Li Linghao, Jiang Qifeng, Li Siying, Li Xin, Sun Shenghe, Wang Xiyi, Sun Chuangqi, Jia Kun, Li Shoujun
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
Guangdong Technological Engineering Research Center for Pet, Guangzhou, China.
Front Vet Sci. 2025 Apr 22;12:1594488. doi: 10.3389/fvets.2025.1594488. eCollection 2025.
The bovine respiratory disease complex poses a significant threat to the cattle industry, necessitating a multifaceted approach to address its occurrence. The syndrome is caused by various pathogens such as bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3), bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BAV3), (Mb), and infectious bovine rhinotracheitis virus (IBRV). The confluence of these pathogens causes substantial economic losses to the cattle industry. Although preventive and control measures have been implemented, containment of bovine respiratory diseases continues to present a formidable challenge, highlighting the need for innovative diagnostic and intervention strategies.
In this study, we designed specific primers targeting six conserved pathogen genes ( of BRSV, of BPIV3, of BVDV, of BAV3, of Mb, and of IBRV). Subsequently, we established a multiplexed fluorescent real-time quantitative PCR (qPCR) assay for simultaneous detection of these pathogens.
The developed method exhibited high specificity and sensitivity, with the lowest detection limits for plasmid DNA standards of BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV being 70.1, 40.4, 15.1, 74.4, 69.6, and 4.99 copies/μL, respectively. The coefficients of variation determined by the assay established in this study were <4%, and the amplification efficiency was 93.84%-111.60%, which showed the reliability and stability of the method.
The detection rates for BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV were 7.59% (17/224), 11.61% (26/224), 8.04% (18/224), 22.32% (50/224), 27.23% (61/224), and 8.04% (18/224), respectively. All 224 cows were cases of natural disease. Fifty-six diseased cattle were infected with a mixture of two or more of the six pathogens at a mixed infection rate of 25% (56/224). Therefore, this study successfully developed a highly efficient, rapid, specific, and sensitive multiplex qPCR method to detect major pathogens associated with bovine respiratory diseases. This advancement is expected to significantly influence the future of the cattle industry and serve as a valuable reference for subsequent research in this field.
牛呼吸道疾病综合征对养牛业构成重大威胁，需要采取多方面方法来应对其发生。该综合征由多种病原体引起，如牛呼吸道合胞病毒（BRSV）、牛副流感病毒3型（BPIV3）、牛病毒性腹泻病毒（BVDV）、牛腺病毒3型（BAV3）、支原体（Mb）和传染性牛鼻气管炎病毒（IBRV）。这些病原体共同作用给养牛业造成了巨大经济损失。尽管已实施预防和控制措施，但控制牛呼吸道疾病仍然是一项艰巨挑战，凸显了创新诊断和干预策略的必要性。
在本研究中，我们设计了针对六种保守病原体基因（BRSV的、BPIV3的、BVDV的、BAV3的、Mb的和IBRV的）的特异性引物。随后，我们建立了一种多重荧光实时定量PCR（qPCR）检测方法，用于同时检测这些病原体。
所开发的方法具有高特异性和高灵敏度，BRSV、BPIV3、BVDV、BAV3、Mb和IBRV的质粒DNA标准品的最低检测限分别为70.1、40.4、15.1、74.4、69.6和4.99拷贝/微升。本研究建立的检测方法所测定的变异系数<4%，扩增效率为93.84%-111.60%，表明该方法具有可靠性和稳定性。
BRSV、BPIV3、BVDV、BAV3、Mb和IBRV的检出率分别为7.59%（17/224）、11.61%（26/224）、8.04%（18/224）、22.32%（50/224）、27.23%（61/224）和8.04%（18/224）。所有224头奶牛均为自然发病病例。56头患病牛感染了六种病原体中的两种或更多种的混合感染，混合感染率为25%（56/224）。因此，本研究成功开发了一种高效、快速、特异且灵敏的多重qPCR方法，用于检测与牛呼吸道疾病相关的主要病原体。这一进展有望对养牛业的未来产生重大影响，并为该领域的后续研究提供有价值的参考。
