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. 2019 Oct;83(4):261–266.

Protective efficacy of a Brucella vaccine using a Salmonella-based delivery system expressing Brucella Omp3b, BCSP31, and SOD proteins against brucellosis in Korean black goats

Won-Kyong Kim 1, Ja-Young Moon 1, Jeong-Sang Cho 1, Jin Hur 1,✉
PMCID: PMC6753886  PMID: 31571725

Abstract

This study aimed to assess the protective efficacy of a novel Brucella vaccine formulation in goats. Twenty black goats were separated into 2 groups. Group A was injected with 3.0 × 109 CFU (colony-forming units)/mL of a Salmonella-based delivery system harboring only vector (pMMP65). Group B was immunized with 3.0 × 109 CFU/mL of the vaccine, a mixture of 3 Brucella vaccine strains (using a Salmonella-based delivery system) expressing each recombinant B. abortus Omp3b, BCSP31, and SOD protein. No Salmonella delivery strain was isolated from all tested lymph nodes and parenchymal organs. Serum immunoglobulin (Ig) G titers and interferon gamma concentrations were significantly higher in group B than those in group A. After intraconjunctival challenge with virulent B. abortus strain 544, 40% of the vaccinated animals in group B were protected against B. abortus infection. The infection index and colonization of B. abortus in tested tissues was significantly lower in group B than group A. We conclude that this Brucella vaccine induces significant antigen-specific immune responses and provides effective protection against B. abortus infection in goats. Further studies are needed to enhance the protection rate of this Brucella vaccine and to discover its practical application in small ruminants.

Introduction

Brucellosis, caused by infection with the Brucella genus, is a contagious disease that causes economic losses for the owners of domestic animals. Brucella melitensis is the most common agent of brucellosis in small ruminants such as goats (1,2). However, other members of the Brucella genus can also infect goats (3,4). Brucellosis in goats is a devastating zoonotic disease causing abortions and other reproductive problems, as well as decreased milk yield and meat production.

As Brucella species invade macrophages within the host immune system and multiply (5,6), cell-mediated immune responses (CMI) are critical to eliminate Brucella species from the host (7). Th1-type immunity derived by interferon gamma (IFN-γ) is useful to protect the host against Brucella infection (8). Many vaccines have been developed to prevent Brucella infection, and both live and attenuated Brucella strains have been used as commercial vaccines in domestic animals. However, these vaccines can revert to pathogenic ones and any strain can potentially interfere with diagnosis; specifically, inoculation with any vaccine strain can cause low levels of abortion in pregnant animals (especially cows) and any vaccinated animal may show reactivity on diagnostic testing due to vaccination, rather than infection (9,10). Because of these limitations a safer and more effective vaccine must be developed.

Some Salmonella strains have been reported as useful delivery systems to carry immunogenic proteins derived from other pathogens (11,12). Reportedly, Omp3b, BCSP31, and Cu/Zn SOD can be used as protective subunit vaccines (4,13–18). Kim et al (17,18) reported that the mixture of the Brucella vaccine strains using a Salmonella-based delivery system (BVSSBDS) expressing each the recombinant Omp3b, BCSP31, and SOD protein of B. abortus were evaluated as a vaccine in mice and beagles. Immunization with the BVSSBDS effectively elicited both antibodies and CMI, and conferred effective protection against experimental infection by virulent B. abortus in mice and beagles (17,18). The current study aimed to appraise the protective efficacy of the BVSSBDS in goats.

Materials and methods

Bacterial strains

Each live and attenuated BVSSBDS expressing each of the recombinant Omp3b (HJL219), BCSP31 (HJL228), and SOD (HJL213) protein of B. abortus was constructed in a previous study (17). The mixture of these 3 BVSSBDS for brucellosis in mice and beagles (17,18). In this study, BVSSBDS were tested as a vaccine for brucellosis in Korean black goats (Table I). Virulent B. abortus strain 544 (HJL254) was used as the challenge strain (19).

Table I.

Bacterial strains used in this study (17).

Strains Description
Escherichia coli
 HJL206 BL21 with pET32a-BCSP31
 HJL204 BL21 with pET28a-Omp3b
 HJL208 BL21 with pET28a-SOD
Salmonella Typhimurium
 HJL229 JOL912 with pMMP65
 HJL228 JOL912 with pMMP65-BCSP31
 HJL219 JOL912 with pMMP65-Omp3b
 HJL213 JOL912 with pMMP65-SOD
Brucella abortus
 HJL254 Brucella abortus strain 544 (ATCC23448)
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Experimental animals

Twenty female South Korean black goats, aged 5 to 6 mo, were separated into 2 groups (n = 10 goats per group). All goats used in this study were purchased from flocks confirmed to be free from brucellosis. The goats were seronegative for brucellosis using the Rose Bengal test and tube agglutination test. All animal experiments were approved (CBU 2016-98) by the Chonbuk National University Animal Ethics Committee according to the guidelines of the Korean Council on Animal Care.

Preparation of each recombinant protein

The HJL204 (Omp3b), HJL206 (BCSP31), and HJL208 (SOD) strains of Brucella were established in previous studies (17,18). The recombinant proteins purified from each strain were used as coating proteins in enzyme-linked immunosorbent assay (ELISA) to measure serum IgG titers and as antigens to re-stimulate peripheral blood mononuclear cells (PBMC).

Preparation of Salmonella delivery strain formulation

The previously described BVSSBDS were also prepared as vaccines of black goats (Table I) (17,18).

Immunization of goats and collection of samples

At 6 mo old, 0 wk post prime immunization (WPPI), all Korean black goats were subcutaneously (SC) primed. At 4 WPPI, all of the goats were boosted SC. Group A goats were injected with about 3.0 × 109 colony-forming units (CFU)/mL of Salmonella-based delivery system harboring only the vector, pMMP65, in 1 mL as a control. Group B goats were vaccinated with about 3.0 × 109 CFU/mL of the mixture of the BVSSBDS (equal CFU from each of the vaccine strains) in 1 mL. The location of the injection site was at the third mean of the neck on the right side for both primed and boosted goats. The lymph nodes (bronchial, mandibular, mediastinal, mesenteric, parotid, portal, retropharyngeal, superficial, and supramammary) and parenchymal organs (heart, kidney, liver, lung, spleen, and uterus) were gathered from all test goats. Serum samples were gathered at 0, 2, 4, 6, and 8 (before challenge) WPPI for evaluation of immune response.

Vaccine safety experiment

The safety evaluation of the BVSSBDS in goats was carried out. Thermometry was done daily on all the goats from 0 WPPI until 8 WPPI. Five goats per group (10 in total) were aseptically euthanized at 8 WPPI. The location of the injection site (third mean of the neck on the right side), lymph nodes (bronchial, mandibular, mediastinal, mesenteric, parotid, portal, retropharyngeal, superficial, and supramammary) and parenchymal organs (heart, kidney, liver, lung, spleen, and uterus) were gathered from all test goats. To isolate BVSSBDS from a total of 15 organs, the tissues were tested using the same method described previously by Kim et al (17,18). The isolates were proved by polymerase chain reaction (PCR) using a Salmonella Typhimurium-specific primer (20) and each of the Brucella vaccine candidate-specific primers described in previous studies (17,18,21).

Serum IgG titers measurement by ELISA

In order to evaluate each of the recombinant protein-specific serum IgG titers from all of the goats, standard ELISA was done in accordance with a slightly modified version of a previously described method (17,18). In brief, the recombinant Omp3b (2 μg/mL), BCSP31 (2 μg/mL), or SOD (2 μg/mL) protein were each coated overnight and blocked for 1 h using phosphate buffered saline (PBS) containing 1% ovalbumin (PBS-OVA). Each serum was diluted at 1:100 in PBS-OVA. The serum was reacted with horseradish peroxidase-conjugated rabbit anti-goat IgG antibody (Bethyl Lab, Montgomery, Texas, USA).

Preparation of peripheral blood mononuclear cells (PBMC)

In order to prepare PBMC, blood samples were drawn from all of the goats at 8 WPPI. The PBMC was prepared in accordance with the same version of a previously described method (18,22).

Cytokine concentration measurement by ELISA

The IFN-γ concentrations in the PBMC culture supernatants were measured by using a goat IFN-γ ELISA kit (MyBioSource, San Diego, California, USA) in compliance with the manufacturer’s instructions.

Challenge experiments

For challenge experiments, virulent B. abortus strain 544, HJL254, was prepared according to a slightly modified method mentioned in a previous study (18). Briefly, the strain was grown in Brucella broth at 37°C for 24 h and resuspended to approximately 1 × 108 CFU/mL. At 8 WPPI, the remaining 10 goats (5 goats per group) were fasted for 16 h before being anesthetized with tiletamine-zolazepam (Zoletil 50; Virbac, Carros, France), 7 to 10 mg/kg of body weight (BW) and xylazine (Bayer Korea, Ansan, South Korea), 2.32 to 3.48 mg/kg BW, both administered intramuscularly. The 10 goats were then intraconjunctivally challenged with approximately 1 × 107 CFU of HJL254/goat (50 μL of inoculum per eye). At 12 wk after challenge, the 10 goats were euthanized. The lymph nodes (bronchial, mandibular, mediastinal, mesenteric, parotid, portal, retropharyngeal, superficial, and supramammary) and parenchymal organs (heart, kidney, liver, lung, spleen, and uterus) were aseptically collected. Isolation of the Brucella challenge strains was attempted from 15 organs per animal using Brucella-selective agar (23) by conventional methods at National Veterinary Services Laboratory (NVSL) (24,25). The challenge strain was proven by PCR using the B. abortus- and IS711-specific primer sets described in previous reports (26,27). The CFU value for each tissue was calculated and a mean value for each tissue count was obtained after logarithmic conversion. Bacteriological examination was done to determine the effectiveness of the vaccination (the number of goats from which no colonies were isolated) and the index of infection (the number of lymph nodes and organs per goat from which the challenge strain was isolated).

Statistical analysis

The significant difference in serum IgG titers and colonization of the challenge strain in tissues between groups A and B were analyzed using a 2-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. In addition, Student’s t-test was conducted to analyze the significant difference in the IFN-γ concentration and the index of infection between groups A and B. All analyses were carried out using computer software (GraphPad Prism software, version 5.0; GraphPad Software, La Jolla, California, USA). A P-value < 0.05 was considered statistically significant.

Results

Safety of the vaccine

The goats immunized SC with the mixture of BVSSBDS showed no clinical signs (such as abnormal behavior or decreased appetite) from 0 WPPI until 8 WPPI. The body temperature of all the immunized goats retained within normal range (38.5°C to 40.5°C, data not shown). No side effects were observed at the vaccine administration site of the immunized goats. All strains of the Salmonella-based delivery system were isolated from each of the 15 tissues of all euthanized goats (5 goats per group) at 8 WPPI.

Serum IgG titers against each protein

Serum IgG titers against each protein are shown in Figure 1. In group B goats, serum IgG titers for each of the Omp3b, BCSP31, and SOD protein were increased from 2 WPPI to 8 WPPI. At 8 WPPI, serum IgG titers were significantly higher in the vaccinated group B goats than in the control group A goats (P < 0.05).

Figure 1.

Figure 1

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Antibody titers against Omp3b, BCSP31, and SOD proteins. Goats in group A were inoculated with attenuated Salmonella typhimurium containing pMMP65 alone as a vector control. Goats in group B were immunized with approximately 3.0 × 109 CFU/mL of the mixture of Salmonella-based B. abortus vaccine strains in 1 mL of sterile PBS. Data represent the means of all goats in each group and error bars represent the standard deviations (SD).

* Significant difference between the values obtained for the immunized group and the vector control group (P < 0.05).

Cytokine analysis

The Omp3b, BCSP31, and SOD protein-specific IFN-γ concentrations following re-stimulation with each the protein were determined by using a commercial ELISA kit. The Omp3b, BCSP31, and SOD protein-specific IFN-γ concentrations from group A goats were 151.5 pg/mL ± 38.31, 122.21 pg/mL ± 50.07, and 157.68 pg/mL ± 28.57, respectively and those from group B goats were 341.73 pg/mL ± 83.43, 370.5 pg/mL ± 56.02, and 324.86 pg/mL ± 15.43, respectively (Figure 2).

Figure 2.

Figure 2

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The interferon (IFN)-γ concentrations (pg/mL) in peripheral blood mononuclear cells (PBMC) at 8 wk post prime immunization (WPPI). Groups A and B are indicated as in Figure 1. Data represent the means of all goats in each group and error bars indicate the standard deviations (SD).

* Significant differences among the values obtained for each group (P < 0.05).

Protection against challenge infection with virulent B. abortus

The protective efficacy of the BVSSBDS against experimental infection by virulent challenge strain was judged by analyzing 3 basic parameters: the effectiveness of vaccination, the index of infection, and the rate of Brucella colonization in tissues [the number of the virulent challenge strain isolated from each tissue (log10 CFU/g of tissue)]. The BVSSBDS offered protection against experimental infection by virulent challenge strain in 40% (2 out of 5) of the goats in group B. The seriousness of experimental infection by virulent challenge strain in group B goats (Table II, Figure 3A), as displayed by the index of infection (2.0 ± 1.87, P < 0.05) and the rate of Brucella colonization in each tissue (0.4 ± 0.89 to 1.0 ± 1.38 log10 CFU/g of tissue, P < 0.05), was significantly lower compared group A goats (index of infection 13.6 ± 1.82; Brucella colonization 1.79 ± 1.79 to 3.44 ± 0.35 log10 CFU/g of tissue; Figure 3B).

Table II.

Lymph nodes and parenchymal organs where B. abortus strain 544 challenge strain was isolated after challenge.

Lymph node Parenchymal organs
Groupa Bronchial Mandibular Mediastinal Mesenteric Parotid Portal Retropharyngeal Superficial Supramammary Heart Kidney Liver Lung Spleen Uterus
A
 A1 +b + + + + + + + + + + + + + +
 A2 + + + + + + + + + + + + +
 A3 + + + + + + + + + +
 A4 + + + + + + + + + + + + +
 A5 + + + + + + + + + + + + + +
B
 B1
 B2
 B3 + + +
 B4 + + + +
 B5 + + +
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a

Goats in group A were subcutaneously (SC) inoculated with approximately 3.0 × 109 colony-forming units (CFU)/mL of a Salmonella-based delivery system containing pMMP65 as a vector control, and group B with approximately 3.0 × 109 CFU/mL of the mixture (equal CFU counts of each strain) of 3 Salmonella-based B. abortus vaccine strains respectively, at 6 mo of age. Goats were boosted with the vector control strain and the vaccine at 7 mo of age.

b

“+” means that the challenged strain was isolated from the tissue.

Figure 3.

Figure 3

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Effectiveness of vaccination (the number of animals from which no colonies were isolated), the index of infection (the number of organs and lymph nodes from the animals in which Brucella was isolated) for goats challenged with B. abortus strain 544 at 8 wk after prime immunization (WPPI) (A), and the rate of Brucella colonization in tissues [the amount of B. abortus strain 544 isolated from tissues (log10 CFU/g of tissue)] (B). Groups A and B are indicated as in Figure 1. All goats in each group were intraconjunctivally challenged with approximately 1 × 107 CFU of virulent wild-type B. abortus 544 at 8 WPPI. The numbers of viable bacteria recovered from each tissue of goats at 12 wk after challenge are shown. Lymph node (LN) colonization and incidence of recovery of B. abortus strain 544 in tissues were significantly higher in all immunized goats compared with in goats in the vector control group (P < 0.05).

Discussion

In this study, a BVSSBDS was evaluated to determine the protective efficacy against experimental infection by a virulent challenge strain in goats. In a previous study (17), BVSSBDS expressing the recombinant Omp3b, BCSP31, and SOD protein of B. abortus were constructed. In a preliminary study to determine the optimal combination of the BVSSBDS expressing each the protein, and the optimal conditions for the vaccine strategy, mice were intraperitoneally primed and boosted with the mixture of 3 BVSSBDS (each Salmonella strain expressing the recombinant Omp3b, BCSP31, and SOD protein of B. abortus) showed complete protection (17). Furthermore, the BVSSBDS is able to induce effective protection against experimental infection by virulent B. abortus in a murine model and a dog model (17,18,21). Therefore, we believed that the BVSSBDS could control brucellosis in small ruminants. The goal of the current study was to estimate the safety and protective efficacy of the BVSSBDS against experimental infection by virulent challenge strain in goats. As expected, this vaccine was found to be safe as determined by body temperature and clinical signs in goats. No inflammatory infiltrates were shown in any immunized goats. Moreover, no Salmonella-based delivery system or Brucella vaccine were isolated from any lymph nodes or tissues of immunized goats at 8 WPPI. Our study results showed that SC immunization with this vaccine is safe in goats.

Each protein-specific serum IgG titer and cell-mediated immune response (CMI) was assessed in the immunized goats. A powerful serum IgG is crucial to protect the host from infections by intracellular bacteria (7,28,29), because it can eliminate bacteria from the blood and enhance the phagocytosis within host via opsonization (29,30). In this study, each protein-specific serum IgG titer was investigated in the SC immunized goats. The serum IgG titers in the vaccinated group B goats were significantly elevated compared with those in the unimmunized group A goats at 8 WPPI. These results indicate that serum IgG titers were significantly increased by each protein expressed from each Brucella vaccine strain, without antagonism among the individually expressed proteins.

The CMI mediated by cytokine IFN-γ plays a critical role in the protection against Brucella infection because Brucella species are facultative intracellular bacteria (5,31–33). Therefore, in this study, we estimated CMI by using IFN-γ cytokine ELISA kits. The CMI induced in the goats vaccinated with the mixture of BVSSBDS was higher than that in the control group. The result indicates that the BVSSBDS could induce the protective level of CMI. The mixture of BVSSBDS provided protection against virulent B. abortus experimental challenge strain in 40% (2 out of 5) of the group B goats. Moreover, the severity of brucellosis among goats infected with the challenge strain in the vaccinated group was significantly reduced compared with that of goats in the control group, as indicated by an index of infection 6.8 times lower and the number of Brucella isolates in each tissue at least 15.5 times lower in the vaccine group goats (P < 0.05) than in the control group. The index of infection in goats vaccinated with the mixture of 3 Brucella vaccine strains was observed as slightly lower protection rate against virulent B. abortus infection compared with that in mice vaccinated with the same vaccine in a previous report (17). Although the mixture of 3 Brucella vaccine strains showed lower levels of protection rates in goats compared with mice, in view of its safety and ability to induce effective serum IgG and CMI in goats, our vaccine might be useful in high-risk herds of small ruminants. Further research is underway to enhance the protection rate of the BVSSBDS against experimental infection by virulent B. abortus in goats, such as immunization with a higher dose of the Salmonella-based delivery system vaccine, and the combination ratio between the Brucella vaccine strains.

Our results showed that SC vaccination with about 3 × 109 CFU of the mixture of BVSSBDS elicited powerful antibody and CMI in goats. The vaccine conferred protection against experimental infection by virulent B. abortus in 40% of the vaccinated goats. Therefore, these results suggest that future work, such as of a higher dose of the vaccine, another inoculation route, and the combination rate between our vaccine strains, is necessary to achieve more powerful protection against brucellosis in small ruminants.

Acknowledgment

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MISP) (No. 2013R1A4A1069486).

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