Article history: Received: 13-09-2017, Accepted: 21-11-2017, Published online: 22-12-2017
Corresponding author: Salik Nazki
E-mail: email@example.comCitation: Nazki S, Wani SA, Parveen R, Ahangar SA, Kashoo ZA, Hamid S, Dar ZA, Dar TA, Dar PA (2017) Isolation, molecular characterization and prevalence of Clostridium perfringens in sheep and goats of Kashmir Himalayas, India, Veterinary World, 10(12):1501-1507.
Aim: The study was conducted to report the occurrence of the Clostridium perfringens in sheep and goats of the Kashmir valley for the 1st time and to characterize them molecularly with respect to toxin genes to determine the prevalence of the various toxinotypes.
Materials and Methods: A total of 177 samples (152 from sheep and 25 from goats) collected from healthy, diarrheic animals, and morbid material of animals suspected to have died of enterotoxaemia were screened for C. perfringens toxinotypes. The presumptive positive isolates were confirmed using 16S rRNA gene-based polymerase chain reaction (PCR). All the confirmed isolates were screened for six toxin genes, namely; cpa, cpb, etx, cpi, cpb2, and cpe using a multiplex PCR.
Results: The PCR amplification of 16S rRNA gene revealed that out of 177 samples collected, 125 (70.62%) were found positive for C. perfringens, of which 110 (72.36%) were from sheep and 15 (60%) were from goats. The highest prevalence of C. perfringens toxinotype D was observed in lambs (56.16%) and kids (46.16%) followed by 3.84% in adult sheep while it was absent in samples obtained from adult goats. The multiplex PCR revealed that 67 (60.90%) isolates from sheep and 8 (53.33%) isolates from goats belonged to toxinotype A, while 43 (39.09%) isolates from sheep and 7 (46.66%) isolates from goats were detected as toxinotype D. None of the isolates was found to be toxinotype B, C, or E. All the C. perfringens toxinotype A isolates from sheep were negative for both cpb2 and cpe genes, however, 27.90% toxinotype D isolates from sheep carried cpb2 gene, and 6.97% possessed cpe gene. In contrast, 12.50% C. perfringens toxinotype A isolates from goats harbored cpb2 and cpe genes while 14.28% isolates belonging to toxinotype D carried cpb2 and cpe genes, respectively.
Conclusion: The high prevalence of C. perfringens was observed, even in day-old lambs. The toxinotypes A and D are prevalent in both sheep and goats. The severity of disease and mortality may be associated with the presence of minor toxins in both the detected toxinotypes.
Keywords: Clostridium perfringens, enterotoxemia, multiplex polymerase chain reaction, toxinotype, 16S rRNA.
1. Uzal, F.A., Songer, J.G., Prescott, J.F. and Popoff, M.R. (2016) Brief description of animal pathogenic clostridia. Clostridial Dis. Anim., John Wiley & Sons, Inc, Hoboken, NJ. [Crossref]
2. Siqueira, F.F., Almeida, M.O., Barroca, T.M., Horta, C.C., Carmo, A.O., Silva, R.O., Pires, P.S., Lobato, F.C. and Kalapothakis, E. (2012) Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity. Vet. Microbiol., 159(3-4): 397-405. [Crossref] [PubMed]
3. Milton, A.A.P., Agarwal, R.K., Bhuvana Priya, G., Saminathan, M., Aravind, M., Reddy, A., Athira, C.K., Ramees, T., Sharma, A.K. and Kumar A. (2017) Prevalence and molecular typing of Clostridium perfringens in captive wildlife in India. Anaerobe, 44: 55-57. [Crossref] [PubMed]
4. Ohtani, K. and Shimizu, T. (2016) Regulation of toxin production in Clostridium perfringens. Toxins (Basel), 8(7), 207. [Crossref]
5. Ashgan, M., Al-Arfaj, A.A. and Moussa, I. (2013) Identification of four major toxins of Clostridium perfringens recovered from clinical specimens. Afr. J. Microbiol. Res., 7, 3658-3664.
6. Silva, R.O., Almeida, L.R., Oliveira Junior, C.A., Lima, P.C., Soares, D.F., Pereira, P.L., Silva, I.J. and Lobato, F.C. (2016) Isolation and genotyping of Clostridium perfringens from free-living South American Coati (Nasua Nasua). J. Zoo Wildl. Med. 47(1): 333-336. [Crossref] [PubMed]
8. Garcia, J.P., Adams, V., Beingesser, J., Hughes, M.L., Poon, R., Lyras, D., Hill, A., McClane, B.A., Rood, J,I. and Uzal FA. (2013) Epsilon toxin is essential for the virulence of Clostridium perfringens Type D infection in sheep, goats, and mice. Infect. Immun., 81(7): 2405-2414. [Crossref] [PubMed] [PMC]
9. Stiles, B.G., Barth, G., Barth, H. and Popoff, M.R. (2013) Clostridium perfringens epsilon toxin: A malevolent molecule for animals and man? Toxins (Basel), 5(11): 2138-2160. [Crossref] [PubMed] [PMC]
10. Songer, J.G. (1999) Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev., 9(2): 216-234.
11. Jemal, D., Shifa, M. and Kebede, B. (2016) Review on pulpy kidney disease. J. Vet. Sci. Technol 7(5): 361. [Crossref]
12. Bokori-Brown, M., Hall, C.A., Vance, C., Fernandes da Costa, S.P., Savva, C.G., Naylor, C.E., Cole, A.R., Basak, A.K., Moss, D.S. and Titball, R.W. (2014) Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia. Vaccine, 32(23): 2682-2687. [Crossref] [PubMed] [PMC]
13. Singh, B. and Prasad S. (2009) A model based assessment of economic losses due to some important diseases in sheep in India. Indian J. Anim. Sci., 79(12): 1265-1268.
14. Markey, B.K. (2013) Clinical Veterinary Microbiology. 2nd ed. Elsevier, Edinburgh. p11, 901.
15. Tonooka, T., Sakata, S., Kitahara, M., Hanai, M., Ishizeki, S., Takada, M., Sakamoto, M. and Benno, Y. (2005) Detection and quantification of four species of the genus Clostridium in infant feces. Microbiol. Immunol., 49(11): 987-992. [Crossref]
16. Ahsani, M.R., Bafti, M.S., Esmailizadeh, A.K. and Mohammadabadi, MR. (2011) Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep. Small Ruminant Res., 95(1): 65-69. [Crossref]
17. van Asten, A.J, Allaart, J.G, Grone, A. and Houwers, D.J. (2008) Application of PCR-based detection of Clostridium perfringens cpb2 in fecal samples. Vet Microbiol., 129(1-2): 215. [Crossref] [PubMed]
18. el Idrissi, A.H. and Ward, G.E. (1992) Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias. Vet. Microbiol., 31(4): 389-396. [Crossref]
19. Greco, G., Madio, A., Buonavoglia, D., Totaro, M., Corrente, M., Martella, V. and Buonavoglia, C. (2005) Clostridium perfringens toxin-types in lambs and kids affected with gastroenteric pathologies in Italy. Vet. J., 170(3): 346-50. [Crossref] [PubMed]
20. Kumar, N.V., Sreenivasulu, D. and Reddy, Y.N. (2014) Prevalence of Clostridium perfringens toxin genotypes in enterotoxemia suspected sheep flocks of Andhra Pradesh. Vet. World, 7(12): 1132-6. [Crossref]
21. Miserez, R., Frey, J., Buogo, C., Capaul, S., Tontis, A., Burnens, A. and Nicolet, J. (1998) Detection of alpha- and epsilon-toxigenic Clostridium perfringens Type D in sheep and goats using a DNA amplification technique (PCR). Lett. Appl. Microbiol., 26(5): 382-386. [Crossref]
22. Redostitis, O.M., Gay, C.C., Hinchcliff, K.H. and Constable, P.D. (2007) Veterinary Medicine. 10th ed. Saunders Elsevier, London.
23. Jabbari, A.R., Tekyei, F., Esmaeilzad, M. and Langroudi, R.P. (2012) Occurrence of Beta2 toxigenic Clostridium perfringens isolates with different toxin types in Iran. Arch. Razi Inst., 67(2): 133-137.
24. Uzal, F.A., Freedman, J.C., Shrestha, A., Theoret, J.R., Garcia, J., Awad, M.M, Adams, V., Moore, R.J., Rood, J.I. and McClane, B.A. (2014) Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol., 9(3): 361-377. [Crossref] [PubMed] [PMC]25. Baums, C.G., Schotte, U., Amtsberg, G. and Goethe, R. (2004) Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet. Microbiol., 100(1-2): 11-16. [Crossref]