Carbúnculo sintomático: abordagem diagnóstica e imunológica

Abstract

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.