The development of a screening protocol for Salmonella spp. and enteropathogenic Yersinia spp. in samples from wild boar (Sus scrofa) also generating MLVA-data for Y. enterocolitica and Y. pseudotuberculosis

Salmonellosis and yersiniosis are notifiable human diseases that are commonly associated with contaminated food. Domestic pigs as well as wild boars and other wild-life have been identified as reservoirs of these bacteria. Methods for cultivation and molecular epidemiological investigations of Salmonella spp. are well established, however, cultivation of enteropathogenic Yersinia spp. is time- consuming and the commonly used method for molecular epidemiological investigations, pulsed-field gel electrophoresis, lack in discriminatory power. The aim of this study was to develop and evaluate a screening protocol well suited for wildlife samples and other highly contaminated samples. The method is based on PCR-screening followed by Multiple Loci Variant number tandem repeat Analysis (MLVA) on enrichment broth to obtain molecular epidemiological data for enteropathogenic Yersinia spp. without the need for pure isolates. The performance of the protocol was evaluated using wild boar samples (n=354) including tonsils, faeces and lymph nodes from 90 Swedish wild boars. The new protocol performed as well as or better than the established ISO-standards for detection and cultivation of Y. enterocolitica and Salmonella spp., however for cultivation of Y. pseudotuberculosis, further development is needed. The selection for motility seems beneficial for the enrichment of Salmonella spp. and Y. enterocolitica. Further, the selective enrichment prior to PCR-analysis eliminates inhibitory factors present in the original sample. In total, ten isolates of Y. enterocolitica of various bio-serotypes were obtained, and the MLVA-profile of these isolates were consistent with the profiles from the corresponding enrichment broth. Further, 22 isolates of Salmonella spp. comprising six different serovars were obtained with S. Fulica, S. Hadar and a monophasic S. Typhimurium being the most common. In conclusion, the presented screening protocol offers a rapid and efficient way to obtain prevalence data from a large sample set as well as MLVA-data within a short time frame. These results can hence improve the knowledge on the epidemiology and distribution of these pathogens and their importance to public health.