41.08±4.86* 204.2±25.53* 76.2±7.22* 20.6±5.55* 2±2.34* 0.8±1.79* group ii (suspected) 13.04±2.46* 17,652±1,964.72* 8.46±1.26* 38.64±6.11* 274±46.78* 76.4±9.20* 22.4±9.21* 1.2±1.79* 0* group iii (seronegative) 14.72±1.03* 14,418±1,538.89** 8.14±0.70* 42.98±7.62* 220.78±51.34* 68.94±2.62** 29.28±2.99* 1.11±1.41* 0.5±1.15* shrivastav et al. 2011 7.8-13.8 6,200-11,050 4.66-9.15 36-45 57-75 18-35 2-6 2-6 values are indicated as mean± standard deviation; *values differing significantly (p < 0.05); hb = haemoglobin level; tec = total erythrocyte count; tlc = total leukocyte count; pcv = packed cell volume; platelets = platelets and components of differential leukocyte count i.e. n = neutrophils; l = lymphocytes; e = eosinophils; m = monocytes. 10 6 7 8 9 5 4 3 2 1 0 ua * * * * ** ** k ca p seropositive suspected seronegative figure 2. uric acid (ua), potassium (k), calcium (ca) and phosphorus (p) values in captive royal bengal tigers and asiatic lions of the mahendra chaudhury zoological park, in chhatbir, punjab, india according to toxoplasma infectious status. superscripts '*' and ‘**’indicate values differing significantly (p < 0.05). 161veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 moudgil et al. toxoplasma gondii antibodies in wild felids beattie 1988, thiangtum et  al. 2006). thus, infected raw buffalo or chicken meat offered to captive wild felids could be responsible for spreading infection. anti-toxoplasma antibodies were detected in the serum sample of 1 rodent. their possible role in the transmission of the parasite to the captive felids could therefore not be neglected. other routes of transmission might have involved mechanical transmission by flies, cockroaches, or dung beetles entering into the living area of the wild felids (thiangtum et  al. 2006). the elephants and leopards displayed negative antibody titres and the comparatively lower titre values of the elephants could be attributed to their herbivorous nature. the values of the serum biochemical parameters (alt, ast, tbil, and alkp) were within the normal range, which is consistent with the findings of mosallanejad and colleagues (mosallanejad et  al. 2007). the increased levels of the globulins in both seropositive and suspected groups could be attributed to the presence of immunoglobulins generated against an ongoing infection (sedlack and bartova 2006). the higher creatinine levels that were recorded were suggestive of t.  gondii infection in the definitive hosts (lappin 1996). toxoplasmosis is usually asymptomatic in cats, however the decreased blood glucose levels that were observed in this study can be correlated with the lethargy observed in t.  gondii seropositive felids (elmore et  al. 2010). the significantly increased creatine kinase levels in seropositive and suspected animals influenced the brain or muscles in animals belonging to these two groups and resulted in lowered response times. the decreased serum ca levels revealed the involvement of the pancreas in chronic toxoplasmosis – a condition that leads to pancreatitis in definitive hosts (advincula et al. 2010). the neutrophilic leucocytosis observed in this study is consistent with the findings of mosallanejad and colleagues (mosallanejad et  al. 2007), who considered it as a major finding relating to toxoplasmosis. other important attributes of the t. gondii infection, including anaemia and jaundice, were not observed in this study. this may be due to the chronic nature of the infection in these animals (advincula et al. 2010). this study is the first report of captive wild felids (of india) exposure to t. gondii. it indicates that captivity (in zoological gardens) fosters stress and further immunosuppression and renders the definitive hosts (wild felids) vulnerable to toxoplasmosis. the haemato-biochemical alterations may prove good indicators for the adverse aftermaths of this intestinal protozoan parasite while undergoing extra-intestinal life cycle. wild felines seropositive for t. gondii risk infecting other animals in the zoological garden as well as the individuals (zoo keepers and veterinarians) associated with the management of (p  <  0.05) higher than the values recorded in the seronegative group (table ii). the tlc values for all the groups were higher than the normal range. only 1 serum sample of wild rodent was seropositive for t. gondii (figure 4). discussion this study highlights the presence of antibodies against t.  gondii infections in captive wild felids (rb tigers and asiatic lions) living in a zoological garden. though the sero-detection of toxoplasmosis has been carried out in domestic ruminants in the past (selvaraj et al. 2007, sharma et al. 2008), there has never been a study that considers wild felids. this investigation recorded the first serological evidence of t.  gondii exposure in rb tigers and asiatic lions in india, and moreover found that seropositivity increased from winter to monsoon. this may be due to the fact that parasites were in incubation stage during the winter with no/fewer detectable anti-t.  gondii antibodies. these levels increased during the monsoon season. this increase in the number of seropositive individuals could be attributed to stressful conditions (environmental stress caused by heat and humidity) encountered by the animals during the summer and monsoon seasons, which would result in the immunosuppression of the animals that would, in turn, cause such an increase (salant and spira 2004). another factor could be the increase in moisture during the monsoon season. the survival of oocysts is prolonged in cool, moist conditions, which in turn would aggravate the infection in these animals (bisson et al. 2000). the management of conditions in the zoological gardens therefore has a direct impact on the susceptibility of animals to contract infections. the most frequent cause of toxoplasmosis infection in definitive hosts is through the ingestion of tissues from an infected intermediate hosts (dubey and infected non-infected suspected 120 100 80 60 40 20 0 r 1 r 2 r 3 r 4 r 5 r 6 r 7 r 8 r 9 r 1 0 r 1 1 r 1 2 r 1 3 r 1 4 r 1 5 r 1 6 r 1 7 r 1 8 r 1 9 r 2 0 r 2 1 r 2 2 r 2 3 r 2 4 r 2 5 r 2 6 r 2 7 r 2 8 r 2 9 r 3 0 r 3 1 r 3 2 r 3 3 r 3 4 r 3 5 r 3 6 r 3 7 r 3 8 r 3 9 r 4 0 figure 4. antibody titres against toxoplasma gondii in wild rodents (r 1 -r 40 ) captured in and around mahendra chaudhury zoological park, in chhatbir, punjab, india. 162 veterinaria italiana 2019, 55 (2), 157-162. doi: 10.12834/vetit.971.5066.3 toxoplasma gondii antibodies in wild felids moudgil et al. singh for his co-operation and help in collecting blood samples from wild felids and elephants in mczp, chhatbir, punjab. thanks are also due to dst for providing an inspire fellowship to the first author for his doctoral programme. thanks are also due to dr. kiran malhotra (associate professor of english (retd.), department of languages and haryanvi culture, ccs haryana agricultural university, hisar) for editing the manuscript for the correct use of the english language. animals. further strategies should be implemented to manage this infection. acknowledgements the authors are thankful to the dean, post graduate studies, gadvasu, ludhiana; chief wildlife warden, punjab and director, mczp chhatbir, punjab for providing every possible facility to undertake this investigation. the authors are indebted to dr. m.p. advincula jkdc., iewida syp. & salibay cc. 2010.serologic detection of toxoplasma gondii infection in stray and household cats and its haematologic evaluation. sci med, 20, 76-82. banerjee k. 2013. decadal change in surface water salinity profile of indian sunderbans: a potential indicator of climate change. j marine sci res development, s11: 002. doi: 10.4172/2155-9910.s11-002. bisson a., maley s., rubaire-akiiki c.m. & wastling j.m. 2000. the seroprevalence of antibodies to toxoplasma gondii in domestic goats in uganda. acta trop, 76, 33-38. dubey j.p. & beattie c.p. 1988. toxoplasmosis of animals and man. crc press, boca raton, fl. dubey j.p. & thulliez p. 1989. serologic diagnosis of toxoplasmosis in cats fed toxoplasma gondii tissue cysts. j am vet med assoc, 194, 1297-1299. elmore s.a., jones j.l., conard p.a., patton s., lindsay d.s. & dubey j.p. 2010. toxoplasma gondii: epidemiology, feline clinical aspects, and prevention. trends parasitol res, 26, 190-196. lappin m.r. 1996. feline toxoplasmosis: interpretation of diagnostic test results. semin vet med surg (small animal), 11, 154-160. liu q., singla l.d. & zhou h. 2012. vaccines against toxoplasma gondii: status, challenges and future directions. hum vaccin immunother, 8, 1305-1308. lopes a.p., cardoso l. & rodrigues m. 2008. serological survey of toxoplasma gondii infection in domestic cats from northeastern portugal. vet parasitol, 155, 184-189. marco i., martinez f., pastor j. & lavin s. 2000. hematologic and serum chemistry values of the captive european wildcat. j wildl dis, 36, 445-449. references miro g., montoya a., jimenez s., frisuelos c., mateo m. & fuentes i. 2004. prevalence of antibodies to toxoplasma gondii and intestinal parasites in stray, farm and household cats in spain. vet parasitol, 126, 249-255. mosallanejad b., malmasi a., mohebali m. & tabatabayi m. 2007. anterior uveitis in a kitten infected with toxoplasma gondii (tehran strain). iranian j vet res, 8, 91-93. salant h. & spira d.t. 2004. a cross sectional survey of anti toxoplasma gondii antibodies in jerusalem cats. vet parasitol, 124, 167-177. sedlack k. & bartova e. 2006.the prevalence of toxoplasma gondii igm and igg antibodies in dogs and cats from the czech republic. vet med, 51, 555-558. selvaraj j., manohar b.m., singh s. & balachandran c. 2007. seroprevalence of toxoplasma gondii in buffaloes. j vet parasitol, 21, 41-42. sharma s., sandhu k.s., bal m.s., kumar h., verma s. & dubey j.p. 2008. serological surveys of antibodies to toxoplasma gondii in sheep, cattle and buffaloes in punjab, india. j parasitol, 94, 1175-1175. tedesco r.c. 2004. ocular toxoplasmosis: the role of retinal pigment epithelium migration in infection. parasitol res, 92, 467-472. tenter a.m., heckeroth a.r. & weiss l.m. 2000. toxoplasma gondii: from animals to humans. int j parasitol, 30, 1217-1258. thiangtum k., nimsuphun b., pinyopanuwat n., chimnoi w., tunwattana w., tongthainan d., jittapalapong s., rukkwamsuk t. & maruyama s. 2006. seroprevalence of toxoplasma gondii in captive felids in thailand. vet parasitol, 136, 351-355. 405 1pan african university life and earth sciences institute. 2viral vaccines production department, national veterinary research institute, vom, nigeria. 3national veterinary research institute, vom, nigeria. *corresponding author at: department of veterinary microbiology, faculty of veterinary medicine, university of ibadan, ibadan, nigeria. e‑mail: ogloryus@yahoo.com. please refer to the forthcoming article as: adeoye et al. 2022. molecular detection of peste des petits ruminants virus (pprv) in goats, sheep and commercial pprv vaccines in ibadan, oyo state, nigeria. vet ital. 10.12834/vetit.2558.15754.1. fiyinfoluwa adeoye1, adedamilola kolapo1, oluyemi ogunmolawa2, adegboyega aluko3, clement meseko4, oluwayelu3 and some wild carnivores, and the measles virus of humans (radostits et al., 2000). peste des petits ruminants is a key transboundary animal disease and has been reported across west, central, east and some parts of north africa, the arabian peninsula, the middle east and asia, although it has been found to be emerging in new areas of the world (banyard et al., 2010, 2014). regions of the world that are endemic for ppr are known to be important sheep introduction peste des petits ruminants (ppr), similarly referred to as ‘syndrome of stomatitis-pneumoenteritis’, ‘goat plague’, ‘ovine rinderpest’ or ‘kata’, is a highly infectious disease of sheep and goats. the disease is caused by ppr virus (pprv), which belongs to the family paramyxoviridae and genus morbillivirus. the virus is tightly linked to the rinderpest virus of buffaloes and cattle, morbilliviruses that affect aquatic mammals, distemper virus that affects dogs keywords goats, ibadan, peste des petits ruminants virus, sheep, vaccine. veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 accepted: 19.07.2021 | available on line: 31.12.2022 summary peste des petits ruminants (ppr) is a vaccine-preventable transboundary animal disease of goats and sheep majorly, and is regarded as a major constraint to small ruminant production especially in developing countries like nigeria. despite different strategies that have been employed to control ppr in nigeria, cases of the disease are still reported in ppr-vaccinated and unvaccinated small ruminant farms. in this study, molecular detection of field ppr virus (pprv) strains was carried out to determine the presence of pprv. a total of 135 samples (45 oculo-nasal swabs and 90 tissue samples) were purposively collected between august and october 2020 from goats and sheep at the akinyele live small ruminant market and at akinyele and amosun abattoirs in ibadan, oyo state, nigeria. using reverse transcriptasepolymerase chain reaction with primers targeting the partial n-gene of pprv, 10 out of the 135 (7.4%) field samples yielded positive results.. the results of this study reveal that pprv currently circulates in ibadan. these findings underscore the need for continuous ppr surveillance, more extensive characterization of circulating pprv strains and the importance of consistent use of quality vaccines in the country to achieve more effective preventive and control strategies against the disease. molecular detection of peste des petits ruminants virus (pprv) in goats and sheep in ibadan, oyo state, nigeria molecular detection of ppr in nigeria adeoye et al. 406 veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 and goat-rearing areas. in nigeria, the population of sheep and goats was projected to be about 50 million, with goats outnumbering sheep (fao, 2006). from 1995 to 2000, a study was carried out which documented ppr seroprevalence in some nigerian states to be 38.34% in goats and 49.26% in sheep. in the same study, ppr was also observed in warthogs and camels (shamaki et al., 2004). furthermore, across various nigerian ecological zones, ppr field outbreaks were reported in 2008 (kazeem et al., 2009). small ruminants are important in providing food and commodities for trade. they provide peasant farmers with basic livelihood, with the meat serving as a chief source of animal protein with an estimation of over 35% (shamaki et al., 2004; adamu et al., 2005). these livestocks sustain the income and employment of many people in the rural communities, contribute draught energy, manure for production of crops and skin for leather industries which also earns foreign exchange. in addition, these animals are used for traditional and religious festivities. however, production of these livestocks which is highly economically important, is extremely hindered by the great morbidity and mortality reportedly associated with pprv. depending on its endemicity in an area, great economic losses have been recorded due to 100% morbidity and 80-90% mortality among affected animals despite the availability of vaccines (adamu et al., 2005). ppr is characterized by diarrhoea, fever, oculo-nasal mucopurulent discharges, enteritis, conjunctivitis, erosive-ulcerative stomatitis, broncho-interstitial pneumonia and fibrino-necrotic tracheitis (kul et al., 2007). in severe cases, death results from severe dehydration caused by acute diarrhea or secondary bacterial pneumonia. abortion has been reported from co-existence of pprv with pestiviruses (abubakar et al., 2008). in some cases, pprv is considered more dangerous in goats than sheep; nonetheless, outbreaks affecting both sheep and goat populations have been described (chauhan et al., 2009; wang et al., 2009). furthermore, in some cases, goats seem uninfected, whereas sheep have great rates of morbidity and mortality (yesilbag et al., 2005). the reason for this is yet unknown although the infecting viral strain(s) and host species seem to play crucial roles. also, although only one pprv serotype is recognized, diverse strains of the virus which differ in virulence when experimentally inoculated into the same breed of goats have been reported (couacy-hymann et al., 2007a). in addition, various breeds of goats are known to react in several ways to disease with the same virus (diop et al., 2005). asymptomatic infection has also been observed in some species (bidjeh et al., 1995, ezeibe et al., 2008). in nigeria, ppr remains an endemic disease of sheep and goats and is the most important single cause of high morbidity and mortality in these animals, thereby limitating livestock production (saliu et al., 2008). hamdy and dardiri (1976) reported the financial loss associated with ppr annually in nigeria to be about usd 1.5 million which is about usd 700 million in recent budget. bearing in mind the economic losses resultant from this infection, the necessity therefore remains for continued monitoring of ppr in nigeria for early detection, and effective management and control. vaccination with homologous ppr vaccines has been the control technique yet sporadic cases on both vaccinated and unvaccinated animals have been reported, thus the disease remains a major problem (luka et al., 2011). furthermore, nigeria shares boundaries with benin republic to the west, cameroon and chad to the east and niger republic to the north. these land borders are porous, permitting continuous influx of pastoralists and their livestock into nigeria. considering this regular cross-border movement of pastoralists and their livestock, and the incessant cases of ppr in small ruminants in nigeria, it is important to regularly carry out surveillance to detect the circulating field ppr virus(es). the study was designed to investigate the presence of pprv in goats and sheep in ibadan, oyo state, nigeria using molecular-based techniques. materials and methods study site and population this study was conducted in ibadan, the capital city of oyo state, south-western nigeria (figure 1). figure 1. map of oyo state showing the study areas in ibadan. adeoye et al. molecular detection of ppr in nigeria veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 407 the commercially available ppr vaccine in ibadan was also included in this study for comparative molecular assay. sampling technique a purposive sampling technique was used for the field sample collection. only sheep and goats for sale or those having clinical signs suggestive of ppr at slaughter were included in this study. a total of 135 samples were purposively collected from akinyele live small ruminant market, and akinyele and amosun abattoirs in ibadan, oyo state between august and october, 2020. the distribution of the animals sampled based on species, breed, sex, age and site of sample collection is presented in table i. the animals are often brought to the market from villages around ibadan and these markets also serve as transit points to other larger markets where the animals are re-sold for profit. table i. distribution of the animals sampled in oyo state, nigeria based on location, species, breed, number, sex and age. location species breed number sex age total number of animals sampled akinyele live small ruminant market caprine red sokoto 9 male >1 year 45 red sokoto 7 female west african dwarf 4 male ovine west african dwarf 3 female west african dwarf 7 male >1 year west african dwarf 14 female sahel 1 female amosun abattoir caprine red sokoto 9 male >1 year 45 red sokoto 5 female west african dwarf 6 male west african dwarf 9 female ovine west african dwarf 5 male >1 year west african dwarf 11 female akinyele abattoir caprine red sokoto 5 male >1 year 45 red sokoto 9 female west african dwarf 8 male west african dwarf 7 female west african dwarf 10 male >1 year west african dwarf 6 female sample collection forty-five oculonasal swabs were collected from akinyele live small ruminant market, and 45 tissue samples each from akinyele and amosun abattoirs. the tissue samples comprised lung, spleen and mesenteric lymph nodes. the samples were properly labeled with designated sample identity tags, site of swab collection or type of tissues collected and date of sample collection. oculo-nasal swabs collected from live small ruminants were transported in viral transport medium containing 2000 units/ml of penicillin, ibadan is located on coordinates 723’47’’n and 3 55’0’’e. it lies in the tropical rainforest zone and has a population of about 3.7 million people as at 2021 with over 6 million living in the metropolis. the city has both tropical wet and dry climates; the wet period extends from march to october while the dry period runs from november through february (wells, 2008). sample size determination using the formula of thrusfield and christley (2005), the size for the field samples (from goats and sheep) collected was calculated to be 135. this was based on an estimated prevalence (pexp) of 9.7% from a previous study (mantip et al., 2016), reliability coefficient (z) of 1.96 at 95% confidence interval and absolute precision (d) of 5%, using the formula n = z² pexp (1-pexp) / d² molecular detection of ppr in nigeria adeoye et al. 408 veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 germany) was added to the spin column and centrifuged at 8000 rpm for 1 min. the spin column was placed in a clean 2 ml collection tube while the tube containing the filtrate was discarded. 500 μl of buffer aw2 (qiagen, hilden, germany) was added to the spin column and centrifuged at 14,000 rpm for 3 min. the qiaamp mini spin column was placed in a clean 1.5 ml microcentrifuge tube while the tube containing the filtrate was discarded. 60 μl of buffer ave (qiagen, hilden, germany) was added to the spin column and incubated at room temperature for 1 min. this was then centrifuged at 8000 rpm for 1 min. finally, the labeled 1.5 ml tube containing the eluted rna was kept at -80°c before moving to the pcr mix room. for positive control, the viral rna was extracted from a previously known positive ppr sample while a non-template control was used as negative control. one-step reverse transcriptase-polymerase chain reaction (rt-pcr) one-step rt-pcr was performed for the amplification of a 350 base pair fragment of the n-gene using the following primers: • forward primer: ppr-np3 (5’-gtc tcg gaa atc gcc tca cag-3’) • reverse primer: ppr-np4 (5’-cct cct cct ggt cct cca gaa-3’), and using a 25 μl final reaction volume, mixtures containing 12.75 μl rnase-free water, 5 μl pcr buffer 5x, 0.5 μl dntps, 0.5 μl one-step rt-pcr enzyme mix, 0.25 μl rnase inhibitor, 5 μl of rna template and 0.5 μl each of the primers. a 96-well thermocycler (applied biosystems) (biopolis, singapore) was used for the amplification. it was set to start at 50°c for 30 minutes, followed by initial denaturation at 95°c for 15 minutes. thereafter, 35 cycles of denaturation, annealing and elongation of the templates were achieved at the cycling conditions of 94°c for 30 seconds, 55°c for 30 seconds and 72°c for 1 minute, respectively. final extension of the templates was achieved at 72°c for 10 minutes. the amplified pcr products were analyzed by electrophoresis in 1% agarose gel stained with ethidium bromide (1 μg/ ml) and run at 120 v for 30 minutes. the bands were visualized under an ultraviolet transilluminator. results agarose gel electrophoresis of amplified products of the 135 field samples tested revealed that only 10 (7.4%) yielded the expected 350 bp band as previously described (couacy-hymann et al., 2002) (figure 2). 2 mg/ml of streptomycin, 0.05 mg/ml of gentamycin and 100 units/ml of mycostatin in a cool flask with ice packs. tissues (lung, spleen and lymph node) were collected from carcasses of slaughtered animals. all samples collected were transported in cool flasks with ice packs to the virology laboratory, department of veterinary microbiology, university of ibadan where they were stored at -20°c under constant power supply for approximately one week before being transported in a cold chain to the national veterinary research institute (nvri), vom, plateau state where they were stored at -80°c until processed for pprv detection. preparation of necropsy tissues necropsy tissues were processed in the as previously described (mantip et al., 2016). briefly, 10% suspensions of the tissues were prepared by pooling 1 g of lung, spleen and lymph node from the same animal and macerating them using a mortar and pestle. thereafter, sterile sand and phosphatebuffered saline were added. the suspension was centrifuged at 2500 g for 5 min and the supernatant pipetted into cryovials, labelled properly and stored at -20°c. the leftover tissues were kept at -80°c for further usage. molecular detection of pprv in goats and sheep viral ribonucleic acid (rna) extraction viral rna was extracted directly from the viral transport medium containing swab samples collected from the live small ruminant market and homogenized tissue samples from the abattoirs. the extraction was performed using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer’s instruction. the procedure was carried out as follows: 560 μl of the prepared buffer avl (qiagen, hilden, germany) containing carrier rna was pipetted into a 1.5 ml microcentrifuge tube. 140 μl of homogenized tissue samples or pulse-vortexed viral transport media containing the swab was pipetted into the buffer avl containing carrier rna in a microcentrifuge tube. this was mixed by pulse-vortexing for 15 seconds and incubated at room temperature for ten minutes after which it was briefly centrifuged to remove drops from the inside of the lid. 560 μl of ethanol (96100%) was added to the mixture which was pulsevortexed for 15 seconds and briefly centrifuged. 630 μl of the solution was aliquoted to the qiaamp mini spin column (in a 2 ml collection tube) and centrifuged at 8000 rpm for 1 min. the tube containing the filtrate was then discarded. thereafter, 500 μl buffer aw1 (qiagen, hilden, adeoye et al. molecular detection of ppr in nigeria veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 409 figure 2. rt-pcr product bands (350 bp) of amplified ppr virus ngene. m = 100 bp molecular weight marker; lanes 1-10 = positive bands for the tissue samples; lane 11 = positive band for nvri ppr vaccine; lane 12 = positive control; lane 13 = negative control. out of the tissue samples collected at amosun abattoir, only one (7.1%) of 14 female west african dwarf (wad) goats, 4 (26.7%) of 15 male wad goats, none (0%) of 11 female wad sheep and one (20.0%) of 5 male wad sheep tested positive for pprv rna, while for the tissue samples collected at akinyele abattoir, one (5.6%) of 18 female wad goats, 3 (23.1%) of 13 male wad goats, none (0%) of 6 female wad sheep and none (0%) of 10 male wad sheep were positive. in addition, none (0%) of the 45 oculo-nasal swab samples collected from goats and sheep at akinyele live small ruminant market was positive for pprv nucleic acid (table ii). analysis of the results based on abattoir location showed that of the 45 of the samples each from amosun and akinyele abattoirs, only six goats and sheep (13.3%) and four goats (8.9%) yielded the expected 350 bp dna band, respectively. table ii. description of the samples that tested positive for pprv by rt-pcr. location species breed sex age sample type collected clinical signs observed rtpcr result amosun abattoir caprine wad male adult lung, spleen nasal discharge, matted eyelid + ve amosun abattoir caprine wad male adult lung, spleen soiled hindquarters nasal discharge + ve amosun abattoir ovine wad male adult lung, spleen nasal and ocular discharges + ve amosun abattoir caprine wad female adult lung, spleen nasal and ocular discharges + ve amosun abattoir caprine wad male adult lung, spleen nasal and ocular discharges + ve amosun abattoir caprine wad male adult lung, spleen soiled hindquarters nasal discharge + ve akinyele abattoir caprine wad male adult lung, spleen, mesenteric lymph node nasal discharge, matted eyelid + ve akinyele abattoir caprine wad female adult lung, spleen, mesenteric lymph node nasal and ocular discharges + ve akinyele abattoir caprine wad male adult lung, spleen, mesenteric lymph node nasal and ocular discharges + ve akinyele abattoir caprine wad male adult lung, spleen, mesenteric lymph node nasal and ocular discharges + ve discussion the results of this investigation show a 7.4% prevalence of pprv in ibadan, oyo state by rt-pcr. the low prevalence can be attributed to the sample size and the season of sample collection as most of the samples were collected between august and october. however, wosu (1994) reported more cases of ppr in south-eastern nigeria throughout the harmattan period (from december to january) compared to the rainy season with the highest rainfall occurring in april. the prevalence of pprv in this study is however below the prevalence (27.3%) reported in adamawa state by ularamu et al. (2002). further, in an earlier ppr survey in the southern states of nigeria by obi and ojeh (1989) using tissue homogenate with dot enzyme-linked immunosorbent assay (elisa) and standard indirect elisa procedures, ppr prevalence rates of 86.8% molecular detection of ppr in nigeria adeoye et al. 410 veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 affect the outcome of rt-pcr which is a limitation with studies conducted in developing countries. the detection of pprv in goats and sheep in ibadan, oyo state, nigeria in this study can probably be attributed to the continuous transportation/ movement of sheep and goats for foraging or commercial purposes from the northern savannah area of nigeria or neighbouring sahelian african countries which are dry, to the southern forest zone of the country, which is wet. nigeria remains a focal point of animal meat consumption in west africa owing to her huge population. in order to satisfy the continually growing local need, there is increased influx of sheep and goats from nearby countries. the sale and transportation of animals and their products from one state to the other and from neighbouring countries into nigeria may serve as a major route of transmission and spread of transboundary animal diseases including ppr. according to studies by cattaneo et al. (1987) and couacy-hymann et al. (2002), it has been proposed that the pprv n-gene is surplus in positive tissue samples. therefore, aiming at the n-gene of pprv for rt-pcr yields good outcome. it has been discovered that the n-gene is responsible for the coding of an inner structural protein. since mrnas of n-gene are copies of the virus, it is thus a conducive target for the development of a diagnostic ppr test, with very high sensitivity and specificity (george, 2002). due to the clinical signs observed in the animals screened in this study and the detection of pprv by rt-pcr technique, it can be concluded that the disease that affected the animals that were positive in this study was caused by pprv. the data obtained from this investigation have thus provided relevant information on the current status and epidemiology of ppr virus in nigeria, particularly in ibadan, oyo state. limitation as at the time of compiling this report, the result of sequencing of the ppr amplicons from the samples that tested positive is yet to be received. the delay is due to changes in protocol associated with the recent coronavirus disease (covid)-19 pandemic. this would have aided the molecular characterization as well as helped to determine whether there are differences between circulating field strains and the locally available vaccine. conclusions the results of this study showed a prevalence of 7.4% for ppr in goats and sheep in ibadan, oyo state, nigeria using rt-pcr detection method. and 81.6%, respectively were recorded. in addition, a prevalence of 51.2% was reported for samples examined in north-central states of nigeria using a set of primers specific for the f gene of pprv (luka et al., 2011). previous studies in some african countries such as morocco revealed ppr prevalence of 44.4% by rt-pcr and a greater prevalence of 80% in sudan (kwiatek et al., 2011). pprv was also confirmed in 33.3% clinical samples tested in algeria, with a group of primers appropriate for the f gene of the pprv (de nardi et al., 2012). in northern and eastern tanzania, pprv was reported in 29.6% and 31.1% of the goats examined, (kgotiele et al., 2014). in ethiopia, a prevalence of 46.4% has been reported (alemu et al., 2019) while in india, a prevalence of 50% was recently reported in an endemic region using rt-pcr (kerur et al., 2008). variations in the prevalence of pprv in the diverse locations reported above could be due to differences in sample size, season of sample collection, detection method used, type of sample(s) employed for ppr diagnosis, phase of infection and the gene targeted for rt-pcr procedure (luka et al., 2012). thus, the detection of pprv in small ruminants in ibadan, oyo state poses a major risk to the neighbouring communities and states as there is a high chance of spread since the disease is transboundary in nature, and considering that these animals are often transported from one market to another and sometimes to farms. this could lead to further epidemics and serious economic impact on the nation. this current investigation revealed a significantly higher rate of ppr in goats compared with sheep using the rt-pcr technique. this finding is in tandem with that reported in ethiopia by alemu et al. (2019). similarly, abubakar et al. (2008) showed ppr cases in pakistan to be more dangerous/virulent in goats compared to sheep. a greater occurrence of ppr was detected in goats when compared with sheep in a survey by mahajan et al. (2013). in a study by abraham et al. (2005) in ethiopia, it was reported that the seeming non-existence of pathogenicity in sheep might be due to a specific resistance of the indigenous species and/or an absence of virulence seen with the ethiopian pprv strains for sheep. also, epidemiological findings from this study revealed that the west african dwarf breed of goats was more predisposed to ppr in comparison with other breeds. this observation is consistent with the position of fao (1999), baazizi et al. (2017) and balogun et al. (2017). the absence of any positive results in the swab samples collected at the live small ruminant market could be due to the method of sample collection and the consideration that despite efforts to maintain cold chain, rna viruses do not store well and this may adeoye et al. molecular detection of ppr in nigeria veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 411 these findings confirm the presence of ppr virus within goat and sheep populations in ibadan. these results show that ppr is still endemic and could be responsible for the intermittent outbreaks of pneumonia and diarrheal disease in small ruminants in nigeria. considering the interstate transportation of sheep and goats in nigeria, this further indicates the potential risk of spread of ppr to regions where the disease is presently absent in the country. statement of animal rights in conducting this study, all relevant institutional, national and/or international guidelines for the care and use of animals were duly followed (world organization for animal health, 2009). acknowledgement the authors acknowledge the technical assistance rendered by members of staff of the national veterinary research institute (nvri), vom, nigeria during this project. grant support the african union commission and the pan african university provided the grant support for this project. molecular detection of ppr in nigeria adeoye et al. 412 veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 radostits o.m., blood d.c. & gay c.c. 2000: in veterinary medicine (a textbook of the diseases of the cattle, sheep, pigs, goats and horses), 9 ed., w.b. saunders, london, uk banyard a., parida s., batten c., oura c., kwiatek o. & libeau g. 2010. global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control. j. gen. virol. 91, 2885–2897. banyard a., wang z. & parida s. 2014. peste des petits ruminants virus, eastern asia. emerg. inf. dis. 20, 2176–2177. f.a.o 2006: faostat database. food and agricultural organisation, rome, italy. www.fao. org/.../ess/publications studies/...yearbook/faostatisticalyearbook2005-2006/en shamaki d., olaleye o.d., obi t. u., diallo a., majiyagbe k.a., lombin l.h. & barrett t. 2004. peste des petits ruminants (ppr) in nigeria: serological and molecular epidemiology. vom j. vet.sci. 1: 18 – 27. kazeem h.m., shamaki d., ibru j.o., fasina f.o., abaadulugba e.p., bitrus y., owolodun o.a., shuaibu s., tyem a.d., ogedengbe m.f., antiabong j.f. & lombin l.h. 2009. 2008 field outbreak of peste des petits ruminants (ppr) in sheep and goats from different geo-ecological zones of nigeria. in 46th annual conference of nig. vet. med. ass. adamu s., ekundayo o.s., useh n.m., neils j.s., bissallah m. & esievo k.a.n. 2005. serum levels of some electrolytes in diarrhoeic savanna nd brown goats in zaria. in 42nd congress of nigerian vet. medical asso. nov. 14 -18, 2005, maiduguri, nigeria. kul o., kabakci n., 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vet. med. 78, 85–88. diop m., sarr j. & libeau g. 2005. evaluation of novel diagnostic tools for peste des petits ruminants virus in naturally infected goat herds. epidemiology infection 133, 711–717. bidjeh k., bornarel p., imadine m. & lancelot r. 1995. first time isolation of the ppr virus in chad and experimental induction of the disease premier isolement au tchad du virus de la ppr et reproduction experimentale de la maladie. rev. elev. med. vet. pays trop. 48, 295–300. ezeibe m., okoroafor o., ngene a., eze j. & eze i. 2008. persistent detection of peste de petits ruminants antigen in the faeces of recovered goats. tropical animal health 40, 517–519. saliu o., audu s., sanda m., aribido s. & olaolu m. 2008. adoption of vaccination and ethnoveterinary treatment for peste des petits ruminnats (ppr) among sheep and goat farmers in ijumu local government area of kogi state. nig. agric. j. 3(5): 404-408. hamdy f.m. & dardiri a.h. 1976. response of whitetailed deer to infection with peste des petits ruminants virus. j. wildl. dis. 12, 516–522. luka p., erume j., mwiine f.n., ayebazibwe c. & shamaki d. 2011. molecular characterization and phylogenetic study of peste des petits ruminants viruses from north central states of nigeria. bmc vet. res. 4, 7. wells j. c. 2008. in longman pronunciation dictionary (3rd ed.). longman. isbn978-1-4058-8118-0 thrusfield m. & christley r. 2005. veterinary epidemiology (vol. 9600). wiley online library. mantip s., quan m., shamaki d. & van vuuren m. 2016. comparison of nucleotide sequences of recent and previous lineages of peste-des-petitsruminants viruses of sheep and goats in nigeria. onderstepoort journal of veterinary research 83(1), a1163. couacy-hymann e., roger f., hurard c., guillou j.p., libeau g. & diallo a. 2002. rapid and sensitive 100, detection of peste des petits ruminants virus by a polymerase chain reaction assay. journal of virological methods 100, 17–25. wosu l.o. 1994. current status of peste des petits ruminants (ppr) disease in small ruminants. a review article’, stud res vet med 2, 83–90. 4. materials and methods study site and population sample size determination sampling technique sample collection . preparation of necropsy tissues molecular detection of pprv in goats and sheep viral ribonucleic acid (rna) extraction one-step reverse transcriptase-polymerase chain reaction (rt-pcr) 5. results 6. discussion tlimitation as at the time of compiling this report, the result of sequencing of the ppr amplicons from the samples that tested positive is yet to be received. the delay is due to changes in protocol associated with the recent coronavirus disease (covid)-19 pandemic. this would have aided the molecular characterization as well as helped to determine whether there are differences between circulating field strains and the locally available vaccine. conclusion the results of this study showed a prevalence of 7.4% for ppr in goats and sheep in ibadan, oyo state, nigeria using rt-pcr detection method. these findings confirm the presence of ppr virus within goat and sheep populations in ibadan. these results show that ppr is still endemic and could be responsible for the intermittent outbreaks of pneumonia and diarrheal disease in small ruminants in nigeria. considering the interstate transportation of sheep and goats in nigeria, this further indicates the potential risk of spread of ppr to regions where the disease is presently absent in the country. 9. acknowledgement 10. grant support 11. references adeoye et al. molecular detection of ppr in nigeria veterinaria italiana 2022, 58 (4), 405-413. doi: 10.12834/vetit.2558.15754.1 413 erume j. 2012. sample type is vital for diagnosing infection with peste des petits ruminants virus by reverse transcription pcr. j vet sci. 13:323–5. mahajan s., agrawal r., kumar m., mohan a. & pande n. 2013. incidence of peste des petits ruminants in nomadic sheep and goat of jammu region. vet world 6(7):384–7. abraham g., libeau g., roger f., laekemariam y., abayneh d. & awoke k.m. 2005. antibody seroprevalences against peste des petits ruminants (ppr) virus in camels, cattle, goats and sheep in ethiopia. prev vet med. 70:51–7. f.a.o. 1999: recognizing peste des petits ruminants. a field manual. prepared by dr. p.l roeder and prof. t.u. obi of food and agriculture organizations of the united nations (fao). http://www.fao.org// docrep/003/x1703e/x1703e00.htm baazizi r., mahapatra m., clarke b.d., aitoudhia k., khelef d. & parida s. 2017. peste despetits ruminants (ppr): a neglected tropical disease in maghreb region of north africa and its threat to europe. plos one 12:e0175461. balogun f.a, fasanmi o.g., oladipo t.a, popoola m.a, olona j.f. & adeoye y.d. 2017. field evaluation and confirmation of acute peste des petits ruminant outbreak in a flock of west african dwarf goats in ibadan,nigeria. international journal of veterinary science and medicine 5:2, 175-180, cattaneo, r., rebmann, g., baczko, k., ter meulen, v. & billeter, m.a. 1987. altered ratios of measles virus transcripts in diseased human brains. virology 160, 523–526. george, a.a. 2002. comparative evaluation of different gene targets for pcr diagnosis of ppr. m. v. sc. thesis, deemed university ivri, izatuagar, india. oie, 2009: oie manual of diagnostic tests and vaccines for terrestrial animals. http://www.oie. int/eng/normes/mmanual/2008/pdf/ 0.01.1b_ foreword. pdf ularamu h.g., owolodun o.a., woma t.y., audu b.j, aaron g.b, chollom s.c. & shamaki d. 2012. molecular diagnosis of recent suspected outbreaks of peste des petits ruminants (ppr) in yola, adamawa state, nigeria. african journal of biotechnology vol. 11(5), pp. 1158-1162, obi t.u. & ojeh c.k. 1989. dot enzyme immunoassay for visual detection of peste despetits-ruminants virus antigen from infected caprine tissues. j clin microbiol, 27(9):2096-2099. kwiatek o., ali y.h., saeed i.k., khalafalla a.i., mohamed o.i., obeida a.a., abdelrahman m.b.,osman h.m., taha k.m., abbas z., el harrak m., lhor y., diallo a., lancelot r., albina e. & libeau g. 2011. asian lineage of peste des petits ruminants virus, africa. emerg infect diseases.17(7):1223–31. de nardi m., lamin saleh s.m., batten c., oura c., di nardo a. & rossi d. 2012. first evidence of peste des petits ruminants (ppr) virus circulation in algeria (sahrawi territories): outbreak investigation and virus lineage identification. transbound emerg dis. 59:214–22. kgotiele t., macha e.s., kasanga c.j., kusiluka l.j.m., karimuribo e.d., van doorsselaere j., wensman j.j., munir m. & misinzo g. 2014. partial genetic characterization of peste des petits ruminants virus from goats in northern and eastern tanzania. transbound emerg dis. 61(1):56–62. alemu b. , gari g., libeau g., kwiatek o., kidane m., belayneh r., siraw b., wieland b., asfaw w. & abdi r. 2019. molecular detection and phylogenetic analysis of peste des petits ruminants virus circulating in small ruminants in eastern amhara region, ethiopia. bmc veterinary research 15:84 kerur n., jhala m.k. & joshi c.g. 2008. genetic characterization of indian peste des petits ruminants virus (pprv) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments. res vet sci 85(1):176-183. luka p.d., ayebazibwe c., shamaki d., mwiine f.n. & 143 ekene vivienne ezenduka* department of veterinary public health and preventive medicine, university of nigeria, nsukka, enugu state, nigeria * corresponding author at: department of veterinary public health and preventive medicine, university of nigeria, nsukka, enugu state, nigeria. tel.: +234 7061032840, e-mail: ev.ezenduka@yahoo.com. veterinaria italiana 2019, 55 (2), 143-148. doi: 10.12834/vetit.395.1870.4 accepted: 12.11.2015 | available on line: 30.06.2019 parole chiave antimicrobici, residui, organi, pollame, matrice. riassunto scopo di questo studio è determinare la prevalenza di residui antimicrobici nel pollame nella città di enugu metropolis in nigeria. rene, fegato, muscolo e ventriglio sono stati prelevati da 100 volatili da allevamento e testati con il three plate test (metodo microbiologico); come prova è stato usato il bacillus subtilis. dei 100 volatili analizzati, 64 sono risultati positivi a residui antibiotici con una prevalenza del 64%. dei 400 campioni, ne sono risultati positivi 155, con valori di prevalenza differenti negli organi indagati. dallo studio emerge un’associazione tra il tipo di organo e l’occorrenza dei residui con una prevalenza maggiore nel fegato (p value:< 0.0001, chi square test). complessivamente, questo lavoro dimostra che il pollame commerciale contiene residui antimicrobici, probabilmente per l’uso indiscriminato nella produzione zootecnica. screening dei residui antimicrobici nel pollame della città di enugu, sud-est della nigeria keywords antimicrobials, residues, organs, poultry, matrix. summary the aim of this study is to determine the prevalence of antimicrobial residues in poultry in enugu metropolis, enugu state, nigeria. four organs – kidney, liver, muscle, and gizzard – were harvested from 100 commercial broiler birds and tested using the three plate test (microbiological method) with bacillus subtilis as test organism. of the 100 sampled birds, 64 were positive for antimicrobial residues, with a prevalence of 64%. out of 400 organs, 155  were positive for antimicrobial residues, with different prevalence values observed in the harvested organs. our findings indicate an association between the type of organ and the occurrence of antimicrobial residues, with the kidney having the highest prevalence (p  value: < 0.0001, chi square test). overall, in this study, commercial poultry were found to contain multiple antimicrobial residues, which strongly suggest the indiscriminate use of antimicrobials in livestock production. screening of antimicrobial residues in poultry meat in enugu metropolis, enugu state, south east nigeria can influence also the microbial composition and metabolic activity of the intestinal micro flora (vollard and clasener 1994, nisha 2008). it is known that a link between the use of antibiotics in food animals and the development of bacterial resistance to these drugs exists (stark 2000, reig and todra 2008). other recorded pathological effects induced by antimicrobial residues in food include autoimmunity, carcinogenicity, mutagenicity, and bone marrow toxicity (pavlov et al. 2008, nisha 2008). to buttress the importance of drug residues, introduction veterinary drugs, specifically antimicrobials, are widely used in poultry production for the treatment of infections, management of stress, and as additives for prophylaxis and growth promotion. these drugs tend to accumulate in tissues forming residues with concentrations above their maximum residue limits (mrl) if withdrawal periods are not observed. antimicrobial residues in food are potential allergens and may result in severe reactions in sensitised individuals. they 144 screening of antimicrobial residues in poultry meat ezenduka veterinaria italiana 2019, 55 (2), 143-148. doi: 10.12834/vetit.395.1870.4 materials and methods the 3-plate test was used for the antimicrobial residues detection with bacillus subtilis as test organism. isolation and identification of bacillus subtilis five grams of soil sample from a cassava waste dump-site was weighed into 45 ml of ringer solution to form a stock mix. this was then heated at 90 °c for 1 hour to encourage the formation of spores as well as for eliminating other unwanted microorganisms. then, 0.1  ml of the mix was inoculated in 0.4% dextrose nutrient agar and incubated at 37  °c for 24 hours. biochemical tests isolated colonies were subjected to api 50 ch biochemical tests (biomeriuex, uk) for identification. the api 50 ch test is a standardised commercial system of 50 biochemical tests in microtubes designed for the study of carbohydrate metabolism, substrate utilisation, and enzyme production of microrganisms. it is used in conjunction with api 50 chb/e medium for the identification of bacillus species. a suspension of the organism was prepared from a 24-hour culture of the isolated b.  subtilis in api 50  chb/e medium and compared with 0.5% mcfarland standard. the organism was inoculated into the 50 tubes and incubated at 24-30  ºc for 24  hours. fermentation was revealed by a colour change in the tube. the api 50 results were read using the api web software (https://apiweb. biomerieux.com/servlet/identify). molecular detection of b. subtilis colonies of b. subtilis were emulsified in 200 µl of tris edta (te) buffer. nextec bacterial dna extraction kit (nextec uk) was used to extract genomic dna from the emulsified isolates. primers specific to a 595 bp amplicon of bacillus subtilis group’s 16s rrna gene were used at a final concentration of 0.4  m to detect the organism genome. the primer sequences and thermal profile we used have already been published (wattiau et  al. 2001). the pcr products were electrophoresed at 100 v for 30 minutes using 2.0% agarose gel and compared with a dna ladder (promega, uk). antimicrobial sensitivity test antibiogram of the b. subtilis isolate was determined using the disc diffusion technique (clsi 2011) with tolerance limits or maximum residue limits (mrls), for antimicrobial residues have been set in most developed countries. countries that do not have mrls have, generally, adopted those established by codex alimentarius, which have also been adopted by the world health organisation (who). several related programmes addressing food safety and drug residues in foods of animal origin have been developed. these include the residue avoidance programme (rap), which was initiated in 1981 by the extension service and food safety and inspection service (fsis) of the united states department of agriculture (usda) with the goal of monitoring food supply in order to ensure that antibiotic residue concentrations do not exceed maximum residue limits. the results from rap are then disseminated to farmers and people working in the livestock industry through an educational programme, thus helping to prevent the occurrence of drug residues. the european union has similarly well-developed abattoir-based programmes for the surveillance and monitoring of antibacterial residues in meat (myllyniemi 2004). in sharp contrast, nigeria has no national programme in place for monitoring drug residues in food animals in farms and abattoirs and no strict regulations on the use of antimicrobials in livestock. several methods to detect antimicrobial residues in different sample matrices are available, but many of these methods are relatively expensive and time consuming. antimicrobial residues in animals are conventionally detected through microbiological tests, including bacillus stearothermophillus disc assay (bsda), the european four plate test (fpt), the german three plate test (tpt), the premi® test, and a number of other commercial kits. these tests, which are essentially qualitative, i.e only able to screen for the presence of antimicrobial residues, are based on bacterial inhibition and are used primarily as screening tools for the presence of antimicrobial residues in meat, milk, and eggs. the tpt, like the fpt, determines the presence of antimicrobials in a sample and also identifies the specific antimicrobial group/class (haasnoot et  al. 1999; javadi et  al. 2009). the test is prepared with bacillus subtilis as test organism at ph 6, 7.2, and 8, hence the name tpt. the ph 6 plates detect, in particular, beta-lactams and oxytetracyclines; ph 7.2 plates detect sulphonamides; while aminoglycosides are detected by ph 8 (chang et  al. 2000). the only variation from the conventional eu fpt is the fourth plate, which contains micrococcus luteus at ph  8 in order to detect beta-lactams and macrolids. the aim of this study is to determine the prevalence of antimicrobial residues in poultry in enugu metropolis, enugu state-south east nigeria, using a conventional microbiological method (tpt) with locally sourced bacillus subtilis. 145veterinaria italiana 2019, 55 (2), 143-148. doi: 10.12834/vetit.395.1870.4 ezenduka screening of antimicrobial residues in poultry meat results cultural and morphological characteristics large, flat undulated colonies with ground glass appearance on nutrient agar, showing gram-positive spore forming small rods, occurred singly and in short chains. these were suspected to be bacillus subtilis. biochemical and molecular tests the organism fermented glycerol, d-glucose, d-fructose, d-manose, d-sorbitol, inositol, and 15 other carbohydrates. the organism was identified as bacillus subtilis with 99.9% accuracy using api web. the pcr amplified a 595 bp product of the 16s rrna, confirming the presence of b. subtilis. antibiogram sensitivity test the isolate, bacillus subtilis, was sensitive to all tested antimicrobials except cefixime, which is not used in poultry production in nigeria. antimicrobial residues in poultry meat and organs out of 100 birds sampled for antimicrobial residues, 45 (64%) were positive while the remaining 25 (36%) were negative. table i shows the ratio of antimicrobial residues in descending order of frequency in the organs as follows: kidney (60%), liver (54%), gizzard (30%), and muscle (11%). a total of 155 organs out of 400  tested positive for antimicrobial residues. a strong association was found between the occurrence of antimicrobial residues and the organ type (χ2  value  =  64.5, p value < 0.0001); the kidney had the most positive cases. out of the 155 positive organ samples, 25 were detected only at ph 6.0 (best detects tetracycline and β-lactams), 26 at ph 7.2 (best detects commercially available discs (oxoid, thermofisher, uk). a total of 16 antibacterial agents belonging to the following classes were used: fluoroquinolones, beta-lactams, aminoglycosides, tetracyclines, amphenicols, glycopeptides, and macrolides. a suspension of a 24-hour fresh culture of the isolate was made in 1 ml of distilled water to correspond to 0.5 mcfarland standards, poured into freshly prepared mueller hilton agar, and then the excess was tilted off. inoculated plates were allowed to dry for approximately 3-5 minutes and the antibiotic discs applied aseptically to the surface of the inoculated agar. the plate was incubated at 37  °c for 24 hours and read afterwards; the diametre of zone of complete inhibition was measured in millimetre scale. sample collection a total number of 100 birds from the 3 major poultry markets in enugu metropolis (artisan market, ogbete main market, and gariki market) in south east nigeria , were randomly selected and purchased. each market was visited twice a week for 4 weeks, and 4 birds were purchased per visit. samples of muscle, liver, kidney, and gizzard were harvested from birds after slaughter. sample preparation for each organ, a 5-g piece was weighed and macerated with equal volume of distilled water at a 1:1 ratio. the mixture was centrifuged at 5,000 rpm for 10 minutes and the supernatant decanted and stored frozen at -20 °c for analysis. the three‑plate test nutrient agar was prepared and adjusted to ph 6, 7.2, and 8 with naoh and hcl; the media were poured onto sterile petri dishes and seeded with bacillus subtilis. each agar plate was bored 5 times. about 100  µl of the organ extracts were then inoculated in 4 holes, each hole representing an organ. the remaining hole was inoculated with 100  µl of distilled water as negative control. plates were incubated at 30 °c for 24 hours. a clear zone of inhibition with annular diameter ≥ 2mm indicated a positive result for antimicrobial residues (myllyniemi et al. 2001). statistical analysis using graphpad statmate 2.0, a sample size of 100 at 90% power was calculated and significance was accepted at p  ≤  0.05. data from the study was analysed in graphpad prism statistical software version 5.02 (www.graphpad.com). table i. antimicrobial residues in the tested organs. matrix (organ) sample no of positive (≥ 2 mm) no of negative (< 2 mm) percentage (%) positive muscle 100 11 89 11 liver 100 54 46 54 kidney 100 60 40 60 gizzard 100 30 70 30 total organs 400 155 245 38.8 ≥ 2 mm = inhibition zones more than or equal to 2 mm; < 2 mm = inhibition zones less than 2 mm. 146 screening of antimicrobial residues in poultry meat ezenduka veterinaria italiana 2019, 55 (2), 143-148. doi: 10.12834/vetit.395.1870.4 and colleagues (aerts et  al. 1995) and myllyniemi and colleagues (myllyniemi et  al. 1999), and are, indeed, used as matrix in many countries in which the level of antibiotics in meat needs to be assessed. kidney is generally used as sample matrix as it is the organ responsible for excreting most drugs. however, other reports show that kidney may give false positive results due to the presence of natural inhibitors of bacterial growth such as lysozymes, which are often present in kidneys (kirbis 2007), and so should be used along with other matrixes, as it was done in this study. the detection of antimicrobials at different ph levels in the same organ implies that different classes of antimicrobials are being administered to a bird at the same time. the concurrent use of different antimicrobials that were observed in this study has been also reported by shareef and colleagues (shareef, et  al. 2009) and ibrahim and colleagues (ibrahim et  al. 2010). bacillus subtilis at ph 6.0 generally detects oxytetracycline (otc), which is the most widely used drug in poultry production in nigeria (kabir et al. 2004, ezenduka et al. 2011), but the detection of tetracyclines (25) alone was lower in this study when compared with sulphonamides (26) detected at ph 7.2 and aminoglycosides (35) detected at ph 8. this could be explained by the fact that most positive ph 6.0 samples may have fallen into multiple ph categories. generally, microbiological methods are basic screening methods for detecting the presence of antimicrobial residues in food and are able to differentiate antibiotic groups. they were the earliest methods used for the detection of antibiotic residues and are still widely used. they are very cost-effective and, in contrast to immunological or receptor-based tests, have the potential to cover the entire antibiotic spectrum in only 1 test. it is however necessary to confirm the presence and concentration of antibiotics and identify specific substances within the different antibiotic groups with chemical or chromatographic methods, particularly high performance liquid chromarography (hplc) and immuno-enzymatic method like elisa (mitchell et al. 1998, kirbis 2007). sulphonamides), and 35 at ph 8.0 (best detects aminoglycosides). these results are shown in table ii. residues were detected in 69 samples with multiple phs: 10 by ph 6.0 and 7.2; 8 by ph 6.0 and 8.0; 10 by ph 7.2 and 8.0, and 41 by the 3 ph levels. discussion up to 64% of the investigated commercial broilers in this study had detectable levels of antimicrobial residues. a previous study done in northern nigeria by kabir and colleagues (kabir et  al. 2004) reports a prevalence of 15.7%. this disparity may be due to the fact that they only used faeces as sample in their study, which can be related to the 11% prevalence of muscle matrix tested in this study. the overall prevalence is also consistent with the findings of ezenduka and colleagues (ezenduka et  al. 2014), who demonstrated a 60% prevalence rate in a similar study at enugu state. a significant presence of different antimicrobials in chickens in the western part of the country has also previously been reported (dipeolu and alonge 2002, dipeolu and dada 2005). similar to other developing countries, a high prevalence of antimicrobial residues in broilers have been recorded: 39.4% and 70% were reported in pakistan by jabbar (jabbar 2004) and muhammad and colleagues (muhammad et al. 2007), respectively; 52% in iraq by shareef and colleagues (shareef et al. 2009), and 70% in tanzania by nonga and colleagues (nonga et  al. 2009). the high prevalence may indicate the excessive prescription, overuse, and abuse of antimicrobial drugs in nigeria, similar scenario observed in most developing countries. this problem may be largely due to the unrestricted availability of antimicrobial drugs and the practice of self-medication by poultry farmers and animal handlers. the unauthorised and unprofessional exposure of poultry to veterinary drugs without adherence to recommended doses and/or withdrawal time promotes an accumulation of residues in meat and eggs. the kidney and liver had the highest concentration of residues, at 60% and 54% respectively. these findings are similar to previous studies by aerts table ii. antimicrobial positive samples according to ph. matrix no positive ph 6.0 7.2 8.0 multiple 6.0/7.2 6.0/8.0 7.2/8.0 all three muscle 11 2 0 5 1 1 0 2 liver 54 6 11 15 2 3 3 14 kidney 60 11 10 9 6 3 3 18 gizzard 30 6 5 6 1 1 4 7 total 155 25 26 35 10 8 10 41 147 ezenduka screening of antimicrobial residues in poultry meat veterinaria italiana 2019, 55 (2), 143-148. doi: 10.12834/vetit.395.1870.4 veterinary practice and livestock production is crucial to establish surveillance programmes for detecting drug residues in meat and other foods of animal origin. based on findings, kidney and liver are recommended as sample matrices for screening drug residues in poultry products. antimicrobial use is widespread in enugu state, nigeria. this behaviour underpins the indiscriminate use of these drugs along with the lack of observation for recommended periods of withdrawal. the implementation of best practices in combination with adequate legislation regarding drug use in aerts m.m.l, hogenboom a.c. & brinkman u.a. 1995. analytical strategies for the screening of veterinary drugs and their residues in edible products. j chromatogr, 667, 1-20. chang c.sh., tai t.f. & li h.p. 2000. 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99-105. references kirbis a. 2007. microbiological screening method for detection of aminoglycosides, β-lactams, macrolides, teteracyclines and quinolones in meat samples. slov ret res, 44 (1/2), 11-18. mitchell j.m., griffiths m.w., mc even s.a., mc nab w.b. & yee j. 1998. antimicrobial drug residues in milk and meat: causes, concerns, prevalence, regulations, tests, and test performance. journal of food protection, 61, 742-756. muhammad a.s., muhammad s. & sajjad u.r. 2007. evaluation of a microbiological growth inhibition assay as a screening test for the presence of antibiotic residues in poultry meat. american j food technol, 2 (5), 457-461. myllyniemi a.l, rintala r., bäckman c. & niemi a. 1999. microbiological and chemical identification of antimicrobial drugs in kidney and muscle samples of cattle and pigs. food additives and contaminants, 16, 339-351. myllyniemi a.l. 2004. development of microbiological methods for the detection and identification of antimicrobial residues in meat. academic dissertation. department of food and environmental hygiene faculty of veterinary medicine university of helsinki helsinki, finland. myllyniemi a.l., nuotio l., lindfors e., rannikko r., niemi a. and bäckman c. 2001. a microbiological six plate method for the identification of certain antibiotic groups in incurred kidney and muscle samples. analyst, 126, 641-646. nisha a.r. 2008. antibiotic residues a global health hazard. veterinary world, 1 (12), 375-377. nonga h.e., mariki m., karimuribo e.d. & mdegela r.h. 2009. assessment of antimicrobial usage and antimicrobial residues in broiler chickens in morogoro municipality, tanzania. pakistan journal of nutrition, 8 (3), 203-207. pavlov a.i., lashev l.i. & vachin rusea v. 2008. residues of antimicrobial drugs in chicken meat and offals. trakia j of science, 6 (1), 23-25. reig m. & todra f. 2008. veterinary drug in meat: concern and rapid methods for detection. meat science, 78, 60-67. riviere j.e. & sundlof s.f. 2001. chemical residue in tissues of food animals. in veterinary pharmacology and therapeutics (adams hr ed). 8th ed., blackwell publishing professional iowa, 1166-1174. shareef a.m., jamel z.t. & yonis k.m. 2009. detection of 148 screening of antimicrobial residues in poultry meat ezenduka veterinaria italiana 2019, 55 (2), 143-148. doi: 10.12834/vetit.395.1870.4 wattiau p., renard m.e., ledent p., debois v., blackman g. & agathos s.n. 2001. a pcr test to identify bacillus subtilis and closely related species and its application to the monitoring of wastewater bio-treatment. appl microbiol biotechnol, 56, 816-819. antibiotic residues in stored poultry products. iraqi journal of veterinary science, 23, 45-48. stark j. 2000. antibiotika nachweis in fleisch. fleischwirtschaft, 80, 46-50. vollard e.j. & clasener h.a.l. 1994. colonization resistance. antimicrobial agent chemotherapy, 38, 409-414. 287 1 department of pathology, faculty of veterinary science, university of agriculture faisalabad, pakistan. 2 department of parasitology, faculty of veterinary science, university of agriculture faisalabad, pakistan. 3 department of clinical medicine and surgery, university of agriculture faisalabad, pakistan. 4 department of poultry production, government of punjab, faisalabad, pakistan. * corresponding author at: department of pathology, faculty of veterinary science, university of agriculture faisalabad-38040, pakistan. tel.: +92 41 9200161-70/3120, e-mail: mtjaved@uaf.edu.pk. parole chiave bufali, elisa, paratubercolosi, ppd, prevalenza, punjab, tubercolina, ziehl-neelsen. riassunto scopo di questo studio è stato valutare la diffusione della paratubercolosi in quattro allevamenti statali in punjab (pakistan). con il test per la tubercolina aviare (ppd) sono stati analizzati 627 animali con più di due anni di età. i risultati sono stati poi validati con elisa indiretta e con il test di ziehl-neelsen (zn). complessivamente è stata riscontrata una prevalenza dell’1,3% ma il valore subisce variazioni significative a seconda dell'allevamento di provenienza ed è compreso tra 0,0 e 3,03%. i risultati della odds ratio hanno suggerito che la probabilità di contrarre l'infezione è di 2,18 volte maggiore quando il peso corporeo dei bufali è inferiore a 500 kg; 1,65 volte in animali all’asciutta, 2,58 volte quando sono presenti piccoli ruminanti, 1,19 volte quando non ci sono bovini. complessivamente sono risultati positivi 12 bufali, di cui 10 confermati con l’elisa e 8 con il test di ziehl-neelsen (zn). considerando l’elisa come “gold standard”, la sensibilità e la specificità relativa della tubercolina aviare ppd sono risultate essere rispettivamente dell’80,0% e dell’89,47%; quelle del test zn, analogamente del 70,0% e del 97,37%. considerando invece come standard il test zn, la sensibilità e specificità relativa della tubercolina aviare ppd sono state rispettivamente del 100% e del 90%. per quanto riguarda i parametri ematologici, negli animali positivi alla paratubercolosi diminuivano significativamente le piastrine mentre aumentava l'mch. prevalenza della paratubercolosi nei bufali indiani negli allevamenti di bestiame statali in punjab, pakistan keywords buffaloes, purified protein derivative, elisa, paratuberculosis, prevalence, punjab, tuberculin, ziehl-neelsen test. summary the present study had the goal to assess the prevalence of paratuberculosis in 4 public livestock farms of the punjab (pakistan). it included 627 total animals of more than 2-year-old tested by avian, purified protein derivative (ppd). the results of the ppd test were confirmed by indirect elisa and by ziehl-neelsen (zn) test. an overall prevalence of 1.3% was recorded. the values were between 0.0% and 3.03%. the results of odds ratio suggested that there are 2.18 time’s higher chances of infection when body weight of buffaloes is less than 500 kg; 1.65 times in dry animals; 2.58 times when small ruminants are present; and 1.19 times when cattle are absent. the total positive buffaloes observed by avian ppd were 12, although only 10 were then confirmed by elisa, and 8 by zn faecal microscopy. the relative sensitivity and specificity of avian ppd by considering elisa as standard test, were 80.0% and 89.47%, respectively. similarly, the relative sensitivity and specificity of the zn faecal test were 70.0% and 97.37%, respectively. the relative sensitivity and specificity of avian ppd by considering zn faecal test as standard, were 100% and 90%, respectively. among haematological parameters, platelets significantly decreased and mch increased in paratuberculosis positive animals. aziz-ur-rehman1, m. tariq javed1*, m. sohaib aslam1, m. nisar khan2, s. misdaq hussain1, khuram ashfaque3 and asim rafique4 prevalence of paratuberculosis in water buffaloes on public livestock farms of punjab, pakistan veterinaria italiana 2018, 54 (4), 287-292. doi: 10.12834/vetit.852.4241.1 accepted: 29.11.2017 | available on line: 31.12.2018 288 veterinaria italiana 2018, 54 (4), 287-292. doi: 10.12834/vetit.852.4241.1 the reactions were read 72 hours after injection. the interpretation was made according to the diameter of swelling at the injection site and increase in thickness of skin folds. animals were considered as positive, doubtful or negative according to the method described by aagaard and colleagues (aagaard et  al. 2003). for the test comparison, about 10-15 g of the faecal sample were collected from all the tuberculin positive animals (n = 12) and 36  randomly selected tuberculin negative animals. the samples were taken directly from rectum using plastic gloves. they were sealed in a plastic container and were numbered with the animal id number. samples were then transported to the lab and used for ziehl-neelsen (zn) microscopy. from these animals blood samples were also taken. five ml of blood were collected in a glass test tube having 0.5 ml of 1% edta from the jugular vein for haematology. similarly, 5 ml of blood were collected in another test tube without anticoagulant for serology. collected sera were stored at - 40°c before being tested for the presence of map antibodies by using a commercial indirect elisa (lsivet ruminant serum paratuberculosis advanced catalogue no. vetptrs2). information on sex, age, body weight, breed, milk production, status of the animal (dry, pregnant, lactating), the stage of lactation, recent culling or purchase, management of calves, feeding, housing, total number of animals at the farm, other animals at the farm, and their number, was recorded. the data were analysed by using winpepi software version 11.43. for each datum the 95% confidence interval was calculated. the fisher chi-square test was also applied to the epidemiological data. the odds ratio was defined for comparing 2 groups. the data on diagnostic tests, including elisa, tuberculin, and zn microscopy were analysed using screening and diagnostic test command in winpepi software. when the performance of elisa, tuberculin and zn microscopy tests were compared, elisa was considered as standard test. when the performance of the zn faecal and tuberculin tests were compared, the zn faecal test was used as standard. the diagnostic techniques mentioned in the oie manual in chapter 2.1.11 on paratuberculosis were used as a guide to compare these tests (oie 2014). data on haematological parameters were analysed by analysis of variance; while means were compared with a bonferroni-t  test by using sas statistical software (sas 2007). results an overall prevalence of 1.3% (8/627) was found in buffaloes at 4 public livestock farms of punjab. the highest prevalence (3.03%) was found in farm  1, while the lowest (0.0%) was in farm 3 (table 1). introduction paratuberculosis is a chronic and progressive infection which leads to debilitating condition and affects a wide range of hosts, mostly ruminants (hermon-taylor et  al. 2004). it is caused by mycobacterium avium subsp. paratuberculosis (map), which has also been found in crohn's disease patients in humans (sechi et  al. 2001). although in developed countries control strategies have reduced its prevalence, paratuberculosis is still widely spread (franken 2005, kennedy 2007, kobayashi et al. 2007, kulkas 2007, nielsen et al. 2007). in recent years, dairy farming has gained a lot of attention in pakistan, which is now number 4th in the world in total milk production. paratuberculosis can then represent a severe threat to this industry and, considering its zoonotic potential, to the public health. economic losses caused by map to the farmers (ott et al. 1999), its probable role in crohn's disease in humans, and the fact that this organism can be present in milk and milk products and can also resist cheese processing and pasteurization, provide compelling reasons to strengthen control measures. animals mostly get infected before 6 months of age, but clinical manifestations show almost after 2 years of age. the causative agent of the disease is horizontally transmitted in young ruminants through contaminated food and water (patterson et al. 1968). in dairy herds of spain, map prevalence has been reported to be 4.03% (diéguez et  al. 2007), while in india its presence was found to be 2.71% (singh et  al. 2004). in lahore slaughterhouse, pakistan, reports on paratuberculosis indicate a prevalence of 14% and 12% in goats and sheep, respectively (chaudhary et  al. 2009). a study on breeding bulls at the semen production unit reported a prevalence ranging from 16.6% to 24.6% (abbas et  al. 2011). another study from pakistan reported a prevalence of 10.63% in sheep (sikandar et al. 2013). these data suggest that map infection is present in different animal species in the country. the present study aims to investigate the prevalence of paratuberculosis in buffaloes at 4 government livestock experimental stations by using avian purified protein derivative (ppd) and the most suitable methods to assess map prevalence in buffaloes. materials and methods the study included 627 buffaloes distributed in 4  government livestock experimental stations, 174 at farm 1, 201 at farm 2, 87 at farm 3, and 165 at farm 4. at the time of selection no animal showed clinical signs of the disease. all animals having more than 2 years of age were screened by delayed hypersensitivity test using an intradermal injection of avian ppd on the left side of the neck. paratuberculosis in water buffaloes in pakistan aziz-ur-rehman et al. 289veterinaria italiana 2018, 54 (4), 287-292. doi: 10.12834/vetit.852.4241.1 no significant differences were found between the prevalence rates observed among farms and similarly none of the variables considered in this study significantly influences the map prevalence. the total positive buffaloes observed by avian ppd were 12, by elisa were 10, and by zn faecal microscopy were 8. the results of avian ppd, while considering elisa as standard test revealed a relative sensitivity of 80.0% and a relative specificity of 89.47% (table 2). similarly, the relative sensitivity and specificity of the zn faecal test were 70.0% and aziz-ur-rehman et al. paratuberculosis in water buffaloes in pakistan table i. prevalence (%) of paratuberculosis in buffaloes at four public livestock farms of punjab on the basis of positive results both by avian ppd (purified protein derivative) test + elisa. parameter negative positive (%) 95% ci odds ratio farms 1 160 5 (3.03) 1.12-6.59 ns 2 173 1 (0.57) 0.03-2.8 3 201 0 (0) 0.00-1.48 4 85 2 (2.29) 0.39-7.39 body weight 300-500 268 5 (1.83) 0.67-4.01 or = 2.18 > 500 351 3 (0.85) 0.22-2.29 age < 4 2 0 (0) 0.00-0.77 ns4-8 370 4 (1.1) 0.34-2.56 > 8 247 4 (1.59) 0.51-3.80 lactation length 0 5 0 (0) 0.00-0.45 ns 1-200 86 1 (1.14) 0.06-5.54 201-300 513 7 (1.34) 0.59-2.64 > 300 15 0 (0) 0.00-0.18 milk yield 0 5 0 (0) 0.00-0.45 ns 1-4.9 194 4 (2.02) 0.65-4.8 5-9.9 417 4 (0.95) 0.30-2.28 10-15 3 0 (0) 0.00-0.63 lactation number < 5 346 6 (1.70) 0.69-3.51 ns5-10 229 2 (0.86) 0.15-2.83 > 10 44 0 (0) 0.00-0.60 status dry 311 5 (1.58) 0.58-3.47 or = 1.65 lactating 308 3 (0.96) 0.25-2.60 total buffalo < 100 85 2 (2.29) 0.39-7.39 ns100-200 333 6 (1.8) 0.72-3.64 > 200 201 0 (0) 0.00-1.48 total sheep 0 286 2 (0.70) 0.12-2.28 ns1-500 160 5 (3.03) 1.12-6.59 > 500 173 1 (0.6) 0.03-2.80 total cattle 0 361 5 (1.36) 0.50-3.00 ns1-100 85 2 (2.35) 0.39-7.39 > 100 173 1 (0.6) 0.03-2.80 total animals < 500 286 2 (0.70) 0.12-2.28 ns500-1,000 160 5 (3.03) 1.12-6.59 > 1,000 173 1 (0.6) 0.03-2.80 small ruminants absent 286 2 (0.70) 0.12-2.28 or = 2.58 present 333 6 (1.8) 0.72-3.64 other animals absent 201 0 (0.5) 0.00-1.48 present 418 8 (1.87) 0.88-3.53 cattle absent 361 5 (1.36) 0.50-3.00 or = 1.19 present 258 3 (1.14) 0.29-3.10 ns = non-significant; or = odds ratio. table ii. sensitivity of avian ppd (purified protein derivative) test, ziehl-neelsen (zn) faecal test based on elisa as standard test, and of avian ppd based on zn faecal test as standard test in buffaloes at the four public livestock farms of punjab. paratuberculosis elisa 95% ci statistics negative positive (%) avian ppd negative 34 2 (5.56) 0.94-17.16 sensitivity = 80.0% specificity = 89.47%positive 4 8 (66.67) 37.69-88.39 zn faecal negative 37 3 (7.5) 1.94-19.07 sensitivity = 70.0% specificity = 97.37%positive 1 7 (87.50) 51.97-99.37 avian ppd zn faecal negative 36 0 (0.00) 0.00-7.98 sensitivity = 100% specificity = 90%positive 4 8 (66.67) 18.41-81.59 table iii. comparison of haematological parameters in avian ppd (protein purified deriviative) positive and negative reactor buffaloes at four livestock experiment station of punjab pakistan. parameter ppd positive animals mean ± sd ppd negative animals mean ± sd rbc 1012/l 7.976 ± 2.766 6.376 ± 0.285 pcv% 46.500 ± 18.185 24.733 ± 3.271 haemoglobin g/dl 18.050 ± 5.8585 13.336 ± 0.317 wbc 109/l 9.600 ± 2.657 7.366 ± 1.171 mch pg 20.366 ± 0.592 a 18.033 ± 2.003 b platelet 109/l 144.500 ± 47.094 b 356.666 ± 70.945 a differential leukocyte count lymphocyte 109/l 6.666 ± 2.344 4.633 ± 1.401 lymphocyte % 59.350 ± 21.756 56.800 ± 1.473 granulocyte 109/l 2.083 ± 2.435 1.333 ± 0.251 granulocyte % 19.266 ± 15.450 15.200 ± 0.264 290 paratuberculosis in water buffaloes in pakistan aziz-ur-rehman et al. veterinaria italiana 2018, 54 (4), 287-292. doi: 10.12834/vetit.852.4241.1 between infection and presence of sheep or small ruminants at the farm: mpa prevalence was lower when small ruminant or sheep were absent and was instead higher when they were present, suggesting some role of small ruminants in maintenance and/or spread of infection in large ruminants or buffaloes. similarly, another study reported a positive association of the prevalence of paratuberculosis with the co-grazing of sheep and cattle (woodbine et  al. 2009). nonetheless, a study on the same farms revealed a protective effect of the presence of sheep on the occurrence of bovine tuberculosis (javed et al. 2011, javed et al. 2012). further studies are required to clarify the role of small ruminants in spreading mpa infection. however this study seems to confirm that keeping multiple species of animals on the farm is a risk factor for both, tuberculosis and paratuberculosis. according to these results, it seems to have more chance of finding mpa infection in animals with lower body weight (or  =  2.18). it is known that the disease causes a decrease in the body weight due to consistent chronic diarrhoea (kudahl and nielsen 2009). when the three tests were compared, results were discordant. if, on the one hand, all animals positive to ppd were not detected either by elisa or zn test, on the other hand, some of the animals which were negative to ppd resulted positive to elisa. these data suggest that tuberculin test may be considered as a screening test along with zn faecal test in resource-poor countries. elisa, instead, can be used as a routine test for screening animals for prevalence studies on paratuberculosis (singh et al. 2014). discordant results were also observed in a study by singh and colleagues where ppd positive animals were not confirmed by elisa and similarly, in a weber and colleagues survey, results obtained by elisa and zn were not fully concordant (weber et al. 2009). the results of haematological parameters revealed no effect of disease on almost all the parameters, with the only exception of the platelet counts and mean corpuscular haemoglobin. these results indicate a decrease in the platelet count, and an increase in mch. another study reported a significant decrease in the haematological parameters including rbc count, wbc count, pcv, mchc, hb, and an increase in monocyte count and platelet in sub-clinically infected camel (salem et  al. 2012). however, another study performed on cattle reported that haematocrit, haemoglobin, and erythrocyte values were within normal ranges in paratuberculosis positive cows, but lower than in control cows (senturk et al. 2009). they also found lower platelet count in infected animals. these changes were linked to decreased serum iron and iron binding capacity. they further reported that as the disease progresses the values of the haematological parameters may 97.37%, respectively, considering elisa as standard test. when the performances of zn faecal and avian ppd tests were compared, the relative sensitivity and specificity of avian ppd were 100% and 90%, respectively. apart from platelet and mean corpuscular haemoglobin, non-significant differences were observed in the the haematological parameters (table 3). in paratuberculosis positive animals, the platelet significantly decreased while mch increased. discussion the present study revealed a prevalence of 1.3% on 4  public livestock farms on the basis of tuberculin testing. studies on mpa are only sporadic in pakistan, a previous study focusing on slaughterhouse reported a prevalence of 6.67% in cattle and 12.5% in buffaloes (sikandar et  al. 2012). the prevalence rate differences found at farm level and in slaughtered animals were expected, as, most of the time, non-productive or low productive animals are taken to a slaughterhouse and culled. the infection may cause chronic diarrhoea which is difficult to treat, and affects the production of animals, thus the animals are culled (ott et al. 1999). a study from iran reported a prevalence between 4.2% and 7.7% (razieh et  al. 2012) while another study from india reported a prevalence of 15.14% in cattle (gupta et  al. 2012). to the best of our knowledge, this is the first report on paratuberculosis in buffaloes on public livestock farms in punjab. it indicates the presence of paratuberculosis in the area. the results of the present study suggests that the mpa prevalence might depend on the number of animals present at the farm. in this study, it was higher in farms with more than 500 animals. another study reported a herd prevalence of 28% of small herds, 53% of medium herds, and 100% of larger herds (kruze et  al. 2013). similarly, lower prevalence in smaller herds (<  100 animals) and higher in larger herds (>  100 animals) has been reported (woodbine et al. 2009). during the present study, the prevalence of disease increased from 0% in animals of less than 4 years to 2.69% in > 4 years of age. these results showed an increase in prevalence with age. a study from india reported an overall prevalence of 29% in bovines, with 13.6% in young, and 29.8% in adults (singh et  al. 2008). another study reported an increase in sero-prevalence of paratuberculosis with the increase in age and reported a higher prevalence at the age between 2 and 3 years (woodbine et al. 2009). however, the prevalence found in bovines in india was much higher than that observed in the present study. this study also revealed a possible association 291 aziz-ur-rehman et al. paratuberculosis in water buffaloes in pakistan veterinaria italiana 2018, 54 (4), 287-292. doi: 10.12834/vetit.852.4241.1 grant support the research grant provided by pakistan science foundation vide project no. psf/res/p-au/bio (431) is acknowledged. acknowledgements special thanks to the administration, livestock and dairy development department, government of punjab, and all the staff of government livestock farms for extending untiring help and support to conduct this study. fall below the reference range. thus, it can be concluded that the haematological changes are likely species-dependent and can vary at different stage of disease. however, more studies are required in buffaloes to have a better idea of the effects of paratuberculosis on these parameters. it can be concluded from the present study that tuberculin testing can be used in conjunction zn faecal test for screening of animals for paratuberculosis in resource-poor countries. the prevalence of paratuberculosis was not high in the farms included in this study, as it was less than 1.3%. aagaard g., govaerts m., limei m.o., andersen p. & pollock j.m. 2003. genomic approach 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fadda g. & zanetti s. 2001. identification of mycobacterium avium sub spp. paratuberculosis in biopsy specimens from patients with crohn’s disease identified by in situ hybridization. j clin microb, 39, 4514-4517. marassi c.d., fraguas s., gonzaga j.s., ristow p., ferreira r., oelemann w.m.r., fonseca l.s. & lilenbaum w. 2005. interference of anti-m. bovis antibodies in serological tests for paratuberculosis. proceeding of the 8th international colloquium on paratuberculosis, copenhagen, denmark 14-18 august, 511515. nielsen s.s., jepsen o.r. & aagaard k. 2007. control programme for paratuberculosis in denmark. bull int dairy fed, 410, 23-29. olsen i., tryland m., wiker h.b. & reitan l.v. 2001. ahpc, ahpd, and a secreted 14-kilodalton antigen from mycobacterium avium subsp. paratuberculosis distinguish between paratuberculosis and bovine 292 paratuberculosis in water buffaloes in pakistan aziz-ur-rehman et al. veterinaria italiana 2018, 54 (4), 287-292. doi: 10.12834/vetit.852.4241.1 singh s.v., singh a.v., singh r., sharma s., shukla n., misra s., singh p.k., sohal j.s., kumar h., patil p.k., misra p. & sandhu k.s. 2008. sero-prevalence of bovine johne’s disease in buffaloes and cattle population of north india using indigenous elisa kit based on native mycobacterium avium subspecies paratuberculosis ‘bison type’ genotype of goat origin. comp immun microb inf dis, 31, 419-433. singh s.v., yadav r.k., gupta v.k., gupta s., chaubey k.k., agarwal n.d. & kumar n. 2014. co-incidence of bovine johne’s disease and bovine brucellosis in young bulls of murrah breed in their native tract (rohtak, haryana, india). adv anim vet sci, 2, 23-25. weber m.f., verhoeff j., van-schaik g. & van-maanen c. 2009. evaluation of ziehl-neelsen stained faecal smear and elisa as tools for surveillance of clinical paratuberculosis in cattle in the netherlands. prev vet med, 92, 256-266. world organisation for animal health (oie). 2014. terrestrial animal health code, vol. ii, chapter 8.11, paratuberculosis. http://www.oie.int/en/ international-standard-setting/terrestrial-code/. woodbine k.a., schukken y.h., green l.e., ramirez-villaescusa a., mason s., moore s.j., bilbao c., swann n. & medley g.f. 2009. seroprevalence and epidemiological characteristics of mycobacterium avium subsp. paratuberculosis on 114 cattle farms in south west england. prev vet med, 89, 102-109. tuberculosis in an enzyme-linked immunosorbent assay. clin diag lab imm, 8, 797-801. ott s.l., wells s.j. & wagner b.a. 1999. herd level economic losses associated with johne’s disease on us dairy operations. prev vet med, 40, 179-192. patterson d.s.p., allen w.m., berrett s., ivins l.n. & sweasey d. 1968. some biochemical aspects of clinical johne’s in cattle. res vet sci, 9, 117-129. razieh s., mostafa j., mirsaed m., ali n. & amin b. 2012. prevalence of mycobacterium avium subsp. paratuberculosis in dairy cattle bred in northern iran by nested-pcr. glob vet, 8, 259-263. salem m., el-sayed a., fayed a. & abo-el-hassan d.g. 2012. subclinical infection of paratuberculosis among camels in egypt. j am sci, 8, 1141-1147. sas 2004. sas statistical software version 9.1. sas institute inc., cary, nc, usa. senturk s., metcitoglu z., ulgen m., borum e., temizel e. & kasap s. 2009. evaluation of serum iron and iron binding capacity in cows with paratuberculosis. tierärztliche praxis, 6, 375-378. sikandar a., cheema a.h., younus m., aslam a., zaman m.a. & rehman t. 2012. histopathological and serological studies on paratuberculosis in cattle and buffaloes. pak vet j, 32, 547-551. singh b.b., gumber s., randhawa s.s., aradhana & dhand n.k. 2004. prevalence of bovine tuberculosis and paratuberculosis in punjab. ind vet j, 81, 1195-1196. 275 1faculty of veterinary medicine, university of teramo, teramo, italy. 2lutria snc, wildlife research and consulting, italy. *corresponding author at: faculty of veterinary medicine,, university of teramo, italy. e-mail: mromanucci@unite.it. parole chiave appendice atriale, cuore, lontra euroasiatica, trauma. riassunto nell'aprile del 2014 un maschio adulto di lontra, trovato morto sul ciglio della strada, è stato sottoposto a necroscopia presso l'unità di patologia veterinaria della facoltà di medicina veterinaria di teramo, nell'ambito del progetto recal [recovery and post mortem analysis of eurasian otters (lutra lutra) in the national park of cilento, vallo di diano and alburni (salerno, italy), and surrounding areas]. l’esame ha rivelato un abbondante emotorace associato a contusioni e lacerazioni polmonari multifocali bilaterali e un grave emopericardio caratterizzato dalla presenza di un coagulo di sangue nel pericardio. alla base dell’auricola di sinistra sono state trovate due piccole lacerazioni, lungo il solco atrioventricolare e sulla parete esterna. poiché i tessuti del miocardio e dell'endocardio non hanno mostrato altre evidenti anomalie istopatologiche, è stata diagnosticata una rottura dell'appendice atriale sinistra causata da trauma toracico. la rottura traumatica cardiaca è segnalata raramente ma nell'uomo è potenzialmente letale. in letteratura veterinaria, per quanto di nostra conoscenza, questo è il primo rapporto su una simile rottura a causa di trauma toracico. in emergenza veterinaria si dovrebbe quindi considerare la possibilità di un trauma cardiaco in conseguenza di una brusca lesione toracica. rottura dell’appendice atriale sinistra per trauma toracico in una lontra (lutra lutra) keywords atrial appendage, blunt trauma, eurasian otter, heart, rupture. summary an adult male eurasian otter, found dead on the roadside, was submitted for post-mortem examination in april 2014 at the veterinary pathology unit of the faculty of veterinary medicine of teramo, as part of the recal [recovery and post mortem analysis of eurasian otters (lutra lutra) in the national park of cilento, vallo di diano and alburni (salerno, italy), and surrounding areas] project. necropsy revealed an abundant hemothorax associated with multifocal, bilateral pulmonary contusions and lacerations, and a severe hemopericardium characterised by the presence of a wide blood clot in the intact pericardial sac. two small laceration wounds of the left auricle were found at the base, along the atrioventricular groove, and on the outer free wall. since myocardial and endocardial tissues showed no other gross and histopathological abnormalities, a left atrial appendage rupture resulting from a blunt chest trauma was diagnosed. blunt traumatic cardiac rupture is a rarely reported, life-threatening condition in humans. to the best of our knowledge, this is the first report on a left atrial appendage rupture due to blunt chest trauma in veterinary literature. the possible occurrence of a cardiac rupture following a blunt thoracic injury should be taken into consideration in veterinary emergency care. mariarita romanucci1*, romina fusillo2, manlio marcelli2, marcella massimini1, daniela malatesta1, laura bongiovanni1 and leonardo della salda1 left atrial appendage rupture due to blunt chest trauma in an eurasian otter (lutra lutra) veterinaria italiana 2019, 55 (3), 275-278. doi: 10.12834/vetit.872.4309.2 accepted: 13.01.2016 | available on line: 30.09.2019 case report 276 blunt traumatic left auricle rupture in an eurasian otter romanucci et al. veterinaria italiana 2019, 55 (3), 275-278. doi: 10.12834/vetit.872.4309.2 rupture due to blunt chest trauma has not been reported in veterinary literature. case description on april 2014, an adult male eurasian otter, found dead on the roadside in policastro bussentino (latitude 40° 4' 12.37", longitude 15° 30' 54.32") (santa marina, salerno, italy), was submitted for post-mortem examination at the veterinary pathology unit, faculty of veterinary medicine, university of teramo (italy). the animal weighed 7.1 kg and was 113 cm long from the nose to the tail tip. this case represents 1 of a series of post-mortem examinations performed, from 2009 to date, as part of the recal [recovery and post mortem analysis of eurasian otters (lutra lutra) in the national park of cilento, vallo di diano and alburni (pncvda, salerno, italy), and surrounding areas] project established for investigating the causes of death, overall health, biometric and demographic parameters, and levels of contamination in the eurasian otter (lutra lutra) italian population, by means of post-mortem analysis. introduction cardiac rupture resulting from blunt trauma to the chest is a rarely reported and often overlooked condition in humans, especially when no external wound is visible. thus, such cases are rarely diagnosed early and most patients die before surgical intervention takes place (salam and frauenhoffer 1996, shalaby et  al. 1999, salooja et al. 2013). about 6%-10% of human patients who undergo blunt chest injury have cardiac rupture. the right side is most commonly affected, whereas left atrial injury accounts for 25% of cardiac rupture cases (ryu et  al. 2013), with left ‘basal’ appendage rupture being particularly rare (tanoue et al. 2008). although all heart chambers are susceptible to traumatic rupture, the atrial appendage (also known as the auricle) is most vulnerable because of its relative thinness (salam and frauenhoffer 1996, salooja et  al. 2013). in particular, the most anatomically weak region in the heart appears to be located at the atrioventricular groove, if previous heart diseases are absent (tanoue et al. 2008). to the best of our knowledge, left atrial appendage figure 1. a. intact pericardial sac with a severe hemopericardium. b. presence of a wide blood clot surrounding the heart base and atria. c. laceration wounds located at the base, along the atrioventricular groove (black arrow), and on the outer free wall (white arrow) of the left auricle. d. higher magnification of the left atrial appendage rupture on the outer free wall (white arrow). 277 romanucci et al. blunt traumatic left auricle rupture in an eurasian otter veterinaria italiana 2019, 55 (3), 275-278. doi: 10.12834/vetit.872.4309.2 with a subaortic paramembranous ventricular septal defect with blood shunting from the left ventricle to the right atrium (gerbode defect), have been described in association with blunt chest trauma in dogs (miller et al. 2004, hezzell et al. 2011). common causes of blunt thoracic injuries in animals include being hit by a car, falling from a height (high-rise syndrome), fights, and human-animal interactions (salci et al. 2010). mechanisms of cardiac rupture by blunt trauma include direct contusion, compression of the chest that crushes the heart between the sternum and vertebral column, rapidly increased hydrostatic venous pressure transferred from the abdomen or lower extremities to the heart, acceleration/ deceleration force, and direct penetration by the sternum or rib fractures (leavitt et  al. 1987, tanoue et al. 2008, ryu et al. 2013, salooja et al. 2013). in the present case, although a car accident could be hypothesised, the exact cause of the otter death remains undetermined. notwithstanding this, the localisation of the heart tears and the presence of pulmonary contusions/lacerations with minimal injury to the chest wall were consistent with the major involvement of a deceleration force in the pathogenesis of these lesions (salooja et  al. 2013). the lack of other gross and microscopical abnormalities in the otter heart also excluded the presence of previous cardiac diseases that may have predisposed the heart to rupture. in this respect, left atrial rupture may occur as a consequence of chronic mitral valve insufficiency, especially in dogs (reineke et al. 2008). to the best of our knowledge, this is the first report on a left atrial appendage rupture due to blunt chest trauma in veterinary literature. the possible occurrence of a cardiac rupture following a blunt thoracic trauma should be taken into consideration in veterinary emergency care. acknowledgements the authors would like to thank dr. laura de riso, national park of cilento, vallo di diano and alburni, as coordinating partner of the recal project. at necropsy, the examination of the external surface and subcutis of the carcass did not reveal macroscopical lesions. on the other hand, a wide haemorrhage involving the ventral paratracheal connective tissue extended from the entrance of the thoracic cavity to the laryngotracheal junction. evaluation of the thoracic cavity also revealed an abundant hemothorax associated with multifocal, bilateral pulmonary contusions and lacerations, as well as a severe hemopericardium characterised by the presence of a wide blood clot into the intact pericardial sac (figure 1 a, b). after careful inspection of the heart, 2 small laceration wounds of the left auricle about 0.3 cm 0.4 cm in size were found at the base, along the atrioventricular groove (figure 1c), and on the outer free wall (figure 1 c, d). the heart showed no other gross abnormalities, as far as both myocardial and endocardial tissues were concerned. other macroscopical findings included a non-displaced, transverse fifth left rib fracture with no pleural laceration and a very mild haemorrhage in the surrounding tissues, as well as a moderate hemoperitoneum associated with multifocal hepatic lacerations. the heart and representative tissues of all other major organs were fixed in 10% neutral buffered formalin, embedded in paraffin, cut in 5  μm-thick sections, stained with haematoxylin-eosin (h&e), and examined by light microscopy. histopathologic examination did not reveal significant microscopical lesions in the heart tissues, nor in any of the other organs that were examined. on the basis of the absence of gross and histopathological, myocardial, or endocardial lesions, a left atrial appendage rupture resulting from a blunt trauma to the chest was diagnosed. discussion review studies concerning dogs and cats indicate that practitioners generally overlook traumatic thoracic injuries, although they are serious and potentially life-threatening conditions (salci et  al. 2010). however, avulsion of the mitral valve from the annulus fibrosus, as well as lesions consistent 278 blunt traumatic left auricle rupture in an eurasian otter romanucci et al. veterinaria italiana 2019, 55 (3), 275-278. doi: 10.12834/vetit.872.4309.2 hezzell m.j., dennis s., lewis d.h. & fuentes v.l. 2011. gerbode defect associated with blunt trauma in a dog. j vet cardiol, 13, 141-146. leavitt b.j., meyer j.a., morton j.r., clark d.e., herbert w.e. & hiebert c.a. 1987. survival following nonpenetrating traumatic rupture of cardiac chambers. ann thorac surg, 44, 532-535. miller l.m., keirstead n.d. & snyder p.s. 2004. traumatic mitral valve avulsion from the annulus fibrosis producing acute left heart failure in a dog. can vet j, 45, 761-763. reineke e.l., burkett d.e. & drobatz k.j. 2008. left atrial rupture in dogs: 14 cases (1990-2005). j vet emerg crit care, 18, 158-164. ryu d.w., lee s.y. & lee m.k. 2013. rupture of the left atrial roof due to blunt trauma. interact cardiovasc thorac surg, 17, 912-913. references salam m.m. & frauenhoffer e.e. 1996. left atrial appendage rupture caused by a seat belt: a case report and review of the literature. j trauma, 40, 642-643. salci h., bayram a.s., celimli n., caliskan g.u., gorgul o.s. & kramer m. 2010. evaluation of thoracic trauma in dogs and cats: a review of seventeen cases. iran j vet res, 11, 325-331. salooja m.s., singla m., srivastava a. & mukherjee k.c. 2013. isolated tear in left atrial appendage due to blunt trauma chest: a rare case report. j saudi heart assoc, 25, 95-97. shalaby r.i., rajendran u. & regunathan r. 1999. blunt traumatic rupture of the heart: case report and selected review. ann thorac cardiovasc surg, 5, 123-129. tanoue k., sata n., moriyama y. & miyahara k. 2008. rupture of the left atrial ‘basal’ appendage due to blunt trauma in an elderly patient. eur j cardiothorac surg, 34, 1118-1119. 413 veterinaria italiana 2022, 58 (4), 413‑423. doi: 10.12834/vetit.2601.17023.2 accepted: 11.02.2022 | available on line: 31.12.2022 1health and animal production master's degree, pitágoras-unopar university. 2unopar. *corresponding author at: istituto zooprofilattico sperimentale dell'abruzzo e del molise "g. caporale". e-mail: rafaelfagnani@hotmail.com. josiane ito eleodoro1, rafael fagnani2* keywords dairy herd, cow, resistance, antibiotic, pathogen. summary considering the high prevalence of subclinical mastitis and its impacts on milk production, thematic studies are need to provide strategic data for its control. this study aimed at in‑ vestigating the most frequent microorganisms associated with subclinical mastitis in dairy cows in brazil through compiling the occurrence of the etiological agents and their sensitivi‑ ty to antibiotics. the systematic review includes articles published between 2009 and 2019. fifty‑seven articles evaluating 22,287 milk samples were selected. the number of publica‑ tions and the sample size were not homogeneous among brazilian regions. most of the stu‑ dies and sampling were conducted in rio grande do sul, whereas no studies were found in some states in the north and mid‑west regions. the most frequent group of pathogens was staphylococcus spp. it was isolated in all studies and had an average prevalence of 49% in the analyzed samples. resistance to penicillin was the most frequent microbial resistance found in brazil, with an average of 66% among the isolates evaluated. moreover, bacterial resistan‑ ce to cephalexin, cefoperazone, erythromycin, gentamicin, neomycin, penicillin, tetracycli‑ ne, and trimethoprim increased over the research period. given the territorial extension, the etiological diversity, and the lack of studies with a representative sample, the compilation of scientific data must be interpreted with caution. regions where a greater number of studies were conducted and with numerous samples, such as the south, provided a comprehensive scenario that is closer to reality. nevertheless, although decision making on the farm cannot be replaced by scientific studies, it can be supported by such efforts. please refer to the forthcoming article as: ito eleodoro et al. 2022. etiological agents and bacterial sensitivity in subclinical mastitis in brazil: a ten‑year systematic review. vet ital. doi: 10.12834/vetit.2601.17023.2 etiological agents and bacterial sensitivity in subclinical mastitis in brazil: a ten-year systematic review treatment period (9%), increased labor (1%), and the premature disposal of animals (14%) (sharma et al., 2012). moreover, there are losses for dairy products owing to the decrease in the quality of the final product, the decrease in the industrial yield for the manufacture of derivatives, and changes in the composition of mastitic milk (ruegg, 2017). antimicrobial drugs are used to treat several diseases that affect dairy cows, and clinical mastitis is one of the main diseases that require the use of these drugs (gomes and henriques, 2016). despite the many benefits of these drugs, from the perspective of public health and food safety, there is concern regarding antibiotic residues in food intended for human consumption from animals treated with introduction the dairy herd occupies a prominent position within the brazilian economic scenario, with milk being one of the main products of national agriculture. in fact, the agribusiness of milk and dairy products plays an important role in the supply of food and the social issue, with the generation of jobs and income for the population, mainly in southern region (beber et al., 2019). however, mastitis is the most frequent and costly infection of dairy farming. this is because intramammary infections result in significant economic losses associated with several factors including the reduction in milk production (more than 70% of cases), cost of treatment and veterinary medical charges (7%), disposal of milk during the subclinical mastitis in brazil ito eleodoro et al. 414 veterinaria italiana 2022, 58 (4), 413-423. doi: 10.12834/vetit.2601.17023.2 to 2019 in scielo, capes periodical portal, google scholar and pubmed. in each of the databases, the keywords were searched in portuguese and english. the terms searched were: • mastitis and subclinical and brazil • etiology and mastitis and subclinical and brazil • antimicrobial resistance and mastitis and brazil • antimicrobials and mastitis and brazil • bovine mastitis and subclinical and brazil • staphylococcus and subclinical and mastitis and brazil • streptococcus and subclinical and mastitis and brazil • corynebacterium and subclinical and mastitis and brazil • milk and subclinical and mastitis and brazil. the data extracted from each article, when available, included: year of publication; journal; first author; brazilian region where the study was conducted; number of animals, number of milk samples analyzed, number of culture‑positive milk samples, description of the etiologic agents isolated from subclinical mastitis, antimicrobials tested and number of resistant samples to each antimicrobial class. the data obtained were tabulated and a descriptive statistical analysis of the absolute and relative frequency of the microbiological findings was performed using microsoft excel® and the combined chi‑square test using biostat® to compare the prevalence of resistance in each year studied. the heterogeneity of the prevalence estimates between studies was quantified by i2 index for the most frequent microorganisms (higgins and thompson, 2002). results our search strategy yielded 41,038 records (sum of all database), from which 75 studies were retained after inclusion/exclusion criteria. following duplicate removal, 69 articles were included in the screening step. during the screening stage, 12 articles were considered as not relevant and were excluded (5 without information regarding the geographic region; 5 without the number of animals and 3 surveys based on interviews). fifty‑ seven articles, published between 2009 and 2018 with 22,287 milk samples evaluated, were selected according to the inclusion criteria. the sampling technique and representativeness of the 57 articles included can be seen in supplementary table 1. antibiotics and the potential development and transmission of antimicrobial resistance which may impact the treatment of diseases (oliver et al., 2020). the appearance of multidrug‑resistant strains has made it difficult to treat mastitis in cows. thus, microbiological diagnosis of mastitis needs to be performed routinely, as it is capable of generating fast and safe results that can identify the problems affecting the herd. according to karach et al., (2015), the isolation and identification of the agent contribute to the most appropriate choice of the drug to be used in therapy, thus avoiding the development of bacterial resistance to antibiotics. one of the strategies to prevent bacterial resistance is knowing the main agents involved in mastitis and their sensitivities. this systematic review aimed at investigating the most frequent microorganisms associated with subclinical mastitis in dairy cows in brazil, compiling data on the occurrence of the etiological agents causing subclinical mastitis and its sensitivity to antibiotics. a critical analysis of the past ten years is justified as it can provide epidemiological data for better control of subclinical mastitis. the results will allow us to develop an overview of the etiological agents and antimicrobial sensitivity of the main agents that cause mastitis, helping to provide a basis to prevent resistance to the condition and address its chronic nature. methods the following systematic review with meta‑analysis was planned according to the preferred reporting items for systematic reviews and meta‑analyses (prisma) network meta‑analysis reporting standards using the start and biostat programs (hutton et al., 2015). observational studies that assessed etiological agents of subclinical bovine mastitis and its antimicrobial resistance/sensitivity were eligible for inclusion. the inclusion criteria were primary studies: (1) related to the proposed topic and available for online consultation in search engines using keyword strings; (2) published between 2009 and 2019; (3) that addressed the bovine species, (4) that used 200.000 cel/ml as somatic cell count threshold for the classification of subclinical mastitis and (5) that were conducted in brazil. the exclusion criteria were (1) primary studies that did not specifically address the etiological agents and its antimicrobial resistance/sensitivity; (2) primary studies published outside the selected period; (3) primary studies that did not address the bovine species; (4) primary studies conducted outside brazil, and (5) secondary studies. the research included articles from journals and annals of scientific events published from 2009 ito eleodoro et al. subclinical mastitis in brazil veterinaria italiana 2022, 58 (4), 413-423. doi: 10.12834/vetit.2601.17023.2 415 supplementary table i. sampling technique and representativeness of 55 articles published between 2009 and 2018 that met the inclusion criteria of the systematic review. reference state type of sampling 1 alencar et al. (2014) rio de janeiro purposive sampling, independent 2 amorim et al. (2016) pernambuco purposive sampling, independent 3 andrade et al. (2010) paraná convenience, random, independent 5 assis et al. (2017) espírito santo convenience, random, independent 6 bandeira et al. (2013) r.grande do sul purposive sampling, independent 7 brito et al. (2014) maranhão convenience, random, independent 8 carvalho et al. 2018 maranhão convenience, random, independent 9 casanova et al. (2016) santa catarina purposive sampling, independent 10 castro et al. (2012) rio de janeiro quota sampling, independent 11 chagas et al. (2012) minas gerais convenience, random, independent 12 costa et al. (2013) minas gerais convenience, random, independent 13 costa et al. (2013) santa catarina snowball sampling, independent 14 costa et al. (2015) santa catarina convenience, dependend 15 cunha et al. (2015) minas gerais convenience, random, independent 16 desantana neres et al. (2015) sergipe convenience, independent 17 dias et al. (2011) minas gerais quota sampling, independent 18 farias et al. (2013) r.grande do sul purposive sampling, independent 19 ferreira et al. (2010) piauí quota sampling, independent 20 filho et al. (2016) paraná convenience, random, independent 21 freitas et al. (2018) r.grande do sul purposive sampling, independent 22 gonçalves et al. (2018) são paulo purposive sampling, independent 23 jardim et al. (2014) paraná convenience, random, independent 24 jobim et al. (2010) paraná convenience, random, independent 24 jobim et al. (2010) r.grande do sul convenience, random, independent 24 jobim et al. (2010) santa catarina convenience, random, independent 25 junior et al. (2015) são paulo purposive sampling, independent 26 kaiser et al. (2015) r.grande do sul purposive sampling, independent 27 karach et al. (2016) paraná convenience, random, independent 28 kolling et al. (2011) r.grande do sul purposive sampling, independent 29 krewer et al. (2013) bahia convenience, random, independent 29 krewer et al. (2013) pernambuco convenience, random, independent 30 lange et al. (2017) paraná convenience, random, independent 31 martins et al. (2010) mato grosso convenience,representative, independent 31 martins et al. (2014) piauí purposive sampling, independent 32 martins et al. (2015) goiás convenience, random, independent 33 melo et al. (2013) pernambuco convenience, random, independent 34 niero 2018 santa catarina purposive sampling, independent 35 oliveira et al. 2009 sergipe convenience, independent 36 oliveira et al. (2010) pará convenience, random, independent 37 oliveira et al. (2012) bahia convenience, random, independent 38 oliveira et al. (2013) paraná simple random sampling, independent 39 peters et al. (2016) r.grande do sul purposive sampling, independent 40 rall et al. (2014) são paulo purposive sampling, independent 41 ribeiro et al. 2009 são paulo purposive sampling, independent 42 ruiz et al. (2011) pernambuco convenience, random, independent subclinical mastitis in brazil ito eleodoro et al. 416 veterinaria italiana 2022, 58 (4), 413-423. doi: 10.12834/vetit.2601.17023.2 the number of publications was not homogeneous (g test = 31.67; p < 0.01) among brazilian states, with a greater occurrence of studies in paraná, rio grande do sul, and santa catarina. thus, 49% of the selected studies were conducted in the southern region (fig. 01). the sample size evaluated in the articles among brazilian states was not uniform (x2 = 8249.88; p < 0.01). the states with the largest number of milk samples analyzed were rio grande do sul, with 23% of the samples, followed by minas gerais (16%), and paraná (12%) (fig. 1). homogeneous among the brazilian states (x2 = 75.40; p < 0.01), with a higher rate in the states of espírito santo (80%), rio de janeiro (77%), and bahia (60%). goiás and são paulo had the lowest rates at 20% and 28%, respectively (fig. 2). figure 1. publications about subclinical mastitis in dairy cows in brazil and sample size (milk samples) retrieved from 57 scientific articles published between 2009 and 2018. figure 2. occurrence of etiologic agents of subclinical mastitis in dairy herds in brazil retrieved from 45 scientific articles published between 2009 and 2018.of the 57 articles selected, 45 studies isolated and identified the etiologic agents that caused subclinical mastitis. the most frequent pathogens were staphylococcus spp., which was isolated in all studies with an average prevalence of 49% in the samples analyzed. there was no significant heterogeneity between 45 studies (q=82.03, df=24, p=0.88), with a heterogeneity index i2 of 19.47%. however, when categorized by region, the distribution of staphylococcus spp. was not the second most frequent pathogens were streptococcus spp., identified in 76% of the identified articles, with an average occurrence of 14% in the analyzed samples and a significant heterogeneity (q=337.70, df=98, p<0.001; i2=70.98) between the studies retrieved. the distribution of streptococcus spp. was not homogeneous among the brazilian states (x2 = 77.62; p < 0.01), with greater prevalence reference state type of sampling 43 saab et al. (2014) paraná convenience, random, independent 44 saeki et al. (2011) são paulo convenience, independent 45 santos et al. (2010) paraná convenience, random, independent 46 senhorelo et al. (2013) espírito santo convenience, random, independent 47 silva et al. (2011) bahia convenience, random, independent 48 silva et al. (2012) pernambuco purposive sampling, independent 49 soethe et al. (2015) paraná convenience, random, independent 50 souza et al. (2016) minas gerais convenience, random, independent 51 ulsenheimer et al. 2018 r.grande do sul purposive sampling, independent 52 valmorbida et al. (2017) santa catarina purposive sampling, independent 53 vesco et al. (2017) r.grande do sul purposive sampling, independent 54 zanette et al. (2010) santa catarina quota sampling, independent 55 zimermann et al. (2017) paraná purposive sampling, independent ito eleodoro et al. subclinical mastitis in brazil veterinaria italiana 2022, 58 (4), 413-423. doi: 10.12834/vetit.2601.17023.2 417 2010 2011 2012 2013 2015 2016 2017 2018 amikacin 0.04a (n=69) 0.04a (n=56) amoxicillin 0.59a (n=453) 0.71a (n=17) 0.05b (n=313) 0.50a (n=30) ampicillin 0.68de (n=289) 0.27ab (n=154) 0.78e (n=242) 0.68de (n=805) 0.47c (n=232) 0.67cde (n=39) 0.18a (n=313) 0.48bcd (n=62) bacitracin 0.41b (n=188) 0.08a (n=194) 0.07a (n=846) cephalexin 0.00a (n=65) 0.01a (n=453) 0.13b (n=56) 0.74c (n=869) 0.19b (n=32) cephalothin 0.13b (n=188) 0.30b (n=50) 0.00a (n=83) 0.01a (n=546) 0.13b (n=153) cefoperazone 0.20a (n=101) 0.50b (n=352) ceftiofur 0.04a (n=101) 0.02a (n=65) 0.01a (n=546) figure 3. occurrence of bacterial resistance to antibiotics in dairy cows in brazil from 28 articles published. in the states of goiás (34%) and paraná (27%) (fig. 2). the third most frequent pathogens were corynebacterium spp., identified in 58% of the studies and with an average prevalence of 8% in the analyzed samples. the heterogeneity index i2 for the prevalence of corynebacterium spp. between those studies was 80.31% (q=497.61, df=98, p<0.001). corynebacterium spp. were not evenly distributed among the brazilian states (x2 = 140.35; p < 0.01), with greater prevalence in the states of pernambuco (31%), mato grosso (27%), and bahia and espírito santo (both 21%) (fig. 2). escherichia coli was the fourth most frequent pathogen, isolated in 47% of the articles and with an average occurrence of 4% in the analyzed samples. a significant heterogeneity was found regarding the prevalence of e. coli among the studies (q=634.75, df=98, p<0.001; i2=84.56). the states of goiás (9%) and rio de janeiro (8%) had the highest prevalence of e. coli. in 87% of the reviewed articles, other microorganisms were also isolated and associated with subclinical mastitis, such as candida spp., micrococcus spp., proteus spp., alcaligenes faecalis, enterobacter aerogenes, klebsiella spp., citrobacter spp., salmonella spp., yersinia spp., pseudomonas spp., nocardia spp., trueperella spp., and serratia spp. this wide range of pathogens was more prevalent in isolates from são paulo (32%) and goiás (30%) (g test = 44.37; p < 0.01) (fig. 2). microbial resistance among the 57 articles reviewed, 28 investigated the occurrence of resistance and sensitivity of isolated microorganisms against the antibiotics tested. only table i. occurrence of bacterial resistance to antibiotics in dairy cows from 57 articles published between 2009 and 2018 (n= number of isolates analyzed). studies from 11 states of the northeast, southeast, and south regions evaluated microbial resistance. these studies were conducted mainly in rio grande do sul, minas gerais, and santa catarina, which together accounted for 43% of the retrieved articles. the most comprehensive samples also came from studies conducted in these three states: rio grande do sul (1876 samples), minas gerais (552), and santa catarina (455) (fig. 3). the most tested antimicrobials were ampicillin, erythromycin, gentamicin, penicillin, and tetracy‑ cline. over the years, there has been an increase in the occurrence of bacterial resistance to cephalexin, cefoperazone, erythromycin, gentamicin, neomycin, penicillin, tetracycline, and trimethoprim (table i). the occurrence of microorganisms resistant to penicillin varied between 34% and 76% between subclinical mastitis in brazil ito eleodoro et al. 418 veterinaria italiana 2022, 58 (4), 413-423. doi: 10.12834/vetit.2601.17023.2 review, most studies and literature reviews also consider streptococcus spp. as the second group of microorganisms of importance in the etiological agents causing mastitis in ruminants. in most herds, streptococcus agalactiae, streptococcus uberis, and streptococcus dysgalactiae are the main isolated species (santos et al., 2018). streptococcus uberis is an important agent of subclinical infections and clinical episodes of bovine mastitis worldwide (hillerton, 2020). santos et al., (2018) reported that the streptococcus dysgalactiae is one of the most common pathogens of bovine mastitis, causing great economic losses. regarding corynebacterium spp., the third most frequent reported in this systematic review, the species isolated the most in bovine mastitis is c. bovis (karach et al., 2015). they have low pathogenicity and high contagiousness, being transmitted mainly during milking, and are considered one of the causes of contagious mastitis. it is detected mainly in the subclinical form of the disease, which in a certain manner guarantees protection to the mammary gland against other more pathogenic cells. the isolation rates of this pathogen are high in herds with problems related to the cleaning of teats, especially post‑dipping (gonçalves et al., 2016). the fourth most frequent pathogen was e.coli. neethan et al., (2017) also indicate e. coli as the main coliform (environmental microorganism) causing subclinical mastitis, with symptoms ranging from mild (with inflammatory signs in the mammary gland) to acute, with systemic signs such as ruminal stasis, dehydration, and shock, which can even lead to the death of the affected animal. although it mainly causes clinical mastitis, the microorganism has also been investigated in cases of subclinical mastitis. they are usually transient infections and are associated with acute or super‑acute clinical 2010 2011 2012 2013 2015 2016 2017 2018 chloramphenicol 0.26b (n=188) 0.14b (n=180) 0.05a (n=194) 0.00a (n=69) 0.25b (n=32) enrofloxacin 0.21b (n=101) 0.02a (n=66) 0.00a (n=83) 0.01a (n=453) 0.03a (n=36) 0.09a (n=32) erythromycin 0.25cd (n=227) 0.16bc (n=116) 0.08ab (n=197) 0.04a (n=275) 0.39d (n=163) 0.23abcd (n=26) 0.20bc (n=120) 0.72e (n=32) streptomycin 0.68b (n=188) 0.12a (n=453) 0.24a (n=46) gentamicin 0.20d (n=101) 0.03ab (n=156) 0.06bc (n=242) 0.02a (n=805) 0.16cd (n=92) 0.75e (n=2100) 0.87e (n=30) neomycin 0.39b (n=101) 0.03a (n=118) 0.03a (n=546) 0.43b (n=56) 0.73c (n=1880) norfloxacin 0.26b (n=188) 0.07a (n=88) 0.03a (n=282) 0.12ab (n=26) 0.09ab (n=32) data for the amoxacillin + clavulanic acid association in 2014 was omitted due to only one entry; a, b,c,d: proportions followed by equal letters did not differ over the years by the chi-square test with 5% significance. 2010 and 2016 and increased to 88% in 2017 (p < 0.05), reaching the highest level of resistance among the isolates (table i). other antimicrobials that started to show increasing values (p < 0.05) of microbial resistance as of 2017 were gentamicin and neomycin, and erythromycin, in 2018. the increase in these three agents may be linked to their widespread use in dairy farms, which results in contributing to the selective pressure of microorganisms resistant to them (tomazi and dos santos, 2020). amikacin and ceftiofur were the antimicrobial drugs with the lowest prevalence of resistance and without variations (p > 0.05) in studies conducted between 2010 and 2016. resistance to amikacin remained at 4% during 2015 and 2016. resistance to ceftiofur was 4% in 2010; 2% in 2011; and 1% in 2015. discussion regarding the most frequent pathogen reported in this systematic review (pooled prevalence of 49%), staphylococci are one of the pathogens most frequently isolated in cases of intramammary infection within dairy herds. this estimate is similar to the study done by ashraf and imran (2020), who conducted a review about the prevalence of various bacterial species worldwide. algharib et al., (2020) stated that s. aureus is an agent that is difficult to treat owing to its high resistance in the udder, which consequently, influences the efficiency of the antibiotics administered. this is due to a mechanism used by the pathogen to invade and colonize the animal’s mammary gland; the microorganism invades the mammary gland through the teat canal and colonizes its epithelium, attaching to the epithelial cells of the mammary gland and forming so‑called “bacterial pockets.” as well as the results of this systematic ito eleodoro et al. subclinical mastitis in brazil veterinaria italiana 2022, 58 (4), 413-423. doi: 10.12834/vetit.2601.17023.2 419 (dyar et al., 2017). one of the facts that may have led to this result is that after 2013, we found no studies evaluating resistance to ceftiofur. in this review, few studies evaluated the effectiveness of amikacin on bovine mastitis isolates. however, authors such as fim junior et al., (2015) and souza et al., (2016) reported 92.3% and 96.0% sensitivity of the isolates, respectively, thus demonstrating the effectiveness of amikacin with their results. the scarcity of studies investigating the use of this antimicrobial drug may be because gentamicin is one of the main aminoglycosides used in veterinary medicine, more specifically in the treatment of mastitis. limitations it is worth noting that analyzing microbial resistance with information obtained from published scientific articles has its limitations. one of them is the temporal and geographical limitation, since, from an epidemiological point of view, the monitoring of publications over the years does not guarantee a significant sample at the national level, and these data are not from a single region of brazil. moreover, the compilation of several studies conducted in one specific period is not representative. furthermore, some geographical areas with lack of studies pose challenges to obtaining high‑quality survey, contributing with bias that can affect the reliability of the findings, mainly when extrapolating the results to other regions. thus, we strengthen the need of more studies at regional level with properly methodologies regarding the sample representativeness. another limitation is related to the seasons, since the time of year is related to antimicrobial treatment in dairy herds (tomazi and dos santos, 2020). the articles selected in this study did not provide enough data to analyze this variable. conclusions with the etiological diversity found in this review, our results strengthen the knowledge of the microbiological agent and antibiotic‑resistance patterns of pathogens isolated from subclinical mastitis in dairy cows at regional level. the spread of bacterial resistance can be prevented using the culture test and antibiogram, and that although decision making in a farm cannot be replaced by scientific studies, it can be supported by such efforts. nevertheless, the identification of the microbiological agent is essential to the most appropriate therapy. when possible, etiology should be determined before treatment to avoid microbial resistance. conditions, which can be fatal. it is important to note that older cows, those at the beginning of lactation, and those with higher yields are most susceptible to the severe manifestation of mastitis by coliforms (byomi et al., 2020; hamali et al., 2017). we observed a high heterogeneity for streptococcus spp, corynebacterium spp and e. coli regarding the prevalence estimates between the retrieved studies, probably due to diversity in farm practices (hygienic milking, dry cow therapy and therapeutic actions) together with herd characteristics (genetic, stage of lactation) and agroclimatic conditions (bangar et al., 2015). regarding microbial resistance, it can be inferred that some of the most used agents in intramammary therapies were evaluated by only a few of the selected studies. an example is the third generation cephalosporins, identified as the second most frequently used class of antimicrobials in brazil (tomazi and dos santos, 2020). we recommend that studies on microbial resistance select the agents most used in the geographic region studied so that the results better reflect reality. penicillin is one of the main antibiotics used for intramammary treatments not only in brazil but also in other countries (tomazi and dos santos, 2020). in the united states, more than 70% of isolates obtained from mastitis caused by s. aureus are resistant to penicillin, whereas in ireland, the level of resistance is around 85% (cazoto et al., 2011). the widespread use of an agent is one of the causes of bacterial resistance (freitas et al., 2018). another cause of resistance to penicillins is owing to staphylococcus spp., the main genus associated with subclinical mastitis, being able to develop resistance to most antimicrobials. resistance to beta‑lactams, as is the case with penicillins, can occur via two main mechanisms: through the production of beta‑lactamases, encoded by the blaz gene and the change in the antimicrobial action site owing to the production of a modified low‑affinity penicillin‑binding protein (pbp2a or pbp2), encoded by the meca gene (soares et al., 2012). indeed, in a systematic review that addressed article from 5 continents, molineri et al. 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(2013). prevalence, etiology, antimicrobial susceptibility and risk factors 425 please refer to the forthcoming article as: sadiq et al. 2022. prevalence and molecular characterization of non-typhoidal salmonella isolated from meat and environmental samples of retail shops of lahore punjab, pakistan. vet ital. 10.12834/vetit.2392.14037.1. 1department of epidemiology and public health, university of veterinary and animal sciences, lahore, pakistan. 2department of environmental sciences, quaid-i-azam university, islamabad, pakistan 3italian national and oie reference laboratory for salmonella. istituto zooprofilattico sperimentale delle venezie (izsve), padua, italy. *corresponding author at: department of epidemiology and public health, university of veterinary and animal sciences, lahore, pakistan. e-mail: shakera.sadiq@uvas.edu.pk. shakera sadiq1*, mamoona chaudhry1, muhammad hassan mushtaq1, junaid sadiq2, saima hasan1, lettini antonia anna3 salmonella is one of the leading causes of bacterial diarrhea worldwide, accounts for one of the four key global causes of diarrheal illnesses (who, 2018). non-typhoidal salmonellae are important foodborne bacterial pathogens that can cause bacteremia, gastroenteritis, and subsequent infection (acheson and hohmann, 2001). every year, non-typhoidal salmonella infections results in approximately 153 introduction foodborne diseases pose a significant impediment to socio-economic development globally and represent a constant threat to public health (devleesschauwer, et al., 2018). almost 1 in 10 people become infected every year due to foodborne diseases. diarrheal diseases are the most common illnesses from contaminated unsafe food. keywords prevalence, zoonotic, salmonella, retail shops. veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 accepted: 28.05.2021 | available on line: 31.12.2022 summary non-typhoidal salmonellae are important foodborne bacterial pathogens that can cause bacteremia, gastroenteritis, and subsequent infection. the aim of the study was to determine the prevalence of salmonella in the live bird market and retail shops of lahore (pakistan). a total of 720 samples of chicken meat, chopping board, cages, hands, and transportation vans were collected. salmonella was recovered from 103 (14.36%) samples. the highest prevalence (33.33%) was found in transportation van samples followed by chicken meat samples (17.26%). in the towns of lahore, the highest prevalence was found in samanabad town (19 %) followed by data ganj bakhsh town (17%) with the lowest in gulberg town (6.9%). salmonella typhimurium was most common (35.92%) followed by s. enteritidis (25.24%), s. dublin (14.56%), s. gallinarum biovar gallinarum (8.74%), and untyped salmonella species (15.53%). this was the first baseline study of the prevalence of non-typhoidal salmonella at the live bird market and retail shops of lahore. implementation of appropriate control measures is required at both the human side and poultry food production chain to reduce the burden and transmission of the zoonotic salmonellae. prevalence and molecular characterization of typhoidal and non-typhoidal salmonella isolated from meat and environmental samples of retail shops of lahore punjab, pakistan non typhoidal salmonellae in of pakistan sadiq et al. 426 veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 million cases of gastroenteritis and 57,000 deaths globally (brunette, 2017). according to the data of the global burden of diseases, among the total deaths due to salmonellosis, 38,500 deaths per year occur in children less than five years of age (delahoy, et al., 2018) poultry is considered as the most important reservoir of salmonella and also considered as one of the common sources of human salmonellosis globally (antunes, et al., 2016; yang, et al., 2011; zwe, et al., 2018). contaminated foods particularly broiler retail chicken meat is considered as the most commonly implicated foods for non-typhoidal salmonellosis in humans (alali, et al., 2012; donado-godoy, et al., 2012; lamas, et al., 2016; li, et al., 2017; shang, et al., 2019). salmonella bacteria own the ability to reside in the healthy chicken without showing any symptoms so it may go unnoticed, therefore poses the risk of human infection through contaminated chicken at retail level (zwe, et al., 2018). salmonella is the leading cause of foodborne disease worldwide especially in the middle east, eastern europe, and southeast asia (ta, et al., 2012). it is also one of the most important zoonotic pathogens both in animals and humans in developing countries (ejo, et al., 2016) important stages for transmission of salmonella to chicken and its products include the status of infection in the host population at the farm after rearing, fecal contamination during transport by vehicles to the slaughtering and processing facility, and cross-contamination between chicken carcasses and equipment during cutting and processing. it is also documented in studies that during transport or shipping, the prevalence of salmonella in positive birds increases due to fecal contamination of feathers and skin by companion birds (mccrea, et al., 2006). in pakistan when the broiler flock is ready after rearing in broiler farms, transport vehicles are used to transport live birds either to processing plants, live bird markets or directly to retail shops of the county. live bird markets or wet markets are common in asian countries (nidaullah et al.2017) including pakistan. these live bird markets are served as wholesale markets and used as an important source of chicken consumed by the population on large scale. live bird markets play an important role in the dissemination of salmonella serovars to the environment and individuals (sharma, et al., 2019). many risk factors have been associated with the presence of  salmonella  spp. at the retail outlets of live bird markets, including the size of live bird market, sources of chicken either from integrated or nonintegrated systems, storage conditions including temperature, geographical location and rearing types of chicken (alali, et al., 2012; jarquin, et al., 2015; khan, et al., 2018; tabo, et al., 2013; yang, et al., 2011), at a worldwide level, foodborne illness is often related to the consumption of poultry meat and its products contaminated with pathogenic bacteria commonly from the retail shop level (shafini, et al., 2017). in pakistan, consumers prefer to purchase meat which has been slaughtered in front of them from retail shops. the important risk factors are contaminated knives, chopping boards, and tables and, contaminated hands of retailers. poor sanitary and hygienic practices are prevalent in these retail outlets in developing asian countries and are not monitored by regulatory authorities (nidaullah, et al., 2017; yang, et al., 2011). according to the author’s knowledge, there is limited data on the prevalence of non-typhoidal salmonella in retail meat shops in lahore. the present study was conducted to check the prevalence of non-typhoidal salmonella at different steps of the poultry chain including sampling from transport vehicle vans, live bird market, and retail shops of nine towns of lahore. material and methods a cross-sectional study was conducted starting from february 2018 to august 2018 in district lahore, punjab. samples were collected (n=720) from the live bird market located in data gunj bakhsh town and retail outlets of lahore. sampling was started from the live bird market by initially taking swabs samples (n=45) from transport vehicles. hand swabs (n=33) were taken after the consent of the drivers. to avoid repetition, the name of the driver and the number of the vehicle were noted on the day of sampling. sterilized cotton swabs were used for sampling the cages of vans and the hands of van drivers. hand swabs were taken by swabbing the palms and fingers with sterilized cotton swabs dipped in 0.1% buffered peptone water (bpw, oxoid) (cm0509) (okareh and erhahon, 2015). swabs were kept in test tubes containing 10 ml buffered peptone water. forty (40) retail shops from the live bird market located in data ganj bashkh town and 90 chicken retail outlets from 9 administrative zones of lahore were selected. a total of 10 shops were selected from each administrative zone of lahore including 9 towns. four types of samples including chicken meat (n=262), chopping board swabs (n=130), cage swabs (n=70), and hand swabs of the retailers (n=180) were collected. meat samples of slaughtered chicken including the thigh, chest meat, and chicken leg were collected. a minimum (approx. 225 gm) of meat was purchased and stored in sterilized polythene bags sadiq et al. non typhoidal salmonellae in of pakistan veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 427 molecular identification of nontyphoidal salmonella enterica by multiplex polymerase chain reaction (pcr) a multiplex pcr was developed for the identification of common serovars of salmonella enterica in lahore. dna extraction four to five pure isolated colonies from fresh culture were first incubated in lysogeny broth (luria bertani medium) (lb broth, oxoid, cm0996) for 24 hours at 37ºc. from lb broth culture, one ml was taken in the eppendorf tube and centrifuged at 10,000 rpm for 10 minutes. the supernatant was discarded, and the pellet was washed by adding 1 ml of ultrapure distilled water and again centrifuged at 10,000 rpm for 10 minutes. the supernatant was discarded, and 180 µl of 10x chelex resin (bio rad) were added. the suspension was mixed properly by vortexing. eppendorf tubes were sealed with parafilm and placed in a water bath for 20 minutes at 100ºc. immediately after water bath, samples were chilled on ice for 2 minutes. samples were centrifuged again at a maximum speed of 14,000 rpm for 10 minutes. the supernatant was used as template dna for pcr. template dna was stored at -20°c until used. multiplex pcr multiplex pcr was developed for simultaneous identification of salmonella serovar, enteritidis, typhimurium, typhi, dublin, and salmonella enterica serovar gallinarum biovar gallinarum (table i). for pcr, 25 µl of the reaction mixture was prepared to contain 1 µl (10 pmol) of each forward and reverse primer, 12.5 µl of master mix (lucigen usa), 2 µl of template dna and remaining nuclease-free water to make final volume. from retail shops. for the sampling of chopping boards and cages, sterilized swabs immersed in 0.1% bpw were used. a few samples containing fecal material and debris from the floor of the cages were also collected in sterilized polythene bags. all the samples were properly labeled and packed and immediately transferred to the laboratory of epidemiology and public health within 24 hours for further microbiological processing. isolation and identification for isolation and cultivation of salmonella, iso 6579 method was used. for meat samples, 25 gr of meat sample were weighed and suspended in 225 ml buffered peptone water for 24 hours at 37°c. for selective enrichment, two types of salmonella selective broths were used. firstly, 0.1 ml of bpw was inoculated into 10 ml of rappaportvassiliadis  enrichment broth (rv broth, oxoid, cm0669). the broth was incubated for 48 hours at 42°c. secondly, one ml of bpw was inoculated in 10 ml of tetrathionate broth (tt broth, oxide cm0671). the broth was incubated for 18-24 hours at 37ºc. for culturing of salmonella, three selective media were used. three loops full (one loop for each media) of inoculum was used for inoculating brilliant green agar (bga, oxoid, cm 0263), salmonella shigella agar (ss agar, oxoid cm0099) and xylose lactose tergitol™ 4 (xlt 4, oxoid cm1061). the plates were incubated for 18-24 hours at 37ºc. three presumptive colonies (pink colonies with red background for bga, round black centered for ss agar, and black centered with the pink background on xlt4) were selected and further streaked for purification on nutrient agar (oxoid, cm0003). suspect colonies showing morphological characteristics of salmonella were further confirmed by the triple sugar iron (oxoid, cm0277) and urease tests (oxoid, cm 057). table i. primer sequences for multiplex pcr for salmonella serovar enteritidis, typhimurium, typhi, dublin, and gallinarum biovar gallinarum. target gene sequence amplification target size bp reference sdf 1 tgtgttttatctgatgcaagagg serovar 304 (de freitas, et al., 2010) tgaactacgttcgttcttctgg enteritidis spy ttgttcactttttacccctga serovar 401 ccctgacagccgttagatatt typhimurium viab cacgcaccatcacaccg serovar typhi 738 aacaggctgtagcgatttagg sed_a1104 gene acgcgaaatctgatggtctt serovar. dublin 203 (stegniy, et al., 2014) gcccaccagttgtgaaaggc sg0266 ccgcacaacacatcagaaag serovar 97 agctgccagaggttacgctg gallinarum non typhoidal salmonellae in of pakistan sadiq et al. 428 veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 figure i. gel image of multiplex pcr to detect salmonella. cycling conditions cycling conditions of the reaction comprised initial denaturation temperature of 95°c for 2 min; (ii) 30 cycles, with 1 cycle consisting of 1 min at 95°c, 1 min at 57°c, and 2 min at 72°c; and (iii) a final elongation step of 5 min at 72°c (alvarez, et al., 2004). the pcr products were electrophoresed in 2.5% (wt/vol) d-1 agarose (oxoid) and stained with 4 µl of ethidium bromide solution. in each pcr run, a non-template control was included to detect possible external dna contamination. the gel was visualized in the gel doc system (syngene, usa). figure ii. map showing prevalence of salmonella in different towns of lahore. table ii. overall prevalence of salmonella in chicken meat, environmental samples, and hand swabs. sample type no of samples tested no of positive samples percentage chicken meat samples 262 46 17.56 hand swabs of retailers 213 21 9.86 cages of birds 70 6 8.57 chopping board samples 130 15 11.54 transportation van sample 45 15 33.33 total 720 103 14.31 results figure i showed results of multiplex pcr with five bands of salmonella enteritidis, typhimurium, dublin, gallinarum and typhi. among the total (n=720) collected samples, 103(14.31%) were found to be positive for salmonella enterica (genus). the highest prevalence was found in transportation vehicle samples (33.3%), chicken meat samples (17.56%), chopping board samples (11.54%), hand swabs of retailers (9.6%) and cage samples (8.57%) (table ii). as the live bird market of lahore is located in data gunj bakhsh town, the samples collected from tollinton market with samples collected from retail shops in streets of data gunj bakhsh town were combined. table iii and figure ii present the area wise prevalence in different towns of lahore. the highest prevalence was found in samanabad town (19%) data gunj bakhsh town (17.0%), ravi town (16%), shalimar town (15.0%), wahga town (15%), alama iqbal town (12%), aziz bhatti town (9%), and gulberg town (7%). table iv shows the serovar distribution sadiq et al. non typhoidal salmonellae in of pakistan veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 429 location of retail outlets total number of shops tested salmonella positive shops no of samples collected no of positive samples percentage of positive samples ravi town 10 6 70 11 15.71 shalimar town 10 7 82 12 14.6 aziz bhatti town 10 4 90 8 8.9 data gunj bakhsh town *50(10+40) 30 §195(75+120) ¶34(15+19) 17.43 samanabad town 10 8 70 13 18.57 gulberg town 10 2 58 4 6.90 wahga town 10 5 55 8 14.55 alama iqbal town 10 6 60 7 11.67 nishter town 10 4 40 6 15 salmonella serovars total number identified percentages salmonella typhimurium 37 35.92 salmonella enteritidis 26 25.24 salmonella dublin 15 14.56 salmonella gallinarum biovar gallinarum 9 8.74 salmonella typhi 0 0 untyped 16 15.53 total 103 100 table iii. area prevalence of salmonella in retail shops of towns of lahore (pakistan). table iv. salmonella serovar identification in retail shops of lahore (pakistan). * total number of shops selected from live bird market and retail shops of data ganj bukhsk town. § total number of samples selected from live bird market and retail shops of data ganj bukhsk town. ¶ total number of salmonella positive samples from live bird market and retail shops of data ganj bukhsk town. among the total isolated samples with the most common serovar being salmonella typhimurium (35.92%) then enteritidis (25.5%), dublin (14.56%), gallinarum (8.74%), typhi (0%), and untyped (15.53%). discussion the present study was conducted for the first time to assess the burden of non-typhoidal salmonella at the retail shops of lahore (no study published earlier). lahore is the largest city of punjab province, pakistan, and the fifth-largest city in south asia. the land area of lahore is 1772 square kilometer with a total population of approximately 12.18 million (2019 statistics). to meet consumer demands, there are more than 9,000 retail shops for red and white meat (nisar, et al., 2018). according to the survey (jalil, et al., 2013), the daily chicken meat consumption in lahore is approx 361,000 kilograms. the chicken is supplied to the whole city through two major markets including the tollinton market (shares 55% of total supply) and the sheranwala market (shares 30% of total supply). the remaining 15% of chicken meat is sold by direct suppliers. the presence of salmonella is a major hindrance to the export of poultry and poultry products from pakistan as well as its zoonotic threat (http://www. livestockpunjab.gov.pk/livestockadmin/uploads/ editor_files/per formance_report_-_directorate_ of_poultry_research_institute_oslln.pdf ). a limited number of studies on non-typhoidal salmonella in poultry have been reported from pakistan. the purpose of this research was to determine the presence of non-typhoidal salmonella in the major live bird market (tollinton market) and retail shops of nine administrative towns of lahore. in the present study, the prevalence rate of salmonella in raw chicken meat is 17.56% which is lower than that reported from all other studies conducted in different areas of pakistan. the reported area wise prevalence of salmonella in chicken meat was 38% in hyderabad (soomro, et al., 2010); (34%) in raw chicken meat in kohat (asif, et al., 2017); (30%) in chicken meat in faisalabad (akhtar, et al., 2010), and (18%) in raw poultry meat in swat (uddin, et al., 2018). the lower prevalence of salmonella in lahore (punjab) as compared to other areas may be due to the role of the poultry research institute (pri, punjab) in surveillance and non typhoidal salmonellae in of pakistan sadiq et al. 430 veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 control of poultry diseases at regional levels. the reported prevalence in raw chicken meat (17.56%) is also lower as compared to other asian countries: 21-65.3% in vietnam (huong, et al., 2006; nguyen, et al., 2016; phan, et al., 2005; ta, et al., 2014; van, et al., 2007); 52.2% in china (yang, et al., 2011); 48% in malaysia (abatcha, et al., 2018). the difference in the prevalence rate of salmonella in asian countries may be due to different farming practices, transport vehicles, slaughtering and processing environment, unhygienic practices at retail shops, and also could be due to differences in isolation methods (sharma, et al., 2019). moreover, in developing nations, the chicken meat is kept at room temperature as no cooling system is installed in wet meat shops and therefore it provides optimum survival conditions for salmonella. the lack of strict hygiene-related laws in retail meat shops and unawareness among butchers are considered the two most important reasons for the increased prevalence of salmonella. the purpose of hand swabbing was to determine the level of hygienic practices of the retailers and their involvement in the dissemination of pathogen to an uninfected chicken carcass. the contamination of the retailer’s hand can impose a greater risk for self-infection as well as the dissemination of pathogens to the family. in the present study, the frequency of the occurrence of salmonella on the retailer’s hands was (9.86%) which is not comparable locally as no study has been conducted before to analyze the level of nontyphoidal salmonella contamination on hands. our reported prevalence varies from that reporte in other studies: 0%-14.29% on meat shop retailers hands in india (sharma, et al., 2019; suresh, et al., 2004); 23.3%-33.3% on hands in ethiopia (abdi, et al., 2017; garedew, et al., 2015). personal hygiene may be the reason for discrepancies in prevalence. poor hand washing practices and use of single cloth for drying hands after every single slaughter are very common in retail shops of lahore. normally, there is one large bowl of water which is used repeatedly for washing of hands without any soap or sanitizer. a large piece of cotton cloth is also commonly seen which is used for drying hands. the prevalence of salmonella in environmental samples was highest in transportation vans samples: 33.3% followed by chopping board samples; 11.54%, and cages; 8.57%. transportation trucks are used to transport live birds from broiler farms to live bird markets and retail shops. these vehicles can be a continuous source of contamination to healthy flocks (beach, et al., 2002; ejo, et al., 2016; gallegos robles, et al., 2009; hald, et al., 2000). the reported prevalence in lahore (33.3%) is lower than reported in korea (62.5%) from vehicles (ha, et al., 2018), where the salmonella prevalence in broiler transporting vehicles was highest in the whole food chain. the difference could be due to uncleanliness of floors of trucks and how frequently these vehicles are washed to reduce contamination. chopping boards are used for cutting and chopping of meat after de-feathering and evisceration process. retailers mostly clean the chopping board with a wet cloth after cutting off one carcass and this cloth is used repeatedly after every carcass cutting. the prevalence of salmonella on chopping boards in the present study was 11.54%, which is higher than the 5.6% reported from ethiopia (garedew, et al., 2015) and lower than the 18.75% reported from india (suresh, et al., 2004). the difference in prevalence could be due to the method of taking samples as the chopping board surface is porous and bacteria sometimes reside inside surfaces which is difficult to collect during swabbing (ak, et al., 1994). studies reported that uncleaned chopping boards are a potential source salmonella cross-contamination (zwe, et al., 2018). cages are compartments mostly placed on one side or the backside of the shop. the floor surface of the cages is covered with droppings and it is cleaned occasionally before the entrance of the next batch of birds. according to previous studies, levels of shedding dramatically increased, while birds were confined in cages before slaughter (corry, et al., 2002; ha, et al., 2018; hald, et al., 2000). a prevalence of 8.57% was reported from cages in the present study. cage samples are considered under environmental samples and prevalence is in the range of that reported from the neighboring country india [7.94% from environmental samples (sharma, et al., 2019)]. in our study, the area wise highest prevalence was found in samanabad town (19%), followed by data gunj bukh town (17%), and gulberg (6.9%). the reason behind the high prevalence in samanabad and data gang bukhsh is the high population congestion rate (22.72 and 25.33 persons/square meter, respectively). conversely, gulberg is a newly established area of lahore with high literacy rate and elite class residents with congestion rate of 20 person/square meter (joshua and ali, 2011; nisar, et al., 2018). the most prevalent serovar in our study was salmonella serovar typhimurium (35.92%), followed by s. enteritidis (25.24%), s. dublin (14.56%), s. gallinarum biovar gallinarum (8.74%). all the samples were negative for salmonella typhi (0%) while the untyped isolates were 15.53%. sadiq et al. non typhoidal salmonellae in of pakistan veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 431 as both, salmonella dublin and gallinarum are host specific serovars, the possible chances of occurrence may be due to infection of birds at farms or the environmental contamination of samples at retail shops. in our study, the predominant serovar was typhimurium, a recent study reported a prevalence of 28.45% from farm samples from faisalabad (punjab) (wajid, et al., 2019). no other study in pakistan reported the prevalence of s. typhimurium. the prevalence of salmonella enteritidis (25.24%) reported in our study was simlar to that reported from kohat (23.3% (asif, et al., 2017), but lower than reported in different districts of punjab (31.34%), (yasmin, et al., 2019). conclusions this is the first baseline study in lahore, punjab for the prevalence of different salmonella serovars. the high prevalence of zoonotic serovars in the retail shops of major city of punjab is a major threat to public health. food and health authorities of pakistan should implement appropriate control strategies to minimize the spread food borne zoonotic pathogens to consumers ethical permission this study was approved by ethical review committee for animals and humans, university of veterinary and animal sciences, lahore. acknowledgments authors acknowledge the poultry retailers for their participation in study. the authors will also like to mr. mubashar hanif laboratory assistant for help in laboratory work and clerk, miss fakhra sarwar for help in formatting of write up. funding statement the current research study was funded by project entitled “molecular detection of emerging and remerging meat borne bacterial pathogens and 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of school food vendors in benin city, nigeria. food non typhoidal salmonellae in of pakistan sadiq et al. 434 veterinaria italiana 2022, 58 (4), 425-434. doi: 10.12834/vetit.2392.14037.1 van t.t.h., moutafis g., istivan t., tran l. t. & coloe p. j 2007. detection of salmonella spp. in retail raw food samples from vietnam and characterization of their antibiotic resistance. appl environ microbiol, 73, 6885-6890.. uddin m.n., farooq m.m. & waqas m. 2018. antibiotic assays of salmonella isolated from poultry chicken of various locations in districts swat. pure appl biol, 7, 78-84. 379 please refer to the forthcoming article as: vilcek et al. 2022. high prevalence of leishmania spp. in canines from central west colombia. vet ital. 10.12834/vetit.2632.16820.2. are pathogenic for humans (akhoundi et al., 2016). these species are grouped by complexes according to the tropism to a specific tissue (akhoundi et al., 2016) and they can infect macrophages in the bone marrow, lymph nodes, spleen, liver, kidneys, gastrointestinal tract, skin, and mucous membranes (reis et al., 2009). this leads to the presentation of three clinical forms: cutaneous, mucocutaneous, and visceral leishmaniasis, characterized by presenting macroscopic lesions such as lymph node hypertrophy, ulcerative dermatitis (periorbital, nasal introduction leishmaniasis is a zoonotic disease caused by the invasion of protozoan parasites of the leishmania genus, transmitted to mammals mainly by the hematophagous activity of sandflies belonging to the lutzomyia and phlebotomus genera (ribeiro et al., 2018; torres et al., 2017). this disease has a worldwide incidence of 0.7 million new cases per year and up to 65,000 deaths are reported annually (alvar et al., 2012). there are around 53 species of leishmania, of which parasitize mammals and 20 veterinaria italiana 2022, 58 (4), 379‑389. doi: 10.12834/vetit.2632.16820.2 accepted: 07.01.2022 | available on line: 31.12.2022 1research group in immunobiology and pathogenesis, faculty of veterinary medicine and zootechnics, university of tolima, ibagué, colombia. 2 municipal health secretary, mayor of ibagué, colombia. *corresponding author at: research group in immunobiology and pathogenesis, faculty of veterinary medicine and zootechnics, university of tolima, ibagué, colombia. e‑mail: lagiraldom@ut.edu.co. leidy alejandra giraldo‑martínez 1*, julieth michel petano‑duque1, heinner fabian uribe‑garcía1, roberto andrés chacón‑novoa1, blanca lisseth guzmán2, iang rondón‑barragán1 keywords canine leishmaniasis, canine, hsp70, its‑, risk factors. summary leishmaniasis is a widespread disease caused by species of the genus leishmania. in colombia, this zoonosis is endemic in rural areas with a high prevalence in the departments of antioquia, santander, meta, tolima and nariño. dogs are the most important domestic reservoirs of the pathogen, given the epidemiological importance of dogs in the control of leishmaniasis is needed to determine the prevalence of leishmania spp. in canines of the rural area of ibagué and to identify potential risk factors related to the presence of this parasite. a cross‑sectional study was carried out in 173 dogs from the rural area of ibagué. leishmania spp. was detected by amplifying the internal transcribed spacer (its‑1) and two regions of the hsp70 gene through pcr. factor associations were calculated through the chi‑square and odds ratio. prevalence of leishmania spp. infection in dogs was of 91.33% (158/173), where 36.71% (58/158) of the leishmania spp. positive dogs showed one or more clinical signs of canine leishmaniasis and 63.29% (100/158) of the dogs were asymptomatic. factors associated with the presence of the parasite did not show significant association. in addition, hsp70d‑pcr was proved to be highly efficient for the detection of leishmania spp. high prevalence of leishmania spp. in from central west colombia leishmania spp. in dogs, central west colombia leidy alejandra giraldo-martínez et al. 380 veterinaria italiana 2022, 58 (4), 379-389. doi: 10.12834/vetit.2632.16820.2 has a population of 569,336 inhabitants (centro de información municipal para planeación participativa, 2021) and a canine population of 40,016 animals (ministerio de salud y protección social de colombia, 2018). rural area is organized into 17 townships with approximately 144 villages. sample size sample size was estimated based on a population of 40,016 canines in the 17 townships of the rural area of ibagué (ministerio de salud y protección social de colombia, 2018) using the formula described by thrusfield (2018), where the expected prevalence was 10%, the level of significance was 95% and the desired absolute precision was 5%. clinical examination, epidemiological survey and blood sampling study was carried out between july and november of 2019. each canine`s owner signed a consent form before any procedure with the animals. clinical examination was performed by a physical inspection to detect signs compatible with leishmaniasis including dermatological lesions (ulcers, alopecia, erythematous lesions), onychogryphosis, apathy, low body condition and abnormal peripheral lymph nodes. a survey was fulfilled in order to find possible risk factors related to the presence of leishmania spp. in the canines, data included canine breed, dwelling features (a type of material of the house and the floor) and dwelling surround characteristics (disposal of garbage, presence of other animals, forest areas, agricultural activities and streams near the home). one hundred seventy‑ three samples of canine blood were collected by peripheral venipuncture in the saphenous or cephalic vein. samples were placed in tubes with ethylenediaminetetraacetic acid and transported in an ice box until storage at ‑20 °c. dna extraction and endpoint pcr total genomic dna extraction was performed from blood samples using phenol‑chloroform‑isoamyl alcohol (25:24:1). dna quality was verified by nanodrop one spectrophotometer (thermofisher, usa) and stored at ‑20 °c until its use (wang et al., 2011). leishmania spp. was detected by amplifying the internal transcribed spacer 1 (its1) and regions c (1545‑1778) and d (711‑1089) of the hsp70 gene (table 1). animals were considered positive to leishmania spp. if one of the three tests was positive. pcr was performed in a t‑100 thermal cycler (bio‑rad, or disseminated), and edema in the limbs (mümtaz, 2018). leishmaniasis has a high prevalence in tropical developing countries and mainly affects the population in low‑income countries (oryan & akbari, 2016). in colombia, it is distributed in rural areas, with high prevalence in the departments of antioquia, santander, meta, tolima, and nariño (mendigaña, 2019), where nine species have been reported: leishmania panamensis, l. braziliensis, l. guyanensis, l. infantum, l. colombiensis, l. amazonensis, l. equatoriensis, l. lansoni and l. mexicana (salgado‑ almario et al., 2019; ramírez et al., 2016). according to the 2019 epidemiological bulletin were reported in the department of tolima, 13 belonging to the municipality of ibagué (incidence rate of 2.26) (sistema de vigilancia en salud pública, 2019). in 2020, 32 cases of cl have been notified with an incidence rate of 5.52% (macedo & urrego, 2020). infection with leishmania spp. in canines can be clinical or subclinical, becoming a public health concern due to the risk of transmission to humans in the presence of hematophagous vectors (chinchilla & vanessa, 2010; flórez et al., 2006; zoghlami et al., 2014). several risk factors have been described related to leishmania spp. infection including canine breed, infestation with ticks, proximity or contact with other animals (e.g. chickens, pigs, horses), improper garbage disposal, dwelling proximity to water sources, forest areas, and informal crops (membrive et al., 2012; oryan & akbari, 2016; silva et al., 2018). a higher prevalence of visceral leishmaniasis has been reported in shorthaired canines and purebreds such as boxer, pit bull terrier, cocker spaniel, and great danish (almeida et al., 2010; belo et al., 2013b; frança‑silva et al., 2003). active epidemiological surveillance based on the detection of infected animals is an initial step to establish appropriate control measures. clinical examination allows the first approach to the diagnosis, however, in asymptomatic animals the use of diagnostic tests such as the polymerase chain reaction (pcr) is required (cavalcanti et al., 2012; reis et al., 2013; zoghlami et al., 2014). in the municipality of ibagué, human leishmaniasis has been reported, nevertheless, the role of canines in the epidemiology of leishmaniasis is unknown. thus, the present study aimed to determine the prevalence of leishmania spp. in canines of the rural area of ibagué and to identify potential risk factors related to the presence of this parasite. material and methods study location study was carried out in ibagué, the capital city of the department of tolima, colombia, which leidy alejandra giraldo-martínez et al. leishmania spp. in dogs, central west colombia veterinaria italiana 2022, 58 (4), 379-389. doi: 10.12834/vetit.2632.16820.2 381 hsp70 gene of leishmania spp. were submitted to genbank in order to obtain accession numbers (mw509745, mw509746, mw509747, mw509748 and mw509749). statistical analysis a database was created from the information collected in the clinical examinations and the epidemiological survey in the epi info software (cdc, atlanta, georgia, usa). analysis of the independent variables in the epidemiological survey was performed using graphpad prism v.8 for macos (la jolla, ca, usa). frequency tables were constructed and their relationship with the presence of leishmania spp. was established through the chi‑square. strength of this relationship was determined by calculating the odds ratio. results characteristics of the canine population a total of 173 dogs were sampled. they were distribuited 65 from totumo, 32 from coello cocora, 14 from el salado, 13 from cay, 11 from san bernardo, 11 from calambeo, 9 from gamboa and 9 from carmen de bulira, 9 from buenos aires. of the 173 sampled dogs, 57.8% were males and 42.2% females. crossbreed dogs were 79.19% and purebred 20.81%, they had short fur (65.32%) with an average age of 48.9 months. all animals were dogs with an owner, but 31.8% did not stay in the dwelling during the day and 22.5% did not stay overnight. regarding the clinical sign compatible with leishmaniasis, 10.4% of the dogs had foci of alopecia, 12.72% skin lesions, 24.85% pruritus, and 2.89% onychogryphosis and lymphadenitis (figure 1). table i. sequences of primers for amplification of its1 and hsp70 genes. gene amplicon size (bp) primer sequence (5´-3´) reference its1 314 f: ctggatcattttccgatg schönian et al. (2003)r: tgataccacttatcgcactt hsp70c 234 f: ggacgagatcgagcgcatggt graça et al. (2012)r: tccttcgacgcctcctggttg hsp70d 379 f: ccgcctcgtcacgttcttcagc graça et al. (2012)r: gttcagctccttgccgccga hercules, california, usa) using a final reaction volume of 25 μl, composed of 14.875 μl of distilled and deionized water (ddw), 5 μl of 5x colorless gotaq flexi buffer, 1 μl of dntps at 1.5 mm, 1 μl of each primer (forward and reverse) [10 pmol/μl], 1 μl mgcl2 at 25 mm, 0.125 μl of gotaq flexi dna polymerase (promega, madison, wisconsin, usa) and 1 μl of the gdna sample as template. amplification steps consisted of initial denaturation cycle at 95 °c for 3 minutes, followed by 35 denaturation cycles at 95 °c for 30 seconds, annealing at 55 °c for 30 seconds, extension at 72 °c for 30 seconds, and final extension at 72 °c for 5 minutes. positive control of l. infantum (mcan/ cn/90/sc) and negative control (ddw) were used. pcr products were revealed by 2% agarose gel electrophoresis, stained with hydragreen (actgene, piscataway, new jersey, usa), for 40 minutes at 100 v in the mygel mini electrophoresis chamber (accuris, edison, new jersey, usa) and visualized under ultraviolet light using an enduro gds (labnet intl, edison, new jersey, usa). dna sequencing and phylogenetic analysis hsp70c‑pcr products of 234 pb were sequenced by the sanger method (macrogen, seoul, korea), sequencing was carried out by the same primer pair (hsp70c). consensus sequences from forward and reverse reads were edited and analyzed using geneious prime software (version 2021.0.3) (biomatters, auckland, new zealand) (kearse et al., 2012), furthermore they were compared with the genbank database (ncbi, usa) by using the blastn tool (version 2.2.28+). bioinformatic analysis included multiple alignments of our sequences and 18 hsp70 sequences from 7 leishmania species reported in genbank database (ncbi, usa), and the construction of phylogenetic trees using neighbor‑joining (nj) method, with a bootstrap consensus inferred from 1000 replicates. it was used as outgroup the region c of hsp70 gene sequence of trypanosoma cruzi (genbank accession no. mf144929.1). sequences of the region c of figure 1. dogs with cutaneous lesions, mucocutaneous lesion, localized alopecia, cutaneous hypopigmentation and onychogryphosis hypopigmentation and onychogryphosis. leishmania spp. in dogs, central west colombia leidy alejandra giraldo-martínez et al. 382 veterinaria italiana 2022, 58 (4), 379-389. doi: 10.12834/vetit.2632.16820.2 the peridomiciliary zone, 56.65% of the ares had a water stream of 56.65%, wooded areas, and in 71.68% there was presence of mosquitoes. of the dog's owners, 67.63% used protective measures for the mosquitoes, like bed nets to sleep (66.96%), and the remaining used repellents or fumigated their household (33.04%). according to the statistical analysis, characteristics of the canine, sociodemographic factors, home conditions and social interactions evaluated did not show associations with the presence of leishmania spp. in the canines of ibagué, and therefore, statistically significant risk factors were not determined. phylogenetic analyses pcr products of hsp70c region (234 bp) from the le_019, le_021, le_022, le_024, le_025, le_029, le_030, le_053 and le_064 samples were sequenced. sequences le_019 and le_029 le_025 have 100% identity with each other, the same as le_021, le_053 and le_064, therefore, 5 partial sequences of the hsp70 gene of leishmania spp. were submitted to genbank. accession numbers of sequences were as follows: le_019 (mw509745), le_021 (mw509746), le_022 (mw509747), le_024 (mw509748) and le_030 (mw509749). phylogenetic analysis bootstrapping provided strong support for all the nodes. leishmania subgenres were separated for one cluster supported by 99.3%. viannia subgenus, this was divided in l. braziliensis complex species and l. equatorensis with a support of 61.5%. leishmania subgenus was divided in l. mexicana complex and l. donovani complex, where sequences of this study cluster with l. donovani complex (figure 2). molecular diagnosis leishmania spp. were detected by amplification of 3 different targets by pcr assay, hsp70c, hsp70d and the its1 of the parasite. one hundred fifty eight of the 173 dogs were positive for leishmania spp. in at least one of the three tests, which represents a prevalence of 91.33%. on the other hand, 63.29% (100/158) of the pcr‑positive dogs did not show any clinical sign compatible with the disease and only 36.71% (58/158) of the dogs presented one or more clinical signs of canine leishmaniasis (table 2). in addition, 4.43% (7/158) of the positive dogs remain during the day in the houses, and 79.75% (126/158) stayed outside of the house at night, in the backyard or in a close neighborhood. efficiency of different pcr assays was compared, taking as a parameter the number of samples detected as positive for the presence of the parasite. one hundred thirty‑eight dna samples were positive for the hsp70 gene of leishmania spp. through the hsp70c‑pcr, 140 by using the hsp70d‑ pcr, and 22 dna samples were positive for the its‑1 of leishmania spp. (table 2). according to which the hsp70d pcr was proven to be highly efficient for the detection of leishmania spp. and its1‑pcr had the fewest detections of the parasite in canine blood. risk factors regarding the housing conditions of the dogs, the wall and floor materials were mostly concrete (65.32% and 64.16%, respectively), the garbage disposal more frequent was in the municipal landfill (41.04%) and burning (35.26%), the 49.71% had agricultural production, specifically poultry. in table ii. distribution of leishmania-positive canines by village. village detection of leishmania spp. clinical signs its1 hsp70d hsp70c positive canines (n) + + + + buenos aires 0 9 9 0 9 0 9 6 3 calambeo 0 11 11 0 11 0 11 4 7 carmen de bulira 4 5 6 3 6 3 9 4 5 cay 0 13 13 0 13 0 13 9 4 coello cocora 5 27 26 6 26 6 28 9 19 el salado 2 12 5 9 4 10 12 5 7 gamboa 6 3 6 3 2 7 9 2 7 san bernardo 0 11 11 0 11 0 11 0 11 totumo 5 60 53 12 56 9 56 19 37 total 22 151 140 33 138 35 158 58 100 leidy alejandra giraldo-martínez et al. leishmania spp. in dogs, central west colombia veterinaria italiana 2022, 58 (4), 379-389. doi: 10.12834/vetit.2632.16820.2 383 solano‑gallego et al., 2001). in brazil, prevalence is reported between 3.2% to 50.3%, depending on the area and methods (marcondes & day, 2019), in this endemic country, serological tests were found to have low capacity for leishmania spp. detection when it is compared with molecular tests in canines (lopes et al., 2017). the most widely methods used for the diagnosis of canine leishmaniasis in latin america are the elisa, ifat and western blot technique as well as pcr (alves & bevilacqua, 2004; trevisan et al., 2015). pcr has high sensitivity and specificity, and its use reduces the costs of the leishmania spp. detection (de paiva et al., 2009; sundar & rai, 2002). on the other hand, pcr can detect the presence of leishmania spp. in discussion molecular diagnosis prevalence of leishmania spp. in canine population in ibagué (91.33%) is highest compared with that reported by molecular methods in chaparral‑ tolima (7.3%) (santaella et al., 2011), sincelejo‑sucre (34.9%), sampúes‑sucre (35.7%) and ovejas‑sucre (11.1%) (paternina‑gómez et al., 2013). in other countries, prevalence values were 24.8% by qpcr in sichuan province‑china, 48.4% by ifat in italy, 26% by elisa and 63% by pcr in mallorca‑spain, and 66% by immunoblot and 80% by pcr in france (berrahal et al., 1996; paradies et al., 2006; shang et al., 2011; figure 2. phylogenetic tree of leishmania species and the studied samples, based in hsp70c region sequence. a bootstrapped nj tree constructed using geneious prime software (version 2021.0.3). numbers at nodes represent the percentage of 1000 bootstrap iterations supporting the branch. t. cruzi was used as outgroup. leishmania spp. in dogs, central west colombia leidy alejandra giraldo-martínez et al. 384 veterinaria italiana 2022, 58 (4), 379-389. doi: 10.12834/vetit.2632.16820.2 different types of clinical samples such as blood, lymph nodes, skin, conjunctival smear and bone marrow aspirates (lachaud et al., 2002; lombardo et al., 2012; manna et al., 2004; reale et al., 1999; reis et al., 2013). several studies reported that amplification of its1 through pcr is highly specific for detection of leishmania spp. and highly sensitive as it is capable of detecting 0.2 parasites per sample (schönian et al., 2003; toz et al., 2013). graça et al. (2012) reported a sensitivity of 81.4% of its1‑pcr and 73.2% of hsp70c‑pcr, using dna extracted from skin biopsies. however, in this study, sensitivity of its1‑ pcr is lower than that obtained with hsp70‑pcr, this may be due to the fact that the blood is less specific (80%) and sensitive (85%) than the aspirates of the lymph nodes (specificity, 100%; sensitivity, 100%) (reale et al., 1999), given that blood tends to have a variable parasite load depending on the stage of infection (manna et al., 2004). nonetheless, the use of blood for the diagnosis of leishmania spp. has shown to be effective in the detection of the parasite mainly in asymptomatic animals (monteiro et al., 2019)” for “… pcr is highly specific for detection of leishmania spp. and highly sensitive as it may detect 0.2 parasites per sample … sensitivity of its1‑ pcr is lower than that obtained with hsp70‑pcr, this may be due to the different matrix (reale et al., 1999; manna et al., 2004).nonetheless, use of blood for the diagnosis of leishmania spp. has shown to be effective in the detection of the parasite mainly in asymptomatic animals (monteiro et al., 2019) as in our study and it is recommended as more suitable compared with aspiration‑based methods (albuquerque et al., 2017). furthermore, blood sampling is a procedure,simple to perform, less stress, with low cost and its repeatability is easier compared to bone marrow sampling or biopsy of lymph nodes (carvalho et al., 2009; lachaud et al., 2002; reale et al., 1999). amplification of the hsp70 gene represents a valuable tool in the detection of leishmania spp., since it contains between five and six hsp70‑i copies followed by one hsp70‑ii copy (ramirez et al., 2011) and its effectiveness in the detection of asymptomatic infections was evidenced in the present study. hsp70 gene region is the most highly conserved in sequence and function in all organisms, with a lower rate of genetic diversity than other markers for instance gp63 gene, rdna genes, or its1 (dabirzadeh et al., 2016), becoming a relevant target for the detection of leishmania species. risk factors differences in clinical manifestations between dogs, such as the severity of clinical signs and the time of onset of the disease, may vary depending on the individual immune response of the infected animals as well as genetic factors, age, nutritional status, and the virulence of leishmania strains (alvar et al., 2004; man et al., 2020; moreno et al., 1999; quinnell et al., 2003). in our study, 63.29% of the pcr‑positive dogs did not present clinical signs of canine leishmaniasis or other symptoms. similarly, it has reported that in endemic areas of visceral leishmaniasis 85% of dogs infected by l. infantum are asymptomatic (dantas‑ torres & brandao‑filho., 2006). nonetheless, it has been described that approximately 46% of infected dogs acquire the infection and develop the disease immediately, another 44% of dogs develop the disease later, and 10% of them can remain without clinical signs of visceral leishmaniasis throughout their lives (moreno & alvar, 2002). dogs have the ability to transmit leishmania spp. to the vector and, consequently, to other canines or humans regardless of whether they present or no clinical signs. the transmission cycle of the parasite could be favored by the asymptomatic infection of leishmania spp. in the canine (dantas‑torres & brandao‑filho, 2006; moshfe et al., 2009; soares et al., 2011). presence of lutzomyia longiflocosa has been reported in the rural zone of ibagué, tolima (guzmán‑barragán et al., 2021). furthermore, in several tolima municipalities (planadas, rovira, casablanca, herveo, ortega, san antonio and chaparral), it has been reported the presence of l. columbiana, l. longiflocosa, l. micropyg, l. rangelina, l. suapensis, l. nuneztovari, l. atroclatava (bejarano et al., 2003; bejarano et al., 2006; cárdenas et al., 1999; contreras et al., 2012; morales et al., 1981; pardo et al., 2006; prado et al., 1999; sierra et al., 2000). transmission potential of infected dogs can differ between symptomatic and asymptomatic animals. by xenodiagnosis methods it has been observed that asymptomatic dogs have a lower capacity to infect sandfly vectors (travi et al., 2001; verçosa et al., 2008), while other studies determined that symptomatic animals are more likely to spread the infection (guarga et al., 2000; molina et al., 1994). some studies have determined that the physical traits of dogs such as height, spaying, purebred, and age (older than 2 years) can have a positive correlation with the occurrence of leishmaniasis (belo et al., 2013a). additionally, associated factors such as the presence of ectoparasites, contact with other animals, dog’s permanence in the backyard, forest areas near dwelling and the presence of birds in the domestic environment, may play a role in attracting sandflies and predispose the infection of dogs (belo et al., 2013b; curi et al., 2014; coura‑vital et al., 2011). however, significant associations were leidy alejandra giraldo-martínez et al. leishmania spp. in dogs, central west colombia veterinaria italiana 2022, 58 (4), 379-389. doi: 10.12834/vetit.2632.16820.2 385 conflict of interest statement the authors declare no conflict of interest. statement of animal rights all the experimental procedures followed the guidelines of the bioethics committee of the central research office of the university of tolima based on law 84/1989 and resolution 8430/1993 and complied with the guidelines for animal care and use in research and teaching. the bioethic committee of the central research office approved bioethics aspects in the agreement 2239 of june 25 at 2019, between the municipal health secretariat and the university of tolima. references akhoundi m., kuhls k., cannet a., votýpka j., marty p., delaunay p. & sereno d. 2016. a historical overview of the classification, evolution, and dispersion of leishmania parasites and sandflies. plos negl trop dis, 10(3), e0004349. https://doi. org/10.1371/journal.pntd.0004349 albuquerque a., campino l. & cardoso l. 2017. evaluation of four molecular methods to detect leishmania infection in dogs. parasit vectors. 10, 57. https://doi.org/10.1186/s13071‑017‑2002‑2 almeida a., mendonça a. & sousa v. 2010. prevalência e epidemiologia da leishmaniose visceral em cães e humanos, na cidade de cuiabá, mato grosso, brasil. cienc rural, 40, 1610‑1615. https://doi. org/10.1590/s0103‑84782010005000102 alvar j., cañavate c., molina r., moreno j. & nieto j. 2004. canine leishmaniasis. adv parasitol, 57, 1‑88. https://doi.org/10.1016/s0065‑308x(04)57001‑x alvar j., vélez i.d., bern c., herrero m., desjeux p., cano j., jannin j., den boer m. & who leishmaniasis control team. 2012. leishmaniasis worldwide and global estimates of its incidence. plos one, 7(5), e35671. https://doi.org/10.1371/journal. pone.0035671 alves w.a. & bevilacqua p.d. 2004. reflexões sobre a qualidade do diagnóstico da leishmaniose visceral canina em inquéritos epidemiológicos: o caso da epidemia de belo horizonte, minas gerais, brasil, 1993‑1997. cad saude publica, 20(1), 259‑265. https://doi.org/10.1590/s0102‑ 311x2004000100043 bejarano e.e. 2006. lista actualizada de los psicódidos (diptera: psychodidae) de colombia. folia entomológica mexicana, 45(1),47‑56. issn: 0430‑8603. bejarano e.e., sierra d. & vélez i.d. 2003. novedades en la distribución geográfica del grupo not found between the risk factors evaluated and the presence of leishmania spp. in the dogs of the municipality. this can be attributed to the immune status of the individual dogs and because of both leishmania‑positive and leishmania‑negative dogs have homogeneous factors, therefore odds ratios did not show significance. additionally, other studies have shown that there are not significant associations between age and the presence of leishmania spp. in the canine population, age is not a strong predictor for leishmania spp. infection and dogs of all ages may be reservoirs in the study areas. moreover, according to belo et al. 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nigeria. 2department of veterinary microbiology, faculty of veterinary medicine, university of ibadan, ibadan, nigeria. 3department of veterinary public health and preventive medicine, university of ibadan, ibadan. nigeria. 4department of veterinary vaccine production and quality control, pan african university institute of life and earth sciences (paulesi), university of ibadan, ibadan. nigeria. *corresponding author at: department of avian medicine, pan african university institute of life and earth sciences (paulesi), university of ibadan, ibadan. nigeria. e‑mail: adedamilolak@gmail.com. adedamilola kolapoi1*, elizabeth amosun2, olufemi olatoye3, fiyinfoluwa adeoye4, omolade oladele1* keywords antibiotics, antimicrobial resistance, ducks, escherichia coli o157, indigenous chicken, live-bird markets, salmonella species. veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 accepted: 15.02.2022 | available on line: 31.12.2022 summary this study investigated the occurrence of escherichia coli o157:h7 and salmonella spp. and their antimicrobial susceptibility from ducks and indigenous chickens in major live-bird markets at ibadan, oyo state, nigeria. thirty-one cloacae samples were each collected from both ducks and indigenous chickens, in three different sample locations for a total of 186 cloaca swab samples. isolation of escherichia coli (e. coli) was done using macconkey agar and sorbitol macconkey agar selective for e. coli o157:h7, while serological latex agglutination test kit was used to confirm isolates. rappaport vassiliadis and xylose lysine deoxycholate agar were used for salmonella spp. antibiotic susceptibility was determined using the disc diffusion method and interpreted using the clsi 2020 standards. data were analyzed using descriptive statistics and fisher’s exact test (p ≤ 0.05). escherichia coli o157:h7 was confirmed in 31 samples (16.7%). e. coli isolates showed high resistance (90.393.5%) to cefuroxime, cefixime, ceftazidime, and amoxicillin, while they were highly susceptible to ofloxacin (96.8%) and gentamycin (80.7%). salmonella was confirmed in 24 samples (12.9%). salmonella showed 100% resistance to cefuroxime, cefixime ceftazidime, and amoxicillin, but was highly susceptible to gentamycin (91.7%) and nitrofurantoin (66.7%). no statistically significant association (p<0.05) was observed between the occurrence of e. coli o157 and salmonella within the three live-bird markets. this study reveals that e. coli and salmonella spp. occur in ducks and indigenous chickens from major live bird markets in ibadan, oyo state with antimicrobial susceptibility. findings from this study underscores the need for further studies on these pathogenic organisms from ducks in nigeria because there is paucity of data on this species of poultry that may serve as reservoir for these zoonotic organisms. please refer to the forthcoming article as: kolapo et al. 2022. occurrence and antimicrobial resistance of escherichia coli o157:h7 and salmonella species isolated from ducks and indigenous chickens in live-bird markets in ibadan, oyo state, nigeria vet ital. doi: 10.12834/ vetit.2553.16733.2 escherichia coli o157:h7 and salmonella species isolated from ducks and indigenous chickens in live-bird markets in ibadan, oyo state, nigeria e. coli o157 and salmonella in live bird markets in nigeria kolapo et al. 370 veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 the spread of zoonotic pathogens such as salmonella spp. and escherichia coli o157:h7 poses threat to public health, as they may be transferred to humans through the food chain (lim et al., 2010; chang et al., 2012) and also antimicrobial resistance (amr) of e. coli and salmonella spp. is a worldwide health concern (peterson and kaur, 2018). different organizations such as the centers for disease control and prevention (cdc), infectious diseases society of america, world economic forum, and the world health organization (who) have declared antibiotic resistance as a global public health concern (michael et al., 2014; spellberg et al., 2016). this study, therefore, investigated the occurrence of e. coli o157:h7 salmonella spp. and their antimicrobial susceptibility in ducks and indigenous chickens from major live bird markets in ibadan, oyo state, nigeria. materials and methods collection of samples three lbms located in shasha, molete, and bode areas of ibadan, oyo state, southwest nigeria were used for this study. the sample size was determined as described by thrusfield, (2005). using a prevalence of 11.4% from previous study from roadside lbms in zaria, kaduna state, nigeria (ejeh et al., 2017) and an absolute error of 5%, the sample size was calculated to be 155, plus 10% non-responsive samples gave a total of 186 samples. one hundred and eighty-six (186) cloacae swab samples were collected; 62 birds per lbms, 31 each from chicken and duck. adequate precaution was taken to avoid contact between cotton swab tips and the surrounding of the anus to avoid crosscontamination. all samples were properly labeled and transported on ice to the bacteriology laboratory of the department of veterinary microbiology, university of ibadan, for bacteriological analysis. escherichia coli o157 identification and isolation samples were pre-enriched using 9mls of buffered peptone water (lab m, uk) and incubated at 37ºc for 24 hours. after which, 0.1ml of the pre-enrichment broth was inoculated onto prepared plates of macconkey agar (oxoid, uk) and incubated at 37ºc for 24 hours. pink, round, medium sized-colonies taken to be e. coli were sub-cultured onto sorbitol macconkey agar, smac, (oxoid, uk) and incubated at 37ºc for 24 hours. colorless colonies (non-sorbitol fermenter) were purified by sub-culturing onto freshly prepared sorbitol macconkey agar. the purified isolates were used for further analyses; introduction escherichia coli o157:h7 is the most widely known shiga-toxin or verotoxin producing organism. it may cause severe illnesses from simple bloody diarrhea to hemorrhagic colitis (hc), hemolytic uremic syndrome (hus), and thrombotic thrombocytopenic purpura in humans (thomas and elliott 2013; atnafie et al., 2017; erickson et al., 2019). the organism was incriminated in an outbreak from an unknown source in march 2021 affecting several people in seven states of the united states (cdc, 2021). enterohemorrhagic o157:h7 is mainly transmitted to humans by the consumption of contaminated food and water, or by direct contact with animals, their feces, and contaminated soil. person-to-person transmission can contribute to disease spread during outbreaks; however, humans do not appear to be a maintenance host for this organism (ahn et al., 2009). salmonella spp. is one of the most important food-borne pathogens. it was the most frequently reported causative agent in bacterial food-borne disease outbreaks and has caused the highest number of deaths due to food-borne illnesses in the european union in 2017 (efsa and ecdc, 2018). poultry meat has been identified as one of the most important sources (fao and who, 2009) of human infections. as the consumption of poultry meat is increasing every year, prevention of salmonella spp. contamination in the poultry meat production chain remains very important. poultry and poultry meat can become contaminated with  salmonella  spp. during the entire  poultry production  chain, that is from the breeder farm, production farm, transportation, slaughterhouse, and retail (hardie et al., 2019; shang et al., 2019). live bird markets (lbms) are termed as a place where live birds are sold to customers (oloso, 2019). in nigeria, about 90% of poultry product marketing is done by sales of live birds with less than 2% comprising processed or frozen chickens. an estimate that over 2 million live birds are sold daily in lbms across nigeria highlights the position of live bird markets in control of avian diseases (muhammed, 2008). the high concentration and interaction of a wide variety of birds coming from different sources make lbms a high-risk area for disease transmission (choi et al., 2005). these processes involve the collection of chickens from multiple households who own few numbers of marketable ages, then mixed at various densities starting from the village markets to the big terminal markets that lead to the spread of infection across a long distance. in addition, multiple species of birds in confined spaces in lbms create a stressful condition, cross-infection, and increased surface contamination (mai et al., 2004). kolapo et al. e. coli o157 and salmonella in live bird markets in nigeria veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 371 previously seeded with an overnight culture of the test organisms using a sterile cotton swab and incubated at 37 oc for 24 hrs, after which zones of inhibition were measured and interpreted accordingly. statistical analysis the ibm® spss® statistical software (spss) was used for data analysis. fisher’s exact test was used to determine the statistically significant association of the prevalence of salmonella spp. and e. coli o157 in ducks and indigenous chickens because the numbers within the two by two categories from our result had values lower than 5, using confidence levels (cl) of 95% and p≤0.05. results a total of 156 (83.9%) escherichia coli isolates were obtained from 186 cloacae swab samples of these, 75 (80.7%) were from ducks and 81 (87.1%) indigenous chickens from three live bird (shasha, molete and bode) markets in ibadan. eighty-seven (46.7%) of the 156 isolates were non-sorbitol fermenters, of which 57 (30.7%) samples showed distinct e. coli characteristics with biochemical test, while 31 (16.7%) were serologically identified as escherichia coli o157:h7 (table i). there was no statistically significant association (p=0.14) in the occurrences of e. coli o157 in the three live-bird markets (p>0.05). a total of 40 (21.5%) salmonella spp. isolates were obtained from 186 cloacae swab samples. of these, 25 (26.9%) ducks and 15 (16.1%) indigenous chickens from three live bird (shasha, molete and bode) markets in ibadan. thirty-three (17.7%) of the 40 isolates were recorded on further purification on xylose deoxycholate agar. using biochemical test, salmonella was confirmed in 24 (12.9%) samples (table ii). gram staining, catalase test, indole test, citrate test, sugar fermentation, and triple sugar iron test, which were performed for e. coli identification. serological latex agglutination test using e. coli o157 antiserum (oxoid, uk), was used for confirmation (according to manufacturer’s instruction) salmonella spp. identification and isolation samples were pre-enriched using 9mls of buffered peptone water (lab m, heywood, uk) and incubated at 37 ºc for 24 hours. after which 0.1ml of the preenrichment broth was inoculated onto 10mls of rappaport vassiliadis (oxoid, hampshire, uk), for selective enrichment of salmonella and incubated at 37 ºc for 24 hours. a loopful from rappaport vassiliadis was inoculated onto xylose lysine deoxycholate, xld, (oxoid, uk) agar, which was incubated at 37 ºc for 24 hours. the presumptive isolates that appeared pinkish, with or without small transparent black centers to predominantly black colonies were subcultured on xylose lysine deoxycholate to obtain a pure culture. the purified isolates were used for further identification; gram staining, catalase test, indole test, citrate test, sugar fermentation, and triple sugar iron test were performed for salmonella identification. antibiotics sensitivity testing confirmed e. coli o157 and purified salmonella spp. isolates were tested for their susceptibility to antimicrobial agents using the agar disc diffusion method (clsi, 2020). commercially available gram-negative antibiotics multi-disc from abtek, liverpool, uk, was used; comprising of ceftazidime (30 μg/disc), cefuroxime (30 μg/disc), gentamicin (10μg/disc), cefixime (5 μg/disc), ofloxacin (5 μg/ disc), amoxicillin (30 μg/disc), nitrofurantoin (5 μg/ disc) and ciprofloxacin (30 μg/disc). the antibiotic discs were placed on muller hilton agar plates table i. e. coli o157 isolation in ducks and indigenous chickens in oyo state, nigeria. sample source (live-bird markets) number of samples presumptive e. coli macconkey agar (mac) presumptive e. coli o157 sorbitol macconkey agar (smac) biochem test e. coli o157 serology e. coli o157:h7 ducks indigenous chickens ducks indigenous chickens ducks indigenous chickens ducks indigenous chickens ducks indigenous chickens molete 31 31 23 27 13 13 7 8 3 2 shasa 31 31 27 30 11 18 6 14 4 11 bode 31 31 25 24 16 16 13 9 7 4 total 93 93 75 81 40 47 26 31 14 17 overall total 186 156 87 57 31 e. coli o157 and salmonella in live bird markets in nigeria kolapo et al. 372 veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 there was no statistically significant association (p= 0.72) in the occurrences of salmonella in the three live-bird markets (p>0.05). table ii. salmonella species isolation in ducks and indigenous chickens in oyo state, nigeria. table iii. antibiotic susceptibility of e. coli o157 isolated in ducks and indigenous chickens in oyo state, nigeria. sample source (live-bird markets) number of samples salmonella spp xylose lysine deoxycholate (xld) salmonella spp (xld) purification biochemical test salmonella spp ducks indigenous chickens ducks indigenous chickens ducks indigenous chickens ducks indigenous chickens molete 31 31 7 6 7 4 5 3 shasa 31 31 15 7 12 5 8 5 bode 31 31 3 2 3 2 1 2 total 93 93 25 15 22 11 14 10 overall total 186 40 33 24 antimicrobial class antimicrobial agent disk potency number of isolates, t=31 sensitive [n (%)] intermediate [n (%)] resistance [n (%)] cephalosporins (2nd generation) cefuroxime 30μg 1 (3.2) 1 (3.2) 29 (93.5) cephalosporins (3rd generartion) cefixime 5μg 2 (6.5) 0 (0.0) 29 (93.5) ceftazidime 30μg 2 (6.5) 0 (0.0) 29 (93.5) penicillins augmentin 30ug 2 (6.5) 1 (3.2) 28 (90.3) aminoglycoside gentamycin 10μg 25 (80.7) 1 (3.2) 5 (16.1) floroquinolone ciprofloxacin 5μg 10 (32.3) 13 (41.9) 8 (25.8) ofloxacin 5μg 30 (96.8) 0 (0.0) 1 (3.2) nitrofuran nitrofurantoin 300μg 18 (58.1) 1 (3.2) 12 (38.7) table iv. antibiotic susceptibility of salmonella spp. antimicrobial class antimicrobial agent disk potency number of isolates, t=24 sensitive [n (%)] intermediate [n (%)] resistance [n (%)] cephalosporins (2nd generation) cefuroxime 30μg 0 (0.0) 0 (0.0) 24 (100) cephalosporins (3rd generartion) cefixime 5μg 0 (0.0) 0 (0.0) 24 (100) ceftazidime 30μg 0 (0.0) 0 (0.0) 24 (100) penicillins augmentin 30ug 0 (0.0) 0 (0.0) 24 (100) aminoglycoside gentamycin 10μg 22 (91.7) 0 (0.0) 2 (8.3) floroquinolone ciprofloxacin 5μg 1 (4.2) 18 (75.0) 5 (20.8) ofloxacin 5μg interpretative break point not available nitrofuran nitrofurantoin 300μg 16 (66.7) 2 (8.3) 6 (25.0) antibiotics susceptibility of e. coli o157 and salmonella isolates the antibiotic susceptibility profile of e. coli o157 (31) and salmonella (24) isolates to the eight antibiotics is shown in table iii and table iv, respectively. kolapo et al. e. coli o157 and salmonella in live bird markets in nigeria veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 373 table v. resistance and multi-drug resistance pattern of e. coli o157 and salmonella spp. isolated in ducks and indigenous chickens in oyo state, nigeria. antibiotics number of resistance e. coli o157 isolates (%) number of resistance salmonella isolates (%) duck indigenous chicken duck indigenous chicken caz 12 (38.7) 17 (54.8) 14 (58.3) 10 (41.7) crx 13 (41.9) 16 (51.6) 14 (58.3) 10 (41.7) cxm 13 (41.9) 16 (51.6) 14 (58.3) 10 (41.7) aug 13 (41.9) 15 (48.4) 14 (58.3) 10 (41.7) nit 9 (29.0) 3 (9.7) 5 (20.8) 1 (4.2) cpr 4 (12.9) 4 (12.9) 2 (8.3) 3 (12.5) gen 1(3.2) 4 (12.9) 2 (8.3) 0 (0) ofl 0 (0) 1 (3.2) 0 (0) 0 (0) aug-nit 9 (29.0) 2 (6.5) 5 (20.8) 1 (4.2) aug-cpr 4 (12.9) 4 (12.9) 2 (8.3) 3 (12.5) aug-gen 1 (3.2) 4 (12.9) 2 (8.3) 0 (0) gen-cpr 0 (0) 1 (3.2) 1 (4.2) 0 (0) aug-nit-cpr 2(6.5) 2 (6.5) 1 (4.2) 0 (0) nit-aug-gen 1 (3.2) 0 (0) 2 (8.3) 0 (0) gen-aug-cpr 0 (0) 1 (3.2) 1 (4.2) 0 (0) escherichia coli o157 showed very high resistance to 4 (90.3-93.5%) of the antibiotics tested, specifically to the cephalosporins: cefuroxime, cefixime and ceftazidime, and also to amoxicillin. intermediate susceptibility to ciprofloxacin (41.9%), while it was highly susceptible to ofloxacin (96.8%) and gentamycin (80.7%), then nitrofurantoin (58.1%), (figure 1 and 2). figure 1. antibiotics resistance pattern of e. coli and salmonella spp. isolated in ducks and indigenous chickens in oyo state, nigeria. figure 2. antibiotics sensitivity pattern of e. coli and salmonella spp. isolated in ducks and indigenous chickens in oyo state, nigeria. salmonella showed 100% resistance to four of the antibiotics tested; specifically, to the cephalosporins; cefuroxime cefixime and ceftazidime and also to amoxicillin. it also showed high intermediate susceptibility to ciprofloxacin (75%) while it was highly susceptible to gentamycin (91.7%) and nitrofurantoin (66.7%), (figure 1 and 2). majority of e. coli o157 and salmonella strains showed resistance to at least two antimicrobial agents. muti-drug resistance (defined as to be non susceptible to at least three or more classes of antimicrobial tested) was highest for e. coli o157 (77.5-80.4%), and observed in cefuroxime-cefixime-ceftazidimeamoxicillin antibiotics, while salmonella spp. had 100% multidrug resistance to the same group of antibiotics. multi-drug resistance was lowest, 0% for e. coli o157 and 4.2% for salmonella spp. to gentamycinamoxicillin-nitrofurantoin-ciprofloxacin combination (table v). key: caz– ceftazidime crx – cefuroxime gen– gentamycin cxm– cefixime oflofloxacin augaugmentin nitnitrofuratoin cpr–ciprofloxacin e. coli o157 and salmonella in live bird markets in nigeria kolapo et al. 374 veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 isolation of e. coli o157 and salmonella spp. from this study with a prevalence of 16.7% and 12.9%, respectively in both duck and indigenous chicken in live bird markets. the presence of these pathogenic organisms predisposes live bird sellers as well as intermediaries having direct contact with birds to be at risk of occupational hazards (ajetombi et al., 2010). others at risk of infection include people who consume raw vegetables because wastes from poultry farms are used for irrigation and fertilizer in vegetable farms in nigeria. pathogenic e. coli from the gut can also contaminate chicken meat and thus may constitute serious public health problems (ejeh et al., 2017). a possible explanation for the high occurrence in indigenous chicken and ducks from lbms in this study may be because live bird markets are high-risk areas for disease transmission due to high concentration and interaction of a wide variety of birds that come from different sources (choi et al., 2005) and multiple species of birds that are kept together in a confined space and common practice of keeping ducks with indigenous chicken which increase the chance of spread of pathogenic organisms (mai et al., 2004; siraju et al., 2016). multiple antibiotics resistance was observed for both e. coli o157 and salmonella spp.. there was 90.3%100% resistance of e. coli o157 and salmonella spp. to cefuroxime, ceftazidime, cefixime, and amoxicillin. this is similar to reports from other studies in poultry in nigeria with salmonella and escherichia coli showing 100% resistance to amoxicillin (adeyanju and ishola, 2014; gbadamosi et al., 2018). escherichia coli o157 and salmonella spp. were also resistant to gentamycin (16.1% and 8.3%), ciprofloxacin (25.8% and 20.8%), and nitrofurantoin (38.7% and 25%), respectively, this is in contrast with other studies from poultry in nigeria that reported a greater (>70%) resistance for these antibiotics (nfongeh et al., 2018; gbadamosi et al., 2018). in this study, e. coli o157 showed the highest sensitivity to ofloxacin (96.8%) and gentamycin (80.7%), while salmonella spp. had the highest sensitivity to gentamycin (91.7%) and nitrofurantoin (66.7%). this is similar to the findings of mohammad et al., 2014 and cvl report 2014/15 in poultry with 100% sensitivity for e. coli and salmonella spp. to gentamycin; greater (>70%) sensitivity of e. coli o157 to gentamycin; mude et al., 2017 (88.46%) and altalhi et al., 2010 (71.7%) in ethiopia, bangladesh and saudi arabia, respectively. the prevalence of antimicrobial-resistant pathogens and the high level of resistance to multiple antibiotics may be due to excessive and uncontrolled use of antibiotics in poultry which may be as a result of these antibiotics being freely available and readily affordable, or from the transfer of resistance gene(s) from another host in the same production environment (swartz 2002; hayes et al., 2004; levy discussion in this study, the cloacal carriage of e. coli o157 and salmonella spp. were investigated amongst indigenous chickens and ducks sold in three selected live-bird markets in ibadan, oyo state, nigeria. escherichia coli had a prevalence of 30.7% and e. coli o157, 16.7%. isolation rate of e. coli o157 was higher in indigenous chicken (18.3%) than in ducks (15.1%), though there was no statistical degree of association in occurrence among the two poultry birds. the 16.7% prevalence of e. coli o157:h7 from this study is similar to reports from aibinu et al., 2007 which reported 10% isolation from 50 cloacal samples of chicken in lagos and 14.5% chicken samples in ogun state. in addition, ojo et al., 2009 confirmed e. coli o157:h7 strains in the faeces of poultry sampled from different farms in nigeria. also, olatoye et al., 2012 reported 13% and 14% isolation of e. coli o157:h7 from lagos and ibadan poultry farms, respectively. furthermore, 13.4% prevalence has been reported from chicken cloacal samples examined in eastern ethiopia (mude et al., 2017). the prevalence observed in this study is however higher compared to the 5.7% in local chicken from zaria (ejeh, et al., 2017) and 6.7% in strayed chicken from calabar (nfongeh et al., 2018). salmonella spp. had a prevalence of 12.9%, the isolation rate in ducks was 15.1% and 10.8% in indigenous chicken, suggesting that there was no significant difference in the occurrence of salmonella spp. in the two poultry species. our finding for salmonella spp. occurrence in local chicken is similar to reports of 12% prevalence in local chickens along the roadside at hanwa, zaria, nigeria (ejeh et al., 2017). while it is lower compared to 59.1% prevalence from poultry cloacal swabs from calabar, (yhiller and bassey, 2015) and 52.5%, from chicken cloacal samples from owerri (umeh and enwuru, 2014), nigeria. salmonella spp. prevalence in ducks from this study is higher than (4.6% and 8.7%) prevalence reported in duck cloacal samples from taiwan (tsai and hsiang, 2005) mekong delta, vietnam (tran et al., 2004), respectively but lower than the prevalence from duck cloacae content from korea (20.7%) (kim et al., 2016), from retail duck meat in vietnam 22.3% of (phan et al., 2005) from ducks from dinajpur, bangladesh (39,58%)(rahman et al., 2016), from duck cloacal samples in sichuan province, china (43,5%) (xinfeng et al., 2020), from duck meat samples in south korea (51,3%) (yoon et al., 2014), from imported day-old duckling in brazil (65,0%) (ribeiro et al., 2003). the variations in prevalence of e. coli and salmonella spp. generally might be due to different sampling techniques, areas, time, and lack of strict hygiene measures among farms and live bird markets (abah et al., 2017). the public health importance is the kolapo et al. e. coli o157 and salmonella in live bird markets in nigeria veterinaria italiana 2022, 58 (4), 369-378. doi: 10.12834/vetit.2553.16733.2 375 poultry farmers and retailers; on the standard of animal production and hygiene practices that allows prevention of pathogenic organisms in poultry and also on the dangers involved in exposure to the various pathogenic organisms that can be associated with poultry such as e. coli o157 and salmonella spp.. there is need to legislate and enforce laws to limit the prescription, dispensing and administration of antibiotics and other drugs to only veterinarians for prophylactic and treatment purposes. in addition, animal health authorities should ban the use of antibiotics as growth promoters in nigeria so as to curb the spread of antimicrobial resistance. more studies should be carried out on the occurrence and antimicrobial resistance of pathogenic organisms in ducks in nigeria because there is paucity of report on this matter. also, studies involving serotype-specific amr patterns and risk factors should be carried out as well as assessment of avian pathogenic e. coli (apec) that has a negative impact on poultry health. acknowledgements the authors are thankful to the director and management team of pan african university institute of life and earth sciences (including health and agriculture) (paulesi). university of ibadan, for providing enabling environments, facilities and funds to carry out this research. and marshall 2004; nelson et al., 2007; usgao 2011a; salihu et al., 2014). it is worthy to note that there is no previously known prevalence of e. coli o157 and salmonella spp. in ducks in nigeria before this study. limitation as at the time of compiling this report, there was non-availability of anti-sera and other reagents for further molecular characterization which was occasioned by recent global coronavirus disease (covid-19) pandemic, which resulted in our change of protocol. conclusions the presence of these pathogenic organisms can cause serious infections that can result in gastroenteritis, hemorrhagic 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(verified december, 2011). 57 parole chiave botulismo animale, clostridium botulinum, bestiame, tossina mosaico dc, italia. riassunto questo articolo descrive l'epidemia di botulismo che ha colpito un allevamento di vacche da latte in toscana nel settembre 2012 . il botulismo animale si presenta raramente in italia ma, come in questo caso, può essere causa di focolai di malattia associati ad alta mortalità. tutte le prove diagnostiche atte ad evidenziare eventuale presenza di batteri, sostanze tossiche o squilibri elettrolitici. sono risultate negative. la multiplex polymerase chain reaction (pcr) per rilevare il gene bont ha individuato nel ceppo di clostridium botulinum tipo dc l'agente patogeno responsabile della morte degli animali. le prove biologiche hanno successivamente confermato i risultati del test pcr. l'elevata mortalità e la presenza di un quadro clinico non caratteristico non hanno facilitato il percorso diagnostico determinando un ritardo che ha invalidato la possibile identificazione della fonte dell'infezione. una diagnosi più veloce avrebbe potuto ottimizzare l'indagine epidemiologica del focolaio, favorendo l’attuazione di misure che prevenissero eventuali casi futuri. la gravità dell'epidemia dimostra che lo screening per il botulismo animale dovrebbe sempre far parte dei protocolli diagnostici utilizzati per indagare i casi di mortalità iperacuta del bestiame senza apparente causa. grave epidemia di botulismo in un allevamento di bovine in toscana keywords animal botulism, clostridium botulinum, cattle, mosaic dc toxin, italy. summary botulism in cattle is rarely reported in italy. this study describes an outbreak of botulism in a dairy herd in central italy in september 2012, and the notably high mortality rate it caused. differential diagnoses involving toxicology and bacteriology, and electrolyte imbalances, all proved negative. a multiplex polymerase chain reaction (pcr) for detecting the bont gene led to the identification of the causative agent as clostridium botulinum type dc. the presence of the toxin was confirmed subsequently via mouse bioassay. initially, the peracute deaths and ambiguous clinical signs delayed the diagnosis and, as a result, impeded identification of the source of the infection on the farm. the severity of the outbreak demonstrates that screening for animal botulism should always form part of the diagnostic protocols used to investigate sudden peracute deaths without apparent cause in livestock. veterinaria italiana 2019, 55 (1), 57-62. doi: 10.12834/vetit.768.3714.2 accepted: 15.07.2016 | available on line: 31.03.2019 1grosseto section, istituto zooprofilattico sperimentale delle regioni lazio e toscana “m. aleandri”, italy. 2laboratory of biotechnology applied to food safety, istituto zooprofilattico sperimentale delle regioni lazio e toscana “m. aleandri”, italy. 3national reference centre for botulism, istituto superiore di sanità. 4laboratory of anatomo-histopathology, istituto zooprofilattico sperimentale delle regioni lazio e toscana “m. aleandri”, italy. 5department of prevention, usl 9 gr, italy. 6agronomist, italy. 7private veterinary consultant, italy. *corresponding author at: istituto zooprofilattico sperimentale delle regioni lazio e toscana, sezione di grosseto laboratorio di diagnostica, via europa 30, grosseto, italy. tel.: +39 0564 456259, fax +39 0564 451990, e-mail: valeria.mariano@izslt.it. valeria mariano1*, alberigo nardi1, sandra gradassi1, paola de santis2, fabrizio anniballi3, stefano bilei2, francesco scholl4, bruna auricchio3, carla bielli5, massimo culicchi6 and giuseppe luca casali de rosa7 a severe outbreak of botulism in cattle in central italy 58 veterinaria italiana 2019, 55 (1), 57-62. doi: 10.12834/vetit.768.3714.2 outbreak of botulism in cattle mariano et al. recumbency (ortolani et al. 1997, bohnel et al. 2001, heider et  al. 2001, braun et  al. 2005, zarenghi et  al. 2006, senturk and cihan 2007). small doses of bonts may not lead to clinical signs for 5-10 days, while high doses, administered experimentally to cattle, lead to recumbency and death in 18-24 hours. thus, the course of illness in an outbreak may vary from 2-30 days, depending on the size of the dose ingested by the animals (whitlock 2004). although rare, cattle botulism outbreaks have been reported from various parts of the world (galey et al. 2000, heider et al. 2001, martin 2003, advisory committee on the microbiological safety of food 2006, aish et al. 2006, senturk and cihan 2007, payne et  al. 2011), including europe. between 2003 and 2009, suspected botulism emerged in ruminants in england and wales; 168 cattle and 19 sheep died, with in-herd mortality rates ranging between 5% and 80% (payne et  al. 2011). outbreaks in france increased significantly in the mid-1990s, peaking at 42 outbreaks in 1995 (afssa 2002); while finland reported its first case of bovine botulism in 2008 (myllykoski et  al. 2009). only 3 outbreaks of cattle botulism have been reported in italy so far. in 1950, bianchi studied an outbreak of botulism amongst cattle in the province of modena (bianchi 1950). the source of the toxin was identified in the carcass of a cat that had contaminated the cattle feed. a strain of c. botulinum type d was subsequently isolated from the viscera of 2 of the dead animals (bianchi 1950). zarenghi and colleagues described a high mortality rate due to botulism in dairy heifers (zarenghi et  al. 2006). during this outbreak, 99  out of 230 (43%) animals died in 1 month, which amalgamated to an 80% morbidity rate (zarenghi et  al. 2006). rosignoli and colleagues described an outbreak of botulism in a herd of cattle in which 8  friesian heifers and a weaned calf were involved (rosignoli et al. 2000). the clinical signs were suggestive of botulism, and the clinical diagnosis was confirmed by the detection of the bont in 4 blood samples, but not in the feed (rosignoli et al. 2000). in the present study we report on an outbreak of botulism in italian cattle. the outbreak yielded a high mortality rate, due to the c. botulinum type dc mosaic toxin. case history, clinical symptoms, pathological findings, and diagnosis in september 2012, an outbreak of botulism affected cattle on a farm in the province of grosseto in the region of tuscany (figure 1). at the time of the outbreak, the dairy herd of 480 friesians included 210 lactating cows. the herd was divided into 2 groups (group a and group b), separated by the foraging line and allocated to separate feeding introduction clostridium botulinum is a gram-positive, spore-forming, obligate anaerobic bacterium, commonly found in soils and aquatic sediments (whitlock 2004, anniballi et al. 2013b). in anaerobic, warm and humid conditions, the microorganism can multiply rapidly, producing the different types of neurotoxins responsible for botulism in humans and animals (senturk and cihan 2007). seven immunologically distinct botulinum neurotoxins (bonts) (serotypes a-g) are recognised. recently, an 8th serotype (bont/h) was identified (barash and arnon 2014, rossetto et al. 2014). this serotype appears to be a genetic arrangement (mosaic) of types a and f toxins, fully neutralised by the type a antitoxin (maslanka et  al. 2015). botulinum neurotoxin types a, b, e, and f are responsible for botulism in humans. types c, d, mosaics cd and dc toxins have been related to animal disease, though type a, b, and e toxins have also been recovered from animals (bianchi 1950, haagsma and ter laak 1979, neill et al. 1989, jean et al. 1995, ortolani et al. 1997, heider et  al. 2001, senturk and cihan 2007, lindström et al. 2010, krüger et al. 2012, payne et al. 2011, anniballi et  al. 2013b). botulin neurotoxin/ dc comprises the l chain (catalytic domain) and hn domain (translocation domain) of serotype d and the hc domain (binding domain) of serotype c, whereas bont/cd consists of the l chain and hn domain of serotype c and the hc domain of serotype d (rossetto et al. 2014). the neurotoxins are produced during the growth and autolytic phases of the bacterium (bohnel et al. 2001, senturk and cihan 2007). literature describes different forms of botulism for both humans and animals. in animals, the most common forms are due to the consumption of contaminated feeds (feed-borne) or as a consequence of toxico-infections. however, wound botulism is also recognised (whitlock 2004, anniballi et  al. 2013b). dead rodents, birds, or reptiles accidentally contained in feeds as well as decaying vegetable material are all substrates in which c. botulinum can proliferate and produce toxins (neill et al. 1989, jean et  al. 1995, wobeser et  al. 1997, galey et  al. 2000, senturk and cihan 2007). furthermore, hay stored in plastic bags, along with high ph forages and silages, seem to encourage the germination of spores (whitlock 2004). different symptoms are described in the literature, and these relate to varying levels of susceptibility to bonts depending on species, breed, and individual (bohnel et al. 2001, anniballi et al. 2013b). the most frequent symptoms reported in cattle are muscular weakness, ataxia, progressive paralysis, dysphagia, loss of tongue tone, decrease in salivation, bradycardia, decreased rumen movement, and 59veterinaria italiana 2019, 55 (1), 57-62. doi: 10.12834/vetit.768.3714.2 mariano et al. outbreak of botulism in cattle lateral recumbency, followed by death. the animals were also hypothermic (min  37.1  °c max  38.3  °c), aware of their surroundings, and able to move their tails and heads (often turned into the flank). muscle fasciculation in the forelimb was also noted. the animals were treated with saline solution (0.9% nacl), antibiotic, cortisone, vitamin b, and methionine, with no signs of improvement. on the second day, a commercial clostridial vaccination against clostridia chauvoei, clostridia novyi, clostridia haemolyticum, clostridia septicum, clostridia perfringens, and clostridia tetani was administered to all animals, the stable was cleaned, and litter and water were changed. during cleaning, 1 of the 5 water troughs was seen to have run dry and smelled of sulphite. by day 6, 7 animals had been necropsied and their organs sent (refrigerated) to different laboratories for testing, along with 7 blood samples (5 from affected animals, and 2 from animals of the healthy group a). animal feed and drinking water were also analysed (1 kg of corn silage, 1 kg of banded alfalfa, 1 tanked milk sample, 8 x 500 ml water samples from 4 drinking troughs used by the affected group (b), and sediments from the water trough found empty). the main post mortem findings in the 7 animals included petechial haemorrhages on the epicardium, haemopericardium, and enteritis of the small intestine. the main histological findings on the liver were steatosis associated with centrolobular congestion, edema, and microhaemorrhages. perivascular edema, and the presence of micro intracerebral haemorrhages were recorded in the brains. the heart showed myocardial congestion and perivascular edema with subepicardial microhaemorrhages; multifocal exudative glomerulonephritis, associated with tubular nephrosis, and numerous haemorrhages alleys. group a consisted of 365 cows, while group b consisted of 115 animals. the 2 groups shared the same external paddock and a common milking compound. all the animals received the same feed ration. the disease was severe, and caused 48 deaths amongst the animals belonging to group b, while the animals in group a showed no signs of illness. the disease manifested on 9 of september 2012 with the sudden death of the first 3 animals (day 1). new cases appeared over the following 12 days (figure 2). the disease evolved over a maximum of 8 days, even though the precise duration could not be assessed in the 24 animals that were euthanised for ethical reasons. the last 4 animals were euthanised on the 15th day of the outbreak (figure  3). after the first 3 asymptomatic deaths, the whole group  b exhibited decreased feed intake, while those showing symptoms reported progressive muscle weakness (characterised by a slow gait), ataxia and figure 1. location of the outbreak. 0 2 4 6 8 10 12 07 /0 9/ 20 12 08 /0 9/ 20 12 09 /0 9/ 20 12 10 /0 9/ 20 12 11 /0 9/ 20 12 12 /0 9/ 20 12 13 /0 9/ 20 12 14 /0 9/ 20 12 19 /0 9/ 20 12 figure 2. daily incidence of new clinical cases occurred in dairy cattle in province of grosseto, italy (september 2012). 0 2 4 6 8 10 12 14 07 /0 9/ 20 12 08 /0 9/ 20 12 09 /0 9/ 20 12 10 /0 9/ 20 12 12 /0 9/ 20 12 13 /0 9/ 20 12 14 /0 9/ 20 12 15 /0 9/ 20 12 17 /0 9/ 20 12 18 /0 9/ 20 12 19 /0 9/ 20 12 21 /0 9/ 20 12 euthanasia death figure 3. daily mortality over 15 days registered during the botulism outbreak occurred in dairy cattle in province of grosseto, italy (september 2012). 60 veterinaria italiana 2019, 55 (1), 57-62. doi: 10.12834/vetit.768.3714.2 outbreak of botulism in cattle mariano et al. accordingly, a commercial vaccine against clostridia was administrated, along with vitamin e and selenium, because of the myopathy seen during necropsy. the initial results from blood tests showed an increase in creatine phosphokinase. this was interpreted as muscular damage caused by recumbency, and the neutrophilia as stress-related. the low bun was interpreted as a sign of hepatosis, linked to high milk production in friesian cattle. a metabolic disturbance was also considered as a possible diagnosis, but later excluded because the blood test results did not show hypomagnesemia, hypokalemia, or hypocalcemia. organophosphate intoxication was excluded because there were no differences in the cholinesterase blood levels between the animals of both groups a and b. this conclusion was subsequently confirmed by the negative toxicological results arising from the assays for organochlorates, carbamates, and organophosphates that were conducted on the liver, kidney, and water samples. the absence of c. perfringens toxins, and the relatively low bacterial counts from the intestine, make it improbable that the bacterium underlays the high mortality rate. once botulism was suspected, 3 of the previously obtained liver samples were analysed using a multiplex pcr for c. botulinum; these tested positive for the mosaic dc gene. for 1 of the samples, the presence of the bont was confirmed via mouse assay. in the 2 remaining samples, the apparent absence of bonts may be due to the integrity of the toxins being compromised following the repeated thawing and freezing of the samples between one laboratory and the next. a belated diagnosis of the cause of this outbreak impeded identification of its origin. various factors may have played a role. for example, it may be worth noting that the animal feeds included newly imported banded alfalfa; it is known that cut-grass that is moist from rain is able to provide an optimal anaerobic environment suited to the production of bonts (braun et  al. 2005). in addition, after being processed in the nearby bio-waste compound, the litter from the neighbouring broiler farm, could have been recycled by being spread over the farm’s adjacent fields. it has been reported that broilers may subclinically carry c.  botulinum type d in their gut flora, so this type of contamination would pass unnoticed in poultry (krüger et  al. 2012, payne et  al. 2011). finally, it is feasible that the original source of the intoxication was the empty waterhole that reeked of sulfite (which is often produced by clostridia). this was suspected during a previous outbreak of botulism in italy (zarenghi et al. 2006). unfortunately, because all feed and water were removed, and the stable was cleaned for biosecurity reasons at the start of the outbreak, nothing could be test for the presence of c.  botulinum and its on the medullary were seen in the kidney; the intestine showed a severe enteritis with areas of de-epithelialisation of the mucosa and microhaemmorrhages, characterised by the presence of extensive lymphocytic and eosinophilic infiltrates in the lamina propria. routine bacteriological analyses were conducted on the organs and biologic liquids (livers, kidneys, intestines, spleens, intracardiac coagulums, pericardiac liquids, and milk) of the deceased animals. unfortunately, they did not reveal the presence of any relevant pathogenic bacterium as a possible causative agent. all the intestine samples were positive for c.  perfringens, but an elisa test (bioxdiagnostic®) revealed no toxins (alpha, beta, gamma, delta). the microbial counts of clostridia were below 108 cfu/g. two spleen and kidney samples were positive for clostridia sordelli, 1 animal resulted positive for klebsiella pneumoniae (brain, lung, and intra-cardiac coagulum), while a further 3 animals were positive for haemolytic escherichia coli. the complete blood cell counts revealed no additional abnormalities other than a slight neutrophilia. serum biochemistry indicated an increase in creatine phosphokinase (cpk) in 3 out of 5 animals tested. two animals showed a slightly low ureic (bun) value, and a further 2 had low iron (fe) levels. the cholinesterase values of the affected group b were similar to those of group a. all liver and kidney samples were negative for organochlorates, carbamates, organophosphates, as well as the associated gastric contents. all water samples chemically matched the parameters of potable water and were negative for pathogenic bacteria, organochlorates, carbamates, organophosphates. the sediments of the empty water trough were negative as well. three liver samples were found positive for c.  botulinum producing mosaic dc toxin using a multiplex pcr method (anniballi et  al. 2013a). the pcr method was performed following the guidelines provided by the italian national reference centre for botulism (iss, rome, italy). the iss confirmed these pcr results, and conducted the mouse bioassay that demonstrated the presence of toxin in 1 of the cow liver samples (iss 2013). discussion in italy, botulism in large animals is a rare occurrence. however, the number of outbreaks in italy and europe has increased in the last decade. the rarity of the disease in combination with the lack of pathognomonic symptoms in animals can lead to a misdiagnosis, or late diagnosis, which results in economic losses for farmers. in the outbreak reported here, the first clinical suspicion consisted of c. perfringens enterotoxaemia. 61veterinaria italiana 2019, 55 (1), 57-62. doi: 10.12834/vetit.768.3714.2 mariano et al. outbreak of botulism in cattle welfare. the high mortality rates characterising this outbreak mirror those reported previously. based on our experience, botulism should form part of the diagnostic protocols that deal with cases of sudden death without apparent cause, especially when encountering laterally recumbent cattle amongst non-periparturient, afebrile, and alert animals. finally, every effort should be made to identify the source of the contamination within the immediate environment in order to circumvent reoccurrence of the disease. in this instance, this was not achievable. aknowledgements financial support for this work was provided by the istituto zooprofilattico sperimentale del lazio e della toscana ‘m. aleandri’. the authors thank the staff of all the involved laboratories for their valuable technical assistance and rudy meiswinkel for helping us with the english version of our study. associated toxins; thus no laboratory data exist on the possible source of contamination. to better understand the epidemiology of these outbreaks, and to circumvent future outbreaks, it is fundamental that an in-depth investigation of the environment is preceded by the rapid and accurate diagnosis of the causative agent. identifying the cause of this outbreak in cattle was hindered by the absence of pathognomic symptoms and the paucity of cases reported in cattle in italy. botulism was suspected based on the high mortality rate, but only after organophosphate, organochlorate, and carbamate poisoning, toxico-infections, and metabolic disturbances had been excluded. the final diagnosis was obtained only after c.  botulinum type dc and related toxins had been detected in the liver samples taken from deceased animals. while the public health risk linked to cattle botulism caused by bont type  c and d is considered to be low (afssa 2002), it remains particularly relevant to animal health and advisory committee on the microbiological safety of food. 2006. report on botulism in cattle. food standard agency. fsa/1112/1206. agence française de sécurité sanitaire des aliments (afssa). 2002. rapport sur le botulisme d’origine aviaire et bovine. agence française de sécurité sanitaire des aliments. nancy, france. aish j., simmons a., livesey c., kennedy s. & gayford p. 2006. change to fsa advice on botulism in cattle. vet rec, 159 (24), 822. anniballi f., auricchio b., woudstra c., fach p., fiore a., skarin h., bano l., segerman b., knutsson r. & de medici d. 2013a. multiplex real-time pcr for detecting and typing clostridium botulinum group iii organisms and their mosaic variants. bio secur bioterror, 11 (s1), s207-s214. anniballi f., fiore a., löfström c., skarin h., auricchio b., woudstra c., bano l., segerman b., koene m. & båverud v. 2013b. management of animal botulism outbreaks: from clinical suspicion to practical countermeasures to prevent or minimize outbreaks. bio secur bioterror, 11 (s1), s191-s199. barash j.r. & arnon s.s. 2014. a novel strain of clostridium botulinum that produces type b and type h botulinum toxins. j infect dis, 209 (2), 183-191. bianchi e. 1950. botulism in cattle in italy. clinica veterinaria, 73, 120-122. bohnel h. & gessler f. 2013. presence of clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism. vet rec, 172 (15), 397. references bohnel h., schwagerick b. & gessler f. 2001. visceral botulism a new form of bovine clostridium botulinum toxication. j vet med a, 48, 373-383. braun u., feige k., schweizer g. & pospischil a. 2005. clinical findings and treatment of 30 cattle with botulism. vet rec, 156, 438-441. galey f., terra r., walker r., adaska j., etchebarne m., puschner b., fisher e., whitlock r., rocke t. & willoughby d. 2000. type c botulism in dairy cattle from feed contaminated with a dead cat. j vet med invest, 12 (3), 204-209. haagsma j. & ter laak e. 1979. first case of type d botulism in cattle in the netherlands. tijdschr diergeneeskd, 15, 609-613. heider l.c., mcclure j.t. & leger e.r. 2001. presumptive diagnosis of clostridium botulinum type d intoxication in a herd of feedlot cattle. canadian vet j, 42, 210-212. istituto superiore di sanità (iss). 2013. metodo per la ricerca di clostridi produttori di tossine botuliniche e per la ricerca di tossine botuliniche (metodo colturale e mouse test). pomiac02, 25. 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89-92. 253 1department of pathophysiology of reproduction and mammary gland, national veterinary research institute in pulawy, bydgoszcz, poland. 2department of immunobiology, institute of experimental biology, kazimierz wielki university in bydgoszcz, bydgoszcz, poland. 3department of large animal diseases with clinic, faculty of veterinary medicine warsaw, university of life sciences, warsaw, poland. 4faculty of animal breeding and biology, utp university of science and technology in bydgoszcz, mazowiecka 28, 85-084 bydgoszcz, poland. *corresponding author at: department of immunobiology, institute of experimental biology, kazimierz wielki university in bydgoszcz, bydgoszcz, poland. e-mail: wierych@o2.pl. parole chiave trattamento antibiotico, mastite clinica, vacca da latte, radiazione laser. riassunto scopo dello studio è stato quello di valutare, ai fini del trattamento della mastite, l'effetto dell'applicazione locale di radiazioni laser stp-99 su mammelle infiammate, confrontandolo con i risultati raggiunti utilizzando le sole infiltrazioni sistemiche o intramammarie di antibiotici. sono state esaminate 124 vacche da latte affette da mastite batterica; un gruppo con segni di infiammazione acuta locale è stato trattato con somministrazione intramammaria di antibiotici autorizzati alla posologia indicata nel foglietto illustrativo (gruppo di controllo). ad un secondo gruppo (gruppo sperimentale), invece, il trattamento antibiotico delle mammelle infiammate è stato supportato da un trattamento radiante con tecnica laser per 5 giorni consecutivi (2 minuti al giorno). le mucche che presentavano anche sintomi sistemici sono state trattate con iniezioni intramuscolari di antibiotici autorizzati alla posologia indicata nel foglietto illustrativo (gruppo di controllo), associando il trattamento laser in quelle oggetto di sperimentazione. nel trattamento intramammario, il tasso di recupero funzionale è stato del 43,7%, in quello sistemico del 46,7%. nel primo caso, la radiazione laser ha aumentato significativamente (p < 0,05%) il valore (+ 32%). sebbene non in modo significativo (p > 0,05%), anche nel secondo caso il trattamento radiante ha aumentato il tasso di recupero (+ 16,6%) delle mammelle. l'aumento dei recuperi funzionali mastite cronica nei bovini da latte: effetto della radiazione laser stp-99 in associazione al trattamento antibiotico keywords antibiotic treatment, clinical mastitis, cow, laser irradiation. summary the aim of the study was to evaluate the effect of stp-99 laser irradiation applied locally to inflamed cow udders on the efficacy of clinical mastitis treatment with either intramammary infusions of antibiotic products or systemic injections of antibiotics. examinations were carried out on 124 milking dairy cows suffering from clinical, bacterial mastitis. cows with signs of local acute inflammation were treated with approved intramammary antibiotic products at labeled doses as control. the exposed cows received the same antibiotic treatment but were also subjected to irradiation of the inflamed udders with a laser for 5  consecutive days (2 minutes a day). cows with local and systemic signs of mastitis were treated with either intramuscular injections of approved antibiotics in label doses alone (controls), or with the same intramuscular treatment protocol and laser irradiation of inflamed glands for 5 consecutive days (2 minutes a day). the recovery rate after intramammary treatment with antibiotics was 43.7%. irradiation with laser significantly (p < 0.05%) increased the recovery rate by 31.2%. the recovery rate in the cow cohort receiving systemic treatment with antibiotics was 46.7%. the laser irradiation resulted in a 16.6% increase in recovery. supportive treatment with laser irradiation increased recovery rates by 24.2%. edward malinowski1†, wiesław krumrych2* and hanna markiewicz3,4 the effect of low intensity laser irradiation of inflamed udders on the efficacy of antibiotic treatment of clinical mastitis in dairy cows veterinaria italiana 2019, 55 (3), 253-260. doi: 10.12834/vetit.818.3989.2 accepted: 11.03.2016 | available on line: 30.09.2019 254 effect of low intensity laser irradiation on clinical mastitis in dairy cows malinowski et al. veterinaria italiana 2019, 55 (3), 253-260. doi: 10.12834/vetit.818.3989.2 prophylaxis, laser therapy has been widely discussed as a promising method in the treatment of bovine clinical mastitis. low-intensity laser irradiation has been shown to affect cell metabolism, stimulate regeneration, and reduce pain and inflammation (huang et  al. 2009). low level laser therapy was used clinically in many areas, including canada, europe, and asia, for the treatment of various neurologic, chiropractic, dental, and dermatologic disorders in humans (posten et al. 2005). stoffel and colleagues, hoedemaker and hackenfort, beneduci and colleagues examined the effect of low power laser in treatment of bovine mastitis (stoffel et  al. 1989, hoedemaker and hackenfort 2003, beneduci et  al. 2007). some authors did not observe any beneficial effects of such a method of a treatment on the affected cows, but other revealed that a laser radiation treatment caused an effective beneficial response of the cows against the mastitis. the aim of the examinations was to evaluate the effect of local stp-99 laser irradiation of inflamed cow udders on the efficacy of clinical mastitis treatment with either intramammary infusions or systemic injections of antibiotics. material and methods examinations were carried out on 124 lactating polish holstein-friesian dairy cows suffering from clinical mastitis. field trials were performed in 2 cowsheds (a 66 cows and b 58 cows) belonging to one dairy farm (free stall boxes, total mixed ratio tmr) located in the north-western part of poland. average milk yield in sheds a and b during 305 days of lactation was 7,100 and 7,500 kg of milk, respectively. laboratory examinations were performed in the department of pathophysiology of reproduction and mammary gland, national veterinary research institute, in bydgoszcz. the study was subdivided into two experiments. experiment 1. effect of laser irradiation on the efficacy of intramammary treatment examinations were carried out on 64 cows (32 control animals and 32 exposed animals) with clinical introduction udder inflammations (mastitis) are still the most frequent and costly diseases affecting dairy cows across the world (bar et  al. 2008, hogeveen et  al. 2011). therapy with antimicrobials is the primary method of combating udder infections and mastitis. the treatment is extremely difficult due to diverse types and sources of pathogens as well as lack of effective, specific methods of prophylaxis. the efficacy depends on clinical course of the disease, etiological agents, type of drugs and the methods and routes of therapy. different authors reported recovery rates from 30% to 70% (bradley and green 2009, roberson et  al. 2004, serieys et  al. 2005, taponen et  al. 2003 a, b), however, in many cases recovery rates exceed 80% (roberson et  al. 2004, serieys et al. 2005). it should be emphasized that antibiotic residue in milk and meat can be harmful to human health. during last decades the efforts have been focusing on decreasing the total selective pressure for antibiotic resistance development in bacteria within the animal population as well as in humans (sheldon 2005, taylor 1999). therefore, the search continues for new methods of treatment and prophylaxis that would allow a reduction in the use of antibiotics. promising results in the treatment of mastitis during the lactating period were obtained with the use of ginseng saponin, herbal extracts, propolis, lysosubtilin, antibacterial proteins, and most prominently lysozyme dimers (malinowski 2002). in addition to the above mentioned compounds, other non-antibiotic methods such as frequent milk-out (roberson et  al. 2004), intramammary ozone infusion (ogata and nagahata 2000) or homeopathatic drugs injection (hektoen et  al. 2004, werner et  al. 2010) were tested. some methods and drugs were examined to improve the efficacy of antibiotics. more or less favorable results were brought about by frequent milk-out (roberson et  al. 2004), injections of the lysozyme dimers (malinowski et  al. 2006a), oxytocin (morin et  al. 1998), non-steroidal anti-inflammatory drugs (krömker et  al. 2011, mcdougall et  al. 2007) or intramammary infusions of lactoferrin (lacasse et al. 2008). in recent years, while searching for new, more efficient and organic methods for treatment and ritorna significativo (p < 0,05%) se si confrontano i risultati ottenuti con il solo trattamento antibiotico (intrammamario + sistemico) con quelli ottenuti con il trattamento antibiotico (intrammamario + sistemico) associato al trattamento laser (+ 24,2%). 255 malinowski et al. effect of low intensity laser irradiation on clinical mastitis in dairy cows veterinaria italiana 2019, 55 (3), 253-260. doi: 10.12834/vetit.818.3989.2 somatic cell count (scc), were also performed on days 7 and 21 after treatment. the first intramammary or systemic treatment of clinical mastitis in control and exposed cows started immediately after diagnosis. quarter milk samples (inflamed secretion) were collected aseptically by field veterinary practitioners or by scientific personnel of the department of pathophysiology of reproduction and mammary gland. samples were cooled and transported to the institute for laboratory examinations. bacteriological examinations were performed according to commonly accepted procedures (malinowski and kłossowska 2002). milk somatic cell count was determined by fossomatic 90 (foss, denmark). criteria of recovery were based on the regression of signs of clinical inflammation in udders, normal appearance of milk, decrease in the scc and negative results of two bacteriological examinations after treatment. the obtained results are presented as arithmetic mean (x) and standard deviation (±  sd). the significance of the differences between mean values was verified using tukey test assuming the differences to be significant if their probability was below 5%. statistical analyses of treatment results were performed using chi-square test. all statistical analyses were performed using statistica v. 6.0 statsoft software. results clinical mastitis cases in each group were caused by camp-negative streptococcus species, coliform bacteria, coagulase-negative staphylococci (cns) and staphylococcus aureus (table i). the number of infected quarters and etiological mastitis agents were very similar in each group. there were no differences in the effectiveness of treatment between cows with a sick quarter and those with two sick quarters. also, the location of quarters (front or rear) had not affected the outcome of treatment (data not shown). hence, the total data of all cows were analyzed together. the course of disappearance of bacteria (etiological agents of mastitis) in infected quarters (negative results of bacteriological examinations), independently on final efficacy of treatment in particular groups, is presented in table ii. it is shown that on days 7 and 21 after treatment less infected quarters were visible in cows treated with supportive laser irradiation compared to groups treated with antibiotics only. all cows from experiment 1 received intramammary antibiotic products (synulox lc) according to the label doses (3 times every 12 hours). the efficacy of treatment is presented in table iii. mastitis and signs characteristic for local acute inflammation (swelling, pain, redness, hardness, and macroscopic changes in milk), without fever. control cows (22 with one sick quarter and 10 with two sick quarters) were treated with approved intramammary antibiotic products synulox lc (pfizer) in label doses (3  times every 12  hours; amoxicillinum trihydrate 200 mg/clavulanic acid 50 mg/prednisolone 10 mg). exposed cows (24  with one sick quarter and 8  with two sick quarters) were treated with the same antibiotic product and with laser irradiation of the inflamed glands. the experimental cohort was exposed daily to one 2  minute session for 5 days consecutively with a low intensity laser device. the laser head was kept at a distance of 10  cm under the treated area and the depth of penetration of the laser beam exceeded 70 cm. experiment 2. the effect of laser irradiation on the efficacy of systemic treatment with antibiotic examinations were carried out on 60 cows (30 control and 30 exposed animals) that in addition to signs characteristic for local acute inflammation (swelling, pain, redness, hardness, macroscopic changes in milk) showed an increase in rectal temperature above 39.5 degrees centigrade. control cows (17 with one sick quarter and 13 with two sick quarters) were treated with intramuscular injections only of approved antibiotics synulox (pfizer) in label doses (once a day for 3 consecutive days; 7  mg/kg amoxycillinum trihydrate /1.75 clavulanic acid). exposed cows (18  with one sick quarter and 12  with two sick quarters) were treated with the same antibiotic and with stp-99 laser irradiation of the inflamed glands according to the protocol described in the experiment 1. the antimicrobial drug was selected on the basis of bacterial sensitivity to antibiotics previously isolated in this herd. in the experiments an stp-99 laser device (stp company, garshino, nizhny novgorod, russia) was used. the laser has 6 unique diodes, which emit low intensity laser pulses in the near-infrared spectrum. the laser radiation wavelength was 870-970 nm. the duration of the pulse wave of variable frequency and length was 1.0 seconds. peak radiated power was a maximum of 1.5 w. the laser devices stp-99 were certified in the eu in 2007. all cows were examined on day zero (at the time of diagnosis) and 7 and 21 days after treatment. the clinical examination of the cows and the udders together with macroscopic evaluation of milk, california mastitis test (cmt) and bacteriological examination of milk samples were carried out on day 0. clinical examinations, bacteriological tests, 256 effect of low intensity laser irradiation on clinical mastitis in dairy cows malinowski et al. veterinaria italiana 2019, 55 (3), 253-260. doi: 10.12834/vetit.818.3989.2 in clinical mastitis caused by non-agalactiae (environmental) streptococci, coliform bacteria and coagulase-negative staphylococci. table v contains the somatic cell count in milk samples taken from recovered quarters of different groups of cows. at the time of the diagnosis, milk was profoundly changed in all inflamed quarters, so scc could not be determined by the fossomatic 90. milk from bacteriologically negative quarters regained its normal appearance on day 7 after treatment. however, scc in milk samples taken 7  days after starting treatment was high in all groups. somatic cell count decreased significantly in the following 14  consecutive days in the groups of cows subjected to intramammary treatment but not significantly in cows belonging to groups subjected to systemic treatment. discussion streptococcus sp., escherichia coli, staphylococcus aureus and coagulase-negative staphylococci were the main etiological agents in cows with clinical forms of mastitis. many authors (bengtsson et  al. 2009, bradley and green 2001, gröhn et  al. 2004, malinowski et al. 2006b, pitkälä et al. 2004, sargeant et  al. 1998, tenhagen et  al. 2009), also isolated the same microorganisms but the percentage of particular species sometimes differed significantly. all treated cows showed local or systemic clinical signs and macroscopic changes in milk typical for acute mastitis (deluyker et  al. 1999, serieys et  al. 2005, wenz et al. 2006). from this table it is visible that the recovery rate after intramammary treatment with antibiotics was 43.7%. the irradiation with stp-99 laser increased the recovery rate by 31.2%. this difference was statistically significant (p  <  0.05). the recovery rate due to systemic treatment with antibiotics was 46.7%. the laser irradiation caused an increase in recoveries by 16.6% (the difference was not statistically significant). when the results of antibiotic treatment (intramammary infusions or intramuscular injections) were taken together, supportive treatment with laser irradiation caused an increase in recovery rates by 24.2% (statistically significant). recovery rates in quarters according to the presence of different species of bacteria are presented in table iv. irradiation with laser increased recoveries table i. etiological agents of mastitis in quarters of cows treated with different methods. etiological agents number of infected quarters in particular groups intramammary intramammary + laser systemically systemically + laser streptococcus sp. 22 23 21 22 staphylococcus aureus 7 5 5 6 coagulase-negative staphylococci (cns) 7 6 9 8 coliforms bacteria 6 6 7 5 trueperella pyogenes 0 0 1 1 total 42 40 43 42 table ii. numbers and percentages of infected quarters before and after antibiotic with or without laser treatment according to groups. days intramammary intramammary + laser systemically systemically + laser n % n % n % n % 0 42 100a 40 100a 43 100a 42 100a 7 22 52.4b 10 25.0b 28 65.1b 14 33.3b 21 22 52.4b 10 25.0b 21 48.8b 12 28.6b difference statistically significant: a,b p < 0.05 table iii. the effect of laser radiation on efficacy of clinical mastitis treatment with antibiotics. treatment method number of treated cows cured cows unhealed cows n % n % intramammary 32 14 43,7 a 18 56.2 intramammary + laser 32 24 74.9 b 8 25.0 systemically 30 14 46.7 16 53.3 systemically + laser 30 19 63.3 11 36.7 antibiotics together 62 28 45.2 c 34 54.8 antibiotics + laser together 62 43 69.4 d 19 30.6 difference statistically significant: a,b p < 0.05; c,d p < 0.01. 257 malinowski et al. effect of low intensity laser irradiation on clinical mastitis in dairy cows veterinaria italiana 2019, 55 (3), 253-260. doi: 10.12834/vetit.818.3989.2 antibiotics alone. the decrease in milk scc could be comparable with data reported by deluyker and colleagues (deluyker et  al. 1999) and taponen and colleagues (taponen et  al. 2003a) for cases cured clinically and bacteriologically. the supportive effect of laser irradiation in treated mammary glands is probably due to the regulatory effect on proand anti-inflammatory cytokines in vivo and in vitro (zhevago et  al. 2006), and with stimulation of the immunological system in vivo (funk et al. 1992, novoselova et al. 2006). this type of light stimulates proliferation of different kinds of cells (pinheiro et al. 2003, shanyfelt et al. 2008), increases growth of cells stressed by nutritional deficits in vitro (eduardo et  al. 2007), and results in inhibition of apoptosis in cells participating in the process of skin regeneration (chyczewski et al. 2010). the use of low levels of visible or near infrared light for reducing pain, inflammation and oedema, promoting healing of wounds, deeper tissues, and nerves and preventing cell death and tissue damage has been reviewed most recently by huang and colleagues (huang et  al. 2009). in non-steroid laser-treated rats, significant acceleration of epithelization and collagen synthesis 2 days and 6 days after surgery was observed in simulated wounds (gál et al. 2009). silveira and colleagues (silveira et al. 2011) recently suggested that low-power laser irradiation of the skin accelerates wound healing due to a reduction of the extent of the inflammatory phase. it seems that laser irradiation stimulates the phagocytic activity of milk granulocytes which the average efficacy of treatment that ranged between 43.5% (intramammary infusions) and 46.7% (intramuscular injections) was not satisfactory. inflammation caused by staph. aureus was particularly refractory to therapy. the results of mastitis treatment with intramammary antibiotic products alone are comparable (bradley and green 2009, deluyker et  al. 1999, malinowski et  al. 2006a, milne et  al. 2005) or lower than data reported by other authors (mcdougall et  al. 2007, roberson et  al. 2004, serieys et  al. 2005, taponen et  al. 2003 a, b). a better effectiveness was found in cases of fresh mastitis with the use of parenteral (barkema et al. 2006, mcdougall et al. 2007, serieys et al. 2005, suojala et  al. 2010) or traditional intramammary therapy (mcdougall 2003). other authors reported a significant increase in recovery as a result of extended (gillespie et  al. 2002, oliver et  al. 2004) or more aggressive (hillerton and kliem 2002) intramammary therapy or when intramammary infusions were combined with either intramuscular antibiotics (taponen et  al. 2003 a, b) or nsaid injections (krömker et al. 2011). irradiation of inflamed udder quarters with the stp-99 laser increased recovery rate from mastitis treated intramammarily or intramuscularly with antibiotics by 31.2% and 16.6%, respectively. irradiated cows showed faster regression of clinical signs such as redness, pain, hardening, faster disappearance of macroscopic changes in milk, and better elimination of intramammary infections compared to mammary glands of cows treated with table iv. quarter recoveries according to etiological agents. etiological agents number and percentage of recoveries intramammary intramammary + laser systemically systemically + laser n/n % n/n % n/n % n/n % streptococcus sp. 12/22 54.5a 20/23 86.9b 11/21 52.4 17/22 77.3 staphylococcus aureus 1/7 14.3a 0/5 0 b 1/5 20.0 1/6 16.7 coagulase-negative staphylococci (cns) 5/7 71.4 5/6 83.3 6/9 66.7 6/8 75.0 coliforms bacteria 2/6 33.3a 5/6 83.3b 4/7 57.1 4/5 80.0 trueperella pyogenes 0 0 0 0 0/1 0 a 1/1 100b total 20/42 47.6a 30/40 75.0b 22/43 51.2 30/42 71.4 difference statistically significant: a,b p < 0.05 table v. numbers and percentages of infected quarters before and after antibiotic with or without laser treatment according to groups. days groups intramammary intramammary + laser systemically systemically + laser 0 not determined changes in milk not determined changes in milk not determined changes in milk not determined changes in milk 7 1,400,920a ± 1,102,190 1,790,682a ± 1,663,660 1,892,000a ± 2,491,270 2,390,700a ± 2,617,800 21 477,560b ± 404,950 837,591b ± 808,625 412,640a ± 400,500 629,950a ± 743,540 a,b values in columns marked with different letters differ significantly (p < 0.05). 258 effect of low intensity laser irradiation on clinical mastitis in dairy cows malinowski et al. veterinaria italiana 2019, 55 (3), 253-260. doi: 10.12834/vetit.818.3989.2 treatment of mastitis but also in the repair process of mammary tissue that was destroyed as a result of inflammation (paape et  al. 2002, silveira et  al. 2011). however, supportive irradiation with stp-99 laser for 5 consecutive days is also too much time consuming. the next problem is a slightly higher milk somatic cell count comparing to quarters that recovered as a result of antibiotic application without irradiation. the higher number of the scc in cows treated with low-intensity laser therapy may be due to a bio-stimulating effect. at the tissue level, laser therapy stimulates the immune system by accelerating blood and lymph circulation. this therapy also stimulates phagocytosis and intracellular generation of active oxygen forms (novoselova et al. 2006). further research is therefore needed to evaluate the effect of irradiation for a shorter time periods, such as 3 days versus 5 days, for example. it seems that shorter irradiation can also increase the efficacy of antibiotics but scc could decrease in a faster manner. further research on the mechanisms of laser action inside cow mammary glands is also necessary. it seems that this treatment method can be realized on organic dairy farming, as in an intensive dairy farming this method may cause organizational problems. conclusion two minutes of daily irradiation for 5 consecutive days with stp-99 laser of clinically inflamed mammary glands in cows treated systemically or with intramammary antibiotics increases recovery rates from clinical bacterial mastitis. then are more active in destroying the etiological agents of mastitis. the activation of the mammary gland immunological system plays a fundamental role both in prophylaxis and treatment of mastitis in cows (burvenich et  al. 2004, paape et  al. 2002). the bactericidal effect of low-intensity laser irradiation that was demonstrated in vitro (žilaitis et  al. 2008) can also be considered. in addition, russian scientists (makarimov et al. 2002) reported that laser irradiation is highly effective in the treatment of endometritis in cows as a sole therapy method or in combination with antibiotics. on the other hand, stoffel and colleagues (stoffel et al. 1989) examined the effects of low-energy laser irradiation with 25  mw on an area of 7.5 cm in diameter on the right front quarter which lasted 30 minutes daily for five consecutive days. parameters measured included milk yield, somatic cell count, conductivity, na/k-ratio in milk whey, and fat, protein and lactose concentrations in milk. no evidence for any stimulation of the healthy mammary gland or therapeutic effects on mastitis could be found. contrary to this study, hoedemaker and hockenfort (hoedemaker and hockenfort 2003) reported a high percentage (84.4%) of clinical recoveries and only 25% of bacteriological recoveries as a result of therapy with the bmsd sport-laser ir 904 nm for the duration of 5-8 min on days 1, 2, 3, 5 and 7. a significant decrease in milk somatic cell count was observed by beneduci (beneduci et  al. 2007) as a result of irradiation of subclinically inflammed mammary glands with a stp-8 laser once a day for 30 sec for 5 days. summarizing, it is known from the literature data that the activity of the immunological system of 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defense of bovine mammary gland by polymorphonuclear neutrophil leukocytes. j mammary gland biol neoplasia, 7, 109-120. pinheiro a.l.b., limeira junior f.a., gerbi m.e.m., romalho l.m.p., marzola c. & ponzi e.a.c. 2003. effect of low level laser therapy on the repair of bone defects grafted with inorganic bovine bone. braz dent j, 14, 177-181. pitkälä a., haveri m., pyörälä s., myllys v. & honkanen-buzalki t. 2004. bovine mastitis in finland 2001 prevalence, distribution of bacteria, and antimicrobial resistance. j dairy sci, 87, 2433-2441. posten w., wrone d.a., dover j.s., arndt k.a., silapunt s. & alam m. 2005. low-level laser therapy for wound healing: mechanism and efficacy. dermatol surg, 31, 334-340. roberson j.r., warnick l.d. & moore g. 2004. mild to moderate clinical mastitis: efficacy of intramammary amoxicillin, frequent milk out, a combined intramammary amoxicillin, and frequent milk out treatment versus no treatment. j dairy sci, 87, 583-592. sargeant j.m., scott h.m., leslie k.e., ireland m.j. & bashiri a. 1998. clinical mastitis in dairy cattle in ontario: frequency of occurrence and bacteriological isolates. can vet j, 39, 33-38. serieys f., raguet y., goby l., schmidt h. & friton g. 2005. comparative efficacy of local and systemic antibiotic treatment in lactating cows with clinical mastitis. j dairy sci, 88, 93-99. 285 introduction the term ‘bacteriophage’ (or phage) refers to viruses that infect bacteria. they are abundant in nature and can be isolated from the same niches where their hosts reside. these small agents were first reported in 1915 by the british bacteriologist frederick w.  twort (twort 1915). in 1917, felix d’herelle named them ‘bacteriophages’, literally the eaters of bacteria, and started to use them in patients with dysentery (d'herelle 1917). since then, phage‑therapy has developed especially in eastern countries, where it was commonly applied with success in animals and in humans (chanishvili 2012). the clinical use of phages declined slowly with the discovery of antibiotics during the 1940s and 1950s. the interest in bacteriophages as an alternative to antibiotic therapy has re‑emerged only recently, and their potential applications are increasingly being istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy * corresponding author at: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy. tel.: +39 0861 332469, e‑mail: g.aprea@izs.it. parole chiave batteriofago, bio‑decontaminante, campylobacter spp., fago‑terapia, patogeno a trasmissione alimentare, listeria monocytogenes, sicurezza. riassunto listeria monocytogenes e campylobacter spp. sono patogeni responsabili di malattie a trasmissione alimentare negli esseri umani. il problema dell’antibiotico‑resistenza e la persistenza dei microrganismi patogeni nell'ambiente, soprattutto nelle produzioni alimentari, sono alcuni dei motivi che hanno portato recentemente alla rivalutazione dei batteriofagi e delle loro lisine come potenziali candidati per il bio‑controllo contro i batteri. in questa monografia l’attenzione è stata focalizzata sul potenziale utilizzo di fagi e relative lisine come strategia alternativa per contenere, in particolare, l. monocytogenes e campylobacter spp. sono stati valutati, inoltre, l’efficacia dell’applicazione nella sicurezza alimentare e nella salute animale, lo sviluppo di fago‑resistenza, la legislazione e le eventuali prospettive future in relazione al loro potenziale impiego. aggiornamento sulle applicazioni dei batteriofagi e delle loro lisine nel biocontrollo di listeria monocytogenes e campylobacter keywords bacteriophage, bio‑decontaminant, campylobacter spp., foodborne pathogen, listeria monocytogenes, phage‑therapy, safety. summary listeria monocytogenes and campylobacter spp. are foodborne pathogens responsible for outbreaks and disease in humans. the emerging problem of bacterial antibiotic resistance and the persistence of pathogens in the environment, especially where foods are processed, are some of the reasons that have led to a re‑emerging interest in bacteriophages and their lysins as potential candidates for bio‑control. this review focuses on the use of bacteriophages and their lysins as alternative strategies for controlling the foodborne pathogens l.  monocytogenes and campylobacter spp. in addition, the application of bacteriophages and their lysins in food safety and animal health, as well as phage‑resistance development, legislation, and future prospects were discussed. giuseppe aprea*, loredana zocchi, mariassunta di fabio, sara de santis, vincenza annunziata prencipe† and giacomo migliorati the applications of bacteriophages and their lysins as biocontrol agents against the foodborne pathogens listeria monocytogenes and campylobacter spp.: an updated look veterinaria italiana 2018, 54 (4), xxx‑xxx. doi: 10.12834/vetit.311.1215.2 accepted: 16.06.2014 | available on line: 31.12.2018 286 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx the public health risk because of their high environmental persistence and the constant development of drug resistance patterns. for these reasons, public health systems reserve a high level of attention for tools that can help to control and minimise these emerging hazards. in this study, the latest findings on bacteriophages specifically active against l.  monocytogenes and campylobacter spp. are reported, focusing on their applications as bio‑decontaminants and bio‑therapeutics. in particular, we investigated phages as a valid, safe, and cost‑effective strategy for eliminating/reducing the levels of specifically targeted bacterial pathogens in foods, with no deleterious effect on the organoleptic properties and without altering the beneficial microflora. phages for biocontrol of listeria monocytogenes many listeria‑phages have been described (dorscht et al. 2009, loessner et al. 2000, schmuki et al. 2012, zimmer et  al. 2003). only b054, a511, and p100 belong to the myoviridae family (carlton et al. 2005, klumpp et al. 2008, schmuki et al. 2012). phages against l.  monocytogenes have been evaluated for their efficacy as biocontrol agents in a variety of foods (e.g., hot dogs, cheese, and salmon fillet) (carlton et  al. 2005, guenther et  al. 2009, soni et al. 2009). among the variants that are determinant for a successful intervention in rte products, 2 are particularly important: the ratio between phage dose and host load and the food chemical composition (guenther et  al. 2009). for this reason there is a need to individually optimise protocols for phage applications with respect to phage characteristics and food matrix (guenther et al. 2009). two phage‑based formulations have been approved. the first one, listshield (intralytix, baltimore, usa), was regulated in usa as a food additive (usg 2006). listshield is a mix of 6 different bacteriophages and its activity has been tested specifically on fruits. in particular, this phage cocktail significantly reduced l.  monocytogenes counts by 2.0‑4.6 log units on melons and by 0.4 log units on apples (leverentz et  al. 2003). in another study, listshield yielded a total bacteria reduction of up to 6.8 log units after 7 days storage when applied onto contaminated honeydew melon tissues (leverentz et  al. 2004). when used in phage‑therapy, listshield effected a preventive reduced concentration of pathogen numbers in the gastrointestinal tract of mice before being infected with l.  monocytogenes. moreover no adverse effects on commensal microbiota composition were observed (loessner et  al. 1995). given the examined for purposes ranging from improving food safety to preventing and treating bacterial diseases, particularly those caused by drug‑resistant pathogens (martinez 2009). in relation to food safety and public health, foodborne disease outbreaks due to bacterial contaminations can occur even if good hygiene practises are mostly applied (holah et  al. 2002). in europe, 2,480 confirmed human cases of listeriosis were reported in 2017, revealing a statistically significant increasing trend over the period 2008‑2017 and a fatality rate of 13.8% (efsa 2018b). after non‑typhoid salmonella spp., l. monocytogenes is responsible for the majority of deaths in usa (da silva and de martinis 2013) and ready‑to‑eat (rte) products have been the most commonly incriminated foods during the last 30 years (d’alton et  al. 1997, fleming et  al. 1985, gottlieb et  al. 2006, graves et  al. 2005, olsen et  al. 2005). the second leading cause of physician visits, hospitalisation, and death in usa is campylobacteriosys (scallan et  al. 2013). the year 2012 registered a 14% increase in the estimated incidence of infection when compared with the period 2006‑2008 (cdc 2013). in europe, campylobacteriosis was the most commonly reported zoonoses in 2017, with 246,158 confirmed human cases (efsa 2018b). bacteria can grow and proliferate in the environment as single or independent cells, or they can organise in aggregates commonly referred to as ‘biofilms’. during biofilm formation, bacteria anchor themselves to surfaces by synthesising extracellular polymeric substances that provide them with protection from environmental stress factors and antimicrobial agents (mclandsborough 2013). in particular, l.  monocytogenes is capable of aggregating on a variety of food processing equipment surfaces, including polystyrene, stainless steel, and teflon. viable cells within biofilms are partially protected from salinity and chemicals as antimicrobials and disinfectants/sanitisers (carpentier and cherf 2011). the bacteria c. jejuni is also known to form biofilm; a correlation between c. jejuni biofilm formation and an increased fluoroquinolone resistance development was recently reported (bae and jeon 2013). in terms of antibiotic responses, β‑lactam penicillin  g and ampicillin are the current drugs of choice for the treatment of listerial infections. many l.  monocytogenes isolates have developed a high resistance to these chemicals over the years (fallah et  al. 2012, krawczyk‑balska et  al. 2012). regarding campylobacter spp., a high resistance to ciprofloxacin, nalidixic acid, and tetracyclines was observed in isolates from fowl, broiler meat, pigs, and cattle, whereas much lower levels were observed for erythromycin and gentamicin (efsa 2018a). foodborne pathogens significantly enhance bacteriophages and their lysins as biocontrol agents aprea et al. veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx 287 et  al. 2005, el shibiny et  al. 2005, loc carrillo et  al. 2005) and the majority of c.  jejuni and c. coli phages are virulent, with the exception of a few temperate bacteriophages. they are classified into 3 groups (groups i, ii, and iii) according to head diameter and genome size (sails et  al. 1998), with long and contractile tails, double‑stranded dna, and icosahedral heads (connerton et  al. 2011). only 8  genomic sequences have been published (janez and loc carillo 2013). the risk that phages could carry unknown genes coding for lysogeny or promoting virulence or resistance properties is therefore still a concern and requires further investigations (carvalho et  al. 2012 a, b). phage studies are mainly focused on preventive/therapeutic applications in animals and as bio‑decontaminants on food and contact surfaces, which demonstrates the priority of reducing campylobacter spp. transmission to humans. wagenaar and colleagues (wagenaar et al. 2005) and loc carrillo and colleagues (loc carrillo et  al. 2005) reported the first phage‑based treatments against campylobacter‑infected livestock. group iii phages administered to chickens challenged with c.  jejuni determined a significant decrease in bacterial colonisation (wagenaar et  al. 2005), while phages cp8 and cp34 determined a decrease in cell count between 0.5 and 5 log cfu/g of cecal content after being administered to infected broilers (loc carrillo et al. 2005). el shibiny and colleagues (el shibiny et al. 2009) tested phage cp220 in birds colonised with c. jejuni and c. coli, producing between 1 and 2 log reductions. more recently, carvalho and colleagues (carvalho et  al. 2010) reported encouraging results against c.  jejuni and c. coli infections after administering, for the first time, a phage cocktail to chickens by oral gavage and in feed. the phage cocktail administered by both routes was able to reduce the titre of c. coli and c.  jejuni in faeces by approximately 2 log cfu/g. in another recent study from kittler and colleagues (kittler et  al. 2013), the authors highlighted the positive effects of administering a phage cocktail to broilers via drinking water from 1 to 4 days prior to slaughter. this led to a reduction of up to 3.2 log cfu in campylobacter  spp. loads. differently from listeria‑phages, phage‑based products against campylobacter  spp. have not yet reached the market, which is potentially due to poor in vivo trial results (loc carrillo et al. 2005). two additional studies used chicken skin tainted with susceptible campylobacter  spp. and treated with phages (atterbury et al. 2003, goode et al. 2003). both groups demonstrated 1  log drop in bacterial loads when samples were stored at 4°c. moreover, atterbury and colleagues (atterbury et  al. 2003) showed a 2 log drop recovery from frozen‑thawed samples, but this result could be due to bacterial good results already yielded on fruits, as a phage cocktail formulation, listshild could be a very good candidate to reduce pathogen contaminations along food chain productions and in foods. the second formulation approved in usa is listex™p100 (micreos food safety, wageningen, the netherlands), which is composed of bacteriophage p100. it was used to control l.  monocytogenes on surface‑ripened red smear soft cheese, yielding a pathogen reduction of at least 3.5 logs (carlton et al. 2005). soni and collaborators also demonstrated its activity on fresh channel catfish fillets (l.  monocytogenes reduction between 1.4 and 2.0 log cfu/g at 4°c, 10°c, and 22°c) (soni et  al. 2009), raw salmon (reductions of 1.8, 2.5, and 3.5 log cfu/g from initial bacterial loads of 2, 3, and 4.5 log cfu/g at 4° and 22°c) (soni et al. 2010), and on queso fresco cheese (initial bacterial reduction of 2‑4 log cfu/ cm2 at 4°c, but a subsequent bacterial regrowth was reported) (soni et al. 2012). more recently, chibeu and colleagues (chibeu et  al. 2013) demonstrated that listex™p100 can enhance rte meat safety (cooked turkey and roast beef ) when used in combination with chemical antimicrobials. guenther and colleagues (guenther et  al. 2009) reported phage p100 ability to reduce bacterial counts to undetectable levels in chocolate milk and mozzarella cheese brine. a reduction of up to 5 log was also observed on various solid foods as rte and vegetables. in this study, phage p100 was used in combination with a511, a lytic phage with a broad host range (the ability to infect almost 95% of l. monocytogenes strains of the major serovar groups 1/2a and 4b) (loessner and busse 1970). bacteriophage a511 was also tested alone on soft ripened white mould and red‑smear cheeses. this led to a reduction of l.  monocytogenes cells below the limit of detection (more than 6 log reduction) (guenther and loessner 2011). the presence of l. monocytogenes in floor drains is a critical issue in the formation of aerosol because it could lead to listeria dispersal in water processing plants (berrang et  al. 2013). nevertheless, some researchers reported the use of competitive exclusion lactic acid bacteria in floor drains against l. monocytogenes viable cells (zhao et al. 2006) and biofilm (zhao et  al. 2013). although there are no scientific reports of similar results obtained with phages, we would like to highlight the potential of phage applications in floor‑drains as an additional means to control this pathogen in food productions. phages for biocontrol of campylobacter spp. the prevalence of campylobacter‑phages in the environment is estimated to be high (atterbury aprea et al. bacteriophages and their lysins as biocontrol agents nick title first author et al. 288 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx thus enabling their biofilm dispersion ability. lu and collins (lu and collins 2007) described the particular activity of a modified t7 bacteriophage that was able to remove 99.97% escherichia coli biofilm cell counts. these results were 2 orders of magnitude better than those observed with a phage that did not produce polysaccharide‑degrading enzymes (lu and collins 2007). this genetically modified phage has been patented in usa (lu and collins 2007). few studies have published the effects of phages against l.  monocytogenes and campylobacter spp. biofilms (table i). briefly, listeriophages limn4l, limn4p, and limn17 were used to test activity against biofilms of l. monocytogenes strains isolated from seafood and grown onto stainless steel and stainless steel coated with fish protein surfaces. the phages produced more than a 3 log reduction. the best lysis was achieved when cells were first slightly dislodged (ganegama‑arachchi et al. 2013). bacteriophage p100 also showed a 3.5‑5.4 log/cm2 reduction of listeria biofilms grown on stainless steel coupon surfaces (soni and nannapaneni 2010a). montanez‑izquierdo and colleagues (montanez‑izquierdo et  al. 2012) confirmed the effectiveness of the p100 biofilm disruption (an average of 5.29 log cfu/cm2 reduction) by comparing classical culture methods and the use of epifluorescence microscopy. roy and colleagues (roy et  al. 1993) investigated the ability of listeriaphages h387, h387‑a, and 2671 to disrupt biofilms grown onto polypropylene surfaces. they observed a reduction of more than 3 log of l. monocytogenes. regarding phage activity against campylobacter spp. biofilms, very little has been reported. siringan and colleagues (siringan et  al. 2011) verified the inactivation by freezing more than to a phage effect. bigwood and colleagues (bigwood et  al. 2008) treated raw and cooked beef meats and compared the results of different phage titres (10‑104 pfu/cm2) against different levels of contamination (< 100‑104 cfu/cm2). significant host inactivations (2  log/ cm2) were achieved using the highest host cell density and the highest phage titres. orquera and colleagues (orquera et  al.2012) described how the application of nctc 12684 and cp81 bacteriophages to raw chicken meat for up to 7 days at 4°c could not produce any relevant reduction in bacterial loads. bacteriophage activity against listeria monocytogenes and campylobacter spp. biofilms bacterial biofilms consist of microorganisms embedded in a glycocalyx that is predominantly composed of exopolysaccharides (costerton et  al. 1994). the glycocalyx provides bacterial protection against environmental stressors, such as desiccation and antimicrobial agents, and may also act as a reservoir for nutrients (allison 1993). the ability of some phages to produce glycanase enzymes (polysaccharide depolymerases) has been reported for over 40 years (adams and park 1956). the role of glycanase enzymes is primarily related to phage‑binding activity to bacterial capsular materials and the degradation of polymers with consequential cell infection. many phages synthetise these enzymes (cornelissen et  al. 2011, cornelissen et  al. 2012) but non‑synthetising bacteriophages can be genetically modified in order to express polysaccharide‑degrading enzyme production, table i. phage application to control listeria monocytogenes and campylobacter spp. biofilms. host(s) phage(s) surface(s) treatment outcome reference listeria monocytogenes (10401 and 8427) h387, h387‑a, 2671 stainless‑steel, polypropylene phage suspensions up to 3.5 x 108 pfu/ml use of single phages and cocktail more than 3 log reduction phage cocktail showed a better efficiency than single phages roy et al., 1993 listeria monocytogenes (serotype ½ a) p100 stainless‑steel coupon phage suspensions of 109 pfu/ml 3.5‑5.4 log/cm2 reduction soni and nannapaneni, 2010b listeria monocytogenes (ccug 15526) p100 stainless‑steel coupon phage suspensions of 5, 6, 7 or 8 log pfu/ml phage concentrations of 6, 7, and 8 log pfu/ml reduced l. monocytogenes in mean 5.29 log cfu/cm2, after 24h montanez‑izquierdo et al., 2012 listeria monocytogenes (sea‑food strains 19co9, 19do3 and 19eo3) limn4l, limn4p, limn17 stainless‑steel, stainless‑steel coated with fish protein phage suspensions of about 9 log 10 pfu/ml on undisturbed and slightly dislodged biofilms use of single phages and cocktail more than 3 log reduction with phages being more effective on dislodged biofilms ganegama‑arachchi et al., 2013 campylobacter jejuni (nctc 11168 and pt14) cp8, cp30 glass single phage suspensions of about 106‑109 pfu/ml 1 to 3 log 10 cfu/cm2 reduction, 24h post‑treatment siringan et al., 2011 first author et al. nick title veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx 289 l.  monocytogenes phage fwllm3 and tested its activity in reducing l. monocytogenes counts in soya milk, with the pathogen concentration reduced by more than 4 log cfu/ml after 3 hours of incubation at 4°c. this was the first report of a listeria phage endolysin tested in foods, and this opens up the possibility of examining the potential use of biocontrol in other rte foods. another lysin, plylm, a putative n‑acetylmuramoyl‑l‑alanine amidase, was shown to be active against l.  monocytogenes strains and other bacteria within the genus level, and above all against l.  monocytogenes biofilm (simmons et al. 2012). in particular, the present study demonstrated a 20% biofilm reduction when plylm was used alone, yielding a complete digestion of the bacterial monolayer when the endolysin was applied in conjunction with a protease. this is the only study dealing with l.  monocytogenes biofilm reduction with the use of endolysins. it is important to highlight that the glycocalyx and cell agglomerates within biofilms play an important role as physical barriers between lysins and bacterial cells, and this produces a consequential reduction of their activity. bacterial resistance to phages bacteria are known to adopt many antiviral mechanisms in order to preserve themselves against phage infection (bikard and maraffini 2012, stern and sorek 2011). one of the most common is based on the modification of cell‑surface molecules (e.g. lipopolysaccharides, pili, and flagella) that are then used as receptors from phages in order to block host recognition and adsorption (hyman and abedon 2010, labrie et al. 2010). these defence mechanisms can be transmitted from resistant to sensitive cells through the transduction of bacterial dna via phage particles, leading to the development of ‘bacteriophage‑insensitive mutants’ (bims) (emond et  al. 1997, garcia and molineux 1995, hudson et  al. 2005). three other phage resistance mechanisms can occur during phage replication within the host cell: the abortive infection, the restriction modification system (golais et al. 2013), and the crispr/cas system (szczepankowska 2012). even if much is known about bacterial anti‑phage immune mechanisms, it is likely that many still remain to be discovered. bacterial resistance to phages does not always work efficiently. sometimes spontaneous mutations that occur in bacteria lead to phage‑resistant strains. this can have deleterious effects on prokaryotes, which does not necessarily confer an evolutive advantage. this could explain the tendency of bim bacteria to revert to sensitive strains once bacteriophages are no longer a threat in their environment (o’flynn et al. 2004). in particular, some authors have observed the dispersal of biofilm matrix from c. jejuni nctc 11168 and pt14 on glass by bacteriophages cp8 and cp30, and reported an average of 2 log cfu/cm2 reduction 24 hours post‑treatment. antimicrobial application of bacteriophage endolysins in food safety peptidoglycan hydrolases (pghs) are a class of enzymes capable of hydrolysing bonds in the peptidoglycan (pg) layer of the bacterial cell wall, resulting in cell death. pghs produced by phages are called ‘endolysins’ (or ‘lysins’), because they lyse the bacterial cells internally (‘lysis from within’). however, suspensions of concentrated endolysins can also be responsible for lysis from outside the cell (‘lysis from without’), and this is the principle that underpins their potential application without using viable phages (abedon 2011). it is important to highlight that their action is principally restricted to gram‑positive bacteria, since gram‑negative prokaryotes have an outer membrane that protects pg from hydrolases (shen et al. 2012). scientists have recently shown a growing interest in lysins because they are safe, biodegradable, non‑corrosive molecules with a high bacterial pg affinity (nelson et  al. 2012) and they are easy to produce on a large scale (zhang et  al. 2012). moreover, this class of enzymes is also believed to be refractory to bacterial resistance development, as we will discuss in the next section. few l.  monocytogenes phage endolyses have been characterised (oliveira et  al. 2013). these include: ply500 (loessner et  al. 1995), plypsa (zimmer et  al. 2003), plyp35 (schmelcher et  al. 2012), ply118 (shen et  al. 2012), ply511 (shen et  al. 2012), plyp40 (schmelcher et  al. 2012), plylm (schmelcher et  al. 2012), and lysz5 (zhang et al. 2012).the ‘chimera’ is an example of a protein‑engineering product where the fusion of phage endolysin ply500 with plyp35 was able to produce a combined enzyme that is active against a broader spectrum of listeria spp. (schmelcher et al. 2012). few studies conducted between 2000 and 2011 reported a reduction of l. monocytogenes cells when treated with lysins plypsa (korndorfer et  al. 2006), ply500 (schmelcher et  al. 2011), ply118, and ply511 (gaeng et al. 2000). the major limitation of these studies is the use of optical density (od600) to measure the bacterial reduction in vitro. in order to achieve more objective results it is necessary to also test lysins activity in vivo and to assay bacterial reductions by plate‑counting methods. zhang and collaborators (zhang et  al. 2012) characterised and purified lysz5 from nick title first author et al. 290 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx possibility of bacteria developing resistance against more anti‑phage infection systems contemporarily (leverentz et  al. 2004). the use of endolysins could also be a good alternative to escaping anti‑phage mechanism development. there is no scientific evidence about the existence of lysine‑resistant bacterial strains and the few studies that were carried out to isolate them in the environment were all unsuccessful (fischetti 2005). the european position on bacteriophages and current relevant international legislation the european food safety authority (efsa) had issued 3 scientific opinions about bacteriophages. the first one was released in 2009 by the panel on biological hazards, and deals with ‘the use and mode of action of bacteriophages in food productions’. this document described the information available on these microorganisms and their potential role as bio‑decontaminants. it recognised the efficacy of some bacteriophages in the elimination of specific pathogens. in this publication, efsa did not approach the issue of safety associated with the use of bacteriophages, and reported few major concerns in relation to bims and efficacy during recontamination (efsa 2009). in 2012, an application dossier by micreos food safety (the netherlands) for the approval of listextmp100 to reduce l.  monocytogenes from food surface contamination led to the publication of a second efsa opinion. the assessment focused on the safety and efficacy of bacteriophage p100 in the treatment of raw fish. authorities expressed scepticism about the absence of industrial‑scale studies, the limited selection of strains used for inoculation, and the lack of results concerning the pathogen reduction in the final fish product. despite the final conclusion that ‘bacteriophages cannot be included on the qualified presumption of safety list of microorganisms intentionally added to food or feed’, efsa agreed that bacteriophage p100 fulfils the safety requirements (efsa 2012). the last efsa opinion on listextmp100 was issued in 2016. safety and efficacy of the phage against l. monocytogenes was recognised for 3 ready‑to‑eat product categories (meat and poultry, fish and shellfish, dairy products). experimental studies indicated that l. monocytogenes strains resistant to listextmp100 could develop, but cleaning of surfaces where the phages are applied together with disposal of unsold treated products could reduce this risk. moreover, it was speculated that phage p100 resistant strains can be accompanied by more sensitivity to some classes of antimicrobials and that phage persistance in the environment is low (efsa 2016). ability of phage‑resistant campylobacter spp. strains to become sensitive after multiplication in chicken guts without exposure to bacteriophages (carvalho et al. 2010, scott et al. 2007). none of l.  monocytogenes phage‑resistant strains was isolated in cheese treated with low concentration of phage p100 (carlton et  al. 2005). on the other hand, other studies showed the isolation of bim l.  monocytogenes isolates from samples taken from a smoked fish processing facility (vongkamjan et al. 2013). unlike l.  monocytogenes, more information is available about campylobacter spp. resistance to phages cp8 and cp30 was observed in surviving cells within c. jejuni nctc 11168 biofilms after phage treatment, while no resistance was observed in c. jejuni pt14 isolates (siringan et al. 2011). loc carrillo and colleagues (loc carrillo et al. 2005) investigated the ability of c. jejuni hpc5 to develop resistance to phages cp8 (8% of the strains) and cp34 (11% of the strains) in vitro, noting that these isolates were able to revert back to sensitive strains. a similar in vivo experiment revealed that only 4% of colonies that recovered after treatment with phage cp34 achieved resistance. coward and colleagues characterised the interaction between c.  jejuni and 16  phages used in the united kingdom as the campylobacter typing scheme (coward et  al. 2006). interestingly, they demonstrated that resistance to this group of phages was associated with motility defects and disruption of capsular polysaccharides (cps) (coward et al. 2006). in 2007, scott and colleagues (scott et al. 2007) evaluated the in vivo competitive colonisation between phage‑resistant and phage‑sensitive strains with and without phage pressure in the environment. their results showed that without a phage predation pressure, the phage‑sensitive strains could out‑compete the phage‑resistant strains. in the presence of phages, the situation was very different, and the phage‑resistant strains were able to out‑compete phage‑sensitive strains. the authors also demonstrated the recovery of phage‑resistant mutants and of poor chicken intestine coloniser strains within a c.  jejuni hpc5 populations of avian gut when treated with phage cp34 (scott et al. 2007). sorensen and colleagues (sorensen et  al. 2012) exposed c.  jejuni nctc 11168 to phage f336 treatment and yielded a large number of phage‑resistant strains characterised by the modification of the capsular polysaccharide's (cps) hypervariable o‑methyl phosphoramidate structure (sorensen et al. 2012). although phage‑resistance development is one of the major concerns for scientists, it can be avoided with a mix of bacteriophages (‘phage cocktails’). in fact, the activity of different phages pulled together against the same host significantly reduces the first author et al. nick title veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx 291 natural product, lysins are molecules purified from a recombinant expression system, which could represent a less problematic issue for approval. future perspectives two of the main pathogens responsible of serious foodborne outbreaks are l.  monocytogenes and campylobacter spp. their insidious persistence in animals is well‑known, as is the increasing development of antibiotic‑resistance patterns and their deleterious effects. these are some of the reasons for an increasing interest in bacteriophages and their potential innovative applications. as natural killers of bacteria, phages are abundant in nature. moreoever, their isolation/replication techniques are relatively easy to perform and cost‑effective when compared with the preparation of new antibiotics. nevertheless, few variants can negatively influence the phage/lysin activity by limiting their delivery to the sites of infection e.g. solid food matrix, biofilm structure (glycocalyx), and anti‑phage detergents. in order to be applied in food productions and therapy, phages should present the following characteristics: strict lytic cycle, broad host range, lack of transduction of bacterial dna, absence of pathogenic genes or allergenic proteins, sequenced genomes, and long‑term stability. these microorganisms are extremely versatile and can be engineered in order to be more efficient in attacking their hosts (lu and collins 2007). other applications in the future may include the production of ‘bio‑food packaging materials’: these novel technologies could be based on encapsulating phage inside electrospun fibres (korehei and kadla 2013) or on immobilising phages onto biological membranes like cellulose (anany et  al. 2011). the inclusion of bacteriophages inside food packaging could be a valid strategy for long‑lasting pathogen control during shelf life. ‘ghost particles’ could be used in order to gain advantages from the ‘lysis from without’, with the application of ‘empty’ phages and without genome implication. phage‑resistant development in host bacteria needs to be monitored. further investigations based on bacteriophage dna sequencing along with more in‑depth research demonstrating phage efficacy at industrial level (trials/challenge tests) are required to better understand phage biology and to assess their potential approval for animal health and in food/ feed productions. the united states food and drug administration (fda) and united states department of agriculture were more tolerant about listextmp100, recognising and granting the product generally recognized as safe (gras) status in 2006 (fda 2006, usda 2006, usg 2006). in 2011, phage p100 was classified as a ‘processing aid’ by usda and the food safety and inspection services (1usda 2011). today, listex™p100 is used in usa, canada, and switzerland. in the eu, the dutch ministry of health issued a formal statement confirming that listex™p100 can be used as a processing aid. unlike l. monocytogenes, no official phage‑based products have been approved against campylobacter spp. eastern european countries and the soviet union are mainly involved in the application of phage‑therapy (kurlenda and grinholc 2012). despite extensive phage use in many reported clinical cases (parfitt 2005, slopek et  al. 1987, sulakvelidze et  al. 2001), today there are no official regulations standardising its use. in fact, a general scepticism still prevails, and the validity of most relevant studies has been questioned, in part due to a relaxed scientific rigour. according to verbeken and colleagues (verbeken et al. 2007), novel therapies must comply with paragraph 32 of the declaration of helsinki and be practiced under the supervision of an ethics committee. in 2005, the polish institute of immunology and experimental therapy of the polish academy of sciences performed experiments under 3 conditions: the approval by an institutional review board, the written informed consent of patients, and health‑insurance (gorski et  al. 2009). the burn wound centre in belgium also performed a clinical trial after approval from an ethics committee. the results revealed that phages are safe for patients and do not cause any adverse side‑effects to eukaryotic cells or natural flora (bruttin and brussow 2005, mai et al. 2010). merabishvili and colleagues (merabishvili et  al. 2009) also addressed the important issue of developing phage‑based compounds under quality control. in their study, they described a small‑scale laboratory‑based production of a phage cocktail designed for the treatment of pseudomonas aeruginosa and staphylococcus aureus infections in burn wound patients. if the process leading to the approval of phage‑use among different countries worldwide seems to be controversial, the situation for phage endolysins could be considered less complicated. in fact, unlike bacteriophages, which are referred to as nick title first author et al. 292 veterinaria italiana 2018, 54 (4), xxx-xxx. doi: 10.12834/vetit.xxxxx abedon s.t. 2011. lysis from without. bacteriophage, 1, 46‑49. adams m.h & park b.h. 1956. an enzyme produced by a phage‑host cell system: ii. the properties of the polysaccharide depolymerase. virol, 2, 719‑736. allison d.g. 1993. biofilm associated exopolysaccharides. microbiol eur, 1, 16‑19. anany h., chen w., pelton r. & griffiths m.w. 2011. biocontrol of listeria monocytogenes and escherichia coli o157:h7 in meat by using phages immobilized on modified cellulose membranes. appl environ microbiol, 77, 6379. atterbury r.j., connerton p.l., dodd c.e., rees c.e. & connerton i.f. 2003. application of host‑specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of campylobacter jejuni. appl environ microbiol, 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(m. lobocka & w.t. szybalski, eds.). academic press, paris, 289‑338. twort f.w. 1915. an investigation on the nature of the ultramicroscopic viruses. lancet, 186, 1241‑1243. united state department of agriculture (usda). 2006. gras notice 000218, gras notification of listextmp100 bacteriophage. www.accessdata.fda.gov/scripts/fcn/ gras_notices/701456a.pdf. united state department of agriculture and food safety and inspection services (usda). 2011. letter of notification on use of listextmp100 as processing aid. www. micreosfoodsafety.com/images/bacteriophage%20 no%20obj%20%20ltr%20%20to%20h ibber t%20 for%20ebi%20as%20processing%20aid%20log%20 no%20%2010‑ing‑0711‑n‑a.pdf. united states government printing office (usg). 2006. listeria‑specific bacteriophage preparation‑21 cfr 172.785. federal register, 71, 47729‑47732. verbeken g., de vos d., vaneechoutte m., merabishvili m., zizi m. & pirnay j.p. 2007. european regulatory conundrum of phage therapy. future microbiol, 2, 485‑491. vongkamjan k., roof s., stasiewicz m.j. & wiedmann m. 2013. persistent listeria monocytogenes subtypes isolated from a smoked fish processing facility included both phage susceptible and resistant isolates. food microbiol, 35, 38‑48. wagenaar j.a., van bergen m.a., mueller m.a., wassenaar t.m. & carlton r.m. 2005. phage therapy reduces campylobacter jejuni colonization in broilers. vet microbiol, 109, 275‑283. zhang h., bao h., billington c., hudson a. & wang r. 2012. isolation and lytic activity of the listeria bacteriophage endolysin lysz5 against listeria monocytogenes in soya milk. food microbiol, 31, 133‑136. zhao t., podtburg t.c., zhao p., schmidt b.e., baker d.a., cords b. & doyle m.p. 2006. control of listeria spp. by competitive‑exclusion bacteria in floor drains of a poultry processing plant. appl environ microbial, 72, 3314‑3320. zhao t., podtburg t.c., zhao p., chen d., baker d.a., cords b. & doyle m.p. 2013. reduction by competitive bacteria of listeria monocytogenes in biofilms and listeria bacteria in floor drains in a ready‑to‑eat poultry processing plant. j food prot, 76, 601‑607. zimmer m., sattelberger e., inman r.b., calendar r. & loessner m.j. 2003. genome and proteome of listeria monocytogenes phage psa: an unusual case for programmed_1 translational frameshifting in structural protein synthesis. mol microbiol, 50, 303‑317. shuffling and module engineering of listeria phage endolysins for enhanced lytic activity and binding affinity. microb biotech, 5, 651‑662. schmelcher m., donovan d.m. & loessner mj. 2012. bacteriophage endolysins as novel antimicrobials. future microbiol, 7, 1147‑1171. schmuki m.m., erne d., loessner m.j. & klumpp j. 2012. bacteriophage p70: unique morphology and unrelatedness to other listeria bacteriophages. j virol, 86, 13099‑13102. scott a.e., timms a.r., connerton p.l., el shibiny a. & connerton i.f. 2007. bacteriophage influence campylobacter jejuni types populating broiler chickens. environ microbiol, 9, 2341‑2353. scott a.e., timms a.r., connerton p.l., loc carrillo c., adzfa radzum k. & connerton i.f. 2007. genome dynamics of campylobacter jejuni in response to bacteriophage predation. plos pathog, 3, e119. shen y., mitchell m.s., donovan d.m. & nelson d.c. 2012. phage‑based enzybiotics. in bacteriophages in health and disease. (p. hyman & s.t. abedon, eds). cab int, wallingford, uk. 217‑239. simmons m., morales c.a., oakley b.b. & seal b.s. 2012. recombinant expression of a putative amidase cloned from the genome of listeria monocytogenes that lyses the bacterium and its monolayer in conjunction with a protease. probiotics antimicro prot, 4, 1‑10. siringan p., connerton p.l., payne r.j., connerton i.f. 2011. bacteriophage‑mediated dispersal of campylobacter jejuni biofilms. appl environ microbiol, 77, 3320‑3326. slopek s., weber‑dabrowska b., dabrowski m. & kucharewicz‑krukowska a. 1987. results of bacteriophage treatment of suppurative bacterial infections in the years 1981‑1986. arch immunol ther exp (warsz), 35, 569‑583. soni k.a., nannapaneni r. & hagens s. 2009. reduction of listeria monocytogenes on the surface of fresh channel catfish fillets by bacteriophage listextmp100. foodborne pathog dis, 4, 427‑434. soni k.a. & nannapaneni r. 2010a. bacteriophage significantly reduces listeria monocytogenes on raw salmon fillet tissue. j food prot, 73, 32‑38. soni k.a. & nannapaneni r. 2010b. removal of listeria monocytogenes biofilms with bacteriophage p100. j food prot, 73, 1519‑1524. soni k.a., desai m., oladunjoye a., skrobot f., nannapaneni r. 2012. reduction of listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic gras antimicrobials. int j food microbiol, 155, 82‑88. sorensen m.c.h., van alphen l.b., fodor c., crowley s.m., christensen b.b., szymanski c.m. & brondsted l. 2012. phase variable expression of capsular polysaccharide modifications allows campylobacter jejuni to avoid bacteriophage infection in chickens. front cell infect microbiol, 2:e11. stern a. & sorek r. 2011. the phage‑host arms race: shaping the evolution of microbes. bioessays, 33, 43‑51. issn 0505-401x 30 il canile sanitario, procedure e protocolli filomena iannino, fabio bellucci, annalisa lombardini, elisabetta finocchi mahne, enzo ruggieri, stefania salucci, greta berteselli, cristina rapagnà, maria luisa danzetta, nicola d’alterio collana di monografie il canile sanitario, procedure e protocolli filomena iannino, fabio bellucci, annalisa lombardini, elisabetta finocchi mahne, enzo ruggieri, stefania salucci, greta berteselli, cristina rapagnà, maria luisa danzetta, nicola d’alterio jan havicksz. steen (leida, 1626 c. leida, febbraio1679) the merry family, l’allegra famiglia, 1668 olio su tela, cm 141 × 110,5 rijksmuseum, amsterdam questa famiglia turbolenta sta facendo molto chiasso: il padre canta a squarciagola mentre alza un bicchiere; la madre e la nonna chiacchierano tra loro; i bambini suonano uno strumento a fiato e fingono di fumare una pipa. il biglietto appeso alla mensola del caminetto recita la morale della storia: “come canta il vecchio, così canterà il giovane”. come si comporteranno i bambini se i loro genitori danno un cattivo esempio? si ringrazia il rijksmuseum di amsterdam per l’immagine di copertina. www.rijksmuseum.nl collana di monografie 30 comitato direttivo silvio borrello, nicola d’alterio, antonia ricci direttore responsabile giovanni savini segreteria di redazione monica bucciarelli, laura ambrogi amministrazione istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale” campo boario, 64100 teramo, italia progetto grafico e impaginazione paola di giuseppe www.veterinariaitaliana.izs.it/index.php/vetit il canile sanitario, procedure e protocolli/ filomena iannino1*, fabio bellucci2, annalisa lombardini3, elisabetta finocchi mahne2, enzo ruggieri1, stefania salucci1, greta berteselli4, cristina rapagnà5, maria luisa danzetta1, nicola d’alterio1 [teramo]: istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale”, ©2022. 52 pp. (collana di monografie; 30). 1istituto zooprofilattico sperimentale dell’abruzzo e molise “g. caporale” 2ministero della salute 3regione emilia-romagna 4libero professionista 5efsa * f.iannino@izs.it isbn 9788893650229 questa rivista è nata nel 1950 con il nome di croce azzurra. dal 1954 si chiamerà veterinaria italiana. campo boario, 64100 teramo, italia telefono +39 0861 3321, fax +39 0861 332251 www.izs.it prefazione il presente manuale fa seguito a “il canile rifugio, procedure e protocolli” pubblicato su veterinaria italiana nel 2020 e ne ripropone l’impostazione. la finalità di questo lavoro e di dare indicazioni circa l’elaborazione di procedure che consentano una corretta organizzazione e gestione della struttura in un’ottica di assoluta trasparenza ed efficienza. la qualità dell’accoglienza dei cani nei canili sanitari è fondamentale anche al fine di ridurre il trauma della cattura e del cambio di luogo di vita e di avviare o ripristinare una buona relazione uomo/animale. introduzione ............................................................................................................................................................................................ 7 scopo del manuale ...................................................................................................................................................................... 9 parte i gestione della struttura requisiti strutturali ................................................................................................................................................................................................................. 10 gestione documenti e archiviazione ............................................................................................................................................. 12 piano di emergenza e di evacuazione ......................................................................................................................................... 13 gestione rifiuti .................................................................................................................................................................................................................................. 14 controllo animali infestanti ................................................................................................................................................................................. 16 sicurezza ........................................................................................................................................................................................................................................................ 17 formazione del personale ...................................................................................................................................................................................... 19 controllo accessi .......................................................................................................................................................................................................................... 19 parte ii aspetti sanitari gestione dei nuovi ingressi ................................................................................................................................................................................. 22 gestione sanitaria ..................................................................................................................................................................................................................... 23 benessere: attività psicofisiche e valutazione ............................................................................................................ 27 alimentazione ................................................................................................................................................................................................................................... 28 adozioni e affidi ............................................................................................................................................................................................................................. 30 eutanasia ....................................................................................................................................................................................................................................................... 32 rinuncia di proprietà del cane ...................................................................................................................................................................... 33 parte iii normativa normativa regionale ........................................................................................................................................................................................................... 37 normativa nazionale .......................................................................................................................................................................................................... 47 normativa europea ................................................................................................................................................................................................................ 48 parte iv bibliografia bibliografia ................................................................................................................................................................................................................................................ 51 collana di monografie 30 veterinaria italiana, collana di monografie, monografia 30, 2022 7 il canile sanitario, procedure e protocolli • la somministrazione delle terapie che si rendano necessarie e l’esecuzione di eventuali esami diagnostici; • l’isolamento sanitario; • l’accudimento e l’ idonea sistemazione per tutti i cani ospitati. le autorità sanitarie locali che presiedono al canile sanitario dovrebbero operare in considerazione dell’obiettivo principale della lotta al randagismo che è quello di non avere cani vaganti sul territorio. per ottenere ciò è essenziale: • diffondere la cultura del possesso responsabile degli animali attraverso la corretta formazione dei proprietari; • aumentare l’identificazione e registrazione in anagrafe degli animali d’affezione; • sterilizzare i cani rinvenuti vaganti; • promuovere la sterilizzazione dei cani di proprietà. si ricorda che tra le cause più importanti del sovraffollamento dei canili vi è il recupero sul territorio di animali vaganti non identificati per i quali non risulta possibile il rintraccio dei proprietari e l’abbandono di cucciolate indesiderate. la normativa vigente stabilisce i requisiti minimi che ogni struttura che ospita animali deve possedere. il ciclo pdca (plan-docheck-act) rappresenta un sistema efficace per il miglioramento continuo della qualità nel lungo periodo. nel canile, in quanto struttura residenziale, gli ospiti rappresentano la componente di maggior rilievo, oggetto di tutela in tutte le attività e nell’intera organizzazione e pertanto, l’applicazione della normativa deve essere considerata solo il primo passo di un percorso di qualità. in italia la lotta al randagismo e la tutela degli animali sono principi cardine definiti dalla legge quadro 14 agosto 1991, n. 281 che ha rappresentato un importante passo in avanti dal punto di vista etico-culturale, riconoscendo agli animali d’affezione il diritto alla vita. la l.q. 281/91 prevede l’esplicito divieto di soppressione dei cani randagi vaganti quale misura di controllo del randagismo e il loro impiego nella sperimentazione. nelle leggi regionali di attuazione della l.q. 281/91 si distinguono principalmente due tipologie di canili, denominate canile sanitario e canile rifugio. per i canili rifugio si rimanda al manuale “il canile rifugio: procedure e protocolli”. nel presente manuale saranno descritti i criteri strutturali e i protocolli operativi dei canili sanitari. il canile sanitario è una struttura di ricovero di prima accoglienza gestita dalle autorità sanitarie territorialmente competenti. al canile sanitario affluiscono solitamente i cani rinvenuti vaganti, cani i cui proprietari hanno rinunciato alla proprietà, e cani sottoposti a provvedimenti speciali dell’autorità competente (p.e. ritenuti pericolosi, affetti da sintomatologia riferibile a malattie infettive denunciabili). il ricovero, nella maggior parte dei casi, è disposto dal sindaco. i canili sanitari sono quindi presidi sanitari e di prevenzione. le funzioni principali dei canili sanitari, previste nelle norme regionali di applicazione della l.q. 281/91 sono: • il soccorso; • l’identificazione e l’iscrizione all’anagrafe canina se non già fatta; • il controllo delle nascite secondo le indicazioni regionali; • i trattamenti profilattici previsti dalle norme nazionali e regionali; introduzione il canile sanitario, procedure e protocolli 8 veterinaria italiana, collana di monografie, monografia 30, 2022 nell’elaborazione dei suddetti protocolli si devono considerare i seguenti punti: • creare un ambiente idoneo dal punto di vista sanitario pur nel rispetto delle esigenze etologiche degli animali; • creare un ambiente accogliente e sicuro dal punto di vista sanitario anche al fine di incrementare le adozioni e gli affidi diretti al canile; • creare un ambiente salubre nel quale si eserciti una adeguata prevenzione e gestione sanitaria; • creare un ambiente sicuro per gli operatori; • stabilire il percorso che porti il canile alla certificazione da parte di un ente terzo indipendente. la finalità di questo manuale è quella di essere una guida facilmente consultabile per gli operatori del settore a qualsiasi livello di responsabilità e per coloro che si accingono alla costruzione o al risanamento di canili con la prerogativa di ottimizzare la gestione delle strutture attraverso un percorso di qualità. poiché ogni canile ha peculiarità diverse che riguardano collocazione, grandezza, situazione epidemiologica del luogo, ecc., devono essere elaborate specifiche procedure per ogni struttura. le indicazioni fornite nel presente manuale dovranno essere adattate e integrate alle singole realtà. tutti i protocolli devono essere sviluppati e dettagliati con chiarezza al fine di ottenere e mantenere standard elevati di gestione del benessere animale e condizioni ottimali di lavoro. scopo del manuale parte i gestione della struttura il canile sanitario, procedure e protocolli 10 veterinaria italiana, collana di monografie, monografia 30, 2022 • un locale di deposito farmaci o attrezzatura sanitaria non accessibile a personale non autorizzato; • una sala lavaggio e disinfestazione; • un magazzino per il cibo; • un magazzino attrezzi; • un adeguato impianto frigorifero per la custodia temporanea degli animali morti; • uno spogliatoio per gli operatori; • servizi igienici; • una sala toelettatura. oltre a quanto già previsto dalle singole leggi regionali, al cui capitolo si rimanda per eventuali approfondimenti, è assolutamente necessario tenere in opportuna considerazione una razionale disposizione delle singole strutture, dei locali e delle relative attrezzature come indicato di seguito: • gli spazi destinati al ricovero dei cani siano adeguati alle loro esigenze fisiche ed etologiche. le esigenze di spazio variano secondo la mole e l’indole dei cani e, pertanto, non è possibile riferire misure ottimali valide in ogni situazione. al solo scopo di dare un’indicazione, si ricorda che la deliberazione n. 353 del 2 aprile 2013 della regione emiliaromagna dispone che box individuali, con area di sgambamento aggiuntiva, abbiano dimensione minima di 9mq, misura che si avvicina maggiormente alle esigenze di spazio degli animali rispetto a quelle previste da numerose leggi regionali. • siano presenti aree o locali che mantengano temperature adeguate per cani con esigenze particolari (per. es. non al di sotto dei 15° gradi per cuccioli e cani di taglia piccola e a pelo raso). • il reparto destinato ai soggetti problematici sia collocato in un’area distante dall’ingresso, lontano da fonti di stress. cespugli e piante basse possono essere utili al cane timoroso o ansioso per sentirsi più al sicuro. i requisiti previsti dalle norme costituiscono la base per la realizzazione di un canile sanitario che rispetti la nuova visione del rapporto uomo/animale e che rappresenti un luogo di lavoro sicuro per tutti gli operatori. i canili sono classificati dal d.m. 5 settembre 1994 “industrie insalubri di i classe” in quanto produttori di cattivi odori, rumori e rifiuti solidi e liquidi, pertanto le strutture di nuova costruzione devono essere collocate lontano dalle abitazioni e dai corsi d’acqua superficiali. molti regolamenti comunali (piani regolatori, regolamenti di tutela igienico sanitaria ecc.) prevedono che queste strutture siano circondate da fasce di verde. è auspicabile comunque che i canili siano circondati da alberi ad alto fusto e siepi, in modo da creare ampi spazi di ombra e integrarli visivamente all’ambiente circostante, creando contemporaneamente un valido isolamento acustico. nella progettazione dei canili di nuova costruzione deve essere considerato con attenzione il raggiungimento di una buona ventilazione ottenuta con accorgimenti strutturali o grazie ad una ben studiata collocazione dell’intera struttura. ciò migliora la situazione igienicosanitaria, allontanando i cattivi odori, mitigando le alte temperature e contribuendo a rendere più gradevole l’ambiente per gli animali e gli operatori. altrettanto importante è l’orientamento della struttura che dovrebbe essere rivolto a sud e nelle regioni più calde a sud-est. nel canile sanitario oltre ai box e alle aree recintate dovrebbero essere previsti almeno i seguenti locali: • un locale adibito ad ufficio; • un ambulatorio veterinario autorizzato dotato di attrezzatura diagnostica; • una sala chirurgica; • un locale infermeria, adeguatamente attrezzato con gabbie di degenza, per la custodia dei cani feriti o in degenza dopo la sterilizzazione; requisiti strutturali veterinaria italiana, collana di monografie, monografia 30, 2022 11 il canile sanitario, procedure e protocolli • le reti di recinzione sovrastino un muretto di cemento o laterizi. tale muretto deve essere adeguatamente interrato per impedire che gli animali scavino gallerie. • i box siano forniti di cucce in materiale lavabile e disinfettabile. è auspicabile che le cucce abbiano una parete o il tetto smontabile in modo da rendere agevole la pulizia. in aggiunta a ciò, l’esperienza di campo indica che sono da preferirsi quelle a tettuccio piano, quindi fruibili dal cane, rispetto a quelle a tettuccio spiovente. • siano previsti dei sistemi di abbeverata automatica o, in alternativa, ci siano delle procedure per garantire il costante rifornimento di acqua pulita. • l’area dedicata alle attività mediche sia suddivisa in ambulatorio, sala chirurgica e area di degenza. • l’ambulatorio e la sala chirurgica abbiano pareti lisce, lavabili e impermeabili fino ad altezza 2 m. e lavandino azionabile preferibilmente a pedale. • sia previsto, laddove possibile, un locale da dedicare alla preparazione chirurgica dell’animale e delle attrezzature necessarie per l’intervento. in alternativa può essere prevista un’area all’interno della sale da dedicare esclusivamente a detta attività. • siano presenti armadi a destinati al deposito della strumentazione e dei farmaci d’uso • l’ambulatorio e la sala chirurgica siano attrezzati con adeguata strumentazione per l’attività chirurgica e clinica (vedi paragrafo “gestione ambulatorio e sala chirurgica”). • nell’ambulatorio sia affisso il nome del direttore sanitario • nell’ambulatorio e nella sala chirurgica siano prontamente disponibili, anche mediante affissione, le procedure perioperatorie e postoperatorie di cui al paragrafo “gestione ambulatorio e sala chirurgica”. • i box destinati ai cani pericolosi siano contigui, separati da doppie porte (a ghigliottina) azionabili dall’esterno in modo che gli operatori possano trasferire i cani da un box all’altro durante le pulizie, lavorando in sicurezza. • le aree pavimentate siano costruite in materiale lavabile, disinfettabile, non sdrucciolevole e lievemente pendenti (la pendenza non deve superare il 3%) in modo da permettere il rapido allontanamento delle urine e delle acque di lavaggio presso i canali di scolo. è da sconsigliare l’uso di piastrelle in quanto possono rompersi per urti meccanici o possono scollarsi con la pressione dell’idropulitrice. una buona soluzione può essere rappresentata da cemento trattato con resine speciali anche colorate (gradevoli alla vista), resistenti in ambienti esterni e che proteggono il cemento evitando la formazione di buche. • gli spigoli e gli angoli siano arrotondati in modo da evitare il ferimento dei cani e degli operatori nonché gli accumuli di sporco. • tutti i locali di servizio (cucine, ambulatori, ecc.) e i box chiusi siano protetti da zanzariere a maglie fitte che impediscano l’ingresso di zanzare e flebotomi. • tutte le parti erbose siano mantenute costantemente rasate al fine di evitare che diventino ricettacolo di parassiti quali zecche. • siano evitati gli accatastamenti di materiali di qualsiasi natura al fine di contenere la riproduzione di alcuni insetti tra cui i flebotomi. • le porte di accesso ai box abbiano requisiti di robustezza e facilità di apertura e chiusura da parte degli operatori ma non dei cani. • i cancelli di accesso ai box abbiano la parte inferiore in materiale resistente agli urti e quella superiore in rete elettrosaldata con maglie sufficientemente fitte da impedire ai cani di infilare gli arti o il muso. il canile sanitario, procedure e protocolli 12 veterinaria italiana, collana di monografie, monografia 30, 2022 grammazione e organizzazione dei seguenti processi: • piano alimentare; • protocollo di igiene ambientale e disinfezione; • procedura per le adozioni e l’affidamento; • procedura per la gestione rifiuti; • procedura di controllo degli animali infestanti; • procedure di biosicurezza per gli operatori; • piano di formazione per l’anno in corso e archivio degli anni precedenti; • piano di emergenza ed evacuazione; • procedura per la corretta tenuta dei registri; • procedure peri e post operatorie. negli stessi uffici devono inoltre essere conservati i documenti e gli atti che registrino il regolare svolgimento dei processi sopra menzionati al fine di monitorare le attività, individuare le eventuali azioni correttive e rendere l’intero processo trasparente. è auspicabile che piani, procedure e tutti gli atti relativi siano gestiti in maniera informatizzata per permettere una gestione più precisa e all’occorrenza una immediata disponibilità dei dati. presso il canile devono essere conservati i seguenti documenti o le relative copie: • organigramma e funzionigramma con l’identificazione del personale organico ed eventualmente dei volontari, laddove prevista la loro presenza; • registro giornaliero degli operatori volontari presenti laddove è consentita la loro frequentazione della struttura; • eventuali convenzioni; • atto di incarico del direttore sanitario laddove previsto; • registro di carico/scarico dei cani (anche informatizzato); • registro farmaci; • registro rifiuti speciali; • documento informativo sulla procedura di adozione e affidamento; • documento di valutazione del rischio per gli operatori; • certificazioni di conformità degli impianti; • schede sanitarie cartacee o informatizzate; • regolamento della struttura con annessi protocolli (sanitario, gestionale, mansionario). presso i locali adibiti ad uffici devono essere inoltre conservati tutti i documenti di progestione documenti e archiviazione veterinaria italiana, collana di monografie, monografia 30, 2022 13 il canile sanitario, procedure e protocolli il canile può essere coinvolto in disastri ambientali di varia natura (dissesti idrogeologici, esondazioni, terremoti, avvelenamento atmosferico, ecc.) che richiedono una immediata evacuazione. in questo contesto due aspetti appaiono rilevanti: • le modalità di evacuazione; • la sistemazione dei cani evacuati. la modalità di evacuazione deve essere descritta da una apposita procedura che differisce a seconda dell’organizzazione del canile. per la sistemazione dei cani evacuati è auspicabile avere delle apposite convenzioni con le strutture ricettive più vicine. strumenti indispensabili per una pronta ed efficiente evacuazione sono: • gli elenchi degli operatori del canile aggiornati e prontamente accessibili che includano i loro indirizzi e recapiti telefonici; • la planimetria del canile (vedi scheda tecnica) affissa all’ingresso della struttura; • le attrezzature per il trasferimento degli animali (guinzagli, gabbie e automezzi) in numero adeguato alla capienza del canile; • elenco dei canili, allevamenti e pensioni più vicini in cui venga anche indicata la capacità di accoglienza delle strutture. è necessario fare particolare attenzione che i percorsi di fuga siano sempre mantenuti liberi e che gli idranti e gli estintori siano sottoposti a controllo periodico, secondo quanto previsto dalla normativa in vigore e dalla casa produttrice. la procedura di evacuazione e i singoli allegati devono essere aggiornati ogni volta che si verifichino le seguenti condizioni: • variazioni negli edifici per quanto attiene sia alle strutture sia agli impianti; • variazioni organizzative; • nuove norme che richiedono modifiche; • mutazioni nelle esigenze di sicurezza; • significative variazioni numeriche degli animali nei box; • variazioni nei recapiti degli operatori; • variazioni nei percorsi di fuga. piano di emergenza e di evacuazione la planimetria deve riportare: i locali e le relative destinazioni di uso; le uscite di emergenza; i percorsi di fuga (colorati); le attrezzature antincendio (estintori, idranti, ecc.); la segnaletica di sicurezza; i punti di erogazione dell’acqua; la posizione del quadro con sganciatore elettrico; la posizione del rubinetto per la chiusura del gas; le cassette di medicazione. scheda tecnica i. planimetria. il canile sanitario, procedure e protocolli 14 veterinaria italiana, collana di monografie, monografia 30, 2022 ai sensi dell’art 183 del dlvo 152/2006, comma 1 lettera a, il rifiuto è “…qualsiasi sostanza od oggetto di cui il detentore si disfi o abbia l’intenzione o abbia l’obbligo di disfarsi.” ai sensi del successivo art. 184, comma 1 “[…]i rifiuti sono classificati, secondo l’origine, in rifiuti urbani e rifiuti speciali e, secondo le caratteristiche di pericolosità, in rifiuti pericolosi e rifiuti non pericolosi”. rifiuti urbani non pericolosi rientrano in questa tipologia i rifiuti che derivano da attività di routine del canile sanitario, non presentano rischio tossico o infettivo e sono assimilabili ai rifiuti urbani per qualità e quantità. la raccolta ed il deposito devono essere organizzati in modo tale da evitare cattivi odori, disordine e inquinamento ambientale. per la raccolta differenziata di vetro, plastica, metallo, carta e cartone devono essere rispettate le specifiche disposizioni comunali. gli scarti di giardinaggio possono essere smaltiti in appositi contenitori da compost ed essere utilizzati sui terreni della stessa struttura oppure, se di modica entità, dopo adeguata riduzione di volume, possono essere depositati presso un contenitore riportante la dicitura “rifiuto organico”. gli indumenti monouso, quando non utilizzati per attività medica o infermieristica, possono essere smaltiti presso gli appositi contenitori recanti la dicitura “raccolta indifferenziata”. rifiuti urbani pericolosi i rifiuti urbani pericolosi sono costituiti da quei rifiuti che contengono al loro interno un’elevata dose di sostanze pericolose e che quindi devono essere gestiti diversamente dal flusso dei rifiuti urbani “normali”. tra questi, quelli di più comune produzione in un canile sanitario sono i medicinali scaduti, i pesticidi e le pile. rifiuti speciali appartengono ai rifiuti speciali i rifiuti sanitari regolamentati da dpr 254/2003. sono rifiuti derivanti dalle attività ambulatoriali, chirurgiche, mediche ed infermieristiche condotte presso gli ambulatori dei canili sanitari e rifugio o comunque praticate sugli animali del canile. i rifiuti sanitari non pericolosi sono costituiti da: farmaci scaduti o di scarto (ad eccezione dei farmaci citotossici e citostatici), sostanze chimiche di scarto non pericolose e non contenenti sostanze pericolose e rifiuti taglienti inutilizzati (aghi, siringhe, lame e rasoi). questi ultimi, tuttavia, devono essere smaltiti in contenitori rigidi come i rifiuti taglienti utilizzati. i rifiuti sanitari pericolosi a rischio non infettivo sono rappresentati da medicinali citotossici e citostatici e dai rifiuti provenienti da laboratorio analisi o radiologico (sostanze chimiche di scarto pericolose o contenenti sostanze pericolose, oli per circuiti idraulici, soluzioni fissative, soluzioni di sviluppo e atgestione rifiuti figura 1. classificazione dei rifiuti (art. 184 del dlvo 152/2006). figura 2. classificazione dei rifiuti sanitari (dpr 254/2003). rifiuti urbani speciali pericolosi non pericolosi pericolosi non pericolosi rifiuti sanitari non pericolosi pericolosi rischio infettivo rischio non infettivo veterinaria italiana, collana di monografie, monografia 30, 2022 15 il canile sanitario, procedure e protocolli la depurazione in loco può essere effettuata tramite sistemi di depurazione biologica (impianto a fanghi attivi, fitodepurazione, percolatore, ecc.). le acque reflue depurate possono essere scaricate in acque superficiali (canali, torrenti o eventualmente fossi poderali). nel caso non sia possibile ricorrere a tali sistemi si possono immettere tutti i reflui (feci e acque di lavaggio) in una vasca di raccolta autorizzata e a tenuta, di capacità adeguata, senza trattamenti, e smaltirli tramite ditte autorizzate. smaltimento feci le feci rimosse con le acque di lavaggio possono essere smaltite come acque reflue (vedi paragrafo acque reflue). le feci raccolte a secco possono essere immesse in contenitori a tenuta e rimosse periodicamente tramite ditte autorizzate. tivanti a base acquosa, materiali isolanti contenenti amianto, lampade fluorescenti ed altri rifiuti contenenti mercurio) i rifiuti sanitari pericolosi a rischio infettivo derivano da attività veterinaria e rispondono ad almeno una delle seguenti caratteristiche: 1. siano contaminati da agenti patogeni per l’uomo o per gli animali; 2. siano venuti a contatto con un qualsiasi liquido biologico, secreto od escreto, per il quale sia ravvisato, dal medico veterinario, un rischio di patologia trasmissibile attraverso tali liquidi. acque reflue le acque reflue di lavaggio del canile sanitario non possono essere destinate a spandimento sul suolo. devono invece essere depurate in loco o avviate a un depuratore. il canile sanitario, procedure e protocolli 16 veterinaria italiana, collana di monografie, monografia 30, 2022 nei canili il controllo di specie animali infestanti (pest) assurge a particolare importanza in considerazione della gravità dei danni che possono causare tra i quali ricordiamo: • danni meccanici alla struttura; • imbrattamento dell’ambiente; • imbrattamento e distruzione delle scorte alimentari; • diffusione di malattie infettive. gli infestanti più comuni sono rappresentati da mosche, zanzare, flebotomi, blatte, coleotteri, ratti, topi, uccelli. i mezzi di controllo degli infestanti sono molteplici e tra questi, negli ultimi anni, sono andate affermandosi strategie di lotta integrata (integrated pest management ipm). i programmi di ipm sfruttano diverse informazioni (ciclo di vita del parassita, interazione parassita/ ambiente, modalità di diffusione, sensibilità ai vari metodi di lotta, ecc.) per raggiungere una maggiore efficacia e una riduzione dell’uso degli agenti chimici. le strategie di lotta integrata comprendono: • monitoraggio e identificazione dei pest • prevenzione • controllo monitoraggio il monitoraggio si basa sull’ispezione visiva e sull’uso di trappole. l’ispezione visiva degli ambienti viene effettuata per rilevare i segni della presenza di specie infestanti (materiali rosicchiati, animali o insetti morti, ragnatele, escrementi, impronte, ecc.). l’uso di trappole specifiche consente di rilevare la presenza di infestanti non rilevabili alla ispezione visiva. prevenzione la prevenzione si basa su: • pulizia dei locali, delle cucce e degli ambienti esterni che deve essere accurata e a cadenza giornaliera e deve essere preceduta dalla rimozione di eventuali ostacoli fisici (scatole, attrezzi, ecc.); • eliminazione di ogni eventuale fessurazione delle strutture murarie, delle porte e degli infissi; • eliminazione di ogni eventuale accatastamento di materiali; • installazione di reti metalliche su tutte le finestre; • cura costante del verde (le zone a manto erboso devono essere rasate e le zone cespugliose devono essere potate). controllo animali infestanti formazione e addestramento degli operatori gli operatori devono essere adeguatamente addestrati sulla rilevazione dei segni di presenza di pest (presenza di materiali rosicchiati, feci, carcasse, impronte, ecc.) e sulle misure di prevenzione e controllo. piano di monitoraggio il piano di monitoraggio deve essere redatto da un esperto e prevedere una appropriata procedura documentabile. piano di prevenzione il piano di prevenzione deve essere redatto da un esperto e prevedere una appropriata procedura documentabile. piano di controllo il piano di controllo deve essere redatto da un esperto e prevedere una appropriata procedura documentabile che deve essere adeguata periodicamente in base ai dati di monitoraggio. scheda tecnica ii. piano di controllo pest. veterinaria italiana, collana di monografie, monografia 30, 2022 17 il canile sanitario, procedure e protocolli setti antagonisti ed agenti patogeni (virus, batteri, protozoi, nematodi). nei canili può essere utile l’utilizzo del bacillus thuringiensis per il controllo delle zanzare. mezzi di controllo chimici i principi attivi utilizzabili nella lotta ai pest sono riportati nel regolamento ue sui biocidi n. 528/2012 mezzi di controllo meccanici i mezzi di controllo meccanici sono rappresentati dalle trappole. in commercio esistono molti tipi di trappole finalizzate alla cattura sia di roditori che di insetti. controllo il controllo degli infestanti è solitamente affidato a ditte specializzate, tuttavia conoscere i principali metodi di controllo è importante per verificare il lavoro svolto. i mezzi di controllo possono essere fisici, biologici, chimici e meccanici. mezzi di controllo fisici • ultrasuoni; • vapore; • congelamento con azoto liquido. mezzi di controllo biologici si basano prevalentemente sull’utilizzo di inil responsabile del canile deve effettuare una corretta valutazione dei rischi per gli operatori e attuare una efficace prevenzione. i rischi più comuni sono rappresentati da: • infortuni; • zoonosi. infortuni possono essere riconducibili a 3 gruppi principali: • infortuni dovuti a caratteristiche strutturali dei locali e degli ambienti del canile; • infortuni dovuti allo svolgimento delle attività; • infortuni dovuti ad aggressioni di animali ospiti. gli infortuni dovuti a caratteristiche strutturali si verificano soprattutto quando le costruzioni non sono ben progettate e ben manutenute. tra le cause più frequenti ricordiamo: • pavimenti sdrucciolevoli e soluzioni di continuità che possono causare scivolamenti e cadute; • angoli, spigoli e rifiniture che possono causare tagli, graffi, escoriazioni; • impianti elettrici che possono causare folgorazioni. le attività lavorative che più frequentemente possono essere causa di infortuni sono rappresentate da: • immagazzinamento e prelievo di fusti (alimenti, agenti chimici, ecc.); • spostamenti di animali. gli infortuni dovuti ad aggressioni di animali ospiti possono essere riconducibili a: • eccessivo affollamento dei box; • presenza di animali con disturbi comportamentali; sicurezza il canile sanitario, procedure e protocolli 18 veterinaria italiana, collana di monografie, monografia 30, 2022 zoonosi per zoonosi si intende qualsiasi malattia e/o infezione trasmessa direttamente o indirettamente dagli animali all’uomo e viceversa; possono essere causate da batteri, miceti, parassiti, virus e veicolate da vettori. i più comuni agenti di zoonosi sono: • campylobacter; • salmonella; • escherichia coli; • giardia; • dermatofiti • leishmania; • leptospira; • echinococco; • toxocara canis; • ancylostoma; • dirofilaria immitis; • dirofilaria repens. le modalità di trasmissione possono essere: 1. morsi o graffi; 2. contatto con sangue infetto e altri liquidi biologici; 3. punture e morsi di artropodi; 4. contatto con liquami; 5. semplice contatto con l’animale. • eccessiva familiarità degli operatori con gli animali e conseguente diminuzione dell’attenzione. prevenzione infortuni gli infortuni legati alle caratteristiche delle strutture possono essere prevenuti attuando le misure riportate nel capitolo “requisiti strutturali”. gli infortuni legati allo svolgimento delle attività possono essere prevenuti predisponendo rigorose procedure per l’esecuzione standardizzata delle attività lavorative. gli infortuni legati a eccessiva familiarità o a errato atteggiamento verso gli animali possono essere ridotti o eliminati con un’adeguata formazione degli operatori. di particolare importanza è l’uso di dispositivi di protezione individuale (dpi). i principali dispositivi sono rappresentati da scarpe e stivali antiscivolo, tuta monouso e guanti. i dpi devono essere adeguati al rischio da prevenire e al luogo di lavoro e adattabili all’utilizzatore. in ogni caso deve essere prevista un’assicurazione per la struttura e/o per gli operatori/ volontari. veterinaria italiana, collana di monografie, monografia 30, 2022 19 il canile sanitario, procedure e protocolli il responsabile deve garantire che tutti gli operatori abbiano una formazione e un addestramento adeguati allo svolgimento delle proprie attività per: • operare in relazione al proprio livello di competenza • costruire un buon rapporto con gli animali, gli altri operatori e i volontari • conoscere adeguatamente le esigenze etologiche e sanitarie degli animali • conoscere le procedure applicate nel canile e la normativa vigente • avere personale esperto e formato in grado di promuovere iniziative volte a diffondere la cultura del possesso responsabile degli animali. oltre ai corsi di formazione e di aggiornamento è opportuno prevedere anche periodi di tirocinio per ogni operatore. formazione del personale il direttore sanitario deve redigere un piano annuale di formazione e aggiornamento per tutto il personale, definendo: gli obiettivi gli argomenti il target dei partecipanti la durata il periodo di erogazione. scheda tecnica iii. piano di formazione. al canile accedono quotidianamente varie categorie di persone (direttore, operatori, veterinari, fornitori) pertanto l’ingresso nelle diverse aree deve essere diversificato secondo le attività e il ruolo svolto. alcune strutture quali ambulatorio, infermeria, sala operatoria, locale di deposito della scorta farmaci, box degli animali malati o pericolosi, possono essere frequentati esclusivamente da personale autorizzato dal responsabile del canile. i visitatori possono recarsi al canile sanitario esclusivamente per il ritiro del proprio cane, qualora sia stato smarrito, ritrovato e condotto al canile e per l’adozione. controllo accessi parte ii aspetti sanitari il canile sanitario, procedure e protocolli 22 veterinaria italiana, collana di monografie, monografia 30, 2022 senta un momento di elevato stress per gli animali e un rischio di introduzione di malattie, per cui è essenziale porre particolare attenzione alle procedure di gestione dei nuovi ingressi. al momento dell’ingresso, ove non già fatto nelle fasi di cattura e consegna dei cani e se non vi è la necessità di effettuare interventi clinici o chirurgici urgenti, deve essere valutata la presenza del microchip e l’iscrizione all’anagrafe al fine dell’accertamento di proprietà. nelle schede tecniche che seguono sono riportati gli adempimenti essenziali da porre in atto. non necessariamente le operazioni devono avvenire nell’ordine descritto in quanto la programmazione può variare secondo l’esito della visita clinica. la prima valutazione clinica e comportamentale del cane è fondamentale per stabilire la collocazione nel canile. i cani che entrano nei canili sanitari possono essere suddivise nelle seguenti categorie: • cani accalappiati vaganti senza identificativo individuale (microchip); • cani accalappiati vaganti con identificativo individuale (microchip) per i quali è possibile rintracciare il proprietario; • cani morsicatori che devono sottostare al periodo di osservazione nei confronti della rabbia di cui all’art.83 del dpr 320/54; • cani sequestrati: • cani ritenuti potenzialmente pericolosi per l’incolumità pubblica e di altri animali a seguito di valutazione del servizio veterinario ufficiale; • cani oggetto di rinuncia di proprietà (previsto in alcune regioni). l’ingresso di nuovi cani nella struttura rappregestione dei nuovi ingressi predisposizione o integrazione della cartella clinica già eventualmente redatta nel corso dell’accalappiamento) esame obiettivo generale valutazione comportamentale identificazione apposizione di microchip e iscrizione all’anagrafe degli animali d’affezione registrazione del cane sul registro di carico e scarico cartaceo o informatizzato valutazione comportamentale predisposizione della cartella clinica nel caso di cani sequestrati o confiscati deve essere ritirato e archiviato il documento di sequestro o confisca scheda tecnica iv. ingresso del cane nel canile sanitario. nel registro di carico e scarico degli animali ospitati devono essere riportate almeno le seguenti informazioni: microchip dell’animale; data di entrata; provenienza; generalità del proprietario (sindaco per i cani vaganti o privato cittadino); data e causa di morte; data di adozione/affidamento; destinazione del cane in caso di affidamento o adozione; data di uscita con specifica della causa (morte, adozione, affidamento, fuga, avvio al canile rifugio) scheda tecnica v. registro di carico e scarico cartaceo o informatizzato. veterinaria italiana, collana di monografie, monografia 30, 2022 23 il canile sanitario, procedure e protocolli cause di morte diventando un indice della qualità di gestione del canile. oltre alla regolare profilassi per gli endo ed ectoparassiti, l’alta incidenza di malattie trasmesse da vettori in alcune zone (per es. leishmaniosi, filariosi, erhlichiosi, borreliosi, ecc.), rende indispensabile il trattamento diretto degli animali con prodotti repellenti e la disinfestazionie ambientale. i protocolli vaccinali variano secondo l’area geografica, il numero di animali ospitati, l’intensità del turn-over, l’anamnesi collettiva e individuale e la situazione del canile e sono stabiliti dal medico veterinario responsabile della struttura. le linee guida per la vaccinazione del cane e del gatto stilate dal vaccination guidelines group (vgg) della world small animal veterinary association (wsava) pubblicate e aggiornate nel 2015 possono rappresentare un ottimo riferimento per la scelta dei la gestione sanitaria di un canile sanitario ha come obiettivi fondamentali: • verificare lo stato sanitario degli animali (anche al fine della salute pubblica); • tutelare il benessere degli animali; • prevenire le patologie degli animali; • curare prontamente le patologie di qualsiasi natura degli animali; • eseguire interventi di sterilizzazione. la gestione sanitaria del canile vede a capo un medico veterinario come direttore sanitario ma a vari livelli e a diverso titolo coinvolge tutti gli operatori. una buona gestione sanitaria porta al miglioramento delle condizioni generali di benessere degli animali e degli operatori e alla riduzione dei costi delle spese mediche riducendo l’incidenza delle malattie e migliorando l’adottabilità. per ottenere ciò, tutte le attività del canile e in maniera particolare l’alimentazione, la gestione corretta delle nuove introduzioni, l’igiene ambientale, la gestione controllata del personale in entrata e in uscita devono essere oggetto di costante monitoraggio. protocolli di prevenzione e cartelle cliniche prevenzione la prevenzione delle malattie infettive nel canile si basa su una conduzione gestionale che limiti l’esposizione ai patogeni e mantenga gli ospiti del canile in uno stato di salute tale da renderli meno inclini ad ammalarsi. risulta di particolare importanza l’eliminazione di contatti tra cani con anamnesi sconosciuta. gli animali morti dovrebbero sempre essere avviati all’esame autoptico. l’accertamento delle cause di morte degli animali ha una funzione di prevenzione di nuovi focolai e contribuisce al monitoraggio delle gestione sanitaria per i trattamenti profilattici obbligatori previsti dalla normativa si rimanda alla legge 281/91, art. 2, comma 5. nei canili italiani sono, di solito, ritenuti “core” le seguenti vaccinazioni: parvovirus-2 canino; virus del cimurro; adenovirus-2 canino. leptospira interrogans sierogruppo canicola leptospira interrogans sierogruppo icterohaemorrhagiae leptospira interrogans sierogruppo australis leptospira kirschneri sierogruppo grippotyphosa solitamente rientrano nelle “non core” le seguenti vaccinazioni: bordetella bronchiseptica; virus parainfluenzale-3; borrelia burgdoferi; herpesvirus-1canino; leishmania infantum; coronavirus canino; microsporum canis; babesia canis. i vaccini devono essere conservati in frigorifero, secondo le indicazioni del produttore e usati entro la data di scadenza. scheda tecnica vi. vaccinazioni. il canile sanitario, procedure e protocolli 24 veterinaria italiana, collana di monografie, monografia 30, 2022 to che riporti i dati e le informazioni illustrati nella scheda tecnica che segue. gestione dell’igiene ambientale e della disinfezione tra i disinfettanti che possono essere utilizzati: • alcool; • composti a base di cloro; • aldeidi; • iodofori; • composti di ammonio quaternario. gli alcool sono battericidi ma non sporicidi e agiscono denaturando le proteine. i composti a base di cloro hanno un ampio spettro di attività, sono economici e sono veloci da utilizzare, ma sono anche corrosivi ed instabili. vaccini di base (core) o facoltativi (non core) e dei protocolli vaccinali. nelle stesse linee guida sono reperibili utili indicazioni circa gli esami sierologici necessari per la documentazione e il monitoraggio delle risposte immunitarie e un paragrafo sulle reazioni avverse. i cicli vaccinali possono non essere portati a termine nel canile sanitario data la natura temporanea del ricovero. in questi casi è necessario che il ciclo sia completato presso le strutture di destinazione quali p. es. canili rifugio e famiglie adottanti. nella scheda che segue sono riportati i trattamenti profilattici e gli esami diagnostici di base da attuare sui nuovi ingressi, fatta eccezione per gli animali di cui è possibile dimostrare l’avvenuto trattamento con relativa tempistica. cartella clinica la cartella clinica, auspicabile in formato informatico, (meglio se integrata al programma di registrazione della banca dati dell’anagrafe degli animali d’affezione) deve contenere tutti la documentazione inerente la storia clinica dell’animale, con documenti di registrazione attestanti i controlli, le diagnosi, le terapie, gli interventi chirurgici, i trattamenti effettuati e devono essere automaticamente aggiornate ad ogni nuovo intervento effettuato. è opportuno inserire nella cartella clinica un prospetfoto nome; microchip; razza; sesso; mantello; taglia; data di nascita (se conosciuta o indicativa); data di ingresso nel canile; motivo di consegna al canile (per es. rinvenuto vagante, sequestrato, consegnato dal proprietario). visite cliniche (tutte le visite effettuate compresa quella presso il canile sanitario) data di effettuazione, nome del veterinario, esito esami di laboratorio data, richiedente ed esito trattamenti antiparassitari data, prodotto usato, veterinario vaccinazioni data, vaccino usato, veterinario terapie data, protocollo terapeutico, veterinario prescrittore interventi chirurgici data, ambulatorio di esecuzione, equipe chirurgica visite comportamentali data, diagnosi, nome del veterinario, terapia prescritta dieta tipo di dieta, durata, veterinario prescrittore scheda tecnica viii. cartella clinica. tipo di intervento esame feci trattamento antiparassitario per parassiti interni trattamento antiparassitario per parassiti esterni esami sierologici (p.e. leishmania, dirofilaria) vaccinazioni secondo protocollo individuale altri esami e altre azioni preventive possono essere inseriti nel piano di prevenzione in base alla situazione epidemiologica o geografica del canile. scheda tecnica vii. trattamenti profilattici e esami diagnostici di base. veterinaria italiana, collana di monografie, monografia 30, 2022 25 il canile sanitario, procedure e protocolli le aldeidi devono essere usate in soluzione acquosa e possono essere utilizzate sia sotto forma gassosa che liquida. possiedono azione battericida, fungicida, virulicida e a determinati valori di ph anche sporicida. l’aldeide più usata è la glutaraldeide. gli iodofori, come soluzioni di iodio, sono utilizzati come disinfettanti della cute e delle mucose. le soluzioni diluite hanno un elevato potere battericida ma la presenza di materiale organico riduce la loro attività. i composti quaternari d’ammonio sono ampiamente utilizzati come disinfettanti e antisettici e normalmente sono inattivati da acqua dura, sapone e residui anionici. possiedono azione fungicida, battericida e virulicida (limitatamente ai virus lipofilici). il loro uso è ottimale per la sanitizzazione ambientale di superfici quali pavimenti, sanitari e muri. ferma restando quindi la possibilità di scegliere i prodotti più confacenti alle esigenze pratiche, si ribadisce che le operazioni di pulizia (con acqua, idropulitrici, ecc.) devono essere eseguite solo dopo aver fatto allontanare il cane dal box. gestione dei farmaci la prescrizione del farmaco può essere fatta solo dal direttore sanitario o da un veterinario da lui delegato. le modalità di tenuta delle scorte dei medicinali deve rispettare quanto indicato dagli art. 80, 82 e 84 del dlgs. 193/2006. i nominativi dei medici veterinari responsabili delle scorte devono essere indicati nella domanda di autorizzazione alla scorta presentata ai servizi veterinari della asl competente per territorio con l’indicazione delle ulteriori far uscire i cani e sistemarli in luogo asciutto. rimuovere tutte le parti mobili. tutti gli oggetti presenti, in maniera particolare ciotole e abbeveratoi, devono essere accuratamente detersi e risciacquati. lavare e disinfettare con prodotti efficaci ed attendere i tempi di azione. la scelta dei prodotti è fatta dal direttore sanitario o da un veterinario da lui delegato. risciacquare abbondantemente con acqua calda in modo da rimuovere i residui di disinfettante. allontanare l’acqua in eccesso. far rientrare i cani solo quando l’ambiente è sufficientemente asciutto. n.b. anche le aree di sgambamento devono essere tenute pulite con la rimozione almeno quotidiana dei rifiuti organici solidi. scheda tecnica ix. gestione dell’igiene ambientale e della disinfezione. strutture presso le quali risultano responsabili della tenuta di scorte. i medicinali devono essere custoditi in idonei locali chiusi o in armadi accessibili solo al veterinario responsabile delle scorte e devono essere conservati, in luogo adeguato, pulito, non esposto a umidità, luce o sbalzi termici. la prescrizione avviene con ricetta elettronica come riportato nel manuale operativo della ricetta elettronica veterinaria di cui al link: https://www.ricettaveterinariaelettronica.it; i documenti d’acquisto dei farmaci devono essere conservati per tre anni. gli operatori che affiancano il veterinario nell’effettuare le terapie devono essere adeguatamente formati ad attenersi rigorosamente alla posologia prescritta. la legislazione vigente prevede che vengano immediatamente segnalate le sospette reazioni avverse ai farmaci al ministero della salute o ai centri regionali di farmacovigilanza (http://www.salute.gov.it/farmacovigilanzavetmodule/farmacovigvetservlet). gestione ambulatorio e sala chirurgica nell’ambulatorio devono essere presenti tutti i presidi utili ad effettuare una visita medica. nella sala chirurgica del canile sanitario, solitamente, vengono effettuati interventi di sterilizzazione e interventi di primo soccorso in cani traumatizzati. la sala pertanto deve essere dotata almeno delle attrezzatura di seguito elencate: • fonte di ossigeno; • dispositivo per l’erogazione dei gas corredato di circuiti e tracheotubi adeguati alle il canile sanitario, procedure e protocolli 26 veterinaria italiana, collana di monografie, monografia 30, 2022 ai fini della corretta gestione del paziente devono essere predisposte almeno le seguenti procedure: • procedure perioperatorie: preparazione paziente: tricotomia, scrub; preparazione chirurgo; preparazione campo operatorio. • procedure postoperatorie: risveglio; monitoraggio; terapie; gestione del dolore. specie trattate; • dispositivi per condurre una procedura anestesiologica (sedazione, anestesia generale/locale); • tavolo chirurgico dedicato; • fonte di luce adeguata; • monitor multiparametro; • elettrobisturi; • aspiratore di liquidi; • sterilizzatrice o autoclave; • teleria sterile; • strumentario chirurgico adeguato al tipo di intervento specialistico. veterinaria italiana, collana di monografie, monografia 30, 2022 27 il canile sanitario, procedure e protocolli stato di salute, tuttavia è possibile individuare riferimenti temporali minimi per ottenere effetti positivi sullo stato di benessere psicofisico dei cani. i cani che hanno accesso ad una zona esterna direttamente dal box, necessitano di attività di movimenti in aree di sgambamento per almeno un’ora al giorno. cani che non hanno accesso diretto ad una zona esterna del box (fermo restando l’obiettivo di dismettere questo tipo di strutture), necessitano dell’accesso alle aree di sgambamento per almeno un’ora, due volte al giorno. il miglioramento della capacità di socializzazione e apprendimento può essere già avviato nel canile sanitario. ciò al fine di favorire gli affidi e le adozioni. per questo motivo è auspicabile che: • gli operatori instaurino un rapporto di scambio comunicativo (visivo, gestuale e vocale) con i cani; • i cuccioli senza madre siano posti in una nursery; • siano impostati percorsi mirati di riabilitazione per i soggetti che manifestino disturbi comportamentali. la collocazione dei cani in canile può essere causa di stress attribuibili a molte cause. le più frequenti sono rappresentate da: • spazi confinati, spesso ristretti; • dinamiche relazionali alterate o assenti sia con l’uomo che con altri cani; • mancanza di esercizio fisico; • mancanza di stimoli ambientali. nel canile sanitario i cani dovrebbero essere ospitati per brevi periodi, tuttavia è importante che non siano in condizioni di disagio psicofisico al fine di evitare traumi e disagi che possano comprometterne il carattere e ridurre l’adottabilità. i segnali di stress possono essere rappresentati da: alterazioni del comportamento alimentare (es. coprofagia), movimenti in circolo prolungati (circling), paure, fobie, intolleranza verso altri animali, iperattività o leccamento, grattamento e vocalizzazioni eccessive. l’attività fisica può ridurre gli effetti avversi del confinamento, soprattutto se associata al contatto con l’uomo e a esercizi mirati alla stimolazione mentale. la necessità di attività motoria varia secondo l’età, la mole, la razza, il periodo fisiologico, lo benessere: attività psicofisiche e valutazione il canile sanitario, procedure e protocolli 28 veterinaria italiana, collana di monografie, monografia 30, 2022 l’alimentazione è fondamentale per un buono stato di salute degli animali e pertanto la dieta deve essere valutata dal direttore sanitario o da un veterinario da lui delegato. la dieta deve rispondere alle esigenze nutritive (caloriche, proteiche, vitaminiche, ecc.) degli animali ma anche rispondere a criteri di appetibilità e per la sua formulazione si devono prendere in considerazione: • l’età dell’animale; • la mole; • la razza; • la stagione; • l’attività fisica; • lo stato fisiologico o parafisiologico (es. gravidanza); • la presenza di patologie. è importante redigere un programma alimentare nel quale vengano definiti: • la tipologia di alimenti (ad esempio preparati industriali o alimenti base per la preparazione dei pasti); • le modalità di preparazione; • le modalità e i tempi di somministrazione; • le quantità; • le misure igieniche da rispettare nella preparazione e nella somministrazione. eventuali esigenze dietetiche particolari riferibili a stati patologici (es. insufficenza renale, diabete ecc.) o a specifici momenti fisiologici (es. allattamento, crescita, ecc.) devono essere annotati nella cartella clinica. alimenti gli alimenti devono essere conservati in ambienti puliti, asciutti e protetti da agenti infestanti (topi, ratti, ecc.). i locali di deposito degli stessi e la cucina devono avere finestre protette da zanzariere. gli spazi degli ambienti e i lavandini devono essere ampi per garantire i movimenti degli operatori e le operazioni di pulizia. i pasti devono essere somministrati a orari fissi e regolari. la dieta può basarsi sull’uso di preparazioni estemporanee di alimenti o di prodotti industriali (crocchette, mangimi umidi inscatolati). nei canili è possibile utilizzare sottoprodotti di origine animale costituiti da rifiuti di cucina e ristorazione come indicato dalla nota congiunta delle direzioni generali della sanità animale e della sicurezza alimentare del 28.12.2015 del ministero della salute. è comunque vietato l’utilizzo per l’alimentazione di olio di cucina esausto e rifiuti di cucina e ristorazione costituiti da residui di alimenti già somministrati al consumatore finale. tali sottoprodotti possono provenire solo da imprese alimentari registrate e riconosciute in base reg.(ce) 852/2004 e reg. (ce) 853/2004, e il loro utilizzo è consentito solo nell’ambito della medesima provincia in cui sono ubicati anche i canili. l’utilizzo di tali sottoprodotti è subordinato a : • registrazione ai sensi dell’art. 23 del reg. ce 1069/2009; • registrazione o riconoscimento ai sensi del reg.(ce) 852/2004 o ai sensi del reg. (ce) 853/2004 dell’industria alimentare produttrice dei sottoprodotti; • trattamento termico; • notifica all’autorità competente locale; • registrazione da parte del proprietario dell’azienda produttrice, in apposito registro. della data di invio e del peso stimato dei sottoprodotti. le preparazioni estemporanee devono essere prodotte sulla base delle indicazioni del direttore sanitario del canile, in considerazione delle esigenze nutritive specifiche degli animali. l’alimentazione con prodotti industriali (crocchette o mangime umido) è sicuramente più agevole ed in molte situazioni consigliabile. alimentazione veterinaria italiana, collana di monografie, monografia 30, 2022 29 il canile sanitario, procedure e protocolli ta un momento di grande importanza nell’interazione cane/uomo e cane/cane. ogni cane deve disporre della propria ciotola in modo da rispettate le prescrizioni quantitative per ogni animale. le mangiatoie a getto continuo fornite di “serbatoi” sono da sconsigliare. queste ultime inoltre riducono l’interazione con gli operatori privandoli anche del ruolo di gestori delle risorse alimentari. un buon sistema di distribuzione dell’acqua può essere costituito da abbeveratoi a riempimento automatico, che garantiscono la costante fornitura d’acqua anche nella stagione calda. in commercio esistono mangimi studiati per soddisfare particolari esigenze in condizioni fisiologiche (crescita, allattamento, gravidanza) o patologiche. è auspicabile che anche nei canili dove sono somministrati esclusivamente prodotti industriali sia conservato un registro di carico e scarico su supporto cartaceo o informatizzato che consenta di tenere sotto controllo sia le scorte che le scadenze. modalità di somministrazione la somministrazione degli alimenti rappresensomministrazione alimenti gli alimenti devono essere riposti in ciotole singole ben pulite; la quantità e la tipologia di alimento deve rispettare quanto disposto dal veterinario nel piano dietetico; a fine pasto le ciotole devono essere ripulite da eventuali avanzi e lavate accuratamente. somministrazione acqua le ciotole per l’abbeveraggio devono essere lasciate a disposizione dell’animale con acqua fresca e pulita. scheda tecnica x. alimenti. il canile sanitario, procedure e protocolli 30 veterinaria italiana, collana di monografie, monografia 30, 2022 su tutti gli aspetti connessi alla presenza di un cane in famiglia. l’adozione dovrebbe essere preceduta da un periodo di affidamento temporaneo. l’affidamento temporaneo deve avvenire in seguito a una dichiarazione scritta dell’adottante che accetta controlli da parte del canile che possono avvenire in qualsiasi momento, e senza preavviso. detti controlli devono essere fatti da personale debitamente formato che utilizza check list validate dal veterinario della struttura o da un veterinario esperto in medicina comportamentale. i dati delle adozioni e degli affidi devono essere registrati e conservati in un apposito registro adozioni (anche informatico) e devono riguardare le seguenti informazioni: • tutte le generalità dell’adottante; • data dell’affido/adozione; • data dell’eventuale restituzione al canile corredata da breve relazione del veterinario o del direttore sanitario sulle condizioni di salute del cane e sulle motivazioni che ne hanno determinato la restituzione. l’adozione deve essere registrata anche sul registro di carico e scarico. il passaggio di proprietà deve essere riportato sull’anagrafe degli animali d’affezione regionale e i documenti aggiornati devono essere forniti all’adottante. all’adottante va consegnata copia della cartella clinica nella quale devono essere riportate eventuali diagnosi di malattie e trattamenti profilattici, terapeutici, chirurgici e comportamentali effettuati. anche l’affidamento deve essere registrato in anagrafe, in quanto comporta un cambio di detentore e l’interruzione da parte del comune dell’erogazione del mantenimento del cane. l’adottante e l’affidatario devono documentare la maggiore età con carta di identità o patente di guida e dichiarare l’assenza di condanne penali per maltrattamenti ad animali con autocertificazione. è consuetudine, in molte regioni, rendere disponibili i cani, per adozione e affidi, solo dopo il trasferimento al canile rifugio. nulla osta, però, a che l’adozione e soprattutto l’affido di cani ritenuti idonei, possano essere effettuati direttamente al canile sanitario dopo i controlli sanitari, l’avvio delle procedure profilattiche e il periodo di osservazione obbligatoria, laddove previsto. per questo motivo riportiamo quanto già scritto nel manuale “il canile rifugio, procedure  e  protocolli”. nel processo di adozione i fattori principali da analizzare sono due: • le aspettative dell’adottante • la capacità dell’adottante di gestire un determinato soggetto (che dipende dall’esperienza e dalla formazione) . conseguenze da evitare sono: • restituzione/abbandono/mal custodia/ maltrattamento; • aggressioni (che a volte possono essere molto gravi). l’iter dell’adozione è particolarmente complesso e deve incentrarsi sull’incrementare il numero di cani adottati e limitarne al massimo il loro ritorno in canile. un cane adottato e poi restituito al canile è sottoposto a stress elevato dovuto ai cambiamenti dei riferimenti sociali e ambientali che richiedono sforzi adattativi che vengono frustrati con possibile insorgenza di stati ansiosi dell’animale. per incrementare le adozioni è necessario utilizzare i social network per pubblicizzare e far conoscere i cani del canile pronti per l’adozione, attraverso schede con caratteristiche sanitarie e caratteriali. il canile come presidio di lotta al randagismo deve essere il punto di riferimento per lo sviluppo e la diffusione del concetto di possesso responsabile. particolare cura dovrà essere data alla distribuzione di materiale divulgativo che informi adozioni e affidi veterinaria italiana, collana di monografie, monografia 30, 2022 31 il canile sanitario, procedure e protocolli restri randagi, (..), perduti, abbandonati o confiscati e il cui stato sanitario potrebbe talvolta non essere noto al momento dell’ingresso nello stabilimento”. ciò corrisponde quindi anche al “canile sanitario”. per la movimentazione dei cani è di fondamentale importanza l’art. 9 che stabilisce che i rifugi per animali destinati a cani, gatti e furetti, da cui tali animali sono spostati in un altro stato membro possono avviare le proprie attività solo dopo il riconoscimento dello stabilimento (ex all’articolo 96, paragrafo 1, del regolamento (ue) 2016/429) da parte dell’autorità competente. per completezza di informazione occorre aggiungere che dal 21 aprile 2021 è in vigore il regolamento delegato (ue) 2019/2035 della commissione del 28 giugno 2019 che integra il regolamento (ue) 2016/429 del parlamento europeo e del consiglio per quanto riguarda le norme relative agli stabilimenti che detengono animali terrestri e agli incubatoi nonché alla tracciabilità di determinati animali terrestri detenuti e delle uova da cova. il suddetto regolamento integra le prescrizioni in materia di tracciabilità (fra l’altro) di cani, gatti e furetti. in questo regolamento la terminologia “rifugio per animali” è usata per indicare “uno stabilimento in cui sono detenuti animali teril canile sanitario, procedure e protocolli 32 veterinaria italiana, collana di monografie, monografia 30, 2022 la legge quadro 281/91, in materia di animali d’affezione e prevenzione del randagismo, ha stabilito che la soppressione di cani e gatti può avvenire solo con eutanasia e soltanto se “gravemente malati o incurabili o di comprovata pericolosità”. alla comprovata pericolosità fa riferimento l’art. 672 c.p. “omessa custodia e malgoverno degli animali”, di competenza degli organi preposti all’ordine pubblico, a difesa dell’incolumità fisica delle persone (minacciata da un cane) e il regolamento di polizia veterinaria, che tra le misure restrittive per contenere la propagazione di malattie infettive e zoonosi comprende l’abbattimento forzato degli animali. la legge n. 189/04 vieta qualunque uccisione degli animali per crudeltà o in assenza di necessità (art. 544 bis c.p.). la stessa legge punisce “chiunque, per crudeltà o senza necessità, cagiona una lesione ad un animale ovvero lo sottopone a sevizie o a comportamenti o a fatiche o a lavori insopportabili per le sue caratteristiche etologiche” (art. 544 ter c.p.). per non incorrere nel reato di maltrattamento, l’eutanasia, così come indicato dall’etimologia di questo termine, non deve provocare alcuna sofferenza all’animale. i cani su cui è esercitata l’eutanasia, in genere, sono già in stato di stress dovuto ad una patologia fisica o comportamentale. è compito del medico veterinario condurre l’eutanasia in maniera da non aggravare lo stato di stress o provocare ulteriori sofferenze quali dolore e ansia. per questo motivo, prima di eseguire le procedure di eutanasia vere e proprie, é indispensabile che il veterinario scelga, in base ad aggiornate conoscenze medico-scientifiche, il miglior protocollo anestesiologico a seconda del caso. i farmaci impiegati per l’eutanasia dell’animale devono essere detenuti e somministrati solo dal veterinario che adotta tutte le precauzioni per evitare rischi da contatto accidentale. gli animali soppressi devono essere distrutti o posti in luoghi inaccessibili ad altri animali; la procedura di eutanasia dei cani riconosce le seguenti fasi: eutanasia valutazione verificare se il cane è in una delle condizioni previste per l’abbattimento (stato di comprovata pericolosità, malattia grave o incurabile). trasporto dell’animale in luogo idoneo e contenimento il contenimento deve essere praticato da personale esperto in maniera da non impartire stress, angoscia o aumentare lo stato di sofferenza del cane. anestesia i protocolli anestesiologici sono scelti in base alle buone pratiche veterinarie. indipendentemente dal protocollo utilizzato, l’anestesia indotta nell’animale deve essere sempre profonda. somministrazione dei farmaci per l’eutanasia i farmaci per l’eutanasia devono essere somministrati da un medico veterinario. compilazione dei documenti e dei registri previsti la morte deve essere riportata nel registro di carico e scarico con indicazione della data di decesso e motivo della morte. smaltimento dell’animale soppresso l’animale deceduto deve essere avviato a un impianto inceneritore. se il canile non possiede un proprio impianto, deve essere stoccato in congelatore sino al ritiro da parte della ditta autorizzata che provvederà al trasporto presso un impianto di incenerimento autorizzato. scheda tecnica xi. eutanasia. veterinaria italiana, collana di monografie, monografia 30, 2022 33 il canile sanitario, procedure e protocolli • somministrazione del farmaco eutanasico; • compilazione dei documenti e registri previsti (certificato di morte, scarico dell’animale dal registro di carico/scarico e comunicazione all’anagrafe canina); • smaltimento dell’animale soppresso secondo la normativa in vigore. • valutazione; • trasporto dell’animale in luogo idoneo e contenimento (da tener presente che questa fase può essere eliminata qualora le condizioni fisiche del cane rendano difficoltoso o stressante lo spostamento); • anestesia profonda, preceduta da eventuale sedazione; in talune regioni è prevista la possibilità di consegnare il proprio cane al canile a titolo definitivo, cedendolo al comune di residenza. tale prassi, che trova giustificazione in caso di decesso o impossibilità (fisico/economica) del padrone di accudire l’animale, deve essere oggetto di controllo da parte del servizio veterinario ufficiale al fine di evitarne un ricorso incontrollato. pertanto dovrebbe essere prevista la registrazione di tale evento in anagrafe per evitare che la stessa persona possa riprendere altri animali nelle medesime condizioni. ai fini della tutela dei cani è auspicabile che il canile tenga traccia di tutti i percorsi di affidamento al fine di valutare e analizzare i percorsi che non si concludono con l’adozione. rinuncia di proprietà del cane parte iii normativa veterinaria italiana, collana di monografie, monografia 30, 2022 37 il canile sanitario, procedure e protocolli regione abruzzo legge regionale n. 47 del 18 dicembre 2013 norme sul controllo del randagismo, anagrafe canina e protezione degli animali da affezione bollettino ufficiale telematico della regione abruzzo speciale n. 127 del 27 dicembre 2013. deliberazione n. 213 del 28 marzo 2011 approvazione, ai sensi dell’art. 2 della l.r. 21 settembre 1999, n. 86, del programma di prevenzione del randagismo della regione abruzzo 2011-2013 bollettino ufficiale regione abruzzo n. 28 del 22 aprile 2011 legge regionale n. 9 del 7 maggio 2007 cimiteri per animali d’affezione bollettino ufficiale regione abruzzo n. 27 dell’11 maggio 2007 legge regionale n. 8 del 23 gennaio 2004 modifiche ed integrazioni alla l.r. 21 settembre 1999, n. 86 bollettino ufficiale regione abruzzo n. 1 (straordinario) dell’11 febbraio 2004 legge regionale n. 86 del 21 settembre 1999 norme sul controllo del randagismo, anagrafe canina e protezione degli animali da affezione bollettino ufficiale regione abruzzo n. 39 del 13 ottobre 1999 legge regionale n. 31 del 9 aprile 1997 finanziamento della costruzione delle strutture di ricovero per cani e gatti nonché per la prevenzione del randagismo bollettino ufficiale regione abruzzo n. 9 del 20 maggio 1997 legge regionale n. 27 del 3 aprile 1995 istituzione del servizio volontario di vigilanza ecologica bollettino ufficiale regione abruzzo n. 10 del 28 aprile 1995. legge regionale n. 34 del 31 maggio 1994 finanziamento costruzione canili sanitari bollettino ufficiale regione abruzzo n. 25 del 24 giugno 1994 legge regionale n. 15 dell’11 febbraio 1992 norme sul controllo del randagismo,istituzione dell’anagrafe canina e sulla protezione degli animali da affezione bollettino ufficiale regione abruzzo n. 8 del 5 marzo 1992 legge regionale n. 26 del 6 aprile 1989 modifiche ed integrazioni alla lr 16.6.87, n. 31 concernente: “tutela e valorizzazione del cane da pastore abruzzese” bollettino ufficiale regione abruzzo n. 17 del 26 aprile 1989 regione basilicata legge regionale n. 46 del 30 novembre 2018 disposizioni in materia di randagismo e tutela degli animali da compagnia di affezione legge n. 35 del 6 dicembre 2017 promozione delle terapie, dell’educazione e delle attività assistite con gli animali. succ. mod. con lr 29 giugno 2018, n. 11 legge regionale n. 11 del 31 maggio 2016 norme in materia funeraria e cimiteriale e di cimiteri per animali d’affezione delibera regionale n. 20160000022 del 12 gennaio 2016 presa d’atto dell’accordo tra il ministero della salute, la regione basilicata e l’ente nazionale protezione animali onlus (enpa) per l’avvio nella regione basilicata del progetto pilota contro il fenomeno del randagismo legge regionale n. 7 del 4 febbraio 2003 disciplina del bilancio di previsione e norme di contenimento e realizzazione della spesa per l’esercizio 2003 normativa regionale il canile sanitario, procedure e protocolli 38 veterinaria italiana, collana di monografie, monografia 30, 2022 decreto del presidente della giunta regionale n. 197 del 20 dicembre 2012 razionalizzazione degli interventi in materia di randagismo: istituzione di una rete di canili sanitari nel territorio della regione calabria. modifiche ed integrazioni bollettino ufficiale della regione calabria n. 2 del 16 gennaio 2013 legge regionale n. 4 del 3 marzo 2000 modifiche ed integrazioni alla legge regionale 5 maggio 1990, n. 41 recante: istituzione anagrafe canina, prevenzione randagismo e protezione degli animali bollettino ufficiale della regione calabria n. 15 dell’11 marzo 2000 legge regionale n. 41 del 5 maggio 1990 istituzione anagrafe canina, prevenzione randagismo e protezione degli animali bollettino ufficiale della regione calabria n. 4 del 12 gennaio 1990 regione campania legge regionale n. 12 dell’ 8 luglio 2019 “modifiche alla legge regionale 11 aprile 2019, n. 3 disposizioni volte a promuovere e a tutelare il rispetto ed il benessere degli animali d’affezione e a prevenire il randagismo legge regionale n. 3 dell’11 aprile 2019 disposizioni volte a promuovere e a tutelare il rispetto ed il benessere degli animali d’affezione e a prevenire il randagismo deliberazione giunta regionale n. 209 del 27 giugno 2014 recepimento dell’accordo tra il governo, le regioni e le province autonome del 24 gennaio 2013 in materia di identificazione e registrazione degli animali d’affezione approvazione del disegno di legge recante “tutela degli animali d’affezione e prevenzione del randagismo” deliberazione n. 2131 del 7 dicembre 2007 priorità, modalità e termini per la concessione dei contributi previsti dalla legge regionale bollettino ufficiale regione basilicata n. 11 del 4 febbraio 2003 legge regionale n. 3 del 24 febbraio 2009 cimiteri per animali d’affezione bollettino ufficiale regione basilicata n. 10 del 01 gennaio 2009 legge regionale n. 6 del 25 gennaio1993 norme sulla prevenzione e sul controllo del randagismo. istituzione anagrafica canina e protezione degli animali di affezione bollettino ufficiale regione basilicata n. 3 del 29 gennaio 1993 provincia autonoma di bolzano decreto del presidente della provincia n. 19 dell’8 luglio 2013 regolamento di esecuzione in materia di protezione degli animali bollettino ufficiale n. 29/i-ii del 16 luglio 2013 legge provinciale n. 9 del 15 maggio 2000 interventi per la protezione degli animali e prevenzione del randagismo bollettino ufficiale della regione bolzano n. 23 del 30 maggio 2000, supplemento ordinario. legge provinciale n. 16 dell’8 luglio 1986 interventi per la protezione degli animali bollettino ufficiale della regione bolzano (prov.) n. 31 del 22 luglio 1986 regione calabria decreto del presidente della giunta regionale n. 32 del 11 maggio 2015 decreto del presidente della giunta regionale n. 51 del 19 maggio 2014, modificativo del dpgr-ca n. 197 del 20 dicembre 2012 razionalizzazione degli interventi in materia di randagismo: istituzione di una rete di canili sanitari nel territorio della regione calabria modifiche e integrazioni decreto del presidente della giunta regionale n. 51 del 19 maggio 2014 veterinaria italiana, collana di monografie, monografia 30, 2022 39 il canile sanitario, procedure e protocolli turali in canili e gattili pubblici. ulteriore proroga dei termini per la presentazione dei progetti delibera giunta regionale n. 1747 del 21 ottobre 2019 programma di formazione e aggiornamento per operatori dei canili e gattili e volontari in tema di tutela degli animali d’affezione e lotta al randagismo. assegnazione e concessione risorse alle aziende unità sanitarie locali deliberazione n. 353 del 2 aprile 2013 approvazione dei requisiti strutturali e gestionali per le strutture di ricovero di cani e gatti, oasi e colonie feline bollettino ufficiale della regione emilia romagna n. 121 dell’8 maggio 2013 legge regionale n. 3 del 29 marzo 2013 modifiche alla legge regionale 17 febbraio 2005, n. 5 (norme a tutela del benessere animale). bollettino ufficiale della regione emilia romagna n. 3 del 29 marzo 2013 delibera giunta regionale 647/2007 indicazioni tecniche in attuazione alla legge regionale 5/05 relativa alla tutela del benessere degli animali. parziale modifica alla delibera 394/06 bollettino ufficiale della regione emilia romagna n. 75 del 5 giugno 2007 legge regionale n. 5 del 17 febbraio 2005 norme a tutela del benessere animale bollettino ufficiale della regione emilia romagna n. 30 del 18 febbraio 2005 legge regionale n. 27 del 7 aprile 2000 nuove norme per la tutela ed il controllo della popolazione canina e felina bollettino ufficiale della regione emilia romagna n. 61 del 10 aprile 2000 regione friuli-venezia giulia legge regionale n. 5 del 13 marzo 2015 modifiche alla legge regionale 11 ottobre 2012, n. 20 (norme per il benessere e la tutela degli animali di affezione) bollettino ufficiale della regione friuli venezia giulia n. 11 del 18 marzo 2015 16/2001 recante: “tutela degli animali d’affezione e prevenzione del randagismo” bollettino ufficiale della regione campania n.1 del 7 gennaio 2008 deliberazione n. 1214 del 23 settembre 2005 modifiche alla delibera di giunta regionale n. 3438 del 12 luglio 2002, concernenti le linee guida interpretative della l.r. 16/01 in materia di tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione campania n. 58 del 9 novembre 2005 deliberazione n. 1276 del 7 ottobre 2005 priorità, modalità e termini per la concessione dei contributi previsti dalla legge regionale 16/2001 recante “tutela degli animali d’affezione e prevenzione del randagismo” bollettino ufficiale della regione campania n. 55 del 31 ottobre 2005 deliberazione n. 3438 del 12 luglio 2002 linee guida interpretative della l.r. 16 del 24 novembre 2001, concernente la tutela degli animali d’affezione e la prevenzione del randagismo bollettino ufficiale della regione campania n. 42 del 9 settembre 2002 legge regionale n. 16 del 24 novembre 2001 tutela degli animali d’affezione e prevenzione del randagismo bollettino ufficiale della regione campania speciale del 29 novembre 2001 legge regionale n. 36 del 2 novembre 1993 tutela degli animali d’affezione e istituzione dell’anagrafe canina bollettino ufficiale della regione campania n. 48 dell’8 novembre 1993 regione emilia romagna accordo per la tutela ed il soccorso degli animali di affezione in caso di calamità naturali e non. prot. n. 165 del 07 luglio 2020 delibera giunta regionale n. 692 del 22 giugno 2020 approvazione delle procedure e modalità per l’ammissione al contributo degli interventi strutil canile sanitario, procedure e protocolli 40 veterinaria italiana, collana di monografie, monografia 30, 2022 norma sulla detenzione, allevamento e commercio di animali esotici bollettino ufficiale della regione lazio n. 4 del 10 febbraio 1997 deliberazione n. 920 del 21 dicembre 2006 revoca della deliberazione di giunta regionale n. 176 del 18 febbraio 2005 e adozione nuove linee guida relative all’applicazione del microchip, quale sistema di identificazione ai fini dell’anagrafe canina ed al rilascio del passaporto europeo per cani, gatti e furetti bollettino ufficiale della regione lazio n. 4 del 10 febbraio 2007 deliberazione n. 487 del 3 luglio 2007 approvazione linee guida per la ripartizione dei fondi regionali per l’attuazione dei piani di controllo delle nascite attraverso la sterilizzazione dei cani randagi catturati e/o a rischio di riproduzione incontrollata e per la costruzione e/o il risanamento dei canili pubblici. revoca della dgr 1370/98. 30-8-2007 bollettino ufficiale della regione lazio n. 24 del 30 agosto 2007 legge regionale n. 34 del 21 ottobre 1997 tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione lazio n. 30 del 30 ottobre 1997 legge regionale n. 89 del 14 dicembre 1990 norme sulla detenzione, l’allevamento ed il commercio di animali esotici bollettino ufficiale della regione lazio n. 36 del 29 dicembre 1990 regione liguria legge regionale n. 23 del 22 marzo 2000 tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione liguria n. 7 del 12 aprile 2000 legge regionale n. 16 del 24 marzo 1994 nuove norme in materia di randagismo bollettino ufficiale della regione liguria n. 9 del 13 aprile 1994 legge regionale n. 20 dell’11 ottobre 2012 norme per il benessere e la tutela degli animali di affezione bollettino ufficiale della regione friuli venezia giulia n. 42 del 17 ottobre 2012 legge regionale n. 134 del 10 giugno 2011 modifiche al decreto del 6 giugno 2002, riformulandone in particolare gli articoli relativi all’anagrafe canina e alla strutture di ricovero bollettino ufficiale della regione friuli-venezia giulia n. 25 del 22 giugno 2011 decreto del presidente della regione n. 171 del 6 giugno 2002 legge regionale n. 39/1990. regolamento di esecuzione della legge regionale 4 settembre 1990, n. 39, in materia di tutela degli animali domestici per il controllo e la prevenzione del fenomeno del randagismo. istituzione dell’anagrafe canina bollettino ufficiale della regione friuli venezia giulia n. 27 del 3 luglio 2002 legge regionale n. 39 del 4 settembre 1990 norme a tutela degli animali domestici per il controllo e la prevenzione del fenomeno del randagismo. istituzione dell’anagrafe canina bollettino ufficiale della regione friuli-venezia giulia n. 108 del 5 settembre 1990 regione lazio deliberazione della giunta regionale n. 621 del 25 ottobre 2016 deliberazione della giunta regionale n. 43 del 29 gennaio 2010 deliberazione n. 394 del 29 maggio 2009 istituzione dell’osservatorio per i diritti degli animali d’affezione e la prevenzione del randagismo. attività di promozione dell’anagrafe canina regionale bollettino ufficiale della regione lazio n. 27 del 21 luglio 2009 regolamento regionale n. 1 del 27 gennaio 1997 regolamento di attuazione della legge regionale n. 89 del 14 dicembre 1990 veterinaria italiana, collana di monografie, monografia 30, 2022 41 il canile sanitario, procedure e protocolli fica alla legge regionale 20 gennaio 1997, n. 10 ‘norme in materia di animali da affezione e prevenzione del randagismo’ e alla legge regionale 18 dicembre 2017, n. 36 ‘modifiche alla legge regionale 20 gennaio 1997, n. 10 ‘norme in materia di animali da affezione e prevenzione del randagismo’”. legge regionale n. 18 del 20 aprile 2015 modifiche alla legge regionale 20 gennaio 1997, n. 10 “norme in materia di animali da affezione e prevenzione del randagismo” bollettino ufficiale della regione marche n. 37 del 30 aprile 2015 delibera della giunta regionale n. 1172/2005 recepimento ed attuazione dell’accordo sancito il 6 febbraio 2003 tra il ministero della salute, le regioni e le provincie autonome di trento e di bolzano in materia di benessere degli animali da compagnia e pet-therapy bollettino ufficiale della regione marche n. 93 del 22 ottobre 2010 regolamento regionale n. 2 del 13 novembre 2001 attuazione della legge regionale 20 gennaio 1997 n.10 norme in materia di animali da affezione e prevenzione del randagismo e succ. modd. bollettino ufficiale della regione marche n. 134 del 22 novembre 2001 legge regionale n. 74 del 29 dicembre 1997 modificazioni alla legge regionale 20 gennaio 1997, n. 10 “norme in materia di animali da affezione e prevenzione del randagismo” bollettino ufficiale della regione marche n. 3 del 9 gennaio 1998 legge regionale n. 25 del 18 marzo 1997 contributo una tantum ad associazioni protezionistiche che gestiscono canili e rifugi per cani. bollettino ufficiale della regione marche n. 22 del 27 marzo 1997 legge regionale n. 10 del 20 gennaio 1997 norme in materia di animali da affezione e prevenzione del randagismo bollettino ufficiale della regione marche n. 8 del 24 gennaio 1997 regione lombardia regolamento regionale n. 2 del 13 aprile 2017 regolamento di attuazione delle disposizioni di cui al titolo viii, capo ii, della l.r. 33/2009 recante norme relative alla tutela degli animali di affezione e prevenzione del randagismo legge regionale n. 15 del 29 giugno 2016 evoluzione del sistema sociosanitario lombardo: modifiche ai titoli v e viii della legge regionale 30 dicembre 2009, n. 33 (testo unico delle leggi regionali in materia di sanità) bollettino ufficiale della regione lombardia n. 27, suppl. del 4 luglio 2016 legge regionale n. 33 del 30 dicembre 2009 testo unico delle leggi regionali in materia di sanità bollettino ufficiale della regione lombardia n. 52, 3° suppl. ord. del 31 dicembre 2009 regolamento regionale n. 2 del 5 maggio 2008 regolamento di attuazione della legge regionale n. 16 del 20 luglio 2006 (lotta al randagismo e tutela degli animali di affezione) bollettino ufficiale della regione lombardia n.19 del 9 maggio 2008 legge regionale n. 16 del 20 luglio 2006 lotta al randagismo e tutela degli animali da affezione bollettino ufficiale della regione lombardia n. 30 del 24 luglio 2006 (supplemento ordinario n. 1 del 25 luglio 2006) legge regionale n. 30 dell’8 settembre 1987 prevenzione del randagismo tutela degli animali e della salute pubblica bollettino ufficiale della regione lombardia n. 36 del 9 settembre 1987 (supplemento ordinario n. 2 del 9 settembre 1987) regione marche legge regionale n. 20 del 05 giugno 2018 modifica alla legge regionale 20 gennaio 1997, n. 10: “norme in materia di animali da affezione e prevenzione del randagismo” e abrogazione della legge regionale 3 aprile 2018, n. 6: “modiil canile sanitario, procedure e protocolli 42 veterinaria italiana, collana di monografie, monografia 30, 2022 bollettino ufficiale della regione piemonte n. 45 del 12 novembre 2009 legge regionale n. 22 del 6 agosto 2009 disposizioni collegate alla manovra finanziaria per l’anno 2009 bollettino ufficiale della regione piemonte n. 31 del 7 agosto 2009 decreto del presidente della giunta regionale n. 10 del 25 giugno 2008 integrazioni al regolamento regionale 11 novembre 1993, n. 2 (regolamento per la tutela e controllo degli animali da affezione) bollettino ufficiale della regione piemonte n. 27 del 3 luglio 2008 deliberazione n. 35-5274 del 12 febbraio 2007 recepimento del d.p.c.m. 28.02.2003 recante “accordo tra il ministro della salute, le regioni e le province autonome di trento e di bolzano, in materia di benessere degli animali da compagnia e pet-therapy” bollettino ufficiale della regione piemonte n. 10 dell’8 marzo 2007 legge regionale n. 9 del 4 luglio 2005 modifiche alla legge regionale 19 luglio 2004, n. 18 (identificazione elettronica degli animali da affezione e banca dati informatizzata. abrogazione della legge regionale 13 aprile 1992, n. 20) bollettino ufficiale della regione piemonte n. 27 del 7 luglio 2005 legge regionale n. 18 del 19 luglio 2004 identificazione elettronica degli animali da affezione e banca dati informatizzata. abrogazione della legge regionale 13 aprile 1992, n. 20 (istituzione dell’anagrafe canina) bollettino ufficiale della regione piemonte n. 29 del 22 luglio 2004 legge regionale n. 39 del 7 aprile 2000 cimiteri per animali d’affezione bollettino ufficiale della regione piemonte n. 15 del 12 aprile 2000 d.c.r. 697/1993 decreto del presidente della regione molise delibera della giunta regionale n. 806 del 18 dicembre 2012 programma 2013-2015 per la prevenzione del randagismo e per la gestione dell’anagrafe canina bollettino ufficiale della regione molise n. 4 del 1 febbraio 2013 legge regionale n.12 del 24 giugno 2011 modifiche ed integrazioni alla legge regionale n. 7 del 4 marzo 2005, recante “nuove norme per la protezione dei cani e per l’istituzione dell’anagrafe canina” bollettino ufficiale della regione molise n. 18 del 1 luglio 2011. legge regionale n. 7 del 4 marzo 2005 nuove norme per la protezione dei cani e per l’istituzione dell’anagrafe canina bollettino ufficiale della regione molise n. 6 del 16 marzo 2005 legge regionale n. 11 del 4 marzo 1992 norme per la protezione dei cani e per l’istituzione dell’ anagrafe canina bollettino ufficiale della regione molise n. 5 del 16 marzo 1992 regione piemonte deliberazione della giunta regionale n. 10-7753 del 30 ottobre 2018 variazione al bilancio di previsione finanziario 2018-2020. finanziamenti statali per attività sanitaria in materia di prevenzione del randagismo di animali. deliberazione della giunta regionale n. 32-7387 del 7 aprile 2014 recepimento dell’accordo rep. n. 5/cu del 24/1/2013 in materia di identificazione e registrazione degli animali da affezione bollettino ufficiale della regione piemonte n. 17s1 del 24 aprile 2014 legge regionale n. 27 del 4 novembre 2009 disciplina del rapporto persone-cani per la prevenzione della salute pubblica e del benessere animale veterinaria italiana, collana di monografie, monografia 30, 2022 43 il canile sanitario, procedure e protocolli legge regionale n. 15 del 31 luglio 1996 integrazione della legge regionale 3 aprile 1995, n. 12 concernente gli interventi per la tutela degli animali di affezione e prevenzione del randagismo bollettino ufficiale della regione puglia n. 86 del 7 agosto 1996 legge regionale n. 12 del 03 aprile 1995 interventi per la tutela degli animali d’affezione e prevenzione del randagismo bollettino ufficiale della regione puglia n. 39 del 18 aprile 1995 regione sardegna deliberazione n. 34/9 del 3 luglio 2018 direttive in materia di lotta al randagismo e protezione degli animali di affezione approvate con la delib.g.r. n. 17/39 del 27 aprile 2010. modifica art. 4 e allegati n. 9, 10, 11 deliberazione n. 17/39 del 27 aprile 2010 delibera della giunta regionale n. 44/35 del 14 dicembre 2010 trasferimenti alle aziende sanitarie locali per l’identificazione elettronica animale e per la gestione anagrafe animale delibera della giunta regionale n. 38/13 del 9 novembre 2010 legge 14 agosto 1991, n. 281 e legge regionale 18 maggio 1994, n. 21. contributi ai comuni per la lotta al randagismo e la gestione dei canili e ripartizione tra le aziende sanitarie locali dei fondi regionali e statali per la prevenzione del randagismo delibera n. 17/39 del 27 aprile 2010 l.r. n. 21/1994 e s.m.i. direttive in materia di lotta al randagismo e protezione degli animali d’affezione ministero della salute circolare 2725/p i.8.d/318 del 27 luglio 2006 revoca dell’obbligo di vaccinazione antirabbica per i cani in ingresso in sardegna decreto del presidente della giunta regionale n. 1 del 4 marzo 1999 giunta regionale n. 4359 regolamento n. 2 dell’11 novembre 1993 regolamento per la tutela e controllo degli animali da affezione bollettino ufficiale della regione piemonte n 47 del 24 novembre 1993 legge regionale n. 34 del 26 luglio 1993 tutela e controllo degli animali da affezione bollettino ufficiale della regione piemonte n. 31 del 4 agosto 1993 regione puglia legge regionale n. 2 del 7 febbraio 2020 norme sul controllo del randagismo, anagrafe canina e protezione degli animali da affezione. abrogazione della legge regionale 3 aprile 1995, n. 12 (interventi per la tutela degli animali d’affezione e prevenzione del randagismo) bollettino ufficiale della regione puglia n.  18 del 10 febbraio 2020 deliberazione della giunta regionale n. 1223 del 4 luglio 2013. linee guida attuative dell’art. 2 della l. 281/91 e degli artt. 6 e 8 della l.r. 12/95 in materia di prevenzione del fenomeno del randagismo bollettino ufficiale della regione puglia n. 102 del 24 luglio 2013 deliberazione della giunta regionale n. 2505 del 27 novembre 2012 tutela degli animali di affezione e prevenzione del randagismo. contributi destinati ai comuni e all’unione dei comuni della regione puglia per la campagna di sterilizzazione di cani padronali e per la realizzazione e/o ampliamento di canili sanitari, di proprietà comunale bollettino ufficiale della regione puglia n. 186 del dicembre 2012 legge regionale n. 34 del 12 dicembre 2006 modifiche e integrazioni alle leggi regionali 9 agosto 2006, n. 26 (interventi in materia sanitaria) e 3 aprile 1995, n. 12 (interventi per la tutela degli animali d’affezione e prevenzione del randagismo) bollettino ufficiale della regione puglia n. 166 del 15 dicembre 2006 il canile sanitario, procedure e protocolli 44 veterinaria italiana, collana di monografie, monografia 30, 2022 decreto 13 dicembre 2007 linee guida per il controllo del randagismo e bandi per la concessione di contributi da destinare al risanamento dei rifugi esistenti e alla costruzione di rifugi sanitari, all’attuazione di piani di controllo delle nascite e al mantenimento di animali bollettino ufficiale della regione sicilia n. 4 del 25 gennaio 2008 circolare n. 300 del 13 febbraio 2007 benessere animale, randagismo, stato di applicazione della legge regionale 3 luglio 2000, n. 15 decreto del presidente della regione siciliana n. 7 del 12 gennaio 2007 regolamento esecutivo dell’art. 4 della legge regionale 3 luglio 2000, n. 15 “istituzione dell’anagrafe canina e norme per la tutela degli animali d’affezione e prevenzione del randagismo” bollettino ufficiale della regione sicilia n. 15 del 6 aprile 2007 decreto del presidente della regione siciliana n. 15 del 27 giugno 2002 regolamento concernente i requisiti dell’albo delle associazioni per la protezione degli animali bollettino ufficiale della regione sicilia n. 47 dell’11 ottobre 2002 legge regionale n. 15 del 3 luglio 2000 istituzione dell’anagrafe canina e norme per la tutela degli animali da affezione e la prevenzione del randagismo bollettino ufficiale della regione sicilia n. 32 del 7 luglio 2000 regione toscana delibera n. 984 dell’11 ottobre 2016 regolamento di attuazione della legge regionale 20 gennaio 2015, n. 9 (disciplina dei cimiteri per animali d’affezione). approvazione definitiva delibera n. 943 del 6 ottobre 2015 linee guida per l’istituzione del soccorso animali delibera n. 1153 del 30 novembre 2015 recepimento dell’accordo stato regioni e province autonome n. 60/csr del 25 marzo 2015, che approva le “linee guida nazionali per gli inregolamento di attuazione della legge 14 agosto 1991, n. 281 e della legge regionale 18 maggio 1994, n. 21 e della legge regionale 1° agosto 1996, n. 35 sulla prevenzione del randagismo bollettino ufficiale della regione sardegna n. 13 del 29 aprile 1999 legge regionale n. 35 del 1 agosto 1996 integrazioni e modifiche alla legge regionale 18 maggio 1994, n. 21, recante: «norme per la protezione degli animali e istituzione dell’ anagrafe canina» bollettino ufficiale della regione sardegna n. 25 dell’8 agosto 1996 legge regionale n. 21 del 18 maggio 1994 norme per la protezione degli animali e istituzione dell’anagrafe canina bollettino ufficiale della regione sardegna n. 17 del 21 maggio 1994 regione sicilia deliberazione n. 468 del 19 novembre 2018 linee guida per il contrasto e la prevenzione nella regione siciliana del fenomeno del randagismo. decreto dell’assessore della salute n. 2164 del 03 novembre 2017 disposizioni per la corretta custodia e per la registrazione nella anagrafe degli animali d’affezione. norme per la corretta movimentazione di cani e gatti decreto dell’assessore della salute n. 2440 del 28 novembre 2011 criteri e modalità per la concessione dei contributi previsti dall’art. 20, commi 1 e 2 della legge regionale 3 luglio 2000 n. 15 legge regionale n. 15 del 3 luglio 2000 istituzione dell’anagrafe canina e norme per la tutela degli animali da affezione e la prevenzione del randagismo direttiva assessorato per la sanità n. 1059 del 12 giugno 2009 controllo del randagismo misure a tutela dell’incolumità pubblica veterinaria italiana, collana di monografie, monografia 30, 2022 45 il canile sanitario, procedure e protocolli tutela degli animali d’ affezione e la prevenzione del randagismo bollettino ufficiale della regione toscana n. 28 del 18 aprile 1995 legge regionale n. 89 del 30 dicembre 1989 lr 4/ 87 istitutiva dell’ anagrafe canina. modifiche ed integrazioni all’art. 14 bollettino ufficiale della regione toscana n. 3 del 10 gennaio 1990 provincia autonoma di trento decreto del presidente della provincia n. 23 del 20 settembre 2013 regolamento recante: “disposizioni regolamentari per l’applicazione della legge provinciale 28 marzo 2012, n. 4 (protezione degli animali di affezione e prevenzione del randagismo)” bollettino ufficiale provincia di trento n. 39 del 24 settembre 2013 legge provinciale n. 4 del 28 marzo 2012. protezione degli animali d’affezione e prevenzione del randagismo bollettino ufficiale provincia di trento n. 14 del 3 aprile 2012 regione umbria legge regionale n.10 del 17 agosto 2016 modificazioni ed integrazioni alla legge regionale 9 aprile 2015, n. 11 (testo unico in materia di sanità e servizi sociali) e alla legge regionale 30 marzo 2015, n. 8 (disposizioni collegate alla manovra di bilancio 2015 in materia di entrate e di spese modificazioni ed integrazioni di leggi regionali). legge regionale n. 11 del 2015 testo unico in materia di sanità titolo xvi, capo iv (norme per il benessere e la tutela degli animali di affezione), capo v (prevenzione e controllo del fenomeno del randagismo) e capo vi (divieto di detenzione e utilizzazione di esche avvelenate) deliberazione n. 255 del 10 giugno 2013 deliberazione della giunta regionale n. 69 del 19 gennaio 2005 terventi assistiti con gli animali (iaa)”, in armonizzazione con la l.r. 59/2009. legge regionale n. 9 del 20 gennaio 2015 disciplina dei cimiteri per animali d’affezione bollettino ufficiale della regione toscana n. 4 del 23.1.2015 delibera n. 1233 del 22 dicembre 2014 linee d’indirizzo per l’accesso degli animali d’affezione in visita a degenti presso strutture sanitarie e ospedaliere pubbliche e private accreditate decreto del presidente della giunta regionale n. 53/r del 1 ottobre 2013 modifiche al d.p.g.r. 4 agosto 2011, n. 38/r regolamento di attuazione della legge regionale 20 ottobre 2009, n. 59 decreto del presidente della giunta regionale n. 38 del 4 agosto 2011 regolamento di attuazione della legge regionale 20 ottobre 2009, n. 59 “norme per la tutela degli animali. abrogazione della legge regionale 8 aprile 1995, n. 43” (norme per la gestione dell’anagrafe del cane, la tutela degli animali d’affezione e la prevenzione del randagismo) bollettino ufficiale della regione toscana n. 39 del 5 agosto 2011 legge regionale n. 59 del 20 ottobre 2009 norme per la tutela degli animali. abrogazione della legge regionale 8 aprile 1995, n. 43 (norme per la gestione dell’anagrafe del cane, la tutela degli animali d’affezione e la prevenzione del randagismo) bollettino ufficiale della regione toscana n. 41 del 26 ottobre 2009 legge regionale n. 90 del 4 dicembre 1998 modifiche ed integrazioni della legge regionale 8 aprile 1995, n. 43 “norme per la gestione dell’anagrafe del cane, la tutela degli animali d’affezione e la prevenzione del randagismo” bollettino ufficiale della regione toscana n. 42 del 10 dicembre 1998 legge regionale n. 43 dell’8 aprile 1995 norme per la gestione dell’anagrafe del cane, la il canile sanitario, procedure e protocolli 46 veterinaria italiana, collana di monografie, monografia 30, 2022 bollettino ufficiale della regione valle d’aosta n. 21 del 10 maggio 1994 regione veneto legge regionale n. 17 del 19 giugno 2014 modifica della legge regionale 28 dicembre 1993, n. 60 “tutela degli animali d’affezione e prevenzione del randagismo” e successive modificazioni bollettino ufficiale della regione veneto n. 62 del 24 giugno 2014 deliberazione della giunta regionale n. 272 del 6 febbraio 2007 linee guida per una regolamentazione uniforme dell’igiene urbana veterinaria nel territorio della regione veneto. completamento del recepimento dell’accordo tra il ministero della salute, le regioni e le province autonome di trento e bolzano in materia di benessere degli animali da compagnia e pet-therapy. modifica d.g.r. 243 del 7 febbraio 2006 deliberazione della giunta regionale n. 243 del 7 febbraio 2006 linee guida per una regolamentazione uniforme dell’igiene urbana veterinaria nel territorio della regione veneto. completamento del recepimento dell’accordo tra il ministero della salute, le regioni e le province autonome di trento e bolzano in materia di benessere degli animali da compagnia e pet-therapy. legge regionale n. 60 del 28 dicembre 1993. tutela degli animali d’affezione e prevenzione del randagismo. bollettino ufficiale della regione veneto n. 111 del 31 dicembre 1993. accordo tra ministero della salute, regioni e province autonome di trento e di bolzano in materia benessere degli animali da compagnia, cimiteri e pet-therapy. recepimento e linee guida vincolanti bollettino ufficiale della regione umbria n. 8 del 23 febbraio 2005 legge regionale n. 19 del 19 luglio 1994 norme per la tutela degli animali di affezione e per la prevenzione ed il controllo del fenomeno del randagismo bollettino ufficiale della regione umbria n. 32 del 27 luglio 1994 regione valle d’aosta dgr n. 1162 del 28 giugno 2013 modifiche ed integrazioni delle linee guida regionali per la tutela degli animali d’affezione linee guida regionali per la tutela degli animali d’affezione approvate con dgr n. 1731 del 24.08.2012 ai sensi art.4 comma 2 della l.r. n. 37/2010 dgr n. 1731 del 24 agosto 2012 linee guida regionali per la tutela degli animali d’affezione linee guida regionali per la tutela degli animali d’affezione ai sensi art. 4 comma 2 della l.r. n. 37/2010. legge regionale n. 37 del 22 novembre 2010 nuove disposizioni per la tutela e per il corretto trattamento degli animali di affezione. abrogazione della legge regionale 28 aprile 1994, n. 14 bollettino ufficiale della regione valle d’aosta n. 51 del 14 dicembre 2010 legge regionale n. 14 del 28 aprile 1994 norme per la tutela e per il corretto trattamento degli animali di affezione veterinaria italiana, collana di monografie, monografia 30, 2022 47 il canile sanitario, procedure e protocolli decreto del presidente della repubblica 8 febbraio 1954, n. 320 regolamento di polizia veterinaria gazzetta ufficiale n. 142 del 24 giugno 1954 legge 14 agosto 1991 n. 281 legge quadro in materia di animali di affezione e prevenzione del randagismo gazzetta ufficiale n. 203 del 30 agosto 1991 ministero della sanità circolare 14 maggio 2001, n. 5 attuazione della legge 14 agosto 1991, n. 281. gazzetta ufficiale n. 144 del 23 giugno 2001 decreto del presidente del consiglio dei ministri 28 febbraio 2003 recepimento dell’accordo del 6 febbraio 2003 recante disposizioni in materia di benessere degli animali da compagnia e pet-therapy gazzetta ufficiale n. 52 del 4 marzo 2003 decreto del presidente della repubblica n. 254 del 15 luglio 2003 regolamento recante disciplina della gestione dei rifiuti sanitari a norma dell’articolo 24 della legge 31 luglio 2002, n. 179 gazzetta ufficiale n. 211 dell’11 settembre 2003 legge 20 luglio 2004, n.189 disposizioni concernenti il divieto di maltrattamento degli animali, nonché di impiego degli stessi in combattimenti clandestini o competizioni non autorizzate gazzetta ufficiale n.178 del 31 luglio 2004 ministero della salute decreto 13 maggio 2005 determinazione dei criteri per la ripartizione dei fondi per la prevenzione e lotta al randagismo, previsti dalla legge del 29 dicembre 2003, n. 376. gazzetta ufficiale n. 169 del 22 luglio 2005 decreto 2 novembre 2006 individuazione delle associazioni e degli enti affidatari di animali oggetto di provvedimento di sequestro o di confisca, nonché determinazione dei criteri di riparto delle entrate derivanti dall’applicazione di sanzioni pecuniarie gazzetta ufficiale n. 19 del 24 gennaio 2007 ministero della salute ministero dell’economia e delle finanze decreto 06 maggio 2008 determinazione dei criteri per la ripartizione tra le regioni e le province autonome delle disponibilità del fondo per l’attuazione della legge 14 agosto 1991, n. 281, recante: «legge quadro in materia di animali di affezione e prevenzione del randagismo». gazzetta ufficiale n. 185 dell’8 agosto 2008 ministero del lavoro, della salute e delle politiche sociali decreto 28 luglio 2009 disciplina dell’utilizzo e della detenzione di medicinali ad uso esclusivo del medico veterinario gazzetta ufficiale n. 230 del 3 ottobre 2009 ministero del lavoro, della salute e delle politiche sociali decreto 26 novembre 2009 percorsi formativi per i proprietari dei cani gazzetta ufficiale n. 19 del 25 gennaio 2010 ministero della salute ordinanza 14 gennaio 2010 proroga e modifica dell’ordinanza 18 dicembre 2008, come modificata dall’ordinanza 19 marzo 2009, recante: «norme sul divieto di utilizzo e di detenzione di esche o di bocconi avvelenati» gazzetta ufficiale n. 33 del 10 febbraio 2010 legge 4 novembre 2010, n. 201 ratifica ed esecuzione della convenzione europea per la protezione degli animali da compagnia, fatta a strasburgo il 13 novembre 1987, normativa nazionale il canile sanitario, procedure e protocolli 48 veterinaria italiana, collana di monografie, monografia 30, 2022 ministero della salute ordinanza 10 agosto 2020 norme sul divieto di utilizzo e di detenzione di esche o di bocconi avvelenati gazzetta ufficiale serie generale n. 222 del 7 settembre 2020 ministero della salute ordinanza 18 luglio 2019 proroga dell’ordinanza contingibile e urgente 6 agosto 2013, e successive modificazioni, concernente la tutela dell’incolumità pubblica dall’aggressione dei cani gazzetta ufficiale n.196 del 22 agosto 2019 nonché norme di adeguamento dell’ordinamento interno gazzetta ufficiale n. 283 del 3 dicembre 2010 accordo 24 gennaio 2013 accordo, ai sensi dell’articolo 9, comma 2, lettera c), del decreto legislativo 28 agosto 1997, n. 281, tra il governo, le regioni e le province autonome di trento e bolzano, le province, i comuni e le comunità montane in materia di identificazione e registrazione degli animali da affezione. (rep. atti n. 5/cu). 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