A <scp>B</scp> tp <scp>B</scp> ‐Deleted <i>Brucella</i> Vaccine Strain Provides Dual Advantages: Enhanced Protection and Diagnostic Differentiation Capability in Mice

The Brucella abortus A19 attenuated live vaccine poses potential infection risks during practical applications and interferes with serological diagnostics, thereby affecting quarantine measures and the establishment of disease-free zones. Consequently, this study aimed to reduce its potential virulence, enhance its protective efficacy and differentiate it from wild-type strains by knocking out the immunosuppressive virulence gene btpB in the A19 strain. Using homologous recombination, we successfully obtained the A19ΔBtpB deletion strain. In a mouse model, the A19ΔBtpB strain demonstrated improved safety and significantly increased TLR2 and TLR4 expression levels in splenic lymphocytes, suggesting attenuated immune suppression. The A19ΔBtpB strain induced Brucella-specific IgG antibodies comparable to those of the A19 strain but exhibited superior performance in promoting cellular immunity. It effectively induced Th1-type cytokine (IL-6 and TNF-α) production, reduced Th2-type cytokine (IL-4 and IL-10) expression and promoted IFN-γ expression in T lymphocytes. Notably, the A19ΔBtpB deletion strain provided better protection against the virulent M28 strain in mice than did the A19 strain. In addition, an indirect ELISA diagnostic method based on BtpB protein has been developed, effectively distinguishing vaccine and wild-type strains in the infection of Brucella. The A19ΔBtpB strain thus represents a promising candidate for a Brucella gene-deletion vaccine, offering both theoretical and empirical support for future vaccine development.