A Triplex Real-Time PCR Assay for Simultaneous Detection of Streptococcus suis, Glaesserella parasuis, and...

Porcine respiratory disease complex is a multifactorial disease syndrome in swine in which bacterial pathogens play important roles. Among them, Streptococcus suis (SS), Glaesserella parasuis (GPS), and Actinobacillus pleuropneumoniae (APP) are important bacterial agents associated with respiratory disease in pigs, underscoring the need for a rapid, accurate, and simultaneous detection method. In this study, we developed a triplex real-time PCR assay for the simultaneous detection of SS, GPS, and APP, and evaluated its performance in tonsil samples from clinically healthy pigs. The assay showed high specificity, sensitivity, and reproducibility. A total of 228 tonsil samples were analyzed in parallel by triplex real-time PCR assay and conventional PCR assay. The triplex real-time PCR assay detected at least one of the three pathogens in 91.23% (208/228) of samples, which was markedly higher than the 73.68% (168/228) detected by PCR assay. Concurrent detection of multiple pathogens was observed in 98 samples (42.98%) by the triplex real-time PCR assay, including 82 samples positive for both SS and GPS (35.96%) and 16 samples positive for SS, GPS, and APP (7.02%). From the triple-positive samples, 12 SS isolates were recovered, of which 91.67% (11/12) were multidrug-resistant. Animal challenge experiments further confirmed the virulence of two representative isolates, SZWUSS183 (cps type 1/2, ST7) and SZWUSS225 (cps type 3, ST117). Overall, the developed triplex real-time PCR assay provides a sensitive and reliable tool for the simultaneous detection of these major bacterial pathogens and may facilitate surveillance of their circulation and co-occurrence in swine herds.