Introduction: Yaks serve as a vital economic and ecological resource in high-altitude regions, but it faces significant health challenges from various pathogens. Among these, Pasteurella multocida and Salmonella are critical pathogens that contribute to severe diseases.Methods: A duplex real-time fluorescence quantitative PCR assay was developed to simultaneously detect Pasteurella multocida and Salmonella. The species-specific genes kmt1 and invA were selected as target regions for primer and probe design. Following rigorous optimization, a duplex assay was established. Recombinant plasmids were constructed to serve as standards for generating standard curves. The detection thresholds were determined using SPSS statistical analysis and receiver operating characteristic curve methods. Furthermore, the assay’s sensitivity, specificity, stability, and clinical applicability were evaluated.Results: The established assay demonstrated high sensitivity, with detection limits of 100 and 10 copies for pMD-kmt1 and pMD-invA, respectively. No cross-reactivity was observed with six pathogens, including Mycoplasma bovis, infectious bovine rhinotracheitis virus and others. The standard curves showed strong linearity, with coefficients of determination of 0.995 and 0.998, and amplification efficiencies of 103.37% and 103.47% for pMD-kmt1 and pMD-invA, respectively. No interference was observed between high- and low-concentration templates during simultaneous detection. The intra- and inter-assay coefficients of variation ranged from 0.23% to 1.51%. Detection thresholds were determined to be cycle threshold values of 41.5 for P. multocida and 40.0 for Salmonella. Clinical evaluation was performed on 226 samples collected from yaks in seven counties of Ganzi Prefecture, Sichuan Province, China. The single infection rates of P. multocida and Salmonella were 20.35% (46/226) and 38.50% (87/226), respectively, while the co-infection rate was 6.19% (14/226).Discussion: This study successfully established a duplex real-time fluorescence PCR assay that enables the simultaneous detection of P. multocida and Salmonella with high sensitivity, specificity, and efficiency. The assay offers a reliable and rapid diagnostic tool that is particularly suited for clinical and epidemiological investigations in yak populations.

Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen that infects cattle and causes significant economic losses. We generated a recombinant adenovirus called rHAd5-F + HN by expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 using the human adenovirus serotype 5 (rHAd5). We evaluated its effects on humoral and cellular immune responses in mice (n = 45) and calves (n = 9). Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), and neutralising antibodies (NAb). After boosting immunity with rHAd5-F + HN, mice produced significantly higher levels of antibodies against the BPIV3 genotype A and genotype C strains. The production of antibodies exceeded those produced by adenoviruses rHAd5-F and rHAd5-HN, which express the F and HN glycoprotein, respectively. The percentages of splenic CD3⁺/CD8⁺T lymphocytes and IL-4⁺ cytokines in rHAd5-F + HN mice were considerably higher than those in the control group. Mice immunised with rHAd5-F + HN exhibited much lower viral loads in the lungs and tracheas compared to the control group. Additionally, the lungs of mice vaccinated with rHAd5-F + HN showed no notable histopathological changes. On the other hand, rHAd5-F + HN produced a humoral immune response in calves. Following the booster intramuscular injection with the rHAd5-F + HN, the serum antibody levels against BPIV3 genotype C strain were 1:20 452, 1:1024, and 1:426 in calves, as detected by ELISA, HI, and NAb, respectively. The HI and NAb levels against the BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicate that rHAd5-F + HN effectively induced immunity against BPIV3 infection.