Michel S. Zygmunt

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export

Background The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Characterization of Heat, Oxidative, and Acid Stress Responses in Brucella melitensis

ABSTRACT Brucella melitensis is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells. Therefore, it has to adapt to a range of different hostile environments. In order to understand the mechanisms of intracellular survival employed by virulent B. melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used. Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis. On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified. Some of them were previously reported forBrucella, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD). Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit. Results indicated thatB. melitensis could bring specific regulatory mechanisms into play in response to stress conditions. For example, the ribosome releasing factor in B. melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B. melitensis in response to heat stress. Some of the identified proteins and their potential specific regulation could be required for the adaptation of B. melitensis to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence.

DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers

The omp-31 gene, encoding a major outer-membrane protein in Brucella melitensis, was PCR-amplified from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-31 published sequence. Amplification of omp-31 was achieved from DNA of all Brucella species with the exception of Brucella abortus, the only Brucella species where expression of omp-31 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (4.4-17 kb) containing B. melitensis 16M omp-31 and adjacent DNA of different sizes, with HindIII-digested total DNA showed that a large fragment, comprising the entire omp-31 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specific markers for B. abortus and B. melitensis. At the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-31 revealed specific markers for Brucella ovis, Brucella canis and Brucella suis bv. 2. Using a combination of omp-31 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1, 3, 4 and 5. Results presented in this paper demonstrate the potential of omp-31 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-31 and flanking DNA. In such a large deletion, other genes in addition to omp-31 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Brucella species.

Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes.

Culture is considered as the reference standard assay for diagnosis of Brucella spp. in humans and animals but it is time-consuming and hazardous. In this study, we evaluated the performances of newly designed real-time PCR assays using TaqMan probes and targeting the 3 following specific genes: (i) the insertion sequence IS711, (ii) bcsp31 and (iii) per genes for the detection of Brucella at genus level. The real-time PCR assays were compared to previously described conventional PCR assays targeting the same genes. The genus-specificity was evaluated on 26 Brucella strains, including all species and biovars. The analytical specificity was evaluated on a collection of 68 clinically relevant, phylogenetically related or serologically cross-reacting micro-organisms. The analytical sensitivity was assessed using decreasing DNA quantities of Brucella ovis, B. melitensis bv. 1, B. abortus bv. 1 and B. canis reference strains. Finally, intra-assay repeatability and inter-assay reproducibility were assessed. All Brucella species DNA were amplified in the three tests. However, the earliest signal was observed with the IS711 real-time PCR, where it varied according to the IS711 copy number. No cross-reactivity was observed in all three tests. Real-time PCR was always more sensitive than conventional PCR assays. The real-time PCR assay targeting IS711 presented an identical or a greater sensitivity than the two other tests. In all cases, the variability was very low. In conclusion, real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella.

Brucella papionis sp. nov., isolated from baboons (Papio spp.)

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T)).

The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB/c mice

Immunogenicity and protective activity against Brucella ovis of detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli, purified rough lipopolysaccharide from B. ovis (R-LPS) and a mixture of rOmp31 extract and R-LPS (rOmp31 extract + R-LPS) were assessed in BALB/c mice. The experimental vaccines were compared with a hot saline extract (HS extract) from B. ovis mainly composed of outer membrane proteins (OMPs) and R-LPS, and known to be protective in mice against a B. ovis infection. Serum antibodies to Omp31 and R-LPS were detected in the corresponding mice using Western blotting with B. ovis whole-cell lysates and ELISA with purified antigens. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. ovis. A significantly lower number of B. ovis colony-forming units in spleens relative to unimmunized (saline injected) controls were considered as protection. Mice immunized with rOmp31 extract or rOmp31 extract mixed with R-LPS developed antibodies that bound to the B. ovis surface with similar titers. Vaccination with rOmp31 extract plus R-LPS provided the best protection level, which was comparable with that given by HS extract. Similar protection was also obtained with rOmp31 extract alone and, to a lesser degree, with R-LPS. Comparisons between groups showed that an extract from E. coli-pUC19 (devoid of Omp31) provided no protection relative to either HS extract, rOmp31 extract or rOmp31 extract mixed with R-LPS. In conclusion, the recombinant Omp31 associated or not with B. ovis R-LPS, could be an interesting candidate for a subcellular vaccine against B. ovis infection.

Outer-membrane protein- and rough lipopolysaccharide-specific monoclonal antibodies protect mice against Brucella ovis

Brucella ovis, a naturally virulent rough Brucella species, is the aetiological agent of ram epididymitis. The identification of protective antigens is necessary to obtain a safe, specific subcellular vaccine. Monoclonal antibodies (MAbs) directed at both brucella outer-membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS) in a mouse protection test were used to identify potential targets for humoral immunity. Mixtures of MAbs directed at the 16.5-, 25-27-, 31-34- and 36-38-kDa OMPs conferred significant protection 7 days after challenge with reference strain B. ovis 63/290 compared with controls receiving either saline or an anti-brucella O-polysaccharide MAb. Furthermore, an anti-R-LPS MAb tested alone conferred protection at a level comparable with that obtained with the mixture of anti-OMP MAbs. The combination of protective OMP MAbs with the anti-R-LPS MAb was also strongly protective. One combination of OMP MAbs, which bound intensely to B. ovis in vitro, was ineffective. These results indicate that B. ovis OMPs and R-LPS are targets for protective antibodies and that they can be regarded as candidates for ram epididymitis subcellular vaccines.

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.

Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep

Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.