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.