al. Development of a real-time reverse transcriptase PCR assay for type A infl uenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40:3256-60. DOI: 10.1128/JCM.40.9.3256-3260.2002 Isolation of Brucella microti from SoilTo the Editor: Brucella microti is a recently described Brucella species (1) that was isolated in 2000 from systemically infected common voles (Microtus arvalis) in South Moravia, Czech Republic. The organism is characterized by rapid growth on standard media and high metabolic activity, which is atypical for Brucella (2). The biochemical profi le of B. microti is more similar to that of Ochrobactrum spp., of which most species are typical soil bacteria.On the basis of the close phylogenetic relationship of Brucella spp. and Ochrobactrum spp. and the high metabolic activity of B. microti, we hypothesized that this Brucella species might also have a reservoir in soil. To test this hypothesis, we investigated 15 soil samples collected on December 11, 2007, from sites in the area where B. microti was isolated from common voles in 2000 (2). Ten of the samples were collected from the surface and at a depth of up to 5 cm near different mouse burrows 5 m apart. The remaining 5 samples were collected from an unaffected area without clinical cases of vole infection. The pH of soil samples ranged from 5.9 to 6.3. No frosts were recorded before the time of collection.To specifi cally detect B. microti in soil samples, we have developed a PCR that targets a genomic island of 11 kb (H.C. Scholz et al., unpub. data) that is unique for B. microti. Briefl y, primers Bmispec_f (5′-AGATACTGGAACATAGCCCG-3′) and Bmispec_r (5′-ATACTCAGGC AGGATACCGC-3′) were used to amplify a 510-bp fragment of the genomic island. PCR conditions were denaturation at 94°C for 5 min, followed by 29 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Total DNA was prepared from 0.5 g of each soil sample by using the MO BIO Ultra Clean Soil DNA Kit (Dianova, Hamburg, Germany). DNA was eluted with 50 μL of double-deionized water of which 2 μL was used in PCRs. Template DNA of B. microti CCM 4915T was used as a positive control. Type strains of all recognized Brucella species, 1 strain of each biovar of all species, and type strains of 11 Ochrobactrum species were used as negative controls.In this PCR, 5 of 15 soil samples and the positive control were positive for the 510-bp fragment; other Brucella spp. and Ochrobactrum spp. were negative. Of the 5 positive samples, 3 were collected from surface soil collected near mouse burrows. However, the remaining 2 positive samples were collected from the unaffected and supposedly negative-control area.For direct cultivation of Brucella spp. from soil, 2 g each of 2 selected PCR-positive samples with the highest amplifi cation rate (both from the affected area) were thoroughly homogenized in 5 mL of phosphate-buffered saline (PBS), pH 7.2, in 50-mL tubes. Of a serial dilution in PBS (10 0 -10 -4 ), 100 μL was plated onto Brucella agar (Merck, Darmstadt, German...

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

Vole population sex ratio varies seasonally. However, population sex ratios have usually been estimated using naïve estimators that do not allow for biases owing to the sex difference in capture probabilities and movement distances (i.e., effective areas sampled). Here we aimed to advance the methodological approach, recognizing that there are two different classes of contributing mechanisms to the pattern which are best addressed separately: (1) those mechan isms imposing a systematic error (bias) in population estimates and (2) those generating the true process variation. Analyzing 7-year capture–recapture data in the common vole (Microtus arvalis (Pallas, 1778)), we quantified both types of biases and revealed that the bias owing to differential capture rates was often severe and less predictable, whereas that owing to differential effective areas was smaller and overestimated male numbers for most of the year. We demonstrated unambiguously that the unbiased population sex ratio indeed varies seasonally, with the males usually being more numerous over winter and spring. By testing predictions from two mechanistic hypotheses to explain the process variability, we found evidence for both the differential recruitment hypothesis and the differential survival hypothesis. From April–May to August, it was the females that were recruited more to the population and that had higher survival rates than males. We suggest that the seasonal variation in the population sex ratio is not merely a result of biasing mechanisms but an important population property driven by the joint effect of differential recruitment and differential survival between sexes.

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Adiaspores of Emmonsia crescens were found in the lungs of 62·1% of 87 adult rodents from 10 windbreaks compared to only 8·2% of 184 adult rodents caught in 10 adjacent arable fields in South Moravia, Czechland. A significantly higher mean weight proportion of plant remnants (predominantly small roots) was present in the soil from windbreaks (0·74%) than from fields (0·24%). In addition, mean relative abundance of rodents was significantly higher in windbreaks than in fields, and the windbreak soil was more acidic (pH 6·2 vs. 6·9, respectively). On the other hand, there was no significant difference in the water content (17·4% vs. 18·5%, respectively). The difference in the infection rate between the two habitats of the agroecosystem could be caused or affected by land use and farming technology.