Novel multiplex PCR approach for the detection of , , and in swine.

, , and are neglected, emerging and zoonotic pathogens in pigs that share common clinical signs and lesions, particularly septicaemia and skin lesions, highlighting the need for differential diagnosis. Currently, no single diagnostic assay is available to test samples for confirmation of the presence of all three pathogens. To address this, a novel multiplex PCR-based detection assay was optimized and evaluated for the simultaneous detection of , , and targeting the, 23S rRNA, , and genes, respectively. The optimized multiplex PCR demonstrated 100% analytical specificity, with a detection limit of 10 copies/μL for and , and 10 copies/μL for . The assay was further validated by comparison with previously established uniplex PCR assays and tested on field-collected pig blood samples to assess real-world applicability. The developed assay is rapid, specific and cost-effective and can serve as a handy diagnostic tool for testing suspect samples for these neglected bacterial pathogens of pigs.