Validation of a combination of pre-enrichment and a yopT-based PCR assay to detect pathogenic Yersinia enterocolitica in organs and lymphoid tissue of slaughtered pigs

Slaughter pigs are the main reservoir for pathogenic Yersinia (Y.) enterocolitica isolates. Particularly meatproducts derived from swine asymptomatically infectedare a potential source of human Yersiniosis. Due to thisfact it is important to have simple, rapid and specificmethods available for the detection of pathogenic Y. enterocolitica in tissue samples from slaughtered pigs.The aim of this study was to validate a 24 h preenrichmentstep in Yersinia-PCR-compatible-enrichment (YPCE) broth followed by a yopT-based PCR-Method forthe rapid detection of pathogenic Y. enterocolitica inorgans and lymphoid tissue of slaughtered pigs. Untilnow, the bacteriological identification of pathogenic Yersinia, described in ISO 10273, is the "goldstandard". To compare the PCR results with the "goldstandard" we choose for the cultural detection a 48hour pre-enrichment step at 25 C in ITC broth followedby plating on CIN agar plates and incubation for 24hours at 30°C. For the validation of the PCR method itwas important to have available samples fromasymptomatically infected pigs. To generate thosesamples Yersinia-free pigs (n = 9) were intragastricallyinfected with a plasmid bearing Y. enterocolitica isolate (DSM 13030). Two additional pigs served asuninoculated controls. Four weeks after infection pigswere slaughtered and 13 samples from each pig weretaken aseptically.In comparison to the conventional cultural examinationthe combination of pre-enrichment in YPCE brothfollowed by yopT-PCR saved about four days ofdiagnostic time. Particularly, in the examined samplesof the palatine tonsil, an organ with the highestcontamination rate in slaughter pigs, demonstrated thePCR method a significantly better sensitivity than thecultural method. In summary, the 24 h pre-enrichmentand subsequent yopT-PCR represents a rapid andsensitiv diagnostic tool for pathogenic Y. enterocolitica in asymptomatically infected pigs at the slaughterhouselevel.