Abstract: The Brucella type IV secretion system (T4SS) effector BtpA plays a significant role as an immunosuppressive molecule in Brucella. This study aims to gain a deeper understanding of the impact of BtpA on the biological properties of Brucella abortus strain A19 and to explore the potential effect of BtpA gene deletion on the immunogenicity of strain A19. The BtpA gene was deleted on a bovine Brucella vaccine strain (strain A19). qRT-PCR was performed to detect the BtpA mRNA expression level in the Brucella A19ΔBtpA deletion strain and to investigate the differences between Brucella A19 and A19ΔBtpA deletion strains with respect to growth curves, intracellular survival, adherence invasion and in vitro stress. In addition, mice were immunized with the A19 and A19ΔBtpA deletion strains, and Brucella-specific antibody levels were measured using iELISA at the 7th, 14th, 21st, 28th and 35th day post-immunization; the expression level of IFN-γ in mouse splenic lymphocytes at day 21 of immunization was detected by ELISpot. IFN-γ-positive CD4⁺ and CD8⁺ T lymphocytes and the classification of CD4⁺ and CD8⁺ T lymphocytes were determined by flow cytometry. The A19ΔBtpA deletion strain was successfully constructed, and the transcript level of BtpA in the A19ΔBtpA deletion strain was significantly lower than that of the A19 strain, and the A19ΔBtpA deletion strain and the parental strain showed a similar trend in terms of the growth curve, intracellular survival, and adhesion invasion. In contrast, the in vitro stress test showed that the surviving bacterial population of the A19ΔBtpA deletion strain was significantly lower than that of the A19 strain under hypertonic stress conditions (P<0.05). In the immunogenicity study, both A19 and A19ΔBtpA groups induced the production of Brucella-specific antibodies in mice very significantly compared with the PBS group (P<0.01); the IFN-γ expression level of lymphocytes in the A19ΔBtpA group was significantly higher compared with that of the A19 group (P<0.05), which resulted in the production of IFN-γ-positive CD4⁺, CD8⁺ T-lymphocytes and CD4⁺/CD8⁺ T-cell ratio was significantly higher (P<0.05). The deletion of the BtpA gene does not impact the in vitro intracellular proliferation of Brucella abortus A19. However, it may be associated with the A19 strain’s ability to withstand hypertonic environments. Furthermore, the deletion strain A19ΔBtpA has shown the ability to induce a more robust Th1-type immune response compared to the A19 strain. Additionally, it stimulates the host to produce Brucella-specific antibody IgG at levels similar to those induced by the A19 strain. These findings suggest that the A19ΔBtpA deletion strain has the potential to be a promising candidate for a Brucella gene deletion vaccine.

Key words: Brucella, BtpA, biological properties, immunogenicity