Veterinary Research Forum
Vet Res Forum

A simple cost-effective method for purification of Clostridium chauvoei cell-surface proteins for detection of antibodies against blackleg disease vaccine

Document Type : Short Communication

Authors

Niusha Adib 1
Azadeh Zahmatkesh 2
Masoumeh Bagheri 3

1 Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Anaerobic Bacterial Vaccines Research and Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

3 Department of Honeybee, Silk Worm and Wildlife Diseases, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

https://doi.org/10.30466/vrf.2024.2023812.4190
Abstract
Cell-surface proteins of Clostridium chauvoei were purified using a simple method. Bacterial cultures were centrifuged and agitated vigorously in phosphate buffered saline with or without further glycine treatment and ammonium sulfate precipitation. Rabbits were immunized subcutaneously with a blackleg disease vaccine twice with a two-week interval. Immunized sera were collected one week after the second injection. Enzyme-linked immunosorbent assay (ELISA) was performed using the proteins purified by the second method as the coating antigen. Bradford assay results showed a higher protein concentration in the second than the first method. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis showed multiple bands for the cell-surface proteins of C. chauvoei in the first method and a sharp band equivalent to flagellin protein in the second method. The ELISA results indicated that the purified proteins were capable of detecting antibodies against Blackleg disease vaccine. The purified protein would be an alternative antigen for indirect ELISA in order to monitor the immune response in vaccinated farm animals.

Keywords

Clostridium septicum
ELISA
Flagellin
Glycine
Immunity

Subjects

  1. Useh NM, Nok AJ, Esievo KA. Pathogenesis and pathology of blackleg in ruminants: the role of toxins and neuraminidase. A short review. Vet Q 2003; 25(4): 155-159.
  2. Prescott JF. Brief description of animal pathogenic clostridia. In: Uzal FA, Songer G, Prescott JF, et al. (Eds). Clostridial diseases of animals. New Jersey, USA: Wiley-Blackwell 2016; 13-19.
  3. Frey J, Johansson A, Bürki S, et al. Cytotoxin CctA, a major virulence factor of Clostridium chauvoei conferring protective immunity against myonecrosis. Vaccine 2012; 30(37): 5500-5505.
  4. Nicholson P, Furrer J, Hässig M, et al. Production of neutralizing antibodies against the secreted Clostridium chauvoei toxin A (CctA) upon blackleg vaccination. Anaerobe 2019; 56: 78-87.
  5. Stevenson E, Minton NP, Kuehne SA. The role of flagella in Clostridium difficile Trends Microbiol 2015; 23(5): 275-282.
  6. Kojima A, Uchida I, Sekizaki T, et al. Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei. Vet Microbiol 2000; 76(4): 359-372.
  7. Lu Y, Swartz JR. Functional properties of flagellin as a stimulator of innate immunity. Sci Rep 2016; 6: 18379. doi: 10.1038/srep18379.
  8. Usharani J, Nagaleekar VK, Thomas P, et al. Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei. Anaerobe 2015; 33: 48-54.
  9. Kitao A, Hata H. Molecular dynamics simulation of bacterial flagella. Biophys Rev 2018; 10(2): 617-629.
  10. Simon R, Curtis B, Deumic V, et al. A scalable method for biochemical purification of Salmonella flagellin. Protein Expr Purif 2014; 102: 1-7.
  11. Arnold F, Bédouet L, Batina P, et al. Biochemical and immunological analyses of the flagellin of Clostridium tyrobutyricum ATCC 25755. Microbiol Immunol 1998; 42(1): 23-31.
  12. Tamura Y, Minamoto N, Tanaka S. Demonstration of protective antigen carried by flagella of Clostridium chauvoei. Microbiol Immunol 1984; 28(12): 1325-1332.
  13. Khani MH, Bagheri M, Zahmatkesh A, et al. Immunostimulatory effects of truncated and full-length flagellin recombinant proteins. Microb Pathog 2019; 127: 190-197.
  14. Kijima-Tanaka M, Ogikubo Y, Kojima A, et al. Flagella based enzyme-linked immunosorbent assay for evaluation the immunity in mice vaccinated with blackleg vaccines. J Microbiol Methods 1998; 32(1): 79-85.
  15. Jayaramaiah U, Singh N, Thankappan S, et al. Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei. Anaerobe 2016; 39: 77-83.
  16. Adib N, Zahmatkesh A, Esmaeilnejad-Ahranjani P, et al. Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity. Anaerobe 2023; 83: 102781. doi: 10.1016/j.anaerobe.2023.102781.
  17. Abdolmohammadi Khiav L, Zahmatkesh A. Vaccination against pathogenic clostridia in animals: a review. Trop Anim Health Prod 2021; 53(2): 284. doi: 10.1007/ s11250-021-02728-w.
  18. Kojima A, Amimoto K, Ohgitani T, et al. Characterization of flagellin from Clostridium chauvoei. Vet Microbiol 1999; 67(3): 231-237.
  19. Tamura Y, Tanaka M. Opsonic activity of anti-flagellar serum against Clostridium chauvoei by mouse polymorphonuclear leucocytes. Vet Microbiol 1987; 14(1): 81-86.
  20. Khani MH, Bagheri M. Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria. Protein Expr Purif 2019; 155: 48-53.
  21. Wright A, Wait R, Begum S, et al. Proteomic analysis of cell surface proteins from Clostridium difficile. Proteomics 2005; 5(9): 2443-2452.
  22. Skountzou I, Martin Mdel P, Wang B, et al. Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine. Vaccine 2010; 28(24): 4103-4112.
  23. Bruxelle JF, Mizrahi A, Hoÿs S, et al. Clostridium difficile flagellin FliC: evaluation as adjuvant and use in a mucosal vaccine against Clostridium difficile. PLoS One 2017; 12(11): e0187212. doi: 10.1371/journal. pone.0187212.
  24. Ziech RE, Farias LDa, Balzan C, et al. Protective efficacy of commercial vaccines against a virulent field strain of Clostridium chauvoei. Semin Cienc Agrar 2019; 40(5): 1837-1848.
Veterinary Research Forum
Volume 16, Issue 1
January 2025
Pages 57-61

  • Receive Date 02 March 2024
  • Revise Date 22 July 2024
  • Accept Date 14 August 2024

Home

Guide for Authors

Submit Manuscript

Reviewers

Contact Us