Effort has been made to identify protective antigens in order to develop a recombinant vaccine against leptospirosis. Several attempts failed to conclusively demonstrate efficacy of vaccine candidates due to the lack of an appropriate model of lethal leptospirosis. The purposes of our study were: (i) to test the virulence of leptospiral isolates from Brazil, which are representative of important serogroups that cause disease in humans and animals; and (ii) to standardize the lethal dose 50% (LD 50 ) for each of the virulent strains using a hamster (Mesocricetus auratus) model. Five of seven Brazilian isolates induced lethality in a hamster model, with inocula lower than 200 leptospires. Histopathological examination of infected animals showed typical lesions found in both natural and experimental leptospirosis. Results described here demonstrated the potential use of Brazilian isolates as highly virulent strains in challenge experiments using hamster as an appropriate animal model for leptospirosis. Furthermore these strains may be useful in heterologous challenge studies which aim to evaluate cross-protective responses induced by subunit vaccine candidates.

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ABSTRACTLeptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species ofLeptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of theEscherichia coliheat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) ofLeptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P< 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

Bovine tuberculosis is a major infectious disease of the cattle. In this study, 85 M. bovis isolates from 162 lymph nodes, obtained from a herd of cattle on a farm in southern Brazil, were evaluated using spoligotyping and VNTR. The strains were grouped into five clusters and five orphans, showing a heterogenic genetic profile, what could represent diverse geographic origins of the introduced cows and/or the frequent movement of cattle between different properties.

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Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

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