Simplicimonas-like DNA in vaginal swabs of cows and heifers cross-reacting in the real-time PCR for T. foetus

Cows on an alpine pasture were presented with severe signs of vaginitis. To rule out infection with Tritrichomonas foetus, vaginal swabs were taken and real-time PCR based on detection via fluorescence resonance energy transfer (FRET) probes and targeting the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) was performed. PCR was positive in 25 of totally 34 assessed cows. However, the melting profiles of the probes targeting the diagnostic PCR products differed from the T. foetus positive control. Subsequent sequencing of the amplicons revealed 91% identity to Simplicimonas sp. sequences deposited in GenBank™. Furthermore, there was no clear association between positive PCR result and presence of vaginitis. To investigate the distribution of this Simplicimonas-like organism in cows, more herds grazing on the same alpine pastures as well as unrelated cows were tested. In total, 133 cows and 16 heifers were sampled, 53 cows and 6 heifers even twice. Vaginitis was evident in 43 cows and 4 heifers. All-over-positivity of PCR was 44%, including nine tests performed on heifers. Melting peak analysis indicated Simplicimonas-like organisms in all positive samples. Culture attempts in bovine InPouch ™ TF failed. No association between a positive PCR result and the presence of vaginitis was found. This is, to the best of our knowledge, the first report on Simplicimonas-like DNA in vaginal swabs of female cattle. Our data suggest that when testing vaginal swabs of cattle by means of T. foetus PCR, false positive reactions due to Simplicimonas-like organisms may occur.