Hao Fuxing, Tao Chunhao, Xiao Ruilong, Huang Ying, Yuan Weifeng, Wang Zhen, Jia Hong
Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China.
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Microorganisms. 2025 Jul 10;13(7):1629. doi: 10.3390/microorganisms13071629.
Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), (M.bovis), (PM), (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes.
牛呼吸道疾病综合征（BRDC）是全球范围内导致牛发病、死亡和经济损失的主要原因之一。准确、快速地鉴定致病病原体对于有效的疾病管理和控制至关重要。在本研究中，开发了一种基于多重荧光定量聚合酶链反应（qPCR）的新型检测方法，用于同时检测与BRDC相关的八种主要病原体。目标病原体包括：牛病毒性腹泻病毒（BVDV）、牛副流感病毒3型（BPIV3）、牛呼吸道合胞病毒（BRSV）、牛冠状病毒（BcoV）、（牛支原体）、（多杀性巴氏杆菌）、（溶血性曼氏杆菌）和传染性牛鼻气管炎病毒（IBRV）。该检测方法经过严格优化，以确保高特异性，各目标之间无交叉反应。所有目标病原体的检测限（LOD）确定为低至每个反应5个拷贝。批内和批间测量的变异系数（CVs）始终低于2%，显示出出色的重现性。为了验证该检测方法的临床实用性，共检测了1012份现场样本，包括来自江苏省A农场的504份鼻拭子和B农场的508份鼻拭子。在A农场检测到了BVDV、BcoV、多杀性巴氏杆菌和溶血性曼氏杆菌，BVDV阳性率为21.63%（109/504），BcoV阳性率为26.79%（135/504），多杀性巴氏杆菌阳性率为28.77%（145/504），溶血性曼氏杆菌阳性率为15.08%（76/504）。此外，在B农场检测到了BcoV、多杀性巴氏杆菌、溶血性曼氏杆菌和IBRV，BcoV阳性率为2.36%（12/508），多杀性巴氏杆菌阳性率为1.38%（7/508），溶血性曼氏杆菌阳性率为14.76%（75/508），IBRV阳性率为5.51%（28/508）。值得注意的是，相当一部分样本显示出混合感染的迹象，突出了BRDC病因的复杂性以及多重诊断方法的重要性。总之，所开发的多重qPCR检测方法为同时检测多种与BRDC相关的病原体提供了一种可靠、快速且经济高效的工具，这对于加强疾病监测、早期诊断和靶向干预策略具有巨大潜力，最终有助于改善BRDC管理和牛的健康状况。
