The aim of the study described in this thesis was to obtain information about the chemical nature of the immunizing antigen of the vaccines against blackleg disease and the mechanism of the immunization. A literature study revealed the existence of much controversy about the reactions caused by Cl. chauvoei germs and toxins. Though the results of serological experiments carried out by various investigators led to the accepted theories about the relationships between homologous lethal toxins and haemolysins~ and between the haemolysis and other reactions of the two different species Cl. chauvoei and Cl. septicum, a comparative chemical study of the toxins had not yet been carried out. A preliminary investigation showed that the soluble immunizing antigen from the Cl. chauvoei cultures had the character of proteins. This was revealed by the loss of activity when pepsin and trypsin were allowed to act upon the antigenic solution and further by the influence of acid and heat upon the antigenic potency. No lethal activity was found in the filtrates of Cl. chauvoei (strain 64) cultures; instead we detected a haemolysin and a hyaluronidase which were also both heat-labile. The isolation procedures for the antigen and haemolysin relied on precipitation with ammonium sulphate and on chromatographic methods. / Thesis (DSc)--University of Pretoria, 1963. / Paraclinical Sciences / DSc / Unrestricted

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Blackleg is an acute and often fatal infection that affects mainly cattle, whose causative agent is Clostridium chauvoei. In this study two experiments were presented, the first with the aim of facilitating the collection and material storage in common filter paper for laboratory diagnosis of blackleg by using PCR (polymerase chain reaction). For this we tested in vitro, the sensitivity, specificity and feasibility of this method over different storage periods at room temperature. For the viability test we used twelve bovine liver tissues which ten samples were impregnated with a suspension containing C. chauvoei inoculum and the tests were performed at 24h, 48h, 72h, and one week of storage. The sensitivity of the technique was not affected by the use of common filter paper. There were no cross-reactions and no amplifications were observed from the negative control samples. Direct PCR by using common filter paper showed amplification in 50% of the samples at 24 h, 100% at 48h, 70% at 72h, and 90% within one week of storage at room temperature. This method demonstred to be a practical and economical alternative for collection, storage and material shipment for molecular diagnosis of blackleg. Second experiment aimed to verify the potency of immunization provided by two polyvalent clostridial vaccines (A and B vaccines) tested against the field strain (SBP 07/09) and the reference strain (MT) of C. chauvoei. The assays were made through of challenge in immunized guinea pigs, officially used by the Ministério da Agricultura, Pecuária e Abastecimento (MAPA, Brazil) (equivalent to Department of Agriculture) and subsequent evaluation of the humoral response of guinea pigs by ELISA (Enzyme Linked Immunosorbent Assay) was done. At the end of the experiment, all vaccinated guinea pigs remained alive and no clinical signs of the disease were observed, while the control group showed characteristic blackleg clinical signs and died within 24 to 48 hours. The vaccines tested were effective in stimulating the humoral response against both field strain SBP 09/07 and the reference strain MT of C. chauvoei. All four vaccinated and challenged groups seroconverted by the ELISA after vaccination and the control group remained seronegative. However, animals immunized with B vaccine had higher levels of antibodies detected when compared with guinea pig immunized with A vaccine. This work has important contributions to the prevention and development of diagnostic methods for blackleg, a relevant disease with economic and animal health impact. / O carbúnculo sintomático é uma infecção aguda e geralmente fatal que acomete principalmente os bovinos, cujo agente etiológico é Clostridium chauvoei. Neste estudo foram apresentados dois experimentos, o primeiro com o objetivo de facilitar a coleta e armazenamento de material em papel filtro comum, para diagnóstico laboratorial do carbúnculo sintomático por PCR (polymerase chain reaction) direta. Para isto, foram testadas, in vitro, a sensibilidade, especificidade e viabilidade do método em decorrência do tempo e à temperatura ambiente. Para o teste de viabilidade, foram utilizados os tecidos hepáticos de doze bovinos, sendo dez impregnados com uma suspensão de C. chauvoei. Os testes foram realizados nos períodos de 24h, 48h, 72h e uma semana após impregnação do papel filtro comum armazenado à temperatura ambiente. A sensibilidade da técnica não foi comprometida com o uso do papel filtro comum. Não foram observadas reações cruzadas e amplificações a partir das amostras controle negativas. A PCR direta do papel filtro comum apresentou amplificação em 50% das amostras nas 24h, 100% nas 48h, 70% nas 72h e 90% no período de uma semana de armazenamento à temperatura ambiente. Este método demonstrou ser uma alternativa prática e econômica para coleta, armazenamento e remessa de material para diagnóstico molecular de carbúnculo sintomático. O segundo experimento teve como objetivo verificar a eficiência da imunização proporcionada por duas vacinas clostridiais polivalentes (vacinas A e B) testadas frente à cepa de campo (SBP 07/09) e a cepa de referência (MT) de C. chauvoei. Os testes ocorreram por meio do desafio em cobaias imunizadas, utilizado oficialmente pelo Ministério da Agricultura Pecuária e Abastecimento (MAPA) e posteriormente foi avaliada a resposta humoral dos animais pelo teste de ELISA (Enzyme Linked Immunosorbent Assay). Ao final do experimento, todas as cobaias vacinadas estavam vivas e sem sinais clínicos da doença, enquanto os animais dos grupos controle apresentaram sinais característicos da doença e morreram no período de 24 a 48 horas. As vacinas testadas foram eficientes em estimular resposta humoral tanto frente à cepa de campo de C. chauvoei quanto frente à cepa de referência MT. Quanto à resposta humoral evidenciada pelo ELISA, os quatro grupos vacinados e desafiados soroconverteram após a vacinação e os animais do grupo controle permaneceram soronegativos. Contudo, os animais imunizados com a vacina B apresentaram maiores níveis de anticorpos detectados quando comparados com os animais imunizados com a vacina A. O presente trabalho trouxe contribuições no processo de prevenção e desenvolvimento de métodos diagnósticos para o carbúnculo sintomático, uma relevante doença, tanto do ponto de vista econômico quanto da saúde animal.

Orientador: Iveraldo dos Santos Dutra / Banca: Samir Issa Samara / Banca: Silvio Arruda Vasconcellos / Resumo: O Clostridium chauvoei é o agente causador do carbúnculo sintomático, doença que acomete bovinos e ovinos. É um dos principais problemas sanitários que ocorre nos rebanhos pecuários em todo o mundo. A vacinação dos animais jovens é a medida profilática indicada, visto que esta medida preventiva nos sistemas de produção é de extrema importância econômica e reconhecida pelos produtores, mesmo sendo de aplicação voluntária. Três grupos de bovinos, contendo 20 animais em cada grupo, foram vacinados com três vacinas comerciais contra o carbúnculo sintomático para a avaliação sorológica dos mesmos por meio do teste de aglutinação rápida em placa. Os animais foram vacinados no dia 0, e o reforço vacinal foi realizado 30 dias após a primeira dose. Após 21 dias do reforço, o sangue foi coletado da veia jugular, e o soro foi obtido a temperatura ambiente quando ocorreu a coagulação sanguínea e separação do soro. Foi comparada a resposta de indução de anticorpos nos grupos de animais reagentes a esse teste com antígenos obtidos de duas cepas, uma de referência (MT) do Ministério da Agricultura, Pecuária e Abastecimento (MAPA) e outra cepa de campo. Os resultados demonstraram que a resposta dos animais imunizados frente à cepa de referência foi satisfatória, não existindo diferença entre as três vacinas testadas. No entanto, quando este mesmo teste foi realizado com antígenos produzidos com a cepa de campo identificada por PCR, verificou-se a ausência de anticorpos aglutinantes na maioria dos bovinos avaliados. Na análise estatística ficou comprovado que existem diferenças significativas nos resultados dos testes de aglutinação rápida em placa quando se utilizam cepas distintas de Clostridium chauvoei, provavelmente em decorrência de diferenças antigênicas existentes entre as cepas. / Abstract: The Clostridium chauvoei is the agent which causes black leg, a disease that attacks the bovine and ovine. It is one of the main sanitary problems that occur with cattle-raising flocks in the world. The vaccination of young animals is the best prevention measure in production systems and is of extreme economic importance and is recognized by producers even if the application is voluntary. Three bovine groups, containing 20 animals each group, were vaccinated using three commercial vaccines against the black leg, for evaluation of bovine serological by tests of plate agglutination. The cattle were treated on day 0 and another dose was given on day 30. After 21 days, the blood was colleted of jugular vein and serum was obtained at 25°C, by blood coagulation. The comparison was made by detection of antibodies response between the animals that react to the test with antigens obtained from two strains which were used, one referred (MT) as Ministério da Agricultura, Pecuária e Abastecimento (MAPA) and another strain isolated from the field cases. The results determined that the response of the immunized animals faced by the referred strain was satisfactory with non-existing differences between the three tested vaccines. However, when the same test was conducted with antigens obtained from the field strain identified by PCR, it was verified the absence of agglutinating antibodies agglutinants in the majority of the bovines evaluated. In the statistics analysis were confirmed that significant differences were obtained in the agglutination plate test when different strains of Clostridium chauvoei were used, maybe because of the occurrence of antigenic difference, between the two strains tested. / Mestre

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-30Bitstream added on 2014-06-13T20:04:34Z : No. of bitstreams: 1 ragazani_avf_dr_jabo.pdf: 399859 bytes, checksum: f93ad8981a9e4aaa051ed6bff03ccacc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium... / The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below)

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-08-05Bitstream added on 2014-06-13T20:35:31Z : No. of bitstreams: 1 lopes_bp_me_jabo.pdf: 2441029 bytes, checksum: 9d91faa58722dae99b55f32ecfbd0b52 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O Clostridium chauvoei é o agente causador do carbúnculo sintomático, doença que acomete bovinos e ovinos. É um dos principais problemas sanitários que ocorre nos rebanhos pecuários em todo o mundo. A vacinação dos animais jovens é a medida profilática indicada, visto que esta medida preventiva nos sistemas de produção é de extrema importância econômica e reconhecida pelos produtores, mesmo sendo de aplicação voluntária. Três grupos de bovinos, contendo 20 animais em cada grupo, foram vacinados com três vacinas comerciais contra o carbúnculo sintomático para a avaliação sorológica dos mesmos por meio do teste de aglutinação rápida em placa. Os animais foram vacinados no dia 0, e o reforço vacinal foi realizado 30 dias após a primeira dose. Após 21 dias do reforço, o sangue foi coletado da veia jugular, e o soro foi obtido a temperatura ambiente quando ocorreu a coagulação sanguínea e separação do soro. Foi comparada a resposta de indução de anticorpos nos grupos de animais reagentes a esse teste com antígenos obtidos de duas cepas, uma de referência (MT) do Ministério da Agricultura, Pecuária e Abastecimento (MAPA) e outra cepa de campo. Os resultados demonstraram que a resposta dos animais imunizados frente à cepa de referência foi satisfatória, não existindo diferença entre as três vacinas testadas. No entanto, quando este mesmo teste foi realizado com antígenos produzidos com a cepa de campo identificada por PCR, verificou-se a ausência de anticorpos aglutinantes na maioria dos bovinos avaliados. Na análise estatística ficou comprovado que existem diferenças significativas nos resultados dos testes de aglutinação rápida em placa quando se utilizam cepas distintas de Clostridium chauvoei, provavelmente em decorrência de diferenças antigênicas existentes entre as cepas. / The Clostridium chauvoei is the agent which causes black leg, a disease that attacks the bovine and ovine. It is one of the main sanitary problems that occur with cattle-raising flocks in the world. The vaccination of young animals is the best prevention measure in production systems and is of extreme economic importance and is recognized by producers even if the application is voluntary. Three bovine groups, containing 20 animals each group, were vaccinated using three commercial vaccines against the black leg, for evaluation of bovine serological by tests of plate agglutination. The cattle were treated on day 0 and another dose was given on day 30. After 21 days, the blood was colleted of jugular vein and serum was obtained at 25°C, by blood coagulation. The comparison was made by detection of antibodies response between the animals that react to the test with antigens obtained from two strains which were used, one referred (MT) as Ministério da Agricultura, Pecuária e Abastecimento (MAPA) and another strain isolated from the field cases. The results determined that the response of the immunized animals faced by the referred strain was satisfactory with non-existing differences between the three tested vaccines. However, when the same test was conducted with antigens obtained from the field strain identified by PCR, it was verified the absence of agglutinating antibodies agglutinants in the majority of the bovines evaluated. In the statistics analysis were confirmed that significant differences were obtained in the agglutination plate test when different strains of Clostridium chauvoei were used, maybe because of the occurrence of antigenic difference, between the two strains tested.

Orientador: Ruben Pablo Schocken-Iturrino / Banca: Maria Luiza Poiatti / Banca: Alessandra Aparecida Medeiros / Banca: Hélio José Montassier / Banca: Antônio Carlos Monteiro / Resumo: Entre as espécies de Clostridium de importância em patologia animal, destaca-se o Clostridium perfringens, o Clostridium botulinum, o Clostridium chauvoei. O objetivo desta pesquisa foi verificar a presença de bactérias anaeróbias esporuladas (Clostridium sp), assim como, identificar as espécies de Clostridium patogênicos para a saúde animal, principalmente de bovinos, no solo de pastagem da micro-região de Jaboticabal, SP. Foram coletadas 250 amostras de solo e realizadas contagens de bactérias esporuladas do gênero Clostridium e identificação das espécies patogênicas presentes. Os resultados permitiram demonstrar a contagem de UFC de Closrtidium sp com média em log 10 igual a 2,79, sendo que os valores mínimo e máximo obtido foram 2,15 e 3,68 respectivamente. Para caracterização e identificação, os resultados permitiram identificar a presença de bactérias anaeróbias esporuladas em 233 amostras (93,2%), entre estas 180 eram do gênero Clostridium...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species of Clostridium of major importance to animal pathology are Clostridium perfringens, Clostridium botulinum, and C. chauvoei. Considering this, the objective of this research was to verify the presence of anareobic sporulate bacteria (Clostridium sp), and also identify the species of pathogenic Clostridium for the animal health, mostly to bovine, in pasture soil of Jaboticabal-SP. A total of 250 samples were collected and used to determine the number of sporulated bacteria from Clostridium genderand identify the pathogenic species present. The results demonstrated that the average number of CFU in log 10 of Closrtidium sp was 2,79, and the minimum and maximum values obtained were 2,15 and 3,68 respectively. After characterization and isolation and identification, the results showed the presence of 233 samples (93,2%) of sporulated bacteria, of these 180 were of Clostridium gender. The biochemical tests were identified in 42 samples, being 23 samples (9,2%) of Clostridium perfringens, 13 samples (5,2%) of Clostridium botulinum and 6 samples (2,4%) of C. chauvoei ...(Complete abstract, click electronic access below) / Doutor

Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in cattle, sheep and many other domestic and wild animals. It is considered the most important Clostridium producing economic losses in livestock. Typically, animals infected with blackleg die rapidly without any signs of illness. Animals quickly die within 12 to 48 hours after contracting the disease. Therefore, the control of this disease is done by commercial vaccines consisting of whole formolized cultures. Immunity against C. chauvoei is associated with whole cell, including its somatic and flagellar antigens while in other clostridial diseases, protective immunity is obtained by the use of vaccines containing toxoids. Moreover, it is essential to obtain new information about the somatic antigens of C. chauvoei. Proteomics is the study of the proteome, the protein complement of the genome. The proteome has been defined as the entire complement of proteins expressed by a cell, organism, or tissue type, and accordingly, proteomics is the study of this complement expressed at a given time or under certain environmental conditions. 2-DE with Immobilized pH Gradients (IPGs) combined with protein identification by Mass Spectrometry (MS) is currently the workhorse for proteomics. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify candidate immunogenic antigens for development of new vaccines. Analyses were performed by Western blot and dot blot techniques against the whole cell extract proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After mice immunization studies with experimental vaccines prepared, sera were obtained for evaluation of the immunoglobulin G antibody level by ELISA. After high level of antibody response determination, 1-DE, 2-DE and immunoblot studies were performed for the characterization of immunogenic proteins. In the study, a total of 460 protein spots could be detected on the 2-DE gels by the help of Delta2D image analysis software and 30 of them were reacted with polyclonal antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and 8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots revealed four different gene products (distinct ORFs). Ornithine decarboxylase, methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has been identified as an immunogenic protein for a pathogenic microbe and in C. chauvoei for the first time. Methionine adenosyltransferase and ornithine decarboxylation were identified as immunogenic for C. chauvoei for the first time. The last defined protein is the flagellin protein FliB(C) which is known to be major immunogenic protein of C. chauvoei.

This study was conducted to compare animal performance, presence of skin reactions and immune response following vaccination of beef calves via needle-free (NF) and needle-syringe (NS) vaccination techniques. Spring-born (Study A) and fall-born (Study B) calves were vaccinated against bovine viral diarrhea virus (BVDV) and Clostridium chauvoei (C. chauvoei) via NF and NS vaccination techniques. The parameters measured in this study included body weight (BW), skin reactions and serum antibodies. Animal performance and antibody levels against BVDV and C. chauvoei did not differ between vaccination techniques. However, NF vaccinated calves had a greater frequency of skin reactions when compared to NS vaccinated calves, except for day 42 of Study B. It can be concluded that a needle-free injection device (NFID) can be used effectively to stimulate an immune response without impacting animal performance, but may cause a greater frequency of skin reactions.

This study was conducted to compare animal performance, presence of skin reactions and immune response following vaccination of beef calves via needle-free (NF) and needle-syringe (NS) vaccination techniques. Spring-born (Study A) and fall-born (Study B) calves were vaccinated against bovine viral diarrhea virus (BVDV) and Clostridium chauvoei (C. chauvoei) via NF and NS vaccination techniques. The parameters measured in this study included body weight (BW), skin reactions and serum antibodies. Animal performance and antibody levels against BVDV and C. chauvoei did not differ between vaccination techniques. However, NF vaccinated calves had a greater frequency of skin reactions when compared to NS vaccinated calves, except for day 42 of Study B. It can be concluded that a needle-free injection device (NFID) can be used effectively to stimulate an immune response without impacting animal performance, but may cause a greater frequency of skin reactions.

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T18:48:02Z : No. of bitstreams: 1 araujo_rf_me_jabo.pdf: 248554 bytes, checksum: e7d4bd27c73efa391fb0fac3082dd622 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O carbúnculo sintomático é um problema sanitário mundial, responsável por elevados coeficientes de mortalidade em bovinos e ovinos. A imunização dos animais jovens, seguida de reforço anual até 2,5 anos de idade, é a principal medida profilática. Foram realizados três experimentos distintos com intuito de avaliar as respostas sorológicas de bovinos vacinados contra o carbúnculo sintomático, pelos testes de aglutinação em placa e Elisa, empregando-se como antígenos a cepa de referência (MT) e uma cepa de campo (SP). No primeiro experimento, os bezerros foram organizados em três grupos (G1, G2 e G3) e submetidos a três protocolos distintos de vacinação empregando-se uma vacina comercial polivalente contra clostridioses. O G1 foi primovacinado aos 4 meses de idade e recebeu o reforço na desmama (8 meses). O G2 recebeu a primeira dose na desmama e reforço 30 dias após. O G3 foi vacinado somente na desmama. As coletas de soro foram realizas aos 4, 8, 9 e 10 meses de idade dos bezerros. O G1 apresentou a melhor resposta sorológica em comparação aos outros dois protocolos. Quando a avaliação dos grupos foi realizada aos 10 meses de idade, independente do protocolo empregado, a resposta sorológica foi similar. No segundo experimento, foi avaliada a imunidade natural passiva de bezerros, filhos de vacas vacinadas até 30 dias antes do parto (2ª dose), empregando-se duas vacinas comercias polivalente contra clostridioses. As coletas de soro foram realizadas aos (±)7, 45 e 90 dias de idade dos bezerros. Independente das vacinas empregadas na imunização ativa das mães, a resposta sorológica passiva dos bezerros avaliados foi similar até os 3 meses de idade. Houve uma correlação linear da resposta sorológica passiva dos bezerros com a data de vacinação das mães e o dia do parto quando empregado o teste de Elisa. No terceiro experimento, as 30 vacas... / Black leg disease is one of the most important sanitary problem, responsible for high levels of mortality observed in bovines and ovines herds. The vaccination of young animals, followed by annual booter until 2,5 years-old, is the major preventive measure against outbreaks. Three distinct experiments were conducted to measure the vaccinal response from bovines. The vaccinal strains used were the reference MT and field Clostridium chauvoei isolated. Sera from vaccinated animals were tested by agglutination and Enzyme Linked Immunosorbent Assay (Elisa), both standardized for the present study. First experiment, calves were divided into three groups (G1, G2 and G3); and submitted to three vaccination schedule with a polyvalent vaccine. The G1 received first vaccine at 4 months of age and a subsequent booster after calving (8 month-old). The G2 received first vaccine dose after calving and booster at 30 days after. The G3 received only one vaccine dose at 8 months. The sera were colleted at 4, 8, 9 and 10 months for all groups studied. The G1 group showed the best serological response at 10 months of age in comparison to G2 e G3 and control. Moreover, at 10 months of age all groups presented similar levels of serological response. The second experiment, the natural immunity of calves, separated from their mothers vaccinated 30 days before calving with two polyvalent vaccines. The respective serum was colleted at (±) 7, 45 and 90 days of age. All calves presented similar serological response at 3 months of age, independent of vaccinal strain used. The third experiment, 30 heifers, Nelore race, aged above 4 years-old, without vaccination against black leg, were vaccinated with two Clostridium strains. When the SP strain was used the serological response was considered good in G3 (first experiment), second and third experiment for agglutination assay. To compare both techniques, agglutination... (Complete abstract click electronic access below)