attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (Δ, Δ and Δ single mutants, and ΔΔ and ΔΔ double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51Δ, RB51Δ, RB51Δ, RB51ΔΔ and RB51ΔΔ mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. A single intraperitoneal immunization with RB51Δ, RB51Δ, RB51ΔΔ or RB51ΔΔ mutants was rapidly cleared from mice within 3 weeks, whereas the RB51Δ mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous strain 2308 or the heterologous strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.


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