Development and application of a triplex real-time PCR method for the detection of <i>Lawsonia intracellularis</i> , <i>Brachyspira hyodysenteriae</i> , and <i>Clostridium perfringens</i>

<i>Lawsonia intracellularis</i> (LI), <i>Brachyspira hyodysenteriae</i> (Bhy), and <i>Clostridium perfringens</i> (Cp) are three common swine enteric pathogens widespread worldwide, leading to huge economic losses to the pig industry. LI, Bhy, and Cp are co-infected and should not be distinguished, and the detection of bacterial nucleic acids in feces allows rapid identification of whether a herd is infected with these three pathogens. In this study, we developed and optimized a TaqMan triple quantitative real-time PCR (qPCR) method for LI, Bhy, and Cp, which was validated and applied to the analysis of actual samples from pigs. This method was then applied to investigate 5,702 fecal swabs, environmental swabs, and ileum in China from 2021 to 2024. The results showed that the positive rates of the three pathogens were 9.54% (544/5,702; LI), 2.42% (138/5,702; Bhy), and 20.78% (1,185/5,702; Cp), respectively. In addition, mixed infections between some pathogens were also found.IMPORTANCEIn this study, we developed a triple real-time PCR method for the detection of <i>Lawsonia intracellularis</i> (LI), <i>Brachyspira hyodysenteriae</i> (Bhy), and <i>Clostridium perfringens</i> (Cp). A total of 5,702 samples from pig farms in China from 2021 to 2024 were detected by this method. The results showed that all three pathogens were detected and mixed infections occurred. This method is rapid, specific, and sensitive and can be used for the monitoring and rapid diagnosis of bacterial diarrhea in pig herds.