Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.     

Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.     

Protective properties of rifampin-resistant rough mutants of Brucella melitensis

Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.     

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.     

Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis.

Brucella group 3 antigens (Ags) are outer membrane proteins (OMPs) with a molecular mass ranging from 25 to 30 kDa. The OMPs are of interest partially because of their potential use as vaccine and diagnostic reagents. We used human convalescent antibody (Ab) to clone a gene that encoded a 28-kDa protein from a lambdagt11 library of Brucella melitensis 16M genomic DNA. DNA sequence analysis revealed a single open reading frame that would encode a protein of 26,552 Da. The 28-kDa protein had a primary amino acid sequence that was 43% similar to a previously described Brucella abortus group 3 Ag, Omp25 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The similarity to a known group 3 OMP, immunoreactivity with Ab prepared against B. abortus group Ags, immunolabeling of whole cells, and Southern hybridization led to our conclusion that the B. melitensis 28-kDa protein was a group 3 protein distinct from B. abortus Omp25. We designated the B. melitensis protein Omp28. Human convalescent sera from patients infected with B. abortus and Brucella suis as well as rabbit antisera prepared against killed B. abortus whole cells recognized B. melitensis Omp28 on Western blots (immunoblots). Furthermore, mice and goats infected with smooth strains of B. melitensis produced Abs against Omp28. Our results may begin to explain the variability in molecular weight seen in Brucella group Ags and point toward their possible use in vaccination against infection as well as diagnosis of the disease.     

Characterization of smooth Brucella lipopolysaccharides and polysaccharides by monoclonal antibodies.

Two mouse monoclonal antibodies (mAb) generated to the M antigen of Brucella melitensis 16M were analysed. Binding profiles of both monoclonals were established by a competitive enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides, O polysaccharides and native hapten polysaccharides from B. melitensis 16M, B. abortus 544 and Yersinia enterocolitica O:9. Using this assay, significant differences in the reactivity of both antibodies were found with A and M antigens from Brucella spp. and the O polysaccharide from Y. enterocolitica O:9. These findings are consistent with the simultaneous expression of A and M epitopes on the lipopolysaccharide of all smooth Brucella strains. Quantitatively similar inhibitory powers were established for the native hapten and O polysaccharide from B. melitensis 16M. However, different behaviour was observed between both antigenic preparations obtained from B. abortus 544. The use of lipopolysaccharide-M-specific mAb in different serological tests instead of polyclonal antisera may be of great practical use for minimizing the risk of the appearance of cross-serological reactions between smooth Brucella strains and Y. enterocolitica O:9.     

Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.

Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.     

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.     

A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody.

A coagglutination test using monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 16M has been developed for the rapid identification of the smooth Brucella species. All reference strains of several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Brucella species, with the exception of B. abortus 45/20, were negative in the assay. Among the different organisms tested not belonging to the genus Brucella, serovar O9 of Yersinia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity, simplicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Brucella strains in diagnostic laboratories.     

Molecular characterization of a Brucella species large DNA fragment deleted in Brucella abortus strains: evidence for a locus involved in the synthesis of a polysaccharide.

A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment.     

Partial protection against Brucella infection in mice by immunization with nonpathogenic alphaproteobacteria

Previous findings indicate that Brucella antigens and those from nonpathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system. We hypothesized that immunization with NPAP would protect mice from Brucella infection. Mice were immunized subcutaneously with heat-killed Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38 (standard positive control) before intravenous challenge with Brucella abortus 2308. Cross-reacting serum antibodies against Brucella antigens were detected at the moment of challenge in all NPAP-immunized mice. Thirty days after B. abortus challenge, splenic CFU counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 than in the phosphate-buffered saline controls (protection levels were 0.80, 0.66, and 1.99 log units, respectively). In mice immunized intraperitoneally with cytosoluble extracts from NPAP or Brucella abortus, protection levels were 1.58 for the latter, 0.63 for O. anthropi, and 0.40 for M. loti. To test whether the use of live NPAP would increase protection further, mice were both immunized and challenged by the oral route. Immunization with NPAP induced a significant increase in serum immunoglobulin G (IgG), but not serum or fecal IgA, against Brucella antigens. After challenge, anti-Brucella IgA increased significantly in the sera and feces of mice orally immunized with O. anthropi. For all NPAP, protection levels were higher than those obtained with systemic immunizations but were lower than those obtained by oral immunization with heat-killed B. abortus. These results show that immunization with NPAP, especially O. anthropi, confers partial protection against Brucella challenge. However, such protection is lower than that conferred by immunization with whole Brucella or its cytosoluble fraction.     

Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.     

Characterization of a Brucella species 25-kilobase DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide

In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysaccharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells.     

Brucella abortus 1119-3 O-chain polysaccharide to differentiate sera from B. abortus S-19-vaccinated and field-strain-infected cattle by agar gel immunodiffusion.

Purified Brucella abortus 1119-3 and Brucella melitensis 16M lipopolysaccharide O-chain polysaccharides were not precipitated in agar gel immunodiffusion by any of 24 sera from vaccinated cattle but were precipitated by 18 of 24 sera from infected cattle. This difference can be used to differentiate sera of cattle vaccinated with B. abortus S-19 from sera of some field-strain-infected cattle.     

Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells

Null cyclic beta-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic beta-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.     

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.     

Deletion of purE attenuates Brucella melitensis 16M for growth in human monocyte-derived macrophages.

We constructed a defined purine-auxotrophic mutant of Brucella melitensis 16M by chromosomal gene replacement. We electroporated B. melitensis 16M with suicide plasmids containing a kanamycin resistance cassette that replaced 226 bp at the carboxyl end of purE, the intergenic region, and 18 bases of the purK open reading frame. Recombinant B. melitensis delta purE201 required exogenous purines for growth on minimal media. Purine auxotrophy was complemented by electroporation of B. melitensis delta purE201 failed to grow in human monocyte-derived macrophages, while the growth of wild-type 16M and the complemented strain, delta purE201 (pSD5), increased by nearly two logs. These results suggest that B. melitensis delta purE201 will be attenuated in animals and humans and thus may be useful as a live attenuated vaccine.