ORIGINAL ARTICLE Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content R. Laukkanen 1 , M. Hakkinen 2 , J. Lunde ´n 1 , M. Fredriksson-Ahomaa 3 , T. Johansson 2 and H. Korkeala 1 1 Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland 2 Microbiology Unit, Department of Animal Diseases and Food Safety Research, Finnish Food Safety Authority Evira, Mustialankatu, Helsinki, Finland 3 Institute of Food Hygiene and Technology, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universita ¨ t Munich, Scho ¨ nleutnerstrasse, Oberschleissheim, Germany Introduction Yersinia enterocolitica is a zoonotic pathogen that can cause yersiniosis, the third most commonly reported zoonosis in European Union countries (EFSA 2007). Pathogenic Y. enterocolitica is frequently isolated from the tonsils and intestinal content of pigs worldwide (Nesbak- ken and Kapperud 1985; Asplund et al. 1990; Fukushima et al. 1990; Letellier et al. 1999; Fredriksson-Ahomaa et al. 2000, 2001a, 2007a; Bhaduri et al. 2005), and similar Y. enterocolitica genotypes have been identified in both pig and human samples (Fredriksson-Ahomaa et al. 2001b, 2006; Fearnley et al. 2005; Ku ¨hni-Boghenbor et al. 2006). In addition, human yersiniosis has been statisti- cally associated with the consumption of pork products in case–control studies (Tauxe et al. 1987; Ostroff et al. 1994; Satterthwaite et al. 1999; Jones 2003; Grahek-Ogden et al. 2007; Boqvist et al. 2008). The high prevalence of pathogenic Y. enterocolitica in pigs, the similarity of genotypes in pig and human infections and case–control studies indicate that pigs and pork products are an important source of human Y. enterocolitica infections. Isolation of pathogenic Y. enterocolitica from samples of animal origin is difficult and time-consuming. How- ever, in many cases, e.g. in outbreak investigations, it is crucial to obtain Y. enterocolitica isolates for further Keywords detection methods, intestinal content, isolation, pig, prevalence, Yersinia enterocolitica. Correspondence Riikka Laukkanen, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, P.O. Box 66, FI-00014 University of Helsinki, Finland. E-mail: riikka.laukkanen@helsinki.fi 2009 ⁄ 0554: received 24 March 2009, revised 16 June 2009 and accepted 15 July 2009 doi:10.1111/j.1365-2672.2009.04494.x Abstract Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intesti- nal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella– Shigella-desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53 ⁄ 68) of the positive samples. Individual isolation steps using cold enrich- ment as the only enrichment or as a pre-enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. entero- colitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica. Journal of Applied Microbiology ISSN 1364-5072 956 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 956–964 ª 2009 The Authors