Optimization of a multiplex PCR assay for simultaneous detection of key bovine respiratory infections

The control of respiratory infections is integral to a healthy livestock farm. The current study was conducted to optimize a multiplex PCR assay for simultaneous detection of Pasteurella multocida (P. multocida), Staphylococcus aureus (S. aureus) and Mycobacterium bovis (M. bovis) in nasal samples of cattle having suspicion of respiratory tract infections. The nasal samples were collected from 10 dairy farms affected with respiratory disease outbreaks in the recent past. These outbreaks were associated with respiratory tract infections caused by bacterial pathogens P. multocida, S. aureus and M. bovis. A multiplex PCR assay was therefore optimized to enable the simultaneous detection of these bacterial pathogens directly from nasal samples. The multiplex PCR assay was optimized using primers already validated for efficient amplification of specific DNA segments in reference strains of targeted bacterial pathogens. The standardized assay was specific and sensitive enough to detect ≥100 genome equivalents of target DNA segments in each of P. multocida, S. aureus and M. bovis. The assay was successfully applied for the detection of the three bacterial pathogens in 50 nasal samples. PCR amplicons were subjected to Sanger dideoxy sequencing followed by phylogenetic analysis to determine if species identification was specific. In short, the optimized multiplex PCR assay may prove as a reliable, economical, and simple tool for the management of bovine respiratory tract infections caused by some key bacterial pathogens.