| Summary: | Background: <i>Brucella abortus</i> is the main causative agent for bovine brucellosis. <i>B. abortus</i> A19 is a widely used vaccine strain to protect cows from <i>Brucella</i> infection in China. However, A19 has a similar lipopolysaccharide (LPS) antigen to that of the field virulent <i>Brucella</i> strain, whose immunization interferes with the serodiagnosis of vaccinated and infected animals. [Aim] To develop a novel <i>Brucella</i> DIVA vaccine candidate. Study design and methods: The <i>B. abortus</i> mutant A19mut2 with the formyltransferase gene <i>wbkC</i> is replaced by an acetyltransferase gene <i>wbdR</i> from <i>E. coli</i> O157 using the bacterial homologous recombination technique, generating a modified O-polysaccharide that cannot induce antibodies in mice against wild-type <i>Brucella</i> LPS. The biological phenotypes of the A19mut2 were assessed using a growth curve analysis, agglutination tests, Western blotting, and stress resistance assays. Histopathological changes and bacterial colonization in the spleens of vaccinated mice were investigated to assess the residual virulence and protection of the A19mut2. Humoral and cellular immunity was evaluated by measuring the levels of IgG, IgG subtypes, and the release of cytokines IFN-γ and IL10 in the splenocytes of the vaccinated mice. ELISA coated with wild-type LPS can distinguish mouse antibodies induced by A19 and A19mut2 immunization. Results: The A19mut2 showed a decreased residual virulence in mice, compared to the A19 strain, but induced significant humoral and cellular immune responses, as the A19 immunization did. The protection efficacy of A19mut2 immunization against <i>B. abortus</i> S2308 Nal<sup>R</sup> infection was similar to that of A19 immunization. Conclusion: The A19mut2 has potential as a novel DIVA vaccine candidate in the future.
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