Brucella abortus RB51 ΔleuB expressing Salmonella FliC conjugated gonadotropins reduces mouse fetal numbers: A possible feral swine brucellosis immunocontraceptive vaccine.

Population and health management of wildlife is a key to environmental health, domestic herd health, and ultimately public health. Many different methods including: surgical sterilization, poison baits, and sponsored hunting programs have been used in the attempt to control populations of various nuisance animal species. Particular interest has been given to immunocontraception through wildlife vaccination protocols. This study specifically looked at the potential immunocontraceptive and protective properties of a Brucella abortus RB51 ΔleuB vaccine expressing Salmonella typhimurium FliC conjugated to porcine follicle stimulating hormone beta subunit (FSHß) or gonadotropin releasing hormone (GnRH) DNA sequences. B. abortus RB51 ΔleuB pNS4-TrcD-FliC- FSH ß (RB51LFSHß) and B. abortus RB51 ΔleuB pNS4-TrcD-FliC-GnRH (RB51LGnRH) were tested in a pilot breeding study with BALB/c mice, and a significant reduction in fertility characteristics was observed in both male and female mice. Ultimately, this study provides support to test these vaccine candidates in feral swine, a destructive invasive species in the United States of America.

Rough Brucella neotomae provides protection against Brucella suis challenge in mice.

Brucellosis is one of the most common zoonotic diseases worldwide. Almost 500,000 new human cases occur each year; yet there is no vaccine for human use. Moreover, there is no universal Brucella vaccine that would provide protection against all pathogenic species of Brucella. We generated a rough, live-attenuated B. neotomae strain by deleting the wboA gene encoding a glycosyltransferase. This strain lacks the O-side chain in its lipopolysaccharide (LPS) and thus the vaccinated animals can be differentiated serologically from the field-infected animals. We tested the efficacy of rough B. neotomae strain to stimulate dendritic cells compared to the smooth wild type strain. Based on TNF-α production, our data suggests that a significantly higher stimulation was obtained when dendritic cells were stimulated with the rough vaccine strain compared to the smooth wild type B. neotomae. Furthermore, the rough mutant was cleared from mice within 6 weeks even at a dose as high as 2 x 108 CFU. Vaccinated mice showed significantly higher level of protection against a virulent B. suis 1330 challenge compared to the control mice. Antibody titers in the mice and cytokine production by the splenocytes from the vaccinated mice showed a Th1 mediated immune response that correlated with the protection.

Correction: Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis.

[This corrects the article DOI: 10.1371/journal.pone.0160350.].

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis.

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.

Vaccination of Elk (Cervus canadensis) with Brucella abortus Strain RB51 Overexpressing Superoxide Dismutase and Glycosyltransferase Genes Does Not Induce Adequate Protection against Experimental Brucella abortus Challenge.

In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area. In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the development of effective disease management strategies for wild elk herds is of utmost importance, not only for the prevention of reintroduction of brucellosis to cattle, but also for the overall health of the Greater Yellowstone Area elk populations. In two studies, we evaluated the efficacy of B. abortus strain RB51 over-expressing superoxide dismutase and glycosyltransferase for protecting elk from infection and disease caused by B. abortus after experimental infection with a virulent B. abortus strain. Our data indicate that the recombinant vaccine does not protect elk against brucellosis. Further, work is needed for development of an effective brucellosis vaccine for use in elk.

Roles of bacterial membrane vesicles.

Outer membrane vesicles (OMVs) are released from the outer membrane of Gram-negative bacteria. Moreover, Gram-positive bacteria also produce membrane-derived vesicles. As OMVs transport several bacterial components, especially from the cell envelope, their interaction with the host cell, with other bacteria or as immunogens, have been studied intensely. Several functions have been ascribed to OMVs, especially those related to the transport of virulence factors, antigenic protein composition, and development as acellular vaccines. In this work, we review some of the recent findings about OMVs produced by specific pathogenic bacterial species.

Proteomic analysis of detergent resistant membrane domains during early interaction of macrophages with rough and smooth Brucella melitensis.

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide the basis for novel ways of treating or diagnosing Brucellosis.

Protection to respiratory challenge of Brucella abortus strain 2308 in the lung.

Brucella is amongst the top 5 causes of zoonotic disease worldwide. Infection is through ingestion, inhalation or contact exposure. Brucella is characterized as a class B pathogen by Centers of Disease Control and Prevention (CDC). Currently, there are no efficacious vaccines available in people. Currently available USDA approved vaccines for animals include B. abortus strain RB51 and B. melitensis Rev1. Protection is mediated by a strong innate and CD4 Th1, CD8 Tc1 immune response. If protective vaccines can be developed, disease in people and animals can be controlled. While strain RB51 protects in cattle, and against intraperitoneal challenge in mice, it does not protect against respiratory challenge. Therefore, we assessed the efficacy of strain RB51 combined with different TLR agonists, and O-side chain from LPS, to enhance protection against respiratory challenge with strain 2308. We hypothesized that TLR agonists and O-side chain would enhance protection. Strains RB51 with TLR2 agonist, RB51 with TLR4 agonist and strain 19 provided significant protection in the lung. Protection using strain RB51 with TLR agonists was associated with increased IgG2a and IgG1 in the (bronchoalveolar lavage) BAL and serum, and increased IgA (serum). Splenocytes from strain RB51 with TLR2 vaccinated mice up-regulated antigen specific interferon-gamma and TNF-alpha production. Vaccination and challenge resulted in significant increases in activated dendritic cells (DCs), and increased CD4 and CD8 cells in the BAL. Overall, this study demonstrates the ability of TLR agonists 2 and 4 to up-regulate strain RB51 mediated protection in the lung to respiratory challenge against strain 2308.

A review of Brucella seroprevalence among humans and animals in Bangladesh with special emphasis on epidemiology, risk factors and control opportunities.

Brucellosis is a neglected bacterial zoonotic disease in many countries affecting both humans and animals. The aim of this paper is to review published reports of the seroprevalence of brucellosis in humans and animals (cattle, buffalo, sheep, goats and dogs) in Bangladesh. The prevalence studies are based primarily on the following serological tests: rose bengal plate agglutination test (RBT), plate agglutination test (PAT), tube agglutination test (TAT), mercaptoethanol agglutination test (MET), standard tube agglutination test (STAT), slow agglutination test (SAT), milk ring test (MRT), indirect enzyme-linked immunosorbant assay (I-ELISA), competitive ELISA (C-ELISA) and fluorescent polarization assay (FPA). Seroprevalences of brucellosis were found to be affected by the sensitivity and specificity of serological tests employed. Brucellosis prevalence varied based on occupations of people (2.5-18.6%) and species of animals (3.7% in cattle, 4.0% in buffalo, 3.6% in goats and 7.3% in sheep). The prevalence of brucellosis in humans was reported in livestock farmers (2.6-21.6%), milkers (18.6%), butchers (2.5%) and veterinarians (5.3-11.1%) who have direct contact with animal and its products or who consume raw milk. According to published reports brucellosis does affect people and livestock of Bangladesh. There is an immediate need for a concerted effort to control and eradicate brucellosis from domesticated animals in Bangladesh.

A history of the development of Brucella vaccines.

Brucellosis is a worldwide zoonosis affecting animal and human health. In the last several decades, much research has been performed to develop safer Brucella vaccines to control the disease mainly in animals. Till now, no effective human vaccine is available. The aim of this paper is to review and discuss the importance of methodologies used to develop Brucella vaccines in pursuing this challenge.

Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages.

Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 µM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 µM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Pluronic P85 enhances the efficacy of outer membrane vesicles as a subunit vaccine against Brucella melitensis challenge in mice.

Brucellosis is the most common zoonotic disease worldwide, and there is no vaccine for human use. Brucella melitensis Rev1, a live attenuated strain, is the commercial vaccine for small ruminants to prevent B. melitensis infections but has been associated with abortions in animals. Moreover, strain Rev1 is known to cause disease in humans and cannot be used for human vaccination. Outer membrane vesicles (OMVs) obtained from B. melitensis have been shown to provide protection similar to strain Rev1 in mice against B. melitensis challenge. In the present work, we tested the efficacy of Pluronic P85 as an adjuvant to enhance the efficacy of Brucella OMVs as a vaccine. P85 enhanced the in vitro secretion of TNF-α by macrophages induced with OMVs and P85. Further, P85 enhanced the protection provided by OMVs against B. melitensis challenge. This enhanced protection was associated with higher total IgG antibody production but not increased IFN-γ or IL-4 cytokine levels. Moreover, P85 alone provided significantly better clearance of B. melitensis compared to saline-vaccinated mice. Further studies are warranted to find the mechanism of action of P85 that provides nonspecific protection and enhances the efficacy of OMVs as a vaccine against B. melitensis.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1(T) and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1(T) and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1(T) and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1(T) maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Effect of exogenous erythritol on growth and survival of Brucella.

Erythritol has been considered as an important factor for the pathogenesis of Brucella abortus 2308 and its ability to cause abortion in ruminants. There is a lack of laboratory models to study the Brucella-erythritol relationship, as commonly used murine models do not have erythritol. We tested the effect of exogenous erythritol on the growth of Brucella in iron minimal medium (IMM), in infected macrophage culture and in infected mice to determine if these models can be used to study the relationship between Brucella and erythritol. An effect of erythritol on Brucella growth was only seen in IMM. There appear to be no effect of erythritol on Brucella growth in macrophage cell cultures or in mice. This shows that administration of erythritol to the mice or macrophages cannot mimic the environment in ruminants during pregnancy and thus cannot be used as models to understand the effect of erythritol on Brucella pathogenesis.

Nanomedicine for intracellular therapy.

Intracellular pathogens like Salmonella evade host phagocytic killing by various mechanisms. Classical antimicrobial therapy requires multiple dosages and frequent administration of drugs for a long duration. Intracellular delivery of antimicrobials using nanoparticle may effectively devise therapies for bacterial infections. This review will address the mechanisms used by Salmonella to avoid host pathogenic killing, reasons for therapeutic failure and advances in nanoparticle drug delivery technology for efficient intracellular bacterial clearance.

Role of TLRs in Brucella mediated murine DC activation in vitro and clearance of pulmonary infection in vivo.

Brucellosis is worldwide zoonoses affecting 500,000 people annually with no approved human vaccines available. Live attenuated Brucella abortus vaccine strain RB51 protects cattle through CD4 and CD8 T-cell mediated responses. However, limited information is known regarding how Brucella stimulate innate immunity. Although the most critical toll like receptors (TLRs) involved in the recognition of Brucella are TLR2, TLR4 and TLR9, it is important to identify the essential TLRs that induce DC activation/function in response to Brucella, to be able to upregulate both vaccine strain RB51-mediated protection, and clearance of pathogenic strain 2308. Furthermore, in spite of the importance of aerosol transmission of Brucella, no published studies have addressed the role of TLRs in the clearance of strain 2308 or strain RB51 from intranasally infected mice. Therefore, we used a (a) bone marrow derived dendritic cell model in TLRKO and control mice to assess the differential role of pathogenic and vaccine strains to induce DC activation and function in vitro, and (b) respiratory model in TLRKO and control mice to assess the critical roles for TLRs in clearance of strains in vivo. In support of the essential TLRs in clearance and protection, we performed challenge experiments to identify if these critical TLRs (as agonists) could enhance vaccine induced protection against pathogenic strain 2308 in a respiratory model. We determined: vaccine strain RB51 induced significant (p≤0.05) DC activation vs. strain 2308 which was not dependent on a specific TLR; strain RB51 induced TNF-α production was TLR2 and TLR9 dependent, and IL-12 production was TLR2 and TLR4 dependent; TLR4 and TLR2 were critical for clearance of vaccine and pathogenic Brucella strains respectively; and TLR2 (p<0.05), TLR4 (p<0.05) and TLR9 (p=0.075) agonists enhanced vaccine strain RB51-mediated protection against respiratory challenge with strain 2308 in the lung.

Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice.

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Proinflammatory caspase-2-mediated macrophage cell death induced by a rough attenuated Brucella suis strain.

Brucella spp. are intracellular bacteria that cause an infectious disease called brucellosis in humans and many domestic and wildlife animals. B. suis primarily infects pigs and is pathogenic to humans. The macrophage-Brucella interaction is critical for the establishment of a chronic Brucella infection. Our studies showed that smooth virulent B. suis strain 1330 (S1330) prevented programmed cell death of infected macrophages and rough attenuated B. suis strain VTRS1 (a vaccine candidate) induced strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774.A1 cells infected with S1330 or VTRS1. In total 17,685 probe sets were significantly regulated based on the effects of strain, time and their interactions. A miniTUBA dynamic Bayesian network analysis predicted that VTRS1-induced macrophage cell death was mediated by a proinflammatory gene (the tumor necrosis factor alpha [TNF-α] gene), an NF-κB pathway gene (the IκB-α gene), the caspase-2 gene, and several other genes. VTRS1 induced significantly higher levels of transcription of 40 proinflammatory genes than S1330. A Mann-Whitney U test confirmed the proinflammatory response in VTRS1-infected macrophages. Increased production of TNF-α and interleukin 1ß (IL-1ß) were also detected in the supernatants in VTRS1-infected macrophage cell culture. Hyperphosphorylation of IκB-α was observed in macrophages infected with VTRS1 but not S1330. The important roles of TNF-α and IκB-α in VTRS1-induced macrophage cell death were further confirmed by individual inhibition studies. VTRS1-induced macrophage cell death was significantly inhibited by a caspase-2 inhibitor but not a caspase-1 inhibitor. The role of caspase-2 in regulating the programmed cell death of VTRS1-infected macrophages was confirmed in another study using caspase-2-knockout mice. In summary, VTRS1 induces a proinflammatory, caspase-2- and NF-κB-mediated macrophage cell death. This unique cell death differs from apoptosis, which is not proinflammatory. It is also different from classical pyroptosis, which is caspase-1 mediated.

Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P<0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.