Protective efficacy of commercial vaccines against a virulent field strain of Clostridium chauvoei

Rosangela Estel Ziech, Luana D’Ávila Farias, Cláudia Balzan, Monique Aldren Burnett da Luz, Joachim Frey, Agueda Castagna de Vargas


Blackleg is an acute and frequently fatal infection that mainly affects cattle and is caused by Clostridium chauvoei. Formalin-killed, whole-cell vaccines are commonly used to control blackleg. The aim of this study was to verify the protective efficacy of two commercial vaccines against the infection of guinea pigs with two strains of C. chauvoei, a virulent field strain (SBP 07/09) and the reference strain used in official tests (Manguinhos-Teixeira or MT). The strains used in the challenge were characterized by whole genome sequencing, and the minimal inhibitory concentrations of 15 antimicrobials were determined. To assess the protective efficacy, guinea pigs were vaccinated and subsequently challenged with C. chauvoei. All four vaccinated and challenged groups seroconverted after vaccination, while the control group remained seronegative, as determined by indirect ELISA. The identical performance of the two C. chauvoei strains in terms of virulence after challenge and their inability to infect vaccinated animals was correlated with their high genetic homology. Both commercial vaccines showed good protective efficacy against both the reference and field strains. Although C. chauvoei vaccination failures have been reported, the results from our study and others reported high similarity among C. chauvoei strains from all over the world, which suggests that the vaccine failures are not due to antigenic variability but inadequate vaccine management.


Blackleg; Clostridial diseases; Mionecrosis; Bacterins; Evaluation

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ARAUJO, R. F.; CURCI, V. C. L. M.; NOBREGA, F. L. C.; FERREIRA, R. M. M.; DUTRA, I. S. Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg. Pesquisa Veterinária Brasileira, v. 30, n. 7, p. 554-558, 2010. DOI: 10.1590/S0100-736X2010000700008

BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Portaria n. 49 de 12 de maio de 1997. Diário Oficial [da] União, Brasília, 16 de maio de 1997. Seção 1, p. 10168-10169.

BRITISH PHARMACOPEIA. Veterinary Antisera and Veterinary Vaccines. 3. ed. Sainte Ruffine: Maisonneuve S.A., 1998.

BUYS, A.; MACDONALD, R.; CRAFFORD, J.; THERON, J. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines. Journal of the South African Veterinary Association, v. 85, n. 1, p. 977, 2014. DOI: 10.4102/jsava.v85i1.977. Available at: Accessed at: 03 mar. 2017.

CHANDLER, H. M. Rabbit immunoglobulin responses to the flagella, somatic, and protective antigens of a highly protective strain of Clostridium chauvoei. Infection and Immunity, v. 12, n. 1, p. 143-147, 1975.

CHANDLER, H. M.; GULASEKHARAM, J. The protective antigen of a highly immunogenic strain of Clostridium chauvoei including an evaluation of its flagella as a protective antigen. Journal of General Microbiology, v. 84, n. 1, p. 128-134, 1974. DOI: 10.1099/00221287-84-1-128

CORPUS, M. D. L. O.; ANDRADE, L. H.; MENDEZ, J. L.; GUTIERREZ, V. T. Prevention of blackleg by an immunogen of Clostridium chauvoei. Annals of the New York Academy of Sciences, v. 1149, n. 1, p. 303-305, 2008. DOI: 10.1196/annals.1428.013

CRICHTON, R.; HARRISS, D. A; MCKAY, D. J. Standards for Clostridium chauvoei vaccine the relationship between the response of guinea-pigs and sheep following vaccination and challenge with virulent Clostridium chauvoei. Australian Veterinary Journal, v. 63, n. 3, p. 68-70, 1986. DOI: 10.1111/j.1751-0813.1986.tb02931.x

CRICHTON, R.; SOLOMON, J.; BARTON, A. M. The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens. Biologicals: Journal of the International Association of Biological Standardization, v. 18, n. 1, p. 49-54, 1990. DOI: 10.1016/1045-1056(90)90070-G

CRUZ, C. D. Programa genes: biometria. Viçosa: UFV, 2006. 382 p.

DUTRA, I. S.; SOUZA, A. M.; BORSANELLI, A. C. Escolha da vacina define a eficácia dos programas preventivos contra clostridioses. In: WORKSHOP VACINAS E VACINAÇÕES NA BOVINOCULTURA, Ribeirão Preto, 2011. Anais... Jaboticabal: Athenas Gráfica e Editora, 2011. v. 1, p. 97-130.

FARIAS, L. D. Carbúnculo sintomático: abordagem diagnóstica e imunológica. 2011. Dissertação (Mestrado em Medicina Veterinária) - Universidade Federal de Santa Maria, Santa Maria, 2011. 61 p.

FREY, J.; FALQUET, L. Patho-genetics of Clostridium chauvoei. Research in Microbiology, v. 166, n. 4, p. 384-392, 2015. DOI: 10.1016/j.resmic.2014.10.013

FREY, J.; JOHANSSON, A.; BÜRKI, S.; VILEI, E. M.; REDHEAD, K. Cytotoxin CctA, a major virulence factor of Clostridium chauvoei conferring protective immunity against myonecrosis. Vaccine, v. 30, n. 37, p. 5500-5505, 2012. DOI: 10.1016/j.vaccine.2012.06.050. Available at: 22749595. Accessed at: 10 mar. 2016.

GACEM, F.; MADADI, M. A.; KHECHA, N.; BAKOUR, R. Study of vaccinal properties of Clostridium chauvoei strains isolated during a blackleg outbreak in cattle in Algeria. Kafkas Universitesi Veteriner Fakultesi Dergisi, v. 21, n. 6, p. 825-829, 2015. DOI: 10.9775/kvfd.2015.13616

GASPAR, E. B.; SANTOS, L. R. Vacinação de bovinos: esclarecendo algumas dúvidas. Bagé: Embrapa Pecuária Sul, 2014. 36 p.

GROSETH, P. K.; ERSDAL, C.; BJELLAND, A. M.; STOKSTAD, M. Large outbreak of blackleg in housed cattle. Veterinary Record, v. 169, n. 13, p. 339-339, 2011. DOI: 10.1136/vr.d4628

HAMAOKA, T.; MORI, Y.; TERAKADO, N. Enzyme-linked immunosorbent assay for evaluation of immunity in mice vaccinated with blackleg vaccine. The Japanese Journal of Veterinary Science, v. 52, n. 1, p. 167-170, 1990. DOI: 10.1292/jvms1939.52.167

HANNA, T. N.; SELIM, A. M.; EBEID, M. H.; HOUMDA, F. K.; ELMENEISY, A. A.; GORGY, S. F. Comparative evaluation of antibody response in sheep vaccinated by bivalent clostridial and pasteurella combined vaccine. Benha Veterinary Medical Journal, v. 27, n. 2, p. 309-316, 2014.

KIJIMA-TANAKA, M.; OGIKUBO, Y.; KOJIMA, A.; SASAKI, Y. Methods Flagella based enzyme-linked immunosorbent assay for evaluation the immunity in mice vaccinated with blackleg vaccines. Journal of Microbiological Methods, v. 32, n. 1, p. 79-85, 1998. DOI: 10.1016/S0167-7012(98)00010-4

KIJIMA-TANAKA, M.; TAMURA, Y.; SUZUKI, S.; NAGAMINE, N.; NAKAMURA, M. Antigenic mimicry of Clostridium chauvoei flagella by polyclonal anti-idiotypic antibodies. Journal of Medical Microbiology, v. 40, n. 1, p. 70-75, 1994. DOI: 10.1099/00222615-40-1-70

KOJIMA, A.; UCHIDA, I.; SEKIZAKI, T.; SASAKY, Y.; OGIKUBO, Y.; KIJIMA, M.; TAMURA, Y. Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei. Veterinary Microbiology, v. 76, n. 4, p. 359-372, 2000. DOI: 10.1016/S0378-1135(00)00256-X

LOPES, A. de A. Avaliação de imunógenos anti-carbúnculo sintomático, em uso no Brasil. 1977. Dissertação (Mestrado em Medicina Veterinária) - Universidade Federal de Minas Gerais, Belo Horizonte.

LOPES, B. P. Resposta sorológica em bovinos vacinados contra o Carbúnculo Sintomático. 2005. Dissertação (Mestrado em Medicina Veterinária) - Universidade Estadual Paulista, Jaboticabal.

MATTAR, M. A.; CORTIAS, T. I.; GUZMÁN, A. M. S. de. Immunogenic protein variations of Clostridium chauvoei cellular antigens associated with the culture growth phase. FEMS Immunology and Medical Microbiology, v. 33, n. 1, p. 9-14, 2002. DOI: 10.1016/S0928-8244(01)00302-9

MATTAR, M. A.; CORTINAS, T. I.; DIGENARO, M. S.; STEFANINI DE GUZMAN, A. M. Immunogenic power of cellular and extracellular antigens of Clostridium chauvoei strains isolated in San Luis, Argentina. Anaerobe, v. 5, n. 3-4, p. 295-296, 1999. DOI: 10.1006/anae.1999.0276

MICALIZZI, B.; GUZMAN, A. M. S. Protective activity of different immunosera against Clostridium chauvoei. Anaerobe, v. 3, n. 23, p. 127-129, 1997. DOI: 10.1006/anae.1997.0088

MIRANDA, A.; FORT, M.; CARLONI, G.; CERVIÑO, M.; BEDOTTI, D. Evaluación de la respuesta serológica de vacunas comerciales frente a Clostridium chauvoei. Veterinaria Argentina, v. 23, n. 221, p. 130-136, 2008.

NAZ, S.; AHMAD, S.; BHATTI, J. Isolation and identification of Clostridium chauvoei from cattle and buffaloes. Pakistan Veterinary Journal, v. 25, n. 2, p. 101-102, 2005.

ORTIZ-ORTEGA, D.; VILLAMIL, L. C.; MARTINEZ, R. Isolation and typing of Clostridium spp. 16S rRNA from soil samples obtained in areas with sudden mortality history in Colombia. Global Advanced Research Journal of Microbiology, v. 1, p. 33-40, 2012.

REED, G. A.; REYNOLDS, L. Failure of Clostridium chauvoei vaccines to protect against blackleg. Australian Veterinary Journal, v. 53, n. 8, p. 393, 1977. DOI: 10.1111/j.1751-0813.1977.tb07961.x. Available at: pubmed/588168. Accessed at: 20 mar. 2017.

REED, L. J.; MUENCH, H. A. A simple method of estimating of fifty percent endpoints. American Journal of Hygiene, v. 4, n. 27, p. 493-497, 1938. DOI: 10.1093/oxfordjournals.aje.a118408

RYCHENER, L.; IN-ALBON, S.; DJORDJEVIC, S. P.; CHOWDHURY, P. R.; NICHOLSON, P.; ZIECH, R. E.; VARGAS, A. C. de.; FREY, J.; FALQUET, L. Clostridium chauvoei, an evolutionary dead-end pathogen. Frontiers in Microbiology, v. 8, n. 1054, p. 1-13, 2017. DOI: 10.3389/fmicb.2017.01054. Available at: Accessed at: 10 mar. 2017.

SALVARANI, F. M.; SILVA, R. O. S.; PIRES, P. S.; CRUZ JÚNIOR, E. C. C.; ALBEFARO, I. S.; GUEDES, R. M. C.; LOBATO, F. C. F. Antimicrobial susceptibility of Clostridium perfringens isolated from piglets with or without diarrhea in Brazil. Brazilian Journal of Microbiology, v. 43, n. 3, p. 1030-1033, 2012. DOI: 10.1590/S1517-83822012000300027

SANTOS, B. A. Avaliação da eficácia em cobaias de imunógenos contra carbúnculo sintomático em uso no Brasil. 2003. Tese (Doutorado em Medicina Veterinária) - Universidade Estadual Paulista, Jaboticabal.

SASAKI, Y.; KOJIMA, A.; AOKI, H. Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene. Veterinary Microbiology, v. 86, p. 257-267, 2002. DOI: 10.1016/S0378-1135(02)00002-0

SEEMANN, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics, v. 30, n. 14, p. 2068-2069, 2014. DOI: 10.1093/bioinformatics/btu153

SILVA, R. O. S.; D’ELIA, M. L.; TEIXEIRA, E. P. T.; PEREIRA, P. L. L.; SOARES, D. F. M.; CAVALCANTI , A. R.; KOCUVAN, A.; RUPNIK, M.; SANTOS, A. L. Q.; OLIVEIRA JUNIOR, C. A.; LOBATO, F. C. F. Clostridium difficile and Clostridium perfringens from wild carnivore species in Brazil. Anaerobe, v. 28, n. 2014, p. 207-211, 2014. DOI: 10.1016/j.anaerobe.2014.06.012

SRINIVASAN, V. A.; REDDY, G. S.; RAO, K. A.; KIHM, U. Serological response of bovines to combined vaccine containing foot and mouth disease virus, rabies virus, Pasteurella multocida and Clostridium chauvoei antigens. Veterinarski Arhiv, v. 71, n. 1, p. 37-45, 2001.

STEVENSON, J. R.; STONGER, K. A. Protective cellular antigen of Clostridium chauvoei. American Journal of Veterinary Research, v. 41, n. 4, p. 650-653, 1980.

TAKEUCHI, S.; HASHIZUME, N.; KINOSHITA, T.; KAIDOH, T.; TAMURA, Y. Detection of Clostridium septicum hemolysin gene by polymerase chain reaction. The Journal of Veterinary Medical Science, v. 59, n. 9, p. 853-855, 1997. DOI: 10.1292/jvms.59.853

TAMURA, Y.; KIJIMA-TANAKA, M.; AOKI, A.; OGIKUBO, Y.; TAKAHASHI, T. Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence. Microbiology, v. 141, n. 3, p. 605-610, 1995. DOI: 10.1099/13500872-141-3-605

TAMURA, Y.; KIJIMA, M.; OHISHI, K.; I; TAKAHASHI, T.; SUZUKI, S.; NAKAMURA, M. Antigenic analysis of Clostridium chauvoei flagella with protective and non-protective monoclonal antibodies. Journal of General Microbiology, v. 138, n. 3, p. 537-542, 1992. DOI: 10.1099/00221287-138-3-537

TAMURA, Y.; TANAKA, M. Opsonic activity of anti-flagellar serum against Clostridium chauvoei by mouse polymorphonuclear leucocytes. Veterinary Microbiology, v. 14, n. 1, p. 81-86, 1987. DOI: 10.1016/0378-1135(87)90055-1

TAMURA, Y.; TANAKA, S. Effect of antiflagellar serum in the protection of mice against Clostridium chauvoei. Infection and Immunity, v. 43, n. 2, p. 612-616, 1984.

TANAKA, M.; HIRAYAMA, N.; TAMURA, Y. Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella. Infection and Immunity, v. 55, n. 8, p. 1779-1783, 1987.

U.S. DEPARTMENT OF AGRICULTURE - USDA, 2003. Clostridium chauvoei Bacterin. EUA. p. 634-635. Available at: Accessed at: 10 mar. 2018.

UZAL, F. A. Evidence-based medicine concerning efficacy of vaccination against Clostridium chauvoei infection in cattle. The Veterinary clinics of North America. food Animal Practice, v. 28, n. 1, p. 71-7, 2012. DOI: 10.1016/j.cvfa.2011.12.006. Available at: Accessed at: 10 mar. 2018.

VALLENET, D.; BELDA, E.; CALTEAU, A.; CRUVEILLER, S.; ENGELEN, S.; LAJUS, A.; LE FÈVRE, F.; LONGIN, C.; MORNICO, D.; ROCHE, D.; ROUY, Z.; SALVIGNOL, G.; SCARPELLI, C.; SMITH, A.A.T.; WEIMAN, M.; MÉDIGUE, C. MicroScope - an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. Nucleic Acids Research, v. 41, n. D1, p. 636-647, 2013. DOI: 10.1093/nar/gks1194

VILEI, E. M.; JOHANSSON, A.; SCHLATTER, Y.; REDHEAD, K.; FREY, J. Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei. Veterinary Research, v. 42, n. 2, p. 1-9, 2011. DOI: 10.1186/1297-9716-42-2

WOOLCOCK, J. B.; FROST, A. J. Failure of Clostridium chauvoei vaccines to protect against blackleg. Australian Veterinary Journal, v. 54, n. 6, p. 319-3319, 1978. DOI: 10.1111/j.1751-0813.1978.tb02476.x


Semina: Ciênc. Agrár.
Londrina - PR
E-ISSN 1679-0359
DOI: 10.5433/1679-0359
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