Resumo

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis and a serious economic problem and public health in Latin America. Vaccine-based control strategies bTB are particularly important in high-prevalence zones where it is economically unfeasible to slaughter animals or eliminate wildlife hosts. Although previous studies in cattle have demonstrated that the protection induced by BCG is not complete, this human attenuated vaccine is a powerful system for heterologous antigens presentation. The 85B antigen (Ag85B) is a M. bovis protective protein and demonstrated improved protection against tuberculosis in animal models. In order to improve the protection efficacy of BCG, we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of a leucine auxotrophic strain, and complementation with leuD. Protective efficacy of candidate vaccine was determined using a current bovine model of challenge. Material and methods: Ag85B encoding gene was amplified by PCR and subsequently cloned into pUP410 vector under control of endogenous promoter. The recombinant vector was introduced into BCG ΔleuD by electroporation and protein expression was demonstrated by Western blot. Three groups of five or six Holstein-Fresian calves were inoculated with 106 CFU of either ∆leuD BCG-85B Pasteur, or BCG Pasteur, or PBS. Eight weeks after vaccination, animals were infected with M. bovis by intratracheal instillation of 106 CFU. Sixteen weeks after infection, the calves were euthanized, head and pulmonary lymph nodes and lungs were analysed looking for granuloma formations. Tissues were fixed in 10% formal saline and processed for histological examination. In order to determinate the candidate vaccine efficacy, macroscopic and histologic pathology was scored and the statistical analysis was performed using Mann-Whitney test. Results and discussion: The recombinant vector pUP410::fbpB was confirmed by PCR and restriction analysis, and Ag85B was readily detectable in rBCG ΔleuD lysates. The ∆leuD BCG-85B strain generated responses resulting in decreased lesion severity on gross and histologic post-mortem examination, significantly lower than in the others groups. Conclusions: Vaccination with rBCG protected cattle against bTB better than BCG Pasteur. Immunological parameters will be studied to reveal how this protection is induced.