A total of 51 strains (including type, reference, vaccine, and field strains) representing all species and biovars of Brucella formed a single deoxyribonucleic acid-deoxyribonucfeic acid hybridization group (Sl nuclease method). Accordingly, we propose that only one species, Bruceila 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). Carmichael and Bruner 1968 (8, 20). These species were proposed on the basis of differential tests and different preferential hosts (8).The identification of these bacteria at the species level is difficult and best suited to reference laboratories.In 1968, Hoyer and McCullough (13,14) studied six strains of Brucella, one of each species, by deoxyribonucleic acid (DNA)-DNA hybridization, using the DNA-agar method and a filter method. These authors (13,14) found that their strains are highly related. Unfortunately, the work of Hoyer and McCullough (13, 14) had a limited impact on the taxonomy of the genus Brucella, probably because DNA hybridization technology and its interpretation had not reached the state of development which it now has, too few strains were studied, and only two of the strains were type strains (B. melitensis 16M and B. canis RM6/66).The purpose of the present study was to apply modern DNA-DNA hybridization methods to 51 strains (including type strains) representing all present species and biovars of Brucella. Our results give no support to the division of the genus Brucella into more than one species. Consequently, we propose that only one species, B. melitensis, be recognized in this genus. MATERIALS AND METHODSBacterial strains. The 51 strains used in this study are listed in Table 1. All strains were kept freeze-dried in the Brucella Culture Collection, INRA-Nouzilly , Monnaie, France. Cultures were grown on Trypcase Soy Agar (Bio-Merieux, Marcy l'Etoile, France) supplemented with 0.1% (wthol) yeast extract (Difco Laboratories, Detroit, Mich.) (TSAYE medium). For fastidious strains (B. abortus biovar 2 and African biovar 3 and B. ovis), sterile equine serum (BioMerieux) was added to TSAYE medium to a final concentration of 5% (vol/vol) (TSAYES medium). The strains were checked for purity and species and biovar characterization by using standard procedures (1).DNA preparation. For each strain, six Roux flasks containing 250 ml of TSAYE or TSAYES medium were inoculated * Corresponding author with a 5-ml suspension in saline (pH 6.8) made with a 1-day culture on TSAYE or TSAYES medium slopes. After incubation for 72 h at 37°C under air (supplemented with 10% [vol/vol] C 0 2 if required), the growth from the six Roux flasks was harvested with 120 ml of 0.05 M tris(hydroxy me thy1)-aminomethane-0.05 M e t hy lenediaminetetraacetic acid-0.1 M NaCl buffer (pH 8). This suspension was checked for purity by streaking onto TSAYE or TSAYES medium. Then 2.6 ml of a 25% (wthol) aqueous solution...
Seventy-seven Bmcella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an N 50 bp deletion a t the 3' terminal end of the gene in all Brucella owis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Bmcella species and within the Bmcella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. owis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Bmcella species as was previously suggested by Ficht et a/. (1990; Mo/ Microbiol4,1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucella species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.
The omp-37 gene, encoding a major outer-membrane protein in Bnrcella melitensis, was PCR-amplif ied from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-37 published sequence. Amplification of omp-37 was achieved from DNA of all Bnrcella species with the exception of Brucella abortus, the only Brucella species where expression of omp-37 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (44-1 7 kb) containing B. melitensis 16M omp-37 and adjacent DNA of different sizes, with Hindllldigested total DNA showed that a large fragment, comprising the entire omp-37 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-specif ic markers for B. abortus and B. melitensis. A t the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-37 revealed specific markers for Bnrcella ovis, Bnrcella canis and Bnrcella suis bv. 2. Using a combination of omp-37 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-37 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-37 and flanking DNA. In such a large deletion, other genes in addition to omp-37 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 Bnrcella species.
U n i t C d I m m u nolog ie-M icro bio log ie, B-5000 Namur, BelgiumIsolation 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-specif ic 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-specif ic probes, and b y 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.
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