Mycoplasma ovis detected in free-living Japanese serows, Capricornis crispus.

Nineteen blood samples collected from free-ranging wild Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the hemoplasma infection by real-time PCR targeting the 16S rRNA gene. Five (26.3%) out of the 19 samples were positive in real-time PCR with an average melting temperature at 85.18 °C. The positive samples in the real-time PCR were reconfirmed by conventional PCR, and one of them was successful for direct DNA sequencing. The nucleotide sequence of the 16S rRNA gene of the representative stain was identical to that of Mycoplasma ovis. This was the first demonstration of hemotropic mycoplasma infections among the free-living Japanese serows in Japan.

Differential detection of hemotropic Mycoplasma species in cattle by melting curve analysis of PCR products.

We developed a real-time PCR procedure followed by melting curve analysis using the green fluorescence dye SYBR Green I for rapid detection and differentiation of hemplasmas in cattle. Analysis of the melting temperature (Tm) of the PCR products allowed for differentiation of the 2 bovine hemoplasmas, Mycoplasma wenyonii and a provisional species, ;Candidatus Mycoplasma haemobos' (a synonym of ;Candidatus M. haemobovis'). The Tm (mean +/- S.E.) of the PCR products from the bovine hemoplasmas were 86.98 +/- 0.12 degrees C for M. wenyonii and 82.04 +/- 0.27 degrees C and 86.98 +/- 0.12 degrees C, respectively; [corrected] Candidatus M. haemobos' in the melting experiments. The protocol described in the present study can decrease the time to results by simultaneous detection and differentiation of the two hemoplasmas in cattle. By using this protocol, we examined hemoplasma prevalence in 109 cattle in Miyagi Prefecture and found that 67 (61.5%) were infected with M. wenyonii, 25 (22.9%) were infected with ;Candidatus M. haemobos' and 14 (12.8%) were infected with both.

Phylogenetic analysis of the 16S rRNA gene of Anaplasma species detected from Japanese serows (Capricornis crispus).

Nineteen blood samples collected from free-living Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the anaplasma infection by PCR amplification of a part of the 16S rRNA gene. Ten (52.6%) out of the 19 samples produced a visible band in electrophoresed agarose gels. Positive PCR products were subjected to DNA sequencing. We found the nucleotide sequences were identical. Almost entire length of the 16S rRNA gene for a representative stain was then sequenced. We found the nucleotide sequence of the 16S rRNA gene distinct from previously established Anaplasma species in phylogenetic analysis. Our data first indicated that anaplasma infection occurred continuously among the free-living Japanese serow populations in northern Japan.