Combined Molecular and Biochemical Identification of Alpha Toxin in Local Isolated Clostridium novyi from the Sheep Liver

Document Type : Original Articles

Authors

1 Department of Pathobiology, Faculty of Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Clostridium novyi (C. novyi) causes deadly Black disease in sheep and rarely in other animals. Alpha toxin (α-toxin), the most apparent pathogen of this disease, is produced by C. novyi type B. Economic damages of C. novyi include sheep mortality costs, depreciation of affected farms, and health problems with infected carcasses. The identification of C. novyi and isolation of its pathogens by conventional methods is a time-consuming process, necessitating a simple and rapid method for isolating and detecting pathogenic C. novyi. Therefore, this study aimed to molecularly identify α-toxin in local C. novyi isolates from the sheep livers. In this study, 75 livers suspected of Black disease were sampled. The samples of the liver were cultured under anaerobic conditions. Some of the cultured colonies were used in biochemical tests. For molecular confirmation, the DNA of isolates was extracted, and the isolates were confirmed by the polymerase chain reaction (PCR) on the liver tissue and cultured samples using specific α-toxin primers. The PCR on α-toxin produced a band in the range of 609 bp, indicating that the samples belonged to C. novyi. According to the results, of 75 isolates, 18 isolates were confirmed as C. novyi. C. novyi type B was isolated from the liver and confirmed by biochemical and molecular characterization. The PCR assay ensured a sensitive and specific tool for the detection of C. novyi in the samples.

Keywords

Main Subjects

References

  1. Aronoff DM. Clostridium novyi, sordellii, and tetani: mechanisms of disease. Anaerobe. 2013;24:98-101.
  2. Smits WK, Lyras D, Lacy DB, Wilcox MH, Kuijper EJ. Clostridium difficile infection. Nat Rev Dis Primers. 2016;2:16020.
  3. Sasaki Y, Takikawa N, Kojima A, Norimatsu M, Suzuki S, Tamura Y. Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences. Int J Syst Evol Microbiol. 2001;51(Pt 3):901-4.
  4. Eklund MW, Poysky FT, Peterson ME, Meyers JA. Relationship of bacteriophages to alpha toxin production in Clostridium novyi types A and B. Infect Immun. 1976;14(3):793-803.
  5. Oakley CL, Warrack GH, Clarke PH. The toxins of Clostridium oedematiens (Cl. novyi). J Gen Microbiol. 1947;1(1):91-107.
  6. Eeckhaut V, Boyen F, Pasmans F, Uzal FA, Ducatelle R, Van Hoorebeke S, et al. Clostridium novyi type B as a causative agent of bovine meat spoilage. Anaerobe. 2012;18(3):286-8.
  7. Ball DW, Van Tassell RL, Roberts MD, Hahn PE, Lyerly DM, Wilkins TD. Purification and characterization of alpha-toxin produced by Clostridium novyi type A. Infect Immun. 1993;61(7):2912-8.
  8. Bhatnagar J, Deleon-Carnes M, Kellar KL, Bandyopadhyay K, Antoniadou ZA, Shieh WJ, et al. Rapid, simultaneous detection of Clostridium sordellii and Clostridium perfringens in archived tissues by a novel PCR-based microsphere assay: diagnostic implications for pregnancy-associated toxic shock syndrome cases. Infect Dis Obstet Gynecol. 2012;2012:972845.
  9. Jeong CG, Seo BJ, Nazki S, Jung BK, Khatun A, Yang MS, et al. Characterization of Clostridium novyi isolated from a sow in a sudden death case in Korea. BMC Vet Res. 2020;16(1):127.
  10. Navarro MA, Uzal FA. Pathobiology and diagnosis of clostridial hepatitis in animals. J Vet Diagn Invest. 2020;32(2):192-202.
  11. Paquet-Bolduc B, Gervais P, Roussy J-F, Trottier S, Oughton M, Brukner I, et al. Detection and Isolation of Clostridium difficile Asymptomatic Carriers During Clostridium difficile Infection Outbreaks: An Exploratory Study. Clin Infect Dis. 2018;67(11):1781-3.
  12. Skarin H, Segerman B. Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens. PLoS One. 2014;9(9):e107777.
  13. Hofmann F, Herrmann A, Habermann E, von Eichel-Streiber C. Sequencing and analysis of the gene encoding the alpha-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. Mol Gen Genet. 1995;247(6):670-9.
  14. Sathish S, Swaminathan K. Genetic diversity among toxigenic clostridia isolated from soil, water, meat and associated polluted sites in South India. Indian J Med Microbiol. 2009;27(4):311-20.
  15. Woo PC, Lau SK, Chan KM, Fung AM, Tang BS, Yuen KY. Clostridium bacteraemia characterised by 16S ribosomal RNA gene sequencing. J Clin Pathol. 2005;58(3):301-7.
  16. Nyaoke AC, Navarro MA, Beingesser J, Uzal FA. Infectious necrotic hepatitis caused by Clostridium novyi type B in a horse: case report and review of the literature. J Vet Diagn Invest. 2018;30(2):294-9.
  17. Zaragoza NE, Orellana CA, Moonen GA, Moutafis G, Marcellin E. Vaccine Production to Protect Animals Against Pathogenic Clostridia. Toxins (Basel). 2019;11(9).
  18. Ardehali M, Moosawi M, Pilehchian R. Characterization of Clostridium oedematiens Strains Isolated from Cases of Black Disease of Sheep in Iran Arch Razi Inst. 1989;40(1):37-45.
  1. Ardehali M, Darakhshan H. Isolation and typing of Clostridium oedematiens (Cl. novyi) from cases of black disease of sheep in iran. Comp Immunol Microbiol Infect Dis. 1979;2(1):107-11.
  2. Pilehchian Langroudi R. Molecular biology of Clostridium perfringens focusing on epsilon and beta toxin genes. J Vet Lab Res. 2013;5(1):5-19.
  3. Ardehali M, Moosawi, Avazpoor J. Isolation, Typing and Rapid Diagnosis of pathogenic clostridia from Infected Animals in Iran. Arch Razi Inst. 1988;38.39(1):35-42.
  4. Abdolmohammadi Khiav L, Jabbari AR, Pilehchian Langroodi R. Molecular identification and evaluation toxin production ofclostridium perfringenstype d strains isolated cases of the enterotoxaemia in the razi institute. J Vet Microbiol. 2018;14(1):71-8.
  5. Songer JG. Clostridial diseases of small ruminants. Vet Res. 1998;29(3-4):219-32.
Archives of Razi Institute
Volume 77, Issue 5 - Serial Number 5
September and October 2022
Pages 1769-1777

Files

Share

How to cite

Statistics