Vaccination of goats with a combination Salmonella vector expressing four Brucella antigens (BLS, PrpA, Omp19, and SOD) confers protection against Brucella abortus infection

Abstract

Salmonella is an intracellular pathogen with a cellular infection mechanism similar to that of Brucella, making it a suitable choice for use in an anti-Brucella immune boost system. This study explores the efficacy of a Salmonella Typhimurium delivery-based combination vaccine for four heterologous Brucella antigens (Brucella lumazine synthase, proline racemase subunit A, outer-membrane protein 19, and Cu/Zn superoxide dismutase) targeting brucellosis in goats. We inoculated the attenuated Salmonella delivery-based vaccine combination subcutaneously at two different inoculation levels; 5 × 10⁹ colony-forming unit (CFU)/mL (Group B) and 5 × 10¹⁰ CFU/mL (Group C) and challenged the inoculations with virulent Brucella abortus at 6 weeks post-immunization. Serum immunoglobulin G titers against individual antigens in Salmonella immunized goats (Group C) were significantly higher than those of the non-immunized goats (Group A) at 3 and 6 weeks after vaccination. Upon antigenic stimulation, interferon-γ from peripheral blood mononuclear cells was significantly elevated in Groups B and C compared to that in Group A. The immunized goats had a significantly higher level of protection as demonstrated by the low bacterial loads in most tissues from the goats challenged with B. abortus. Relative real-time polymerase chain reaction results revealed that the expression of Brucella antigens was lower in spleen, kidney, and lung of immunized goats than of non-immunized animals. Also, treatment with our combination vaccine ameliorated histopathological lesions induced by the Brucella infection. Overall, the Salmonella Typhimurium delivery-based combination vaccine was effective in delivering immunogenic Brucella proteins, making it potentially useful in protecting livestock from brucellosis.

Keywords: Brucella vaccine; Salmonella vector; brucellosis; goats; protective efficacy.

Fig. 1. Humoral immune responses. The systemic humoral responses elicited by the four vaccine strains were investigated by measuring the relative levels of plasma immunoglobulin G (IgG) from samples obtained from non-immunized negative control (NC) group, Group A (vector control containing pJHL65), Group B (Salmonella delivery vaccine inoculated at 5 × 10⁹ CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10¹⁰ CFU/mL). Serum IgG titers against Brucella lumazine synthase (BLS), Brucella superoxide dismutase (SOD), Brucella outer-membrane protein 19 (Omp19), and Brucella proline racemase subunit A (PrpA) in Group C goats were significantly increased compared to those of the non-immunized NC and vector control groups at 3 and 6 weeks post-immunization. Antibody levels are expressed as mean optical density at 492 nm (OD₄₉₂) ± SE values. Bars labeled with a different letter on the graph are significantly different from each other (p < 0.05) based on results of one-way ANOVA with Duncan's multiple range test.

Fig. 2. Bar graphs of the enzyme-linked immunosorbent assay analysis results for the levels of goat interferon (IFN)-γ in peripheral blood mononuclear cells re-stimulated with each antigen at 6 weeks after immunization. The levels of IFN-γ (pg/mL) from the non-immunized negative control (NC) group, Group A (vector control containing pJHL65), Group B (Salmonella delivery vaccine inoculated at 5 × 10⁹ CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10¹⁰ CFU/mL) were significantly elevated in Group C, and Group B levels were higher than those of Group A and the NC group. Optical density of each well was measured by using a microplate reader set to 450 nm. Values are presented as mean ± SE. Bars labeled by different letters on the graph are significantly different from each other (p < 0.05) based on one-way ANOVA and Duncan's multiple range test. BLS, Brucella lumazine synthase; Omp19, outer-membrane protein 19; PrpA, proline racemase subunit A; SOD, superoxide dismutase.

Fig. 3. Protective efficacy of the Salmonella delivery vaccine. Group A (vector control containing pJHL65), Group B (Salmonella delivery vaccine inoculated at 5 × 10⁹ CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10¹⁰ CFU/mL) goats were challenged with virulent Brucella abortus strain 544 at 6 weeks after immunization and were euthanized at 8 weeks post-challenge. The challenge-bacteria load recovered from various organs and lymph nodes (LN) reflects the potency of immunization. The number of CFUs was counted to assess bacterial proliferation in tissues. Bars labeled by different letters on the graph are significantly different from each other (p < 0.05) based on one-way ANOVA and Duncan's multiple range test.

Fig. 4. Relative expression of Brucella abortus antigens in various tissues of goats. The expression levels of Brucella antigens in the non-immunized negative control (NC) group, Group B (Salmonella delivery vaccine inoculated at 5 × 10⁹ CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10¹⁰ CFU/mL) were lower than that in Group A (vector control containing pJHL65) in the spleen, liver, kidney, and lung. No significant differences were noted between Groups B, C, and NC. Values are presented as mean ± SE. Bars labeled by different letters on the graph are significantly different from each other (p < 0.05) based on one-way ANOVA and Duncan's multiple range test.

Fig. 5. Microphotograph of liver and spleen sections of from the non-immunized negative control (NC) group, Group A (vector control group), Group B (Salmonella delivery vaccine inoculated at 5 × 10⁹ CFU/mL; CFU, colony-forming unit), and Group C (Salmonella delivery vaccine inoculated at 5 × 10¹⁰ CFU/mL) at 8 weeks post-challenge. (A) The liver sections revealed aggregation of inflammatory cells (arrow) that consisted predominantly of lymphocytes (microgranulomas). Spleen sections revealed distinctly visible trabeculae (arrowhead), suggesting a response to septicemia in the vector control group (Group A). H&E stain. Scale bars = 50 µm. (B) Comparison of microgranulomas based on counting the number of the foci in 10 microscopic fields per tissue sample showed that the microgranulomas per 100× fields in the vector control group was significantly higher than that in the immunized groups. Bars labeled by a different letter on the graph are significantly different from each other (p < 0.05) based on one-way ANOVA and Duncan's multiple range test.