Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.     

Identification of Brucella by Ribosomal-Spacer-Region PCR and Differentiation of Brucella canis from Other Brucella spp. Pathogenic for Humans by Carbohydrate Profiles

Molecular and chemical characteristics often provide complementary information in the differentiation of closely related organisms. The genus Brucella consists of a highly conserved group of organisms. Identification of the four species pathogenic in humans (Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis) is problematic for many clinical laboratories that depend primarily on serology and phenotypic characteristics to differentiate species. PCR amplification of the 16S-23S ribosomal DNA interspace region was evaluated for species-specific polymorphism. B. abortus, B. melitensis, B. suis, and B. canis produced identical PCR interspace profiles. However, these PCR products were unique to brucellae, allowing them to be readily distinguished from other gram-negative bacteria (including Bartonella spp. and Agrobacterium spp.). Carbohydrate profiles differentiated B. canis from the other three Brucella species due to the absence of the rare amino sugar quinovosamine in the three other species. PCR of the rRNA interspace region is useful in identification of the genus Brucella, while carbohydrate profiling is capable of differentiating B. canis from the other Brucella species.     

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Effect of P39 Gene Deletion in Live Brucella Vaccine Strains on Residual Virulence and Protective Activity in Mice

The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could provide a means for differentiating vaccinated from infected animals.     

Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1–Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC‐vaccinated mice displayed a strong recall proliferative response with high amounts of IL‐4, IL‐12 and IFN‐γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.     

Development and Characterization of Mouse Models of Infection with Aerosolized Brucella melitensis and Brucella suis

There is a need to identify vaccines that can protect against Brucella, a potential bioterrorism agent. We have developed mouse models of infection with aerosolized Brucella melitensis and Brucella suis and demonstrated their utility for the evaluation of vaccines using the model live B. melitensis vaccine strain Rev.1.Brucellosis is a zoonotic disease that is caused by Brucella species. Four species, Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis, are currently known to be pathogenic to humans (1, 5, 14). In animals, brucellosis can have a huge economic impact, since infection can lead to abortions, stillbirths, and the loss of fertility in livestock. In comparison, brucellosis in humans is a debilitating disease characterized by fever, sweats, and aches. In approximately 5% of cases it can be fatal when complications, usually endocarditis, arise (4). The illness can last a number of weeks, and even with antibiotic treatment, relapses can occur. Brucellosis is common in developing countries and areas without effective animal disease control policies. In these countries, the microorganisms are usually transmitted through ingestion, inhalation, or direct skin contact. Unpasteurized milk is a common source of infection, as is inhalation from carcasses among abattoir workers (5, 11). There are several live attenuated vaccines licensed for use in animals. Of these, the most widely used are B. melitensis Rev.1 and B. abortus S19 or RB51 (reviewed in reference 17). These vaccines are unsuitable for use in humans since they are insufficiently attenuated and still cause disease (1, 17).Brucellosis is one of the most-common laboratory-acquired infections; it is readily aerosolized and highly infectious. Brucella species have been considered potential biological warfare agents and are classed as category B threat agents (15). B. suis was the first agent weaponized by the United States, in 1952 (6). Furthermore, there are also claims that Brucella bacteria were used by the Japanese Manchuria Unit and were developed by the former Soviet Union Biopreparat offensive biological weapon program (15). Since it is possible that a bioterrorist attack with Brucella bacteria would result in aerosolized bacteria causing inhalational infection, there is a requirement to develop and utilize appropriate animal models of aerosolized Brucella infection in order to evaluate the efficacy of vaccines or therapies for human brucellosis.A small-rodent model of brucellosis offers advantages over the use of larger animals for preliminary studies, including the relative ease of use, ethical acceptability, and cost. Here we describe the development and characterization of mouse models of infection with aerosolized B. melitensis and B. suis bacteria and demonstrate the utility of these models in evaluating vaccines and therapeutics for inhalational brucellosis using the model live attenuated B. melitensis Rev.1 vaccine. A mouse model of intranasal (i.n.) infection with B. melitensis has previously been described (12), and since we started this work, two other laboratories have described studies of infection of mice with aerosolized Brucella bacteria. Kahl-McDonagh et al. described aerosol infection of BALB/c mice with B. abortus 2308 and B. melitensis 16 M and the use of these models for the evaluation of protective efficacy of deletion mutants (10). Olsen et al. similarly described the infection of BALB/c mice with aerosolized strain 16 M or 2308 and demonstrated that vaccination with the live attenuated animal vaccine B. abortus RB51 provided protection against intraperitoneal but not aerosolized B. abortus challenge (13). Our findings both support and extend the data generated in these studies.     

Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen

Brucellosis is one of the most common zoonotic diseases caused by species of Brucella. At present, there is no commercially available vaccine for the human brucellosis. Brucella melitensis and Brucella abortus are the main causes of human brucellosis, worldwide. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved among human Brucella pathogens. The purpose of the current study was to evaluate and compare the immunogenicity and protective efficacy of the L7/L12-TOmp31 construct administered as DNA/DNA and DNA/Pro vaccine regimens. Vaccination of BALB/c mice with the DNA/Pro regimen provided more protection levels against B. melitenisis and B. abortus challenge than did the DNA/DNA regimen. IgG1 and IgG2a titers were higher in the sera from DNA/Pro-immunized mice than in those from mice immunized with DNA alone. Moreover, splenocytes from DNA/Pro-immunized mice produced significantly higher levels of IFN-γ than did those from mice given DNA alone. The pcDNA-L7/L12-TOmp31 priming followed by rL7/L12-TOmp31 boosting led to improved protection against B. abortus or B. melitensis infection.     

Aerosol infection of BALB/c mice with Brucella melitensis and Brucella abortus and protective efficacy against aerosol challenge

Brucellosis is a zoonotic disease with a worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. In order to engineer superior vaccine strains against Brucella species for use in animals as well as in humans, the possibility of challenge infection via aerosol needs to be considered to properly evaluate vaccine efficacy. In this study, we assessed the use of an aerosol chamber to infect deep lung tissue of mice to elicit systemic infections with either Brucella abortus or B. melitensis at various doses. The results reveal that B. abortus causes a chronic infection of lung tissue in BALB/c mice and peripheral organs at low doses. In contrast, B. melitensis infection diminishes more rapidly, and higher infectious doses are required to obtain infection rates in animals similar to those of B. abortus. Whether this difference translates to severity of human infection remains to be elucidated. Despite these differences, unmarked deletion mutants BAΔasp24 and BMΔasp24 consistently confer superior protection to mice against homologous and heterologous aerosol challenge infection and should be considered viable candidates as vaccine strains against brucellosis.     

Protective Live Oral Brucellosis Vaccines Stimulate Th1 and Th17 Cell Responses

Zoonotic transmission of brucellosis often results from exposure to Brucella-infected livestock, feral animals, or wildlife or frequently via consumption of unpasteurized milk products or raw meat. Since natural infection of humans often occurs by the oral route, mucosal vaccination may offer a means to confer protection for both mucosal and systemic tissues. Significant efforts have focused on developing a live brucellosis vaccine, and deletion of the znuA gene involved in zinc transport has been found to attenuate Brucella abortus. A similar mutation has been adapted for Brucella melitensis and tested to determine whether oral administration of ΔznuA B. melitensis can confer protection against nasal B. melitensis challenge. A single oral vaccination with ΔznuA B. melitensis rapidly cleared from mice within 2 weeks and effectively protected mice upon nasal challenge with wild-type B. melitensis 16M. In 83% of the vaccinated mice, no detectable brucellae were found in their spleens, unlike with phosphate-buffered saline (PBS)-dosed mice, and vaccination also enhanced the clearance of brucellae from the lungs. Moreover, vaccinated gamma interferon-deficient (IFN-γ^−/−) mice also showed protection in both spleens and lungs, albeit protection that was not as effective as in immunocompetent mice. Although IFN-γ, interleukin 17 (IL-17), and IL-22 were stimulated by these live vaccines, only RB51-mediated protection was codependent upon IL-17 in BALB/c mice. These data suggest that oral immunization with the live, attenuated ΔznuA B. melitensis vaccine provides an attractive strategy to protect against inhalational infection with virulent B. melitensis.     

Some structural and biological properties of Brucella endotoxin

Hot phenol-water extraction of smooth Brucella abortus and B. melitensis cells yielded a toxic fraction which was recovered from the phenol phase (fraction 5). Chemically, fractions 5 from both Brucella species were lipid-carbohydrate-protein-2 keto-3-deoxyoctulosonic acid complexes which were stable to heat and resistant to Pronase digestion. Electron micrographs of the Brucella toxins were morphologically indistinguishable from those of enterobacterial endotoxins. Biologically, Brucella toxins were lethal for mice and immunogenic for rabbits. An intravenous injection of Brucella toxin induced severe leukopenia with subsequent leukocytosis in mice. Cross-tolerance experiments with mice demonstrated that pretreatment with B. abortus toxin lessened the hypoferremia produced by challenge with Escherichia coli endotoxin. Furthermore, fractions 5 from B. abortus and B. melitensis were able to form hybrids with E. coli and Salmonella enteritidis endotoxins and also with each other. Although Brucella toxins possess many structural and biological properties in common with endotoxins from the Enterobacteriaceae, some quantitative differences in their biological potencies were observed. Brucella toxins were relatively innocuous in tests for pyrogenicity in rabbits and lethality for chick embryos. In nonspecific protection tests, Brucella toxin had only 1/75 the potency of E. coli endotoxin in protecting mice against challenge with virulent S. typhi. However, on the basis of the data presented and on the work done previously, we concluded that the heat-stable toxins of B. abortus and B. melitensis were endotoxins.     

VirB12 is a serological marker of Brucella infection in experimental and natural hosts

The Brucella species type IV secretion system, encoded by the virB1-12 locus, is required for intracellular replication and persistent infection in vivo. The requirement of VirB proteins for infection suggests that they are expressed in vivo and may therefore represent serological markers of infection. To test this idea, we purified recombinant VirB1, VirB5, VirB11, and VirB12 and tested for their recognition by antibodies in sera from experimentally infected mice and goats by using an indirect enzyme-linked immunosorbent assay. Antibody responses to VirB12 but not to VirB1, VirB5, or VirB11 were detected in 20/20 mice experimentally inoculated with Brucella abortus and 12/12 goats experimentally infected with Brucella melitensis. The potential use of VirB12 as a serological tool for the diagnosis of brucellosis was evaluated in the natural bovine host. Serum samples from 145 cattle of known serology (29% negative and 71% positive) were analyzed for the production of antibody responses to VirB12. One hundred two cattle samples (70.3%) were positive for antibodies to VirB12, while 43 samples were negative (29.7%). A positive serological response to VirB12 correlated with positive serology to whole B. abortus antigen in 99% of samples tested. These results show that VirB12 is expressed during infection of both experimental and natural hosts of Brucella species, and they suggest that VirB12 may be a useful serodiagnostic marker for brucellosis.     

Epitope Mapping of the Brucella melitensis BP26 Immunogenic Protein: Usefulness for Diagnosis of Sheep Brucellosis

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.     

Influence of Brucella endotoxins on the initiation of antibody-forming spleen cells in mice immunized with sheep red blood cells

Changes in the formation of antibodies to sheep red blood cells (sRBC) in the presence of Brucella extracts was studied in mice whose spleen cells were assayed by the Jerne procedure. Two strains of female mice were employed. Brucella extracts were prepared: (i) by trichloroacetic acid extraction (LPSN), (ii) by phenol extraction (LPS), and (iii) by hot acetic acid hydrolysis (Ps). B. abortus LPSN and B. melitensis LPSN or LPS, administered with sRBC, stimulated the specific response to sRBC, but only at high doses of endotoxins. B. abortus LPSN and B. melitensis LPSN suppressed nonspecific responses against horse red blood cells (hRBC), in contrast to the typical events following administration of Serratia marcescens endotoxin (or endotoxins from other ubiquitous organisms). In CD-1 mice, B. abortus Ps depressed the specific anti-sRBC response. Attempts to presensitize mice with abortus LPSN resulted in a stimulation of the response to sRBC, but pretreatment with B. melitensis LPSN had an inhibitory effect. When injected alone, Brucella endotoxins activated anti-sRBC antibody-forming cells but not anti-hRBC cells. B. abortus Ps was unable to modify the background number of anti-sRBC cells and inhibited the hRBC response. These data suggest (i) that there exists a “common antigen” between Brucella cells and sRBC and (ii) that the so-called primary response to endotoxins from ubiquitous organisms represents a secondary response to already naturally sensitized animals.     

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.     

Identification of a new immunogenic candidate conferring protection against Brucella melitensis infection in Mice

Identification of bacterial proteins that contribute to the replication and survival of the engulfed bacteria within phagolysosome is critical in the pathogenesis of intracellular bacteria. Heat shock proteins (HSPs) are molecular chaperones that prevent unwanted protein aggregation and protect the bacteria against cell stress. In order to study the potential of HspA for development of a Brucella subunit vaccine, immunogenicity and protective efficacy of recombinant HspA (rHspA) from Brucella melitensis was evaluated in BALB/c mice. The hspA gene was cloned in pDEST42 and the resulting recombinant protein was used as subunit vaccine. rHspA elicited mixed TH1/TH2 immune responses with higher titers of specific IgG1 than IgG2a. In lymphocyte transformation assay, splenocytes of immunized mice exhibited a strong recall proliferative response with high amounts of IFN-γ, IL-12, IL-10 and IL-6 and very low levels of IL-5 and IL-4 production. The protective effect of rHspA was evaluated by administering rHspA to mice that resulted in a significant reduction in bacterial load and high degree of protection against B. melitensis challenge compared to control mice (p < 0.001). These results suggest that rHspA may be a useful candidate for the development of subunit vaccine against brucellosis.     

Crucial Role of Gamma Interferon-Producing CD4+ Th1 Cells but Dispensable Function of CD8+ T Cell,B Cell,Th2, and Th17 Responses in the Control of Brucella melitensis Infection in Mice

Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4⁺ T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8⁺ T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.     

Validation of a simple universal IELISA for the diagnosis of human brucellosis

The definitive diagnosis of brucellosis requires isolation of the agent, although negative isolation does not rule out the infection. In contrast, serological testing is more sensitive and, therefore, preferred in clinical practice. The majority of reported cases around the world were caused by Brucella melitensis, B. abortus, B. suis and B. canis. The first three species contain O-polysaccharide (OPS) on the cell surface, but B. canis contains no measurable OPS on the rough lipopolysaccharide (R-LPS). A universal indirect enzyme immunoassay for the detection of serum antibody to smooth and rough Brucella spp. in both normal (u-IELISA®) and rapid forms (R-u-IELISA®) has been developed, and, therefore, the potential use of this method was assessed in comparison to cELISA, conventional tests, IELISA and RSAT on a total of 478 sera. The 77 sera from blood donors with no clinical or epidemiological evidence of brucellosis and negative serological tests showed a specificity of 100 % for both u-IELISA® and R-u-IELISA®, with a cut-off value of %P 24 and %P 18, respectively. Sera from 49 culture-positive cases (16 B. suis, 15 B. abortus, 12 B. melitensis and 6 B. canis) yielded a sensitivity of 98 % for u-IELISA® and 95.9 % for R-u-IELISA®. In general, u-IELISA® showed good correlation with cELISA and IELISA for the detection of antibodies to smooth and rough Brucella strains, as well as for monitoring patients during treatment, but R-u-IELISA® seems to need additional optimisation. u-IELISA® is simple to perform and could be a suitable test for field laboratories and hospitals lacking skilled personnel.     

Molecular characterization of Brucella melitensis Rev.1 strain in aborted sheep and goats in Iran

Live attenuated Brucella melitensis Rev.1 strain is currently used in some countries against caprine and ovine brucellosis. Although live B. melitensis Rev.1 strain is considered the best vaccine available for the prophylaxis of brucellosis in sheep and goats, its residual abortifacient potential in pregnant animals is still a problem. We used a specific PCR–RFLP method to distinguish B. melitensis Rev.1 from other Brucella field isolates derived from aborted fetuses of sheep and goats in Iran. Molecular typing of 46 Brucella spp. isolates revealed that five strains were B. melitensis Rev.1 vaccine strain.     

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.