Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases

SUNAGA, Fujiko; TSUCHIAKA, Shinobu; KISHIMOTO, Mai; AOKI, Hiroshi; KAKINOKI, Mari; KURE, Katsumasa; OKUMURA, Hanako; OKUMURA, Maho; OKUMURA, Atsushi; NAGAI, Makoto; OMATSU, Tsutomu; MIZUTANI, Tetsuya

PRDC is one of the most important health concerns for pig producers and involves multiple viral and bacterial pathogens. PRDC is multifactorial, with both infectious and non-infectious factors contributing to respiratory disease seen in pigs between the ages of 3 and 6 months. The interaction of viral and bacterial pathogens, environmental factors, pig-specific factors and management conditions all contribute to the development and impact the severity of PRDC [20] . The most commonly isolated pathogens are PRRSV, SIV, PCV2, and M. hyopneumoniae. The other pathogens associated with PRDC are S. suis, A. pleuropneumoniae, P. multocida, P. multocida toxin, B. bronchiseptica, H. parasuis, M. hyorhinis, M. hyosynovie, PRV, PRCV, PCMV [7, 8, 15, 20] . However, no single-reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Routine diagnostic methods for detection of viruses implicated in PRDC include virus isolation in cell culture, antigen detection by direct fluorescent antibody staining, and enzyme immunoassay [5] and culture-based methods for bacteria [23] . These methods are time-consuming and require independent tests for each pathogen. Furthermore, the detection of bacterial pathogens typically depends on culture-based methods that can take several days to obtain results. Due to their high sensitivity and ease of use, PCR and real-time PCR tests have been developed for several agents implicated in the PRDC; however, these tests typically target single pathogens [24] . A multiplex PCR assay capable of detecting five porcine viruses including two porcine respiratory viruses was developed [4] . However, to date, there are no diagnostic tests capable of simultaneous detection of multiple major viral and bacterial porcine respiratory pathogens in a single reaction.