Abstract

Bovine viral diarrhea virus (BVDV), Pasteurella multocida (PM), and Brucella spp. are major pathogens that significantly compromise the global cattle industry. To address the need for accurate molecular detection, a novel triplex TaqMan qPCR assay was established targeting three conserved genetic markers: BVDV 5’UTR, PM Kmt1 , and Brucella Bcsp31 . The method achieved detection limits of 18.9, 3.19, and 3.32 copies/µL for BVDV, PM and Brucella spp. , with acceptable amplification efficiencies (93–101%). Additionally, the assay was highly specific, as no cross-reactions were detected with a panel of non-target bovine pathogens. Performance evaluation confirmed high reliability, with intra- and inter-assay CVs consistently under 1.34%. Large-scale epidemiological surveillance of cattle suspected of BVDV, PM, and Brucella spp. infections in Guizhou Province of China from 2022 to 2025 revealed overall detection rates of 18.47% for BVDV, 20.76% for PM, and 3.63% for Brucella spp. from nasal swabs. Co-infections were also observed, most notably BVDV and PM (3.37%), followed by PM and Brucella spp. (0.67%), and BVDV and Brucella spp. (0.31%). Annual detection rates across the four-year period remained relatively stable, with slight upward fluctuations for all three pathogens. This study developed an efficient triplex qPCR platform for the surveillance and differential detection of these high-impact cattle pathogens.