Veterinary World

     Open access and peer reviewed journal  

ISSN (Online): 2231-0916

ISSN (Print): 0972-8988


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: 19-10-2014)

18. Polymerase chain reaction amplification and cloning of immunogenic protein NAD-dependent beta hydroxybutyryl CoA dehydrogenase gene of Clostridium chauvoei - Saroj K. Dangi, Ajay P. Singh, Satyaveer S. Dangi, Prasad Thomas, Santosh K. Gupta, Rajesh K. Agarwal and K. N. Viswas

Veterinary World, 7(10): 848-851



   doi: 10.14202/vetworld.2014.848-851



Saroj K. Dangi: Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

Ajay P. Singh: Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

Satyaveer S. Dangi: Division of Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

Prasad Thomas: Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

Santosh K. Gupta: Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

Rajesh K. Agarwal: Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;

K. N. Viswas: Division of Bacteriology & Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India;


Received: 28-06-2014, Revised: 14-09-2014, Accepted: 20-09-2014, Published online: 19-10-2014


Corresponding author: K. N. Viswas, e-mail:

Aim: The present study was aimed at polymerase chain reaction (PCR) amplification and cloning of NAD-dependent betahydroxybutyryl coenzyme A dehydrogenase (BHBD) gene of Clostridium chauvoei.

Materials and Methods: C. chauvoei was cultured and confirmed by 16-23S rDNA spacer region primers. The primers for nad-bhbd gene of C. chauvoei were designed to aid in cloning into pRham-N-His SUMO-Kan vector, and nad-bhbd gene was amplified by PCR. The amplified nad-bhbd gene was purified and cloned into pRham-N-His SUMO-Kan expression vector. The recombinant plasmid was transformed into E. cloni 10 G cells and the clone was confirmed by colony PCR using the pRham-SUMO-NAD-For and pRham-SUMO-NAD-Rev primers and also by sequencing.

Results: PCR amplification of nad-bhbd gene yielded a product length of 844 base pairs which was cloned into pRham-NHis SUMO-Kan vector followed by transformation into E. cloni 10G chemically competent cells. The recombinant clones were characterized by colony PCR, sequencing, followed by basic local alignment search tool (BLAST) analysis to confirm the insert.

Conclusions: Immunogenic protein NAD- dependent BHBD of C. chauvoei was cloned and the recombinant clones were confirmed by colony PCR and sequencing analysis.

Keywords: black quarter, Clostridium chauvoei, NAD-beta-hydroxybutyryl coenzyme A dehydrogenase.

1. Groseth, P.K., Ersdal, C., Bjelland, A.M. and Stokstad, M. (2011) Large outbreak of blackleg in housed cattle. Vet. Rec., 169(13): 339.
2. Anonymous. (2013) Species-Wise Incidence of Livestock Diseases in India during 2011 (January-December). Annual Report 2012-13. Dept. of Animal Husbandry, Dairying & Fisheries, Government of India. p98-99.
3. Scott, J.P. (1928) The aetiology of black leg and methods of determining Clostridium chauvoei from other anaerobic organisms found in cases of blackleg. Cornell. Vet., 18: 249-271.
4. Sterne, M. and Edwards, J.B. (1955) Black leg in pigs caused by Clostridium chauvoei. Vet. Rec., 67: 314-315.
5. Clay, H.A. (1960) A case of "black quarter" in the pig. Vet. Rec., 72: 265-266.
6. Rahman, H., Chakraborty, A., Rahman, T., Sharma, R., Shome, B.R. and Shakuntala, I. (2009) Clostridial myonecrosis clinically resembling black quarter in an Indian elephant (Elephas maximus). Rev. Sci. Tech. Off. Int. Epiz., 28: 1069-1075.
7. Nagano, N., Isomine, S., Kato, H., Sasaki, Y., Takahashi, M., Sakaida, K., Nagano, Y. and Arakawa, Y. (2008) Human fulminant gas gangrene caused by Clostridium chauvoei. J. Clin. Microbiol., 46: 1545-1547.
PMid:18256217 PMCid:PMC2292918
8. Weatherhead, J.E. and Tweardy, D.J. (2012) Lethal human neutropenic entercolitis caused by Clostridium chauvoei in the United States: Tip of the iceberg? J. Infect., 64(2): 225-227.
9. Hatheway, C.L. (1990) Toxigenic clostridia. Clin. Microbiol. Rev., 3: 66-98.
PMid:2404569 PMCid:PMC358141
10. Songer, J.G. (1996) Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev., 9: 216-234.
PMid:8964036 PMCid:PMC172891
11. Useh, N.M., Nok, A.J. and Esievo, K.A. (2003) Pathogenesis and pathology of blackleg in ruminants: The role of toxins and neuraminidase. A short review. Vet. Q., 25: 155-9.
12. Uzal, F.A. (2012) Evidence-based medicine concerning efficacy of vaccination against Clostridium chauvoei infection in cattle. Vet. Clin. North Am. Food Anim. Pract., 28(1): 71-77.
13. Chandler, H.M. and Gulasekharam, J. (1974) The protective antigen of a highly immunogenic strain of Clostridium chauvoei including an evaluation of its flagella as a protective antigen. J. Gen. Microbiol., 84(1): 128-134.
14. Tamura, Y., Kijima-Tanaka, M., Aoki, A., Ogikubo, Y. and Takahashi, T. (1995) Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence. Microbiology, 141: 605-610.
15. Tanaka, M., Hirayama, N. and Tamura, Y. (1987) Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella. Infect. Immunol., 55: 1779-1783.
PMid:3301677 PMCid:PMC260601
16. Useh, N.M., Ajanusi, J.O., Esievo, K.A. and Nok, A.J. (2006) Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain). Cell Biochem. Funct., 24(4): 347-352.
17. Verpoort, J.A., Joubert, F.J. and Jansen, B.C. (1966). Studies on the soluble antigen and hemolysin of Clostridium chavoei strain 64. S. Afr. J. Agric. Sci., 9: 153-172.
18. Hang'ombe, B.M., Kohda, T., Mukamoto, M. and Kozaki, S. (2006) Purification and sensitivity of Clostridium chauvoei hemolysin to various erythrocytes. Comp. Immunol. Microbiol. Infect. Dis., 29(4): 263-268.
19. Vilei, E.M., Johansson, A., Schlatter, Y., Redhead, K. and Frey, J. (2011) Genetic and functional characterization of the Nan A sialidase from Clostridium chauvoei. Vet. Res., 42: 2.
PMid:21314964 PMCid:PMC3031224
20. Frey, J., Johansson, A., Burkina, S., Vilei, E.M. and Redhead, K. (2012) Cytotoxin CctA, a major virulence factor of Clostridium chauvoei conferring protective immunity against myonecrosis. Vaccine, 30: 5500-5505.
21. Liu, Z.X., Liu, G.Y., Li, N., Xiao, F.S., Xie, H.X. and Nie, P. (2012) Identification of immunogenic proteins of Flavobacterium columnare by two-dimensional electrophoresis immunoblotting with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes). J. Fish Dis., 35: 255-263.
22. Wilson, K. (1997) Preparation of genomic DNA from bacteria. In: Taylor, G.P., editor. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York. p2.4.1-2.4.5.
23. Sasaki, Y. Yamamato, K. Kojima, A. Norimatsu, M. and Tamura, Y. (2001) Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region. Res. Vet. Sci., 71: 289-294.