Jean-Michel Verger

Brucella, a Monospecific Genus as Shown by Deoxyribonucleic Acid Hybridization

A total of 51 strains (including type, reference, vaccine, and field strains) representing all species and biovars of Brucella formed a single deoxyribonucleic acid-deoxyribonucleic acid hybridization group (S1 nuclease method). Accordingly, we propose that only one species, Brucella melitensis, be recognized in the genus. We recommend that other specific epithets formerly associated with the generic name Brucella be used in a vernacular form for biovar designation (e.g., Brucella melitensis biovar Abortus 1).

Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus

A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale. These strains were identified as brucellae by conventional typing tests. However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called Brucella maris. In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species. This observation was also recently made for a minke whale Brucella isolate. The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals. By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates. Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals. Therefore, grouping the marine mammal Brucella isolates into one species Brucella maris seems inappropriate unless the currently recognized Brucella species are grouped. With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B. pinnipediae and B. cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus.

Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale (Balaenoptera acutorostrata)

Isolation of Brucella spp. in marine mammals has been reported during the past several years. A Brucella strain from the spleen and liver of a minke whale (Balaenoptera acutorostrata) was isolated. Conventional typing methods indicated that this isolate was related to the genus Brucella but did not match the profiles of any known Brucella species or biovar. Successful PCR amplification of the Brucella rrs-rrl spacer sequence and of the insertion sequence IS6501 also indicated that the minke whale strain was related to the genus Brucella. In addition, the rrs gene of this strain shared a very high degree of nucleotide identity (>98%) with published Brucella spp. rrs sequences. However, RFLP studies using an IS6501-specific probe showed a unique profile for this strain in comparison with the profiles of the six known Brucella species. Moreover, analysis of the omp2 locus by PCR-RFLP, by Southern hybridization using omp2a- and omp2b-specific probes, and by DNA sequencing showed that the minke whale isolate possesses two copies of the omp2b gene instead of one omp2a and one omp2b gene copy or two copies of the omp2a gene described in the six known Brucella species. Thus, molecular typing methods showed that this isolate is clearly distinct from all other known Brucella species and strains. The specific molecular features of this minke whale Brucella isolate raise questions about the lineage between the Brucella strains isolated from marine mammals and the Brucella species isolated from terrestrial mammals.

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.

DNA polymorphism in the genus Brucella

The genus Brucella has been described as consisting of six species, three of them including several biovars, which display a high degree of DNA homology by DNA-DNA hybridization. However, DNA polymorphism able to differentiate the six Brucella species and some of their biovars has been shown to exist. This work reviews the DNA variability in the genus Brucella and discusses the relationships between its members according to this genetic variability and a proposal for their evolution based on genetic diversity of the omp2 locus.

Molecular Typing of Brucella With Cloned Dna Probes

Brucella constitutes a single genomic species (B. melitensis); however, for epidemiological studies, methods are needed for discriminating strains within this genomic species. DNA samples from 112 Brucella strains were cleaved by restriction endonucleases and the fragments separated by agarose gel electrophoresis and transferred to nylon membranes. When the DNA fragments on the membranes were probed with 32P-labelled 16 + 23 S rRNA from Escherichia coli, a single rRNA gene restriction pattern was obtained after cleavage with all endonucleases tested (HindIII, EcoRI, SmaI, and XhoI) except BamHI. This indicated high genomic homogeneity within the single Brucella species. Of 30 probes consisting of random Brucella DNA fragments cloned into lambda EMBL3, 20 yielded a single BamHI restriction pattern per probe when applied to 112 Brucella DNA tested. However, 7 probes yielded 3 to 12 different patterns among DNA tested. These patterns more-or-less correlated with the classification of strains into biogroups (Melitensis, Abortus, Suis, Neotomae, Ovis and Canis) and biovars (18 biovars represented). Probe A was capable of separating biogroup Melitensis from the other biogroups. Probe C separated the set of biogroups Melitensis-Abortus-Ovis from the other biogroups. By reference to the patterns obtained using 1 to 7 probes, the most frequently occurring biovars (Melitensis 1, Melitensis 3, Abortus 1, Abortus 3, Suis 2 and Ovis) could be distinguished from each other. Eight biovars showed more than one pattern with 1 to 7 probes. The proposed typing system should be useful for epidemiological subtyping and does not pose safety problems once the DNA has been extracted.

Comparison of the efficacy of Brucella suis strain 2 and Brucella melitensis Rev. 1 live vaccines against a Brucella melitensis experimental infection in pregnant ewes

The comparative efficacy of Brucella suis strain 2 (S2) and Brucella melitensis strain Rev. 1 (Rev. 1) live vaccines in protecting sheep against B. melitensis infection was evaluated by clinical and bacteriological examination of ewes vaccinated conjunctivally with a dose of 1 x 10(9) c.f.u. when 4 months old and then challenged with 5 x 10(7) c.f.u. of the B. melitensis virulent strain 53H38 (H38) at the middle of the first or second pregnancy following vaccination. Animals were considered to be protected when no abortion, no excretion of the challenge strain and no infection at slaughter occurred. The percentages of protection in Rev. 1-vaccinated groups challenged during either first (80%) or second (62%) pregnancy were significantly different (p < 0.001 and p < 0.05, respectively) compared with those of the relevant unvaccinated control groups. In contrast no significant difference in protection was found between the S2-vaccinated and control groups.

Classification of Brucella strains isolated from marine mammals using DNA-DNA hybridization and ribotyping

DNA-DNA hybridization showed that the Brucella strains recently isolated from marine mammals belong to the monospecific genus Brucella (more than 77% DNA relatedness). Ribotyping (HindIII rDNA restriction patterns) showed that they may represent a separate subgroup (marine type) specifically associated with marine mammals.

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

ABSTRACT 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.

Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp.

Seven genes of the wb locus of Brucella melitensis 16M involved in the biosynthesis of the lipopolysaccharide O-side chain have been recently identified, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC, coding, respectively, for proteins homologous to mannosyltransferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABC-type transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) the seven O-chain biosynthetic genes for polymorphism among Brucella spp. PCR-RFLP showed that the seven genes are highly conserved and occur even in the naturally rough species B. ovis and B. canis and also in rough strains of B. abortus and B. melitensis. Nevertheless, the few polymorphisms that were observed consisted of absence of additional restriction sites sometimes allowing differentiation at the species level (e.g. B. ovis) or at the biovar or strain level. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described.