Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg

Pesquisa Veterinária Brasileira

Araujo, Rafael F.¹  Dutra, Iveraldo S.³  Ferreira, Rosa M.M.³  Curci, Vera C.L.M.⁴  Nobrega, Fabiana L.C.² 

[1] Universidade Estadual Paulista, Jaboticabal, Brazil;Instituto Butantan, São Paulo, Brazil;Unesp, Araçatuba;Unidade de Pesquisa e Desenvolvimento de Araçatuba, Araçatuba, Brazil

关键词: Clostridiosis;    Clostridium chauvoei;    vaccine;    antibody;    beef cattle.;   

DOI  :  10.1590/S0100-736X2010000700008

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

The serological response of beef calves was evaluated with different vaccination regimens against blackleg, using an official strain (MT) and a field-collected strain of Clostridium chauvoei as antigens. Sixty calves were randomly allocated to four different groups and were submitted to distinct vaccination protocols with a commercial polyvalent vaccine. Group G1 was first vaccinated at four months of age and a booster shot was given after weaning, at eight months. Group G2 was given the first dose at eight months and a booster shot 30 days later. Group G3 was vaccinated only once at eight months and the control group was not vaccinated. These alternative vaccination regimens were proposed in an effort to adequately protect cattle under open-field farming conditions. Serological evaluations were made by Elisa at 4, 8, 9 and 10 months of age. Both groups receiving booster shots had a significantly increased serological response 30 days later. However, the serum IgG levels against C. chauvoei were significantly higher in the calves that were first vaccinated at four months. At 10 months, the two booster shot groups (G1 and G2) had similar serological responses, while the calves that were treated with a single dose of vaccine at weaning (G3) had a response that was similar to that of the control group. The serological response of the calves was significantly inferior at several of the evaluation times when the field strain of the bacteria was used as a challenge antigen instead of the official MT strain. The serological response of calves that are vaccinated twice was found to be satisfactory, independent of the first injection being made at four or eight months of age. It was also concluded that it would be useful to include local bacterial strains in commercial vaccine production. Index terms: Clostridiosis, Clostridium chauvoei, vaccine, antibody, beef cattle. RESUMOFoi avaliada a resposta sorológica de bezerros de corte submetidos a diferentes esquemas de vacinação contra o carbúnculo sintomático, empregando-se como antígenos duas cepas distintas de Clostridium chauvoei: uma oficial (MT) e a outra uma cepa de campo. Os animais (n=60) foram randomizados em quatro grupos (G1, G2, G3 e Controle) e submetidos a três protocolos distintos de vacinação com um produto comercial polivalente. O G1 foi primovacinado aos 4 meses de idade e recebeu o reforço após desmama (8 meses de idade). O G2 recebeu a primeira dose aos 8 meses de idade e reforço 30 dias após. O G3 foi vacinado somente aos 8 meses de idade e o Controle não foi vacinado. As avaliações sorológicas pelo ensaio imunoenzimático (Elisa) foram realizadas aos 4, 8, 9 e 10 meses de idade dos bezerros. Nos dois esquemas em que os animais receberam o booster (G1 e G2), houve um aumento significativo (p<0,05) da resposta sorológica quando foram avaliados 30 dias após. No entanto, os valores séricos de IgG contra C. chauvoei foram significativamente maiores nos animais primovacinados aos 4 meses de idade. Aos 10 meses, os dois grupos que receberam o reforço vacinal (G1 e G2) não diferiram entre si na resposta sorológica e os bezerros que receberam uma única dose de vacina na desmama (G3) não diferiu do Controle. A resposta sorológica dos bezerros foi significativamente inferior (p<0,05) em diversos momentos da avaliação, quando a cepa de campo foi empregada como antígeno e quando comparada à da cepa MT. Pode-se deduzir que a resposta sorológica dos bezerros vacinados aos 4 e 8 meses de idade foi satisfatória e que existiram diferenças significativas nos valores séricos de anticorpos contra C. chauvoei quando na avaliação foi empregada cepa de campo.Termos de indexação: Clostridiose, Clostridium chauvoei, vacina, anticorpos, bovinos de corte.     INTRODUCTIONBlackleg is a necrotizing myositis that is caused by the activation of latent spores of Clostridium chauvoei in the muscles of bovines. This clostridiosis is a wide-spread sanitary problem that has the potential to cause serious economic losses. This disease mainly affects 6 to 30-month old bovines, when there apparently is a reduction in antibody levels, making the animals susceptible to this disease (Kriek & Odendaal 2004).Immunization against blackleg is one of the most important prophylactic measures for cattle rearing throughout the world and has been used since the 19th century (Blobel & Schliesser 1980). Though vaccination is done voluntarily, cattle producers recognize the importance of this preventative measure. In Brazil, according to current data provided by the National Syndicate of the Production of Animal Products (Sindan), about 150 million doses of vaccine are sold per year; these include monovalent and polyvalent formulations, all containing C. chauvoei.Since the 1980s, the Ministry of Agriculture, Livestock and Food Supply (MAPA) has evaluated commercial lots of vaccine against blackleg, determining their efficiency in tests made with guinea pigs challenged with a lineage of C. chauvoei denominated MT (Brasil 1997). During this period, substantial changes have been made in the formulation of commercial vaccines against blackleg, evolving from monovalent imunogens produced in the country, to complex polyvalent formulations, in order to protect against various clostridioses and multiple species, with importation of part of the vaccine material. The prescription for the use of these commercial bacterins and polyvalent toxoids in this country indicates a need for a booster shot 30 days after the first injection (Sindan 2010). However, operational difficulties in the open-field beef cattle rearing system used in Brazil makes it difficult to apply the recommended booster shots. Consequently, it is common practice in the southeastern and central west regions of the country to apply a single vaccination dose at weaning (eight months of age).There is very little information available concerning the serological response of cattle against C. chauvoei stimulated by vaccination; that and the increasing production and use of complex polyvalent vaccines, the need to objectively understand the normal vaccine protocols and the economic importance of blackleg, prompted us to evaluate the serological response of beef calves submitted to different vaccination regimens against blackleg. A commercial polyvalent vaccine was used, with two different bacterial strains as antigens.  MATERIALS AND METHODSVaccine. The calves were vaccinated with an imported polyvalent vaccine that is approved by the Ministry of Agriculture, Livestock and Food Supply (MAPA). This vaccine against blackleg contained, along with Clostridium chauvoei, bacterins and toxoids of other clostridial bacteria (aluminum hydroxide adjuvant); it was stored in a refrigerator and applied according to the manufacturer's instructions in aliquots of 5ml, or according to the research protocol.Vaccination and serological evaluation schemes. Serological evaluations were made of 60 mixed breed calves (Nelore, Aberdeen Angus, Brahman and Red Angus), born during the first 20 days of June 2007, the mothers of which were multiparous Nelore cattle that had been submitted to the normal sanitary management of the farm, which consisted of two annual vaccinations against blackleg (May and November). The calves that were to be immunized were randomly divided into three groups (n = 15 each) and were submitted to one of three different vaccination regimens. Group 1 (G1) was vaccinated at four months and a booster shot was given at weaning (8 months). Group 2 (G2) was first vaccinated at weaning and received the booster shot 30 days later. Group 3 was vaccinated with a single dose of the vaccine at weaning. The control group (n=15) was not vaccinated. The blood samples were collected from the external jugular vein with Vacutainer® tubes, which were maintained at room temperature until complete retraction of the coagulum. They were then centrifuged to obtain the serum, which was maintained in a freezer (-20ºC) until the serological tests. The serum collections were made at 4, 8, 9 and 10 months of age.Clostridium chauvoei strains. The serological evaluation was made with antigens that consisted of two different strains of C. chauvoei: the reference strain, denominated MT and a field isolate that came from a disease epidemic associated with vaccine failure (Santos 2003). The reference strain (MT) was supplied by MAPA. The natural epidemic isolate was identified by PCR and maintained in the bacterial library of the Infectious Diseases Laboratory of the Department of Animal Production, Health, and Husbandry, Araçatuba Campus of Unesp.Immunoenzymatic test (Elisa). The positive serum used to standardize the serological test was obtained from two 18-month-old bovines that had been immunized with three doses of commercial polyvalent vaccine against clostridioses, with a 15-day interval between vaccinations. The negative serum sample used in the Elisa test was obtained from a new-born calf, before it had fed on colostrum. The antigens from the reference strain (MT) and from the field-collected strain were standardized as recommended by Crichton et al. (1990).The 96-hole microplates (Nunc®) were sensitized with 100ml/well of antigen diluted in carbonate-bicarbonate buffer, diluted 1:50, and incubated in a humid chamber for 12 hours at 4ºC. Soon after this step, the plates were submitted to three rinses with 0.05% PBS Tween (PBS-T) in an automatic pipette washer. The reaction was then blocked with 200ml/well of reconstituted 10% powdered milk (Molico®) diluted in the carbonate-bicarbonate buffer. The plates were then incubated in a humid chamber at 37ºC for 45 minutes, and submitted to the rinse cycle. Then 100ml of the test and control sera diluted 1:100 in PBS + 10% powdered milk. Two wells were left without sera; only the buffer was added. This was the plate control (blank). After this step, the plates were incubated at 37ºC for 60 minutes, and they were then run through the rinse cycle. Then, 100ml of commercial immunoenzyme conjugate SIGMA®) was added to each well (this consisted of anti-bovine IgG rabbit serum conjugated with peroxidase, diluted 1:10,000 in PBS + 10% powdered milk); the plates were incubated at 37ºC for 90 minutes and were again rinsed three times. Immediately afterward, 50ml of ortho-phenylenediamine substrate, diluted in citrate-phosphate buffer, was added. The reaction was interrupted after 15 minutes with 2 M HCl (50 ml/well), and the readings made with a microplate spectrophotometer (Labsystem - Multiskan EX®), with a 492nm filter.Statistical analysis. The Elisa data was transformed by log (x+1) and submitted to analysis of variance with repeated measures and residual analysis to check for normality and homogeneity of the variance, which are prerequisites for analysis of variance. The means were compared with the Tu

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