Protective efficacy of a canine brucellosis vaccine candidate based on live attenuated Salmonella expressing recombinant Brucella BCSP31, Omp3b and SOD proteins in Beagles.

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.

Canine brucellosis is usually caused by Brucella canis (B. canis). In some countries, such as U.S.A. and Canada, brucellosis in dogs, kept in close association with cattle, can also be caused by B. abortus [6, 7, 10, 15, 29]. The most likely means of cattle to dog transmission may be by contact with aborted fetus or placental membranes infected with B. abortus [5, 15, 29]. In addition, dog to cattle transmission of B. abortus has been reported in experiments, using B. abortus-infected dogs imprisoned with Brucella free cattle and under field conditions [15, 29].

Cell-mediated immune (CMI) response is crucial to eradicate Brucella strains from the host organisms [33], because Brucella strains invade and multiply intracellularly in macrophages within the host immune system [4, 9]. In particular, the Th1-type immune cells mediated by interferon gamma (IFN-γ) is crucial to defend Brucella infection [39]. Many various types of vaccines have been developed to prevent brucellosis by B. abortus, but live, attenuated Brucella strains such as B. abortus strain 19 and strain RB51 have been used as commercially available vaccines in field. However, these strains can revert into pathogenic strains and some strains may interfere with the diagnosis [2, 28]. Therefore, there is necessary to develop better and safer vaccine.

Live, attenuated Salmonella strains have been known to be a useful delivery system for carrying protective antigens of other pathogens [16, 17]. A protein, BCSP31, purified from B. abortus strain 19 designated has been reported to serve as a protective subunit vaccine [34, 35]. Outer membrane protein 3b (Omp3b) has been known to be one of the most abundant and immunogenic Brucella proteins [14, 38]. Cu/Zn superoxide dismutase (SOD) has been also reported as one of virulence factors scavenging harmful oxygen radicals mediated during phagocytosis by macrophages within host immune system [36].

In a previous study [21], S. Typhimurium strains expressing the recombinant B. abortus BCSP31, Omp3b, and SOD proteins were evaluated a vaccine candidate in a murine model. Intraperitoneal immunization with the mixture of the S. Typhimurium-based B. abortus vaccine strains induced adequate humoral and CMI responses and effectively protected against B. abortus infection in mice. Although the most important agent for canine brucellosis is B. canis, B. abortus is known to infect dogs. Especially, the protective efficacy of the S. Typhimurium-based strains against B. abortus infections were mainly evaluated as B. abortus vaccine candidate [21, 24, 35, 36]. At the first time, the present study aimed to establish a proof-of-concept of the protective efficacy of S. Typhimurium delivery strains expressing recombinant B. abortus BCSP31, Omp3b, and SOD proteins as a B. abortus vaccine candidate in Beagles. Beagles were subcutaneously immunized with the mixture of the live, attenuated S. Typhimurium strains expressing recombinant B. abortus BCSP31, Omp3b, and SOD proteins. The vaccinated Beagles exhibited robust humoral and CMI responses, thereby vaccine Beagles were effectively protected from virulent B. abortus infection.

MATERIALS AND METHODS

Animals, and ethics statement

A total of 20 female Beagles aged 6 months were distributed into two equal groups. All Beagles were purchased from tested brucellosis-free flocks to use in this study. All Beagles used for this study were again confirmed seronegative for brucellosis in the laboratory by Rose Bengal test (RBT) for B. abortus, and 2-mercaptoenthanol rapid slide agglutination test (2ME-RSAT) for B. canis. All animal experiments performed in this study received ethical approval (CBU 2015-052) from the Chonbuk National University Animal Ethics Committee in accordance with the guidelines of the Korean council on Animal Care.

Bacterial strains, and growth conditions

Live, attenuated S. Typhimurium strains expressing the recombinant B. abortus BCSP31, Omp3b, and SOD proteins, which have been constructed and used as a vaccine candidate for mouse brucellosis in a previous study [21], were evaluated as a vaccine candidate for canine brucellosis in the present study. In addition, attenuated S. Typhimurium delivery strain containing pMMP65 vector only, was used as vector control strain [21]. B. abortus strain 544 was used as the virulent challenge strain [23]. The recombinant BCSP31, Omp3b, and SOD proteins were overexpressed and purified from HJL906, HJL904, and HJL908 strains, respectively [21]. Except the virulent challenge strain, all strains were grown in Luria–Bertani broth (LB; Becton, Dickinson and Co., Sparks, MD, U.S.A.) or on LB agar. Virulent challenge strain was grown on Brucella broth or Brucella agar. All strains were cultured at 37°C.

Preparation of individual recombinant proteins

The recombinant BCSP31, Omp3b, and SOD proteins purified from HJL906, HJL904, and HJL908, respectively [21], and each recombinant protein was used as coating antigens of ELISA to evaluate serum IgG titers and as re-stimulating protein for peripheral blood mononuclear cells (PBMCs) in the present study.

Preparation of S. Typhimurium-based B. abortus vaccine candidate

Live, attenuated S. Typhimurium strains expressing the recombinant B. abortus BCSP31, Omp3b, and SOD proteins of B. abortus were prepared as the method described in previous study [19]. The mixture of three S. Typhimurium strains was as S. Typhimurium-based B. abortus vaccine candidate in Beagles.

Immunization of Beagles and sample collection

Twenty 6 months of age female Beagles were divided into two equal groups (n=10 Beagles per group). All Beagles were subcutaneously (SC) primed at 6 months of age [0 weeks post prime immunization (WPPI)], and were SC boosted at 3 WPPI. Group A Beagles were inoculated with approximately 3.0 × 109 colony-forming units (CFU)/ml of S. Typhimurium delivery strain containing pMMP65 only in 1 ml as the vector control. Group B Beagles were immunized with approximately 3.0 × 109 CFU/ml of the mixture (equal CFU counts of each strain) of three S. Typhimurium-based B. abortus vaccine strains in 1.0 ml. Blood samples were collected at 0, 3 (before booster vaccination), and 6 (before challenge) WPPI to evaluate the serum IgG titers. All samples were stored at −70°C until use.

Assessment of vaccine safety

The safety of the live and attenuated Salmonella-based B. abortus vaccine candidate in Beagles was assessed. Daily clinical thermometry observation of the Beagles in the immunized group was performed during 6 WPPI. At 6 WPPI, 5 Beagles per group were euthanized aseptically, from which samples of the lymph nodes (mandibular, prescapular, retropharyngeal, and superficial inguinal), and parenchymal organs (spleen, kidney, and uterus) were collected. In total, 7 organs were sampled from each animal. The tissue homogenates were plated onto Brilliant Green Agar (Becton, Dickinson and Co.) and incubated under aerobic conditions for 48 hr at 37°C. Isolation of Salmonella delivery strains was confirmed by PCR using OMPC- and TYPH-specific primer set described in a previous study [18], and BCSP31-, Omp3b-, and SOD-specific primers described in a previous study [20, 21].

Immune response measurement by ELISA

Standard ELISA was performed to evaluate the immune responses against the recombinant BCSP31, Omp3b, and SOD proteins in serum samples obtained from Beagles according to slightly modified method described previously [21]. Briefly, Ninety-six well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with pretitrated recombinant BCSP31 (4 µg/ml), Omp3b (2 µg/ml), or SOD (4 µg/ml) proteins in phosphate-buffered saline (PBS), blocked for 1 hr using PBS containing 1% bovine serum albumin (diluent; 200 µl/well), and washed with PBS containing 0.05% Tween-20. Serum samples were diluted as 1:100 in diluent. The plates were treated with horseradish peroxidase-conjugated goat anti-dog IgG antibody (Bethyl Lab Inc., Montgomery, TX, U.S.A.). Enzymatic reactions were carried out by adding substrate containing o-phenylenediamine (Sigma-Aldrich, St. Louis, MO, U.S.A.) and were measured through an automated ELISA spectrophotometer (Thermo Scientific Multiskan GO, Thermo Fisher Scientific Oy, Ratastie, Vantaa, Finland) at 492 nm. The ELISA results are expressed as mean ± standard deviation.

Preparation of Peripheral blood mononuclear cells (PBMCs)

At 6 WPPI, blood was obtained from all Beagles and placed into an acid-citrate dextrose solution. PBMCs were enriched by density centrifugation using a Ficoll sodium diatrizoate gradient (Sigma Diagnostics, Inc., St. Louis, MO, U.S.A.). PBMCs were diluted in RPMI 1640 medium to 2.5 × 106 viable cells per ml as determined by trypan blue dye exclusion. The PBMCs (5 × 105 cells/well) were stimulated in vitro as previously described [21, 37] with media as unstimulated control, Concanavalin A (0.5 µg/well) as positive control, or with each recombinant BCSP31, Omp3b, and SODC protein (8 µg/well), and incubated at 37°C under 5% CO2. Culture supernatants were collected after 72 hr of re-stimulation and stored at −70°C till used for cytokine measurement.

Cytokines measurement by ELISA

ELISA was used to measure the concentration of IL-4, TNF-α, and IFN-γ in the culture supernatants using DuoSet canine cytokine reagent set according to the manufacturer’s instructions (R & D system, Minneapolis, MN, U.S.A.). Results of the ELISA are expressed as the mean concentration ± standard deviation.

Challenge experiments

For challenge experiments, the challenge strain, strain 544, was prepared. Briefly, the strain was grown in Brucella broth at 37°C for 24 hr and resuspended to approximately 2 × 108 CFU/ml. At 6 WPPI, the Beagles were fasted for 16 hr prior to being anesthetized with carfentanil (Wildnil; 0.007 to 0.008 mg/kg of body weight; Wildlife Pharmaceuticals, Ft. Collins, CO, U.S.A.) and xylazine (0.10 to 0.13 mg/kg; Mobay Corp., Shawnee, KS, U.S.A.) administered intramuscularly. At 8 weeks after intraconjunctival challenge with approximately 2 × 107 CFU of B. abortus strain 544/Beagle (50 µl of inoculum per eye), all Beagles were euthanized by intravenous administration of sodium pentobarbitol. Beagle samples obtained at necropsy included lymph nodes (mandibular, prescapular, retropharyngeal, and superficial inguinal), spleen, kidney, and uterus. Tissue samples were triturated in 0.15 M NaCl by using a tissue grinder and plated on Farrell’s medium containing 5% bovine serum, antibiotics and ethyl-violet (Brucella-selected agar) [30]. Isolation of Brucella bacteria from lymph nodes and subsequent biochemical identification of isolates were performed by conventional methods [29]. Briefly, each node was trimmed of excess fat, dipped in 95% ethanol, and flamed to remove surface contaminants. The lymph nodes were trimmed into small pieces, were weighed and were homogenized with an equal volume of sterile PBS in a stomacher blender for ≥2 min. The resulting homogenate was then swabbed onto Farrell’s medium with 5% bovine serum, antibiotics and ethyl-violet. All agar plates were incubated at 37°C and 10% CO2 for ≥10 days. B. abortus bacteria were identified on the basis of colony morphology, growth characteristics, and were confirmed by PCR using the B. abortus-specific primer (5′-GACGAACGGAATTTTTCCAATCCC-3′) and IS711-specific primer (5′-TGCCGATCACTTAAGGGCCTTCAT-3′) described in previously study [10, 11]. If no colony detected on the Brucella-selected agar, then the number of viable challenge strain from each tissue of the Beagles means <102 CFU.

Statistical analysis

To observe differences among different vaccinated groups, absorbance data values of ELISA were used for the analysis of variance with post-hoc turkey for pair-wise comparison using SPSS version 16.0 (SPSS Inc., Chicago, IL, U.S.A.). Kruskal–Wallis followed by Dunn’s procedure for multiple comparisons between groups was carried out to compare the log transformed CFU values in organs obtained from each immunized group of Beagles with the respective control group for bacterial challenge experiments. Statistical significance was determined at (P<0.05).

RESULTS

Safety of the vaccine

SC immunization of Beagles with the mixture of S. Typhimurium-based B. abortus vaccine strains did not cause any negative impact on the overall clinical status (behavior, appetite, etc.) during our study period (6 WPPI). The body temperature of all the Beagles remained within normal limits (38.0–39.0°C) (data not shown). Importantly, no side effects and residual numbers of strain were observed in the inoculation site of the immunized beagles. Furthermore, no Salmonella delivery strains were isolated from 7 tissues of Beagles in groups A and B tested at 6 WPPI.

Antibody response to recombinant BCSP31, Omp3b or SOD protein

The antibody titters against each antigen in the sera are presented in Fig. 1. In Group A, serum IgG titers against each recombinant BCSP31, Omp3b and SOD protein were 0.22 ± 0.035, 0.10 ± 0.015, and 0.19 ± 0.007, respectively, at 0 WPPI. The serum IgG titers against each recombinant protein of group A Beagles at 2 and 4 WPPI were maintained similar to those of group A at O WPPI. However, the serum IgG titers against each recombinant BCSP31, Omp3b and SOD protein of group B Beagles were 0.20 ± 0.066, 0.12 ± 0.010, and 0.21 ± 0.056, respectively, at 0 WPPI. The serum titers were increased from 2 WPPI till the end of this study. At 6 WPPI, serum IgG titers to each recombinant protein of group B were 0.61 ± 0.049, 0.66 ± 0.080, and 0.84 ± 0.117, respectively.

Fig. 1.

Antibody titers (optical density) against each recombinant BCSP31, Omp3b, and SOD protein. Group A Beagles were inoculated with attenuated Salmonella Typhimurium containing pMMP65 only as a vector control. Group B Beagles were immunized with approximately 3.0 × 109 CFU/ml of the mixture of S. Typhimurium delivery strains expressing recombinant BCSP31, Omp3b, and SOD proteins in 1 ml. Data represent the means of all Beagles in each group, and error bars represent the standard deviations (SDs). Asterisks indicate a significant difference between the values obtained for the immunized group and those obtained for the vector control group (*P<0.05).

Cytokine analysis

IL4, TNF-α, and IFN-γ concentrations in response to recombinant BCSP31, Omp3b, and SOD proteins after re-stimulation with the individual proteins were measured using ELISA kit at 6 WPPI. IL4, TNF-α, and IFN-γ concentrations to each recombinant BCSP31, Omp3b, and SOD protein in the culture supernatant from group B Beagles when PBMCs were re-stimulated with each recombinant protein in vitro, were significantly higher than those from group A Beagles (Fig. 2) (P<0.05).

Fig. 2.

IL-4, TNF-α, and IFN-γ concentrations (pg/ml) in peripheral blood mononuclear cells at 6 weeks post prime immunization. Groups A, and B are indicated as in Fig. 1. Data represent the means of all Beagles in each group, and error bars indicate the standard deviations (SDs). Asterisks indicate significant differences among the values obtained for each group (*P<0.05).

Protective efficacy against B. abortus infection

Protective efficacy of the S. Typhimurium-based B. abortus vaccine candidate against B. abortus strain 544 challenge was evaluated by assessing three basic parameters: the effectiveness of vaccination (or degree of protection against infection), the index of infection (the number of B. abortus strain 544-infected tissues), and the rates of Brucella colonization in tissues. Among 5 Beagles of group B, the S. Typhimurium-based B. abortus vaccine candidate provided complete protection (effectiveness of vaccination) against B. abortus strain 544 challenge in 40% of Beagles. The severity of B. abortus strain 544 challenge in group B Beagles as indicated by the index of infection (1.2 ± 1.10, P<0.05) (Table 1) (Fig. 3A) and rates of B. abortus colonization in tissues [0.0 ± 0.0 to 2.5 ± 216 log10 CFU/g of tissue, P<0.05 (Fig. 3B)] were significantly lower than that of the vector control group [index of infection 5.6 ± 0.55 (Table 1) (Fig. 3A); B. abortus colonization 1.45 ± 2.05 to 4.72 ± 0.76 log10 CFU/g of tissue (Fig. 3B)].

Table 1.

Summary of B. abortus isolation from culture positive Beagles after challenge with B. abortus strain 544 in vector control and S. Typhimurium-based vaccinated groups

Group		Specimen
		Kidney	Spleen	Uterus	Mammary	Mandibular LNa)	Prescapular LN	Retropharyngeal LN	Superficial inguinal LN
A	A1	-b)	+c)	+	-	+	+	+	+
	A2	-	+	-	-	+	+	+	+
	A3	-	+	+	-	+	+	+	+
	A4	-	+	+	-	+	+	+	+
	A5	-	+	-	-	+	+	+	+
B	B1	-	-	-	-	+	-	+	-
	B2	-	-	-	-	-	-	-	-
	B3	-	-	-	-	+	-	+	-
	B4	-	-	-	-	+	-	+	-
	B5	-	-	-	-	-	-	-	-

a) LN means lymph node. b) “-” means that no colony detected on the Brucella-selected agar. c) “+” means that at least one colony of B. abortus strain 544 was isolated on the Brucella-selected agar.

Fig. 3.

Index of infection for Beagles challenged with B. abortus strain 544 at 6 weeks post prime immunization (A), colonization and incidence of recovery of B. abortus strain 544 in tissues (B). Groups A, and B are indicated as in Fig. 1. All Beagles in each group were conjunctivaly challenged with approximately 2 × 107 CFU of virulent B. abortus 544 at 6 weeks post prime immunization. The numbers of viable bacteria recovered from each tissues of Beagles at 8 weeks post challenge are shown. LN, lymph node.

DISCUSSION

In this study, we experimentally evaluated the protective efficacy of live, attenuated S. Typhimurium expressing recombinant BCSP31, Omp3b, and SOD proteins of B. abortus against B. abortus infection as a B. abortus vaccine candidate in a dog model. In a previous study [21], live, attenuated S. Typhimurium delivery strains expressing recombinant B. abortus BCSP31, Omp3b, and SOD proteins were constructed as a B. abortus vaccine candidate. Furthermore, effective protection of the S. Typhimurium-based B. abortus vaccine candidate against B. abortus infection were observed in murine model [21]. Accordingly, we thought that the S. Typhimurium-based B. abortus vaccine candidate could also help in control canine brucellosis caused by B. abortus, although brucellosis by B. canis in dogs is mainly caused. Therefore, in this study, our goal was to evaluate safety and protective efficacy of S. Typhimurium-based Brucella vaccine candidate against brucellosis by B. abortus but not B. canis in Beagles. As expected S. Typhimurium-based B. abortus vaccine candidate was found safe in clinical thermometry in Beagles. Notably, any inflammatory infiltrates were not observed in Beagles immunized SC with S. Typhimurium-based B. abortus vaccine candidate. Moreover, Salmonella-based B. abortus vaccine strains were not isolated from any tissues of all Beagles tested at 6 WPPI. These results revealed that our vaccine candidate is safe in Beagles.

We analyzed antigen-specific humoral and CMI responses in Beagles immunized SC with the S. Typhimurium-based B. abortus vaccine candidate. Strong serum IgG is necessary to protect hosts from intracellular pathogens infections [1, 12, 31], because serum IgG helps to eradicate the intracellular pathogens from the blood and to improve the phagocytosis efficacy within their immune system through opsonization [12, 25]. In present study, serum IgG titers were investigated in Beagles immunized SC with the S. Typhimurium-based B. abortus vaccine candidate. The serum IgG titers from the candidate-immunized Beagles were significantly increased compared to those from the un-immunized Beagles at 6 WPPI. The result shows that protective serum IgG titers against each recombinant BCSP31, Omp3b, and SOD protein can be induced by each recombinant protein delivered from each S. Typhimurium-based B. abortus vaccine strain without antagonism between expressed-recombinant proteins. Furthermore, induction of cytokines to each recombinant protein from PBMCs collected from all Beagles immunized with the S. Typhimurium-based B. abortus vaccine candidate, and re-stimulated in vitro with each the recombinant protein indicated a powerful Th2 type immunity (IL-4 levels represent the Th2 bias of immunity). These results show that serum IgG, and IL-4, which are associated with enhancing serum IgG response, were significantly increased by the S. Typhimurium-based B. abortus vaccine candidate.

CMI cells play a major role in clearance of B. abortus species, because B. abortus is a facultative intracellular pathogen [4, 19, 27, 40]. Therefore, in present study, CMI using TNF-α, and IFN-γ cytokines ELISA kits were evaluated. After all, showed high TNF-α, and IFN-γ concentrations to each recombinant protein in the culture supernatant when PBMCs re-stimulated with each recombinant protein in vitro, giving an indication of CMI response, which was reflected in levels of protection induced by S. Typhimurium-based B. abortus vaccine candidate. These results show that SC immunization with the S. Typhimurium-based B. abortus vaccine candidate effectively produces the cytokines, which are related to CMI response.

Brucella canis cause abortions, stillbirths, epididymitis, orchitis, and sperm abnormalities in dogs [8]. However, criteria for clinical signs of brucellosis by B. abortus in dogs are not well-settled [3, 15, 29], uncommon, and variable [22, 29], but abortion, epididymitis, and arthritis have been described [7, 13, 26, 32]. A main difficulty that has interrupted the development of vaccines to protect host from Brucella infections, is the shortage of the precise proof of effective protection. Isolation of challenge strain, B. abortus strain 544, was attempted to in kidney, spleen uterus, and lymph nodes, such as mandibular, prescapular, retropharyngeal, and superficial inguinal lymph nodes, at 8 weeks after challenge. Among 5 Beagles of immunized group, challenge strain was isolated from only mandibular and superficial inguinal lymph nodes of 3 dogs, whereas the challenge strains were detected from spleen, uterus (except two Beagles) and mandibular, prescapular, retropharyngeal, and superficial inguinal lymph nodes of all unimmunized Beagles. Infection of these sites, such as spleen, and retropharyngeal lymph node, does not result in shedding of B, abortus strain in urine, feces or estrual discharges [7]. Furthermore, a number of the challenge strains were isolated from uterus of 3 dogs among unimmunized Beagles, whereas no isolates were observed from 5 immunized Beagles. The Brucella strain in this region can result in abortion when the Beagles become in pregnancy. Thankfully, S. Typhimurium-based B. abortus vaccine candidate provided complete protection against B. abortus strain 544 infection in 2 dogs among 5 Beagles. Furthermore, the severity of brucellosis even among diseased Beagles vaccinated with the S. Typhimurium-based B. abortus vaccine candidate was significantly reduced, indicated by infection index 4.7 times-lower in Beagles with B. abortus colonization in tissue at least 1.9 log10 times lower in S. Typhimurium-based B. abortus vaccine candidate-immunized group Beagles (P<0.05) compared to vector control group Beagles. This infection index in Beagles immunized with the S. Typhimurium-based B. abortus vaccine candidate showed slightly low levels of protective efficacy than that in mice immunized with same vaccine strains in a previous study [21]. Though the S. Typhimurium-based B. abortus vaccine candidate in Beagles provided lower levels of efficacy than in mice, considering its safety and ability to elicit enhanced protective humoral and CMI responses in Beagles, it might be useful to use in Beagles in high risk herds. Further investigations are in progress to improve the protective efficacy of the Salmonella-based B. abortus vaccine candidate in Beagles, such as use of higher dose of the vaccine, different inoculation route, and administration of the combination of the Salmonella-based B. abortus vaccine candidate with suitable potent adjuvants.

In conclusion, the results of this study demonstrated that SC immunization with approximately 3 × 109 CFU of the mixture of S. Typhimurium-based B. abortus vaccine strains induced robust serum IgG and CMI responses in Beagles. In addition, the Beagles immunized with the vaccine candidate displayed more efficient protection than unimmunized Beagles. Therefore, our S. Typhimurium-based B. abortus vaccine candidate elicits antibodies to B. abortus each recombinant BCSP31, Omp3b, and SOD protein, which are effective at protecting the Beagles from brucellosis. Thus, this study suggests that future study such as the combination of our vaccine strains with appropriate potent adjuvants is necessary for more powerful protection in Beagles.

CONFLICT OF INTEREST

The authors do not have competing interest.