Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus

We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 108 CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis.