Mycoplasma bovis is a major bovine pathogen causing respiratory disease, arthritis, otitis media, mastitis, and genital disorders in cattle worldwide. M. bovis infections tend to persist in affected herds and are often resistant to antibiotics. Spread by close contact of infected animals or a heavily contaminated environment, M. bovis can colonize mucosal surfaces and disseminate to other organ sites. Rapid identification and isolation of diseased animals is critical to prevent the spread of infection.
Artificial insemination with M. bovis contaminated semen is a common source of infection of the female bovine genital tract. In this study, we report the development and validation of a dual target TaqMan real-time PCR system for the rapid detection of M. bovis in bovine semen. The assays target the housekeeping genes fusA and oppD/F. Both assays exclusively amplified M. bovis when tested against a panel 15 bovine mycoplasma species including 59 field and laboratory strains of M. bovis. Quantification was determined by testing serial dilutions of plasmids containing the target sequences; both the fusA and oppD/F assays demonstrated a detection limit of 1 plasmid copy/uL and efficiencies of 1.975 and 1.977 respectively.
Bovine semen was spiked with serial dilutions of M. bovis in order to compare the detection limits of the real-time PCR assays and culture in clinical samples. Culture demonstrated a detection limit of 310 organisms/µl, while the fusA and oppD/F assays detected 3.1 organisms/µl. Fresh semen ejaculates from 26 commercial bulls were obtained from an artificial insemination center and evaluated for M. bovis by real-time PCR and culture. All samples were negative for the organism by both real-time PCR and culture.
Although real-time PCR systems have been described for the detection of M. bovis in other bovine fluids and tissues, no PCR assays have been developed to address the specific evaluation of M. bovis in bovine semen. In this study, we present two highly specific and sensitive TaqMan real-time PCRs for the rapid detection and quantification of M. bovis in bovine semen.