Differentiation of mycoplasma bovis, mycoplasma bovigenitalium, mycoplasma californicum and identification of ureaplasma diversum by real-time PCR

Abstract

Mycoplasmas and ureaplasmas are important etiological agents of mastitis, pneumonia and reproductive disorders in cattle, which cause significant economic damage to cattle farming. The most significant species are Mycoplasma bovis, M. bovigenitalium, M. californicum and Ureaplasma diversum. Commercial diagnostic PCR systems for the detection of bacteria of the genus Mycoplasma in different biological samples are described, but no PCR kits have been developed to address the identification of Mycoplasma species. In this work, real time PCR assays for differentiation of pathogenic mycoplasmas (Mycoplasma bovis, M. bovigenitalium, M. californicum) and detection of Ureaplasma diversum in biological material (semen, milk, vaginal swabs, tissues) are developed. UvrC gene for M. bovis, 16S rRNA gene for M. bovigenitalium and U. diversum, and rpoB gene for M. californicum were chosen as target genes. The PCR assays included a system of primers and probes for detection of exogenous noncompetitive internal control sample. The specificity of the developed techniques was tested on a panel of samples containing viral and bacterial pathogens causing diseases in cattle, as well as cow genomic DNA. To assess the sensitivity of each PCR assay, positive control samples were developed based on genetically engineered constructs containing the region of the corresponding specific DNA. Analytical sensitivity of the PCR assays was evaluated separately for each pathogen, for which we used 10-fold dilutions of the corresponding control samples in negative samples of biological material, i.e. semen, milk, vaginal swabs and tissues. The sensitivity (detection limit) of the assays for different types of biological species was 5½103 copies per ml on average. The efficiency of PCR was 99 % for M. bovis, 87 % for M. bovigenitalium, 94 % for M. californicum, and 98 % for U. diversum. A total of 410 samples of bovine semen intended for artificial insemination from local and foreign breeding centers were tested to detect M. californicum, M. bovigenitalium, M. bovis and U. diversum. DNA of M. bovis was found in 2.5 % of semen samples from foreign centers. In samples of Russian origin M. bovis DNA was not detected. DNA of M. bovigenitalium was identified for 60.7 % of local and 25.1 % of foreign semen samples; DNA of M. californicum was detected in 51.7 % and 25.1 % samples, respectively. Ureaplasma diversum DNA was found in 55.0 % of semen samples from Russian bulls and in 12.1 % of semen samples of foreign origin. Co-infection of M. californicum/M. bovigenitalium was detected in 97 samples (23.7 %), M. bovigenitalium/U. diversum in 86 cases (21.0 %). Simultaneous infection of M. bovigenitalium, M. californicum and U. diversum was observed in 52 samples (24.6 %) of semen from domestic bull sires and in 4 samples (2.0 %) from foreign breeding centers. Novel PCR assay tests can be used for monitoring of semen quality as well as control and prevention of the pathogens distribution.