Brucella Microti

[Emmanuelle Thaller]

TheB. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 μg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9 % identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile.

Given that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity in this Brucella species.

Hubalek et al. [6] reported brucellosis in common voles (Microtus arvalis) in the Czech Republic, and the pathogen was described as B. microti [7]. Since then it has been detected in soil samples [8] and lymph nodes of red foxes [9] in the Czech Republic and Austria. The whole genome sequence of the type strain CCM 4915T was published in 2009 [10], its pathogenic potential was investigated in murine models [11], and the intraspecies biodiversity of the species was investigated by Al Dahouk and colleagues [12]. As B. microti is the only Brucella species known to persist in soil, it is of particular interest as an environmental reservoir and data on the distribution, ecology, zoonotic potential, genomic organization of this bacterium and relatedness to other strains are urgently needed [12].

A B. microti strain was isolated from the submandibular lymph node of a female wild boar (Sus scrofa) in Hungary near Rajka town (47.9 N, 17.2 E) in close vicinity to the Austrian border in September, 2014. This is the first isolation of B. microti from wild boar. The aim of the study was to describe the isolation conditions and to determine the morphological, biochemical, and genetic characteristics of the isolate and compare it with previous B. microti isolates originating from different host species.

The submandibular lymph node sample of the wild boar did not show any gross pathological or histological changes and was negative with B. abortus, B. suis and B. canis specific sera in the immunohistochemical examinations. The directly inoculated Brucella selective agar remained negative during the incubation period but colonies appeared in pure culture after two days incubation on the Brucella selective agar inoculated with the enriched Brucella selective broth. The colonies were small, translucent, had creamy consistency and displayed a brownish pigmentation after 3 days of incubation. The Gram-negative and modified acid-fast, small coccobacilli demonstrated oxidase, catalase, and urease activity, but failed to produce H2S. The isolate grew in the presence of 20 μg/ml thionin and basic fuchsin and did not require CO2 for its growth. The isolate showed strong agglutination with sera A and M, but agglutination was not observed with serum R. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9 % identity, with positive reactions in nitrate reduction, urease activity, D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, adipic acid and malic acid assimilation tests and negative reactions for indole production, D-glucose fermentation, arginine dihidrolase tests, esculin and gelatin hydrolysis, β-galactosidase activity, and in D-mannitol, potassium gluconate, capric acid, trisodium citrate and phenylacetic acid assimilation reactions.

The hunting area in Hungary where the wild boar was harvested is close to Austria where several previous B. microti isolates originated. Hungary is located in Central Europe and current data indicate that countries in this area are enzootic foci of B. microti. We hypothesize that the wild boar acquired the B. microti strain while eating a rodent, a carcass (e.g., dead fox), or simply rooting in the soil. The hunters did not report any gross pathological lesions in other parts of the carcass, so it seems likely that the wild boar did not develop any obvious clinical disease. Thus we suspect that wild boar merely serves as a reservoir species capable of maintaining and spreading the pathogen in nature.

The negative results of both the S and R type Brucella-specific immunohistochemical reactions imply that the amount of B. microti in the lymph node was below the detection limit of this method, which is also supported by the finding that the strain could only be isolated after enrichment in Brucella selective broth. These results and the lack of histological lesions in lymph node further support the hypothesis that the wild boar was only an asymptomatic carrier of this bacterium.

The morphological, growth and biochemical characteristics of the isolated B. microti strain were congruent with the description of Scholz et al. [7], including the described brownish pigmentation of the colonies after 3 days incubation. The isolate displayed a unique agglutination reaction, agglutinating with anti-A and anti-M but not with anti-R monospecific sera. The first common vole strains only agglutinated with anti-M serum [7], but Al Dahouk et al. [12] reported several different agglutination patterns; despite the M antigen dominancy a fox isolate only agglutinated with anti-A serum, while the soil isolates agglutinated all three monospecific sera.

The observed 30 SNPs between the whole genome sequence of the Czech common vole and Hungarian wild boar strain is surprisingly few, suggesting relatively high genetic homogeneity within B. microti in Central Europe. We omitted potential sources of variation such as multi-copy genetic elements, insertion sequences, and repeat regions from the SNP analysis. Thus, our estimate of the number of SNPs is a conservative one but is more likely to represent biologically meaningful differences. Moreover, SNPs from homologous loci have been demonstrated as reliable for phylogenetic analysis of Brucella species because of their coverage of the entire genome, relative stability over evolutionary time, ease of comparison, and inclusion of intergenic regions [21]. Therefore the primary aim of SNP analysis was to phylogenetically (at large evolutionary scale level) compare the present Hungarian B. microti isolate with the Czech B. microti strain.

The highly mutable genetic markers, VNTRs, are not captured in the re-sequenced draft genome. However, our amplicon sequencing of these loci allowed for placement of our isolate within a MLVA framework, indicating that the Hungarian wild boar B. microti isolate is clearly differentiated from the common vole, fox and soil isolates originated from Austria and the Czech Republic. The question regarding the source of infection in wild boar (i.e., common vole, fox, soil or other host) remains open and additional efforts to better understand the epidemiology and ecology of B. microti are needed.

This is the first isolation of Brucella microti from a wild boar, and also the first isolation of this species in Hungary. The information gained through bacteriological, histological, immunohistological and molecular analysis with emphasis on whole genome re-sequencing highly contribute to our knowledge of the host range, geographic distribution and genomic organization of B. microti.

ZR isolated the strain, performed the morphological, biochemical tests, multiplex PCRs and wrote the manuscript. ZK performed MLVA. D, KD, JTF analyzed the whole-genome sequence data. KB and SM performed the whole-genome re-sequencing. LS performed the pathological, histological and immunohistochemical examinations. SJ performed morphological tests. MG designed the study, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated.

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Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

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