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...

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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 Abbreviations: MLSA, multilocus sequence analysis; MLVA, multilocus variable number of tandem repeat analysis; RTD, routine test dilution.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, omp2a and omp2b gene sequences of strains F8/08-60 T and F8/08-61 are HG932316 and HG932317 (16S rRNA gene), KJ493822 and KJ493823 (omp2a) and KJ510540 and KJ510541 (omp2b), respectively.A supplementary table and a supplementary figure are available with the online Supplementary Material.

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Abstract. More than 10,000 Caspian seals (Phoca caspica) were reported dead in the Caspian Sea during spring and summer 2000. We performed necropsies and extensive laboratory analyses on 18 seals, as well as examination of the pattern of strandings and variation in weather in recent years, to identify the cause of mortality and potential contributory factors. The monthly stranding rate in 2000 was up to 2.8 times the historic mean. It was preceded by an unusually mild winter, as observed before in mass mortality events of pinnipeds. The primary diagnosis in 11 of 13 seals was canine distemper, characterized by broncho-interstitial pneumonia, lymphocytic necrosis and depletion in lymphoid organs, and the presence of typical intracytoplasmic inclusion bodies in multiple epithelia. Canine distemper virus infection was confirmed by phylogenetic analysis of reverse transcriptase-polymerase chain reaction products. Organochlorine and zinc concentrations in tissues of seals with canine distemper were comparable to those of Caspian seals in previous years. Concurrent bacterial infections that may have contributed to the mortality of the seals included Bordetella bronchiseptica (4/8 seals), Streptococcus phocae (3/8), Salmonella dublin (1/8), and S. choleraesuis (1/8). A newly identified bacterium, Corynebacterium caspium, was associated with balanoposthitis in one seal. Several infectious and parasitic organisms, including poxvirus, Atopobacter phocae, Eimeria-and Sarcocystis-like organisms, and Halarachne sp. were identified in Caspian seals for the first time.