Molecular typing divides marine mammal strains of Brucella into at least three groups with distinct host preferences.

In order to investigate the genetic relationships within Brucella isolated from marine mammals, two genome-based typing methods, variable number of tandem repeats (VNTR) typing and multilocus sequence analysis (MLSA), were applied to a selection of 74 marine mammal isolates. All isolates were examined by VNTR and data were compared with multilocus sequencing data from a subset of 48 of these. Marine mammal brucellae are distinct from classically recognized species by these methods and appear to correspond to three major genetic groups, which reflect distinct preferred hosts. One group contains isolates predominantly found in pinnipeds (seals) and corresponds to the previously proposed species 'Brucella pinnipediae'. However, isolates corresponding to the previously proposed species 'Brucella cetaceae' fall into two distinct groups that appear to have different preferred cetacean hosts (porpoises and dolphins). Furthermore, these two groups appear less closely related to each other than either group is to 'B. pinnipediae' isolates. The groups identified by VNTR typing and MLSA are completely congruent. The relevance of these findings to current proposals to recognize two species of marine mammal Brucella is discussed.

Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp.

Members of the genus Brucella infect many domesticated and wild animals and cause serious zoonotic infection in humans. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. We report here on a molecular subtyping system based on 21 variable-number tandem-repeat (VNTR) loci consisting of 13 previously unreported loci and 8 loci previously reported elsewhere. This approach was applied to a collection of 121 Brucella isolates obtained worldwide and representing all six classically recognized Brucella species. The size of repeats selected for inclusion varied from 5 to 40 bp giving VNTR loci with a range of diversities. The number of alleles detected ranged from 2 to 21, and Simpson's diversity index values ranged from 0.31 to 0.92. This assay divides the 121 isolates into 119 genotypes, and clustering analysis results in groups that, with minor exceptions, correspond to conventional species designations. Reflecting this, the use of six loci in isolation was shown to be sufficient to determine species designation. On the basis of the more variable loci, the assay could also discriminate isolates originating from restricted geographical sources, indicating its potential as an epidemiological tool. Stability studies carried out in vivo and in vitro showed that VNTR profiles were sufficiently stable such that recovered strains could readily be identified as the input strain. The method described here shows great potential for further development and application to both epidemiological tracing of Brucella transmissions and in determining relationships between isolates worldwide.