Development and preliminary evaluation of multiplex PCR assays for the detection of bacterial respiratory pathogens in bovine and ovine

Bovine and ovine respiratory diseases are commonly associated with multiple bacterial pathogens, including Mycoplasma bovis (Mb), Mycoplasma ovipneumoniae (Mo), Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), and Mycobacterium bovis (Tb). Among these, Mb and Mo are the primary causative agents of mycoplasma pneumonia in cattle and sheep, respectively. Based on this difference, two multiplex PCR assays were developed for the detection of bacterial respiratory pathogens in cattle and sheep, differing mainly in the primers targeting Mycoplasma spp. The bovine assay simultaneously detected Mb, Mh, Pm, and Tb, while the ovine assay detected Mo, Mh, Pm, and Tb, with expected amplicon sizes of 319/320 bp, 390 bp, 528 bp, and 274 bp. After optimization, both assays were able to simultaneously detect the target pathogens and showed good specificity, sensitivity, and reproducibility, with detection limits of 1.0 × 10³ copies/μL for the bovine assay and 1.0 × 10²–10³ copies/μL for the ovine assay. Preliminary testing using clinical samples demonstrated that the assays could detect the target bacterial pathogens. These results indicate that the developed multiplex PCR assays provide a basis for further validation and potential application in the detection of bacterial respiratory pathogens in cattle and sheep.