Deletion of gshB virulence factor attenuates Brucella canis and confer protection against wild-type challenge = Deletion of gshB virulence factor attenuates Brucella canis and confer protection against wild-type challenge

다국어 초록 (Multilingual Abstract)

Introduction: Canine brucellosis, caused by Brucella canis, is worldwide bacterial disease that affects dogs has been shown to constitute a risk for humans. However, there has been little research conducted on the development of vaccine against canine brucellosis. Unfortunately, efforts to develop an effective vaccine against B. canis have been unsuccessful to date. Consequently, the task for development of potential vaccines for control and eradication of canine brucellosis is of great importance. In our previous investigations, we have identified gshB gene in B. canis using transposon mutagenesis that required for macrophage survival and virulence in mice. In an effort to develop safe and effective vaccine, we constructed gshB gene-deletion mutant in virulent B. canis strain 26 (BC26), and described the effect of these deletions on virulence and protective immunity in mice.
Methods: Gene-deletion (Δ) of gshB gene in BC26 was constructed and named as BC26Δgsh ::KM. To complement the BC26Δgsh ::KM mutant, the intact copy of gshB gene including the flanking DNA regions was cloned into the broad host range plasmid pBBR1-MCS4. The mutant was evaluated for virulence in RAW264.7 and HeLa cells and BALB/c mice, and for protective immunity against BC26 challenge. Immune response were evaluated using several parameters IgG, IgG1, IgG2a, IFN-γ and IL-10.
Results: Deletion of gshB gene leads to significant attenuation of B. canis virulence either in vitro and in vivo. The BC26Δ gsh ::KM mutant was found to be more sensitive to oxidative stress, and virtually incapable of intracellular replication in both RAW264.7 and the HeLa cells. The virulence of mutant at either dose 10⁶ or 10⁸ CFU was highly attenuated in BALB/c mice. Mice inoculated parental BC26 strain induced splenomegaly as a consequence of inflammatory responses but not in mice inoculated with mutant. The complementation of BC26Δgsh ::KM mutant restored their ability to survive in vitro and in vivo to a level comparable with that of wild type. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of this mutant to confer protective immunity in mice against challenge with virulent BC26. Vaccination of mice with mutant induced strong Th1-type biased immune response, and provided significantly better protection against challenge with virulent BC26 than with either inactivated B. canis vaccine or the strain RB51 vaccine.
Conclusion: This finding indicates that the gshB gene plays important role in the virulence of B. canis . These results suggest that BC26Δgsh ::KM mutant could be ideal live attenuated vaccine candidates and deserves further evaluations in animals for vaccine development.