Aim: Blackleg disease is caused by Clostridium chauvoei in ruminants. Although virulence factors such as C. chauvoei toxin A, sialidase, and flagellin are well characterized, hyaluronidases of C. chauvoei are not characterized. The present study was aimed at cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of C. chauvoei.

Materials and Methods: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR) using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct.

Results: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringens and Clostridium paraputrificum.

Conclusion: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing.

Keywords: black quarter, Clostridium chauvoei, hyaluronoglucosaminidase.

1. Frey, J. and Falquet, L. (2015) Patho-genetics of Clostridium chauvoei. Res. Microbiol., 166: 384-392. [Crossref] [PubMed]

2. Ayele, B., Tigre, W. and Deressa, B. (2016) Epidemiology and financial loss estimation of blackleg on smallholder cattle herders in Kembata Tambaro zone, Southern Ethiopia. SpringerPlus, 5: 1822. [Crossref] [PubMed] [PMC]

3. Anonymous. (2017) Species-Wise Incidence of Livestock Diseases in India Annual Report 2016-17. Department of Animal Husbandry, Dairying and Fisheries, Government of India, New Delhi.

4. 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. [Crossref] [PubMed]

5. Vilei, E.M., Johansson, A., Schlatter, Y., Redhead, K. and Frey, J. (2011) Genetic and functional characterization of the nanA sialidase from Clostridium chauvoei. Vet. Res., 42: 2. [Crossref] [PubMed] [PMC]

6. 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 my necrosis. Vaccine, 30: 5500-5505. [Crossref] [PubMed]

7. 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. [Crossref] [PubMed]

8. Hynes, W.L. and Walton, S.L. (2000) Hyaluronidases of gram-positive bacteria. FEMS microbial. lett., 183: 201-207.

9. 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.

10. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol., 30: 2725-2729. [Crossref] [PubMed] [PMC]

11. Sophia, I., Viswas, K.N., Usharani, J., Thomas, P. and Gupta, S.K. (2013) PCR amplification and cloning of immunogenic region of cctA gene of Clostridium chauvoei. Indian J. Compost. Microbiol. Immunol. Infect. Dis., 34(2): 15-18.

12. Dangi, S.K., Singh, A.P., Dangi, S.S., Thomas, P., Gupta, S.K., Agarwal, R.K. and Viswas, K.N. (2014) Polymerase chain reaction amplification and cloning of immunogenic protein NAD-dependent beta hydroxybutyryl coA dehydrogenase gene of Clostridium chauvoei. Vet. World, 7(10): 848-851. [Crossref]

13. Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S. and Hayashi, H. (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. PNAS, 99(2): 996-1001. [Crossref] [PubMed] [PMC]

14. Nakai, K. and Horton, P. (1999) PSORT: A program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci., 24: 34-36. [Crossref]

15. Li, H., Morimoto, K., Kimura, T., Sakka, K. and Ohmiya, K. (2003) A new type of p-n-acetylglucosaminidase from hydrogen-producing Clostridium paraputrificum M-21. J. Biosci. Bioeng., 96: 268-274. [Crossref]