Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60 T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60 T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of .80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60 T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60 T (5BCCN 09-2 T 5DSM 101715 T ).Abbreviations: AAI, average amino acid identity; ANI, average nucleotide identity.The GenBank/EMBL/DDBJ accession numbers for the genome sequences (chr1 and chr2) of strains F60 T and F965 are LN997863-LN997864 and LN998033-LN998034, respectively, and can be retrieved from the European Nucleotide Archive under

The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed. The detection limit of the assay was 10 3 to 10 4 bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.Within the genus Francisella there are two known species, Francisella tularensis and Francisella philomiragia, which have a 16S rRNA gene sequence similarity of more than 98% (6). F. tularensis, a gram-negative, small (0.2 to 0.7 by 0.2 m) facultative intracellular bacterium,

From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration. Brucella species are facultatively intracellular pathogens responsible for one of the world's most widespread zoonotic diseases. The bacteria may cause reproductive failure and abortion in domestic animals and a potentially debilitating multiorgan infection in humans. Like Agrobacterium and Rhizobium spp., brucellae belong to the order of Rhizobiales of the ␣-2 subgroup of Proteobacteria. Members of the class Alphaproteobacteria include organisms that are either mammalian or plant pathogens or symbionts (12). Within the family Brucellaceae, Ochrobactrum, a genus comprising soil-associated facultative human pathogens, contains the closest phylogenetic neighbors to Brucella. Ochrobactrum intermedium and Brucella spp. are 98.8% identical in their 16S rRNA gene sequences (14). Furthermore, Brucella species are closely related to each other (monophyletic genus), showing 98 to 99% similarity in most of the coding sequences. Despite this high genetic homology, brucellae differ widely in host tropism, phenotypic characteristics, and pathogenicity (38).The phylogeny of Brucella species does not always match that of their nominal mammalian hosts (36). Currently, the genus Brucella...

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