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.

The aim of the present study is to estimate the protective efficacy of Brucella abortus lysed cells by GI24 against brucellosis in Beagles. Group A was subcutaneously (sc) immunized with sterile phosphate-buffered saline, and group B was sc immunized with approximately 3 × 109 of the lysed cells. Brucella-LPS-specific serum IgG titers and IL-4, TNF-α and IFN-γ concentrations were investigated by enzyme linked immunosorbent assay. All dogs were intraconjunctivally challenged with B. abortus strain 544 at 6 weeks post-prime immunization. The serum IgG titers were considerably higher in group B than in group A. The levels of IL-4, TNF-α and IFN-γ in group B than in group A were significantly higher. Following challenge, no challenge strain was observed from all tissues of three dogs of group B. However, challenge strain was detected from spleen, uterus (except one Beagle) and inguinal and retropharyngeal lymph nodes of all group A Beagles. The results of this study demonstrated that sc immunization with the lysed cells induced robust antibody and cell-mediated immune responses in Beagles. The lysed cells also conferred protection against infection with B. abortus. These results suggest that sc immunization with B. abortus lysed cells by GI24 is a good vaccine candidate against brucellosis in dogs.

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.

Background Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. Objectives The study aimed to determine the protective efficacy of attenuated Salmonella strain expressing highly conserved Brucella immunogens in goats. Methods Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 10 9 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. Results Among these vaccines inoculated at 5 × 10 9 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Real-time polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the non-vaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. Conclusions These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 108 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 108 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 109 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B–D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus.

The aim of this study was to establish a proof-of-concept of protective efficacy of Salmonella-based B. abortus vaccine candidate in Beagles. Group A Beagles (n=10) were subcutaneously (SC) inoculated with S. Typhimurium delivery strain containing pMMP65 (vector to deliver antigens) only as vector control. Group B Beagles (n=10) were SC vaccinated with the mixture of the three Salmonella delivery strains expressing the recombinant B. abortus BCSP31, Outer membrane protein 3b (Omp3b), and superoxide dismutase (SOD) proteins, respectively. No Salmonella delivery strains were isolated from all tissues tested. Serum IgG, interleukin-4, tumor necrosis factor-alpha, and interferon-gamma concentrations were significantly higher in group B than in group A. Following intraconjunctival challenge with B. abortus 544, among 5 group B Beagles, the challenge strain was isolated from mandibular, and retropharyngeal lymph nodes of three Beagles, and no isolates were observed from all tissues of two Beagle. However, the challenge strains were detected from spleen, uterus (except two Beagles), and mandibular, prescapular, retropharyngeal, and superficial inguinal lymph nodes of all group A Beagles. These results suggest that the mixture of three S. Typhimurium delivery strains be a good vaccine candidate against brucellosis by B. abortus in dogs. Further investigations are needed to improve the protective efficacy of the Salmonella-based B. abortus vaccine candidate and explore its practical application in dogs.