Protective efficacy by various doses of a new brucellosis vaccine candidate based on Salmonella strains expressing Brucella abortus BSCP31, Omp3b and superoxide dismutase against brucellosis in murine model

Abstract

Brucella species are important etiological agents of zoonotic diseases. Attenuated Salmonella strains expressing Brucella abortus BCSP31, Omp3b and superoxide dismutase proteins were tested as vaccine candidates in this study. In order to determine the optimal dose for intraperitoneal (IP) inoculation required to obtain effective protection against brucellosis, mice were immunized with various doses of a mixture of the three vaccine strains. Fifty BALB/c mice were divided into five equal groups (groups A-E). Group A mice were intraperitoneally inoculated with 100 μL of sterile phosphate-buffered saline. Group B, C, D and E mice were intraperitoneally immunized with approximately 1.2 × 105 colony-forming units (CFU) mL-1 of Salmonella containing pMMP65 in 100 μL and with 1.2 × 104 CFU mL-1, 1.2 × 105 CFU mL-1 and 1.2 × 106 CFU mL-1 of the mixture of the three strains in 100 μL, respectively. Serum IgG, tumor necrosis factor alpha and interferon gamma concentrations were significantly higher in group E than in groups A-D. Following challenge with B. abortus 544, the challenge strain was not detected in the spleen of any mouse from group E. Thus, IP immunization with 1.2 × 106 CFU mL-1 of the mixture of the three vaccine strains induced immune responses and provided effective protection against brucellosis in mice.

Keywords: Brucella abortus; attenuated Salmonella Typhimurium; brucellosis; immunization; public health.

Figure 1.

Antibody responses (ng mL⁻¹) against BCSP31, Omp3b and SOD antigens. Group A mice were inoculated with sterile phosphate-buffered saline as a control. Group B mice were immunized with Salmonella Typhimurium containing pMMP65 only as a vector control. Group C, D and E mice were inoculated with approximately 1.2 × 10⁴ CFU mL⁻¹, 1.2 × 10⁵ CFU mL⁻¹ and 1.2 × 10⁶ CFU mL⁻¹ of the mixture of the three delivery strains in 100 μL, respectively. Data represent the means of all mice in each group, and error bars represent the standard deviations. * P < 0.05 vs Group A; # P < 0.05 vs Group B; ¶ P < 0.05 vs Group C; § P < 0.05 vs Group D.

Figure 2.

Cytokine concentrations (pg mL⁻¹) in splenocytes at 3 WPI. Groups A, B, C, D and E are indicated as in Fig. 1. Data represent the means of all mice in each group, and error bars indicate the standard deviations. * P < 0.05 vs Group A; # P < 0.05 vs Group B; ¶ P < 0.05 vs Group C; § P < 0.05 vs Group D.

Figure 3.

Bacterial proliferation in the spleens of mice challenged with wild-type Brucella abortus strain 544. Groups A, B, C, D and E are indicated as in Fig. 1. All mice in each group were intraperitoneally challenged with 1 × 10⁴ CFU of virulent wild-type B. abortus 544 at 3 WPI. The numbers of viable bacteria recovered from the spleens of mice on day 14 post-challenge are shown. The lines represent the means (n = 5 mice per group). * P < 0.05 vs Group A; # P < 0.05 vs Group B; ¶ P < 0.05 vs Group C; § P < 0.05 vs Group D.