Veterinary World

     Open access and peer reviewed journal  

ISSN (Online): 2231-0916


Home l Editorial board l Instructions for authors l Reviewer guideline l Open access policy l Archives l FAQ

Open Access

Copyright: The authors. This article is an open access article licensed under the terms of the Creative Commons Attribution License

( which permits unrestricted use, distribution and reproduction in any medium, provided the work is properly cited.

Research (Published online: 26-12-2014)

19. Prevalence of Clostridium perfringens toxin genotypes in enterotoxemia suspected sheep flocks of Andhra Pradesh - N. Vinod Kumar, D. Sreenivasulu and Y. N. Reddy

Veterinary World, 7(12): 1132-1136



   doi: 10.14202/vetworld.2014.1132-1136



N. Vinod Kumar: Department of Veterinary Microbiology, College of Veterinary Science, Tirupathi, Andhra Pradesh, India;

D. Sreenivasulu: Department of Veterinary Microbiology, College of Veterinary Science, Tirupathi, Andhra Pradesh, India;

Y. N. Reddy: Department of Veterinary Microbiology, College of Veterinary Science, Tirupathi, Andhra Pradesh, India;


Received: 31-07-2014, Revised: 11-11-2014, Accepted: 19-11-2014, Published online: 26-12-2014


Corresponding author: N. Vinod Kumar, e-mail:

Aim: To identify the Clostridium perfringens toxin genotypes prevailing in enterotoxemia suspected sheep flocks in Andhra Pradesh by using multiplex PCR.

Materials and Methods: Intestinal scrapings were collected from lambs below three months of age from flocks with similar management from different Districts of Andhra Pradesh, in India. A total of 208 samples were collected with 140 from enterotoxemia suspected flocks and 68 from healthy flocks. Following processing and culture of the samples, colonies were identified by morphological and biochemical tests. All the clostridial isolates were analyzed by multiplex PCR.

Results: C. pefringens were isolated from 97 out of 140 enterotoxemia suspected flocks (69.29%) and 27 out of 68 healthy flocks (39.71 %). Genotyping of the 97 iolates by multiplex PCR from enterotoxemia suspected flocks indicated C. perfringens type A, C and D 67.01% (65 out of 97); 11.34% (11 out of 97) and 21.65% (21 out of 97) respectively. Isolates from healthy flocks indicated the presence of type A and D 92.59% (25 out of 27) and 7.40% (2 out of 27) respectively. Number of isolates from enterotoxaemia suspected flocks were significantly high (P<0.001) than healthy flocks. Type A is found to be predominant in both enterotoxemia suspected and healthy flocks (67.01% and 92.59%).

Conclusions: Prevalence of C. perfringens type C was reported for the first time in India. Clostridium perfringens type D and type C were found to be the major causative types for enterotoxemia.

Keywords: Clostridium perfringes, enterotoxaemia, lamb mortality, multiplex PCR, Toxin typing.

1. Uzal, F.A., Vidal, J.E., McClane, B.A. and Guraj, A.A. (2014) Clostridium perfringens toxins involved in mammalian veterinary diseases. Open Toxicol. J., 2: 24-42.
2. Rood, J.I. and Cole, S.T. (1991) Molecular genetics and pathogenesis of Clostridium perfringes. Microbiol. Rev., 55(4): 621-648.
PMid:1779929 PMCid:PMC372840
3. Carter, G.R. (1984) Diagnostic Procedure in Veterinary Microbiology. Charles C Thomas, Springfield, Illinois.
4. Naylor, R.D. and Barker, L.T. (1997) Detection of Clostridium perfringens alpha toxin by enzyme-linked immunosorbent assay. Res. Vet. Sci., 63(1): 101-102.
5. 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): 346-350.
6. Kalender, H., Ertas, H.B., Cetinkaya, B., Muz, A., Arslan, N. and Kilic, A. (2005) Typing of isolates of Clostridium perfringens from healthy and diseased sheep by multiplex PCR. Vet. Med. Czech., 50(10): 141-148.
7. Petit, L., Gibert, M. and Popoff, M.R. (1999) Clostridium perfringens: Toxinotype and genotype. Trend Microbiol., 7(3): 104-110.
8. Songer, J.G. and Meer, R.R. (1996) Genotyping of Clostridium perfringens by polymerase chain reaction is a useful adjunct to diagnosis of clostridial enteric disease in animals. Anae. 2: 197-203.
9. Van Asten, A.J.A.M., Wiel, C.W., Van Nikolaou, G., Houvers, D.J. and Grone, A. (2009) A multiplex PCR for toxin typing of Clostridium perfringens. Vet. Microbiol., 136(3-4): 411-412.
10. 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.
11. Josephine, W., Zang, W., Xie, B., Wu, M., Tong, X., Kalpoe, J. and Zhang, D. (2009) Detection and toxin typing of C. perfringens in formalin-fixed, paraffin-embedded tissue samples by PCR. J. Clin. Microbiol., 47(3): 807-810.
PMid:19109478 PMCid:PMC2650962
12. Bedagliacca, P., Provvido, A.D., Scattolini, S., Pompei, G. and Giannatale, E.D. (2010) Toxin genotyping of Clostridium perfringens strains using a polymerase chain reaction protocol. Vet. Ital. 46(1): 113-8, 107-12.
13. 13. Ahsani M R, bafti M S, Esmailizadeh A K and Mohammadabadi M R (2011) Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep. Small Ruminant Research 95 : 65-69. .
14. Gurumu, E.B., Hazarika, R.A., Borah, P. and Barua, A.G. (2013) Presence of enterotoxegenic Clostridium perfringens in food of animal origin, Gowahati, India. J. Environ. Occup. Sci., 2(1): 45-50.
15. Ahsani, M.R., bafti, M.S., Esmailizadeh, A.K. and Mohammadabadi, M.R. (2010) Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep. Small. Rumin Res., 95: 65-69.
16. Ramadoss, P. (2006) DNA isolation from bacteriological culture using high salt method, ICAR workshop on emerging biotechnological methods on disease diagnosis. Department of Animal Biotehcnology, MVC, Chennai, 19th April to 9th May, 2006.
17. Cato, E.P., George, W.L. and Finegold, S.M. (1986) Genus clostridium prazmowski 1880, 23 AL. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., editors. Bergey's Manual of Systematic Bacteriology. Vol. 1. The Williams & Wilkins Co., Baltimore. p1141-1200.
18. Popoff, M.R. (1984) Bacteriological examination in enterotoxaemia of sheep and lamb. Vet. Rec., 114(13): 324.
19. Hadimli, H.H., Ergamis, O., Sayin, Z. and Aras, Z. (2012) Toxinotyping of Clostridium perfringens isolates by ELISA and PCR from lambs suspected of enterotoxemia. Turk. J. Anim. Sci., 36(4): 409-415.
20. Sawires, Y.S. and Songer, J.G. (1995) Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe, 11(5): 262-272.
21. Gkiourtzidis, K., Frey, J., Bourtzi-Hatzopoulou, E., Iliadis, N. and Sarris, K. (2001) PCR detection and prevalence of alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes in Clostridium perfringens isolated from lambs with Clostridial dysentery. Vet. Microbiol., 82(1): 39-43.
22. Popoff, M.R. (2011) Epsilon toxin is fascinating pore forming toxin. FEBS J., 278(23): 4602-4615.
23. Uzal, F.A. and Songer, G. (2008) Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. J. Vet. Diagn. Invest., 20(3): 253-265.