Bovines play a crucial role in global agriculture and livestock production. However, outbreaks of infectious diseases in cattle pose significant economic losses and pose significant risks to the cattle farming industry. Among the myriad of bovine pathogens, Bovine Ephemeral Fever Virus (BEFV), Bovine Herpesvirus 4 (BoHV4), Bovine Rotavirus (BRV), and Clostridium perfringens (CP) are particularly prevalent, causing respiratory, gastrointestinal, and systemic infections (Lee, 2019, Wathes et al., 2020, Kostanić et al., 2024, Ba et al., 2024).

BEFV, an RNA virus, is the causative agent of bovine ephemeral fever, characterized by high fever, muscle pain, joint stiffness, and anorexia, leading to decreased production performance (Walker and Klement, 2015). BoHV4, a DNA virus, is associated with various bovine diseases, including reproductive disorders, mastitis, and immunosuppression, often manifesting as asymptomatic infections and facilitating co-infections with other pathogens (Dağalp et al., 2020). BRV, a double-stranded RNA virus belonging to the Reoviridae family, is a major cause of diarrhea in calves, which can result in severe dehydration, electrolyte imbalance, and even death, particularly in young stock (Parashar et al., 2006). CP, a Gram-positive anaerobic bacterium, produces toxins such as alpha and beta that can induce acute enterotoxemia or necrotic enteritis, commonly affecting intensively farmed cattle, causing substantial mortality (Songer, 1996).

Co-infections involving BRV and CP frequently result in severe diarrhea and mortality in calves, while the synergistic effect of BEFV and BoHV4 may exacerbate respiratory symptoms (Bartels et al., 2010, Chung et al., 2016). The transmission risks of BEFV and BoHV4 peak during the mosquito season in summer and autumn, whereas BRV and CP outbreaks are more prevalent during winter in confined feeding conditions (Lavon et al., 2023, Klamminger et al., 2017, Wang et al., 2023, Moustafa et al., 2022). The high prevalence of mixed infections among these pathogens underscores the importance of early detection, isolation, and intervention strategies for effective disease control.

Current diagnostic methods for bovine pathogens include virus isolation, serological assays, and molecular techniques such as PCR (Huang et al., 2025, Chakraborty et al., 2019). However, these methods are often time-consuming, labor-intensive, or lack the ability to simultaneously detect multiple pathogens. Fluorescence quantitative PCR (qPCR) has emerged as a powerful tool due to its high sensitivity, specificity, and speed, allowing for the detection of multiple targets in a single reaction (Bustin et al., 2025). Despite its advantages, the development of a qPCR assay that can simultaneously detect BEFV, BoHV4, BRV, and CP remains lacking.

To address this gap, the present study aimed to develop a multiplex qPCR assay for the simultaneous detection of these four prevalent bovine pathogens. By targeting conserved genes or virulence factors within each pathogen's genome, this assay aims to provide a rapid, accurate, and cost-effective diagnostic tool for the bovine industry, contributing to enhanced disease control and prevention strategies.