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.Brucellosis is a zoonotic disease of major public health, animal welfare, and economic significance worldwide. In humans, infection with Brucella can lead to a chronic debilitating infection; in domesticated animals, the main symptom is reproductive failure. Disease in humans usually reflects occupational exposure or the consumption of unpasteurized dairy products. Brucellosis remains a major problem in many parts of the world, particularly Mediterranean regions, western Asia, and parts of Africa and Latin America (11), although in many developed countries it has been eradicated or severely curtailed by a combination of strict veterinary hygiene measures, monitoring programs, and improved food safety measures. Brucella species have also long been considered potential biological warfare agents, and in 1954 Brucella became the first biological agent to be treated as a weapon and field tested on animals under the old U.S. offensive biological weapons program. Recent history has raised awareness in this area (28), and the organism remains on the list of Centers for Disease Control and Prevention category B potential biological warfare agents (25).Classical Brucella taxonomists developed a classification system that recognized six species based on subtle phenotypi...

LETTERSgenotyping M. bovis strains isolated from farm animals to help elucidate the source of infection and transmission of M. bovis in Taiwan.

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. INTRODUCTIONThe genus Brucella contains Gram-negative, facultatively intracellular pathogens that can infect many species of animals of economic importance, such as cattle, sheep, goats and pigs . Humans usually become infected through ingestion of unpasteurized dairy products or via occupational activities such as veterinary work, farming and butchery. Whilst animal brucellosis can present few symptoms and infection is often only recognized on abortion, in humans the disease causes flulike symptoms, including recurrent fever, chills, night sweats and nausea, as well as chronic fatigue and depression if the infection is not cleared early.Brucella can also infect wild animals (Davis, 1990), and in recent years it has also been isolated from various marine mammal species. In 1994, two reports described the first isolation of Brucella from marine mammals. These strains originated from four common seals (Phoca vitulina), two harbour porpoises (Phocoena phocoena) and a common dolphin (Delphinus delphis) in Scotland (Ross et al., 1994), and a captive bottlenose dolphin (Tursiops truncatus) in California, USA (Ewalt et al., 1994). Since these initial isolations, marine mammal Brucella have been isolated from a range of animals, including Atlantic white-sided dolphins (Lagenorhynchus acutus), striped dolphins (Stenella coeuleoalba), grey seals (Halichoerus grypus), hooded seals (Cystophora cristata), European otters (Lutra lutra) (Foster et al., 1996), Pacific harbour seals (Phoca vitulina richardsi) (Garner et al., 1997), minke whales (Balaenoptera acutorostrata) (Clavareau et al., 1998), harp seals (Phoca groenlandica), ringed seals (Phoca hispida) (Forbes et al., 2000) and whitebeaked dolphins (Lagenorhynchus albirostris) (Foster et al., 2002).Six Brucella species are currently recognized based on pheno...

Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of Brucella isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of Brucella isolates. The technique could readily differentiate Brucella spp. from all Ochrobactrum spp. representing the group of organisms most closely related to Brucella spp. Application of AFLP highlighted the genetic homogeneity of Brucella. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including Brucella abortus, B. melitensis, B. ovis, B. neotomae, marine mammal isolates (no species name), B. canis, and B. suis, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only B. suis and B. canis isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to Brucella and enhance the value of this approach.Brucella spp. comprise a closely related group of organisms that classical taxonomists divided into six species based on subtle phenotypic and antigenic differences and host specificity: Brucella abortus (bovine), B. melitensis (caprine and ovine), B. ovis (ovine), B. canis (canine), B. suis (porcine), and B. neotomae (only seen in the desert wood rat). The situation has recently been complicated by the identification of Brucella isolates in marine mammals that do not fit into any of the recognized species and themselves show intragroup diversity (4,14). Some of the species are classically divided into biovars such that several B. abortus, B. melitensis, and B. suis biovars are recognized. In addition, host specificity is not absolute; thus, B. melitensis and B. suis are important causes of bovine disease in some countries, and B. suis biovars 2, 4, and 5 have been associated with hares, reindeer, and rodents, respectively. The traditional view on Brucella taxonomy was challenged some time ago on the basis of the high degree of homology indicated by DNA hybridization experiments (26) and the inability to differentiate Brucella spp. by 16S rRNA sequencing. Although these findings conform better with the view of a single species within which the six classical spec...