Yersinia enterocolitica is a foodborne pathogen that causes illness in humans and animals. The biotype 4/O:3 has been commonly associated with yersiniosis and is characterized by the presence of chromosomal and extra-chromosomal virulence genes. Molecular typing methods have been successfully used to characterize Y. enterocolitica genetic heterogeneity and to study the epidemiology of the bacteria from different origins. In this study, 320 Y. enterocolitica biotype 4/O:3 isolates originating in pigs and slaughterhouses were characterized according to the virulence profile, and 61 isolates were typified through SE-AFLP, ERIC-PCR, and PFGE techniques. The majority of the isolates originated from pigs, and the predominant virulence profile was ail+ virF+ rfbC+ ystA+, representing 83.4% of the tested isolates. All of the Y. enterocolitica 4/O:3 isolates were positive for at least ystA gene. The SE-AFLP and ERIC-PCR patterns were highly homogeneous. The SE-AFLP was more discriminative than the ERIC-PCR and tended to cluster isolates according to the slaughterhouse. Despite the limited genetic diversity of Y. enterocolitica 4/O:3, PFGE was shown to be the most discriminative technique considering one band of difference. Fattening pigs proved to be an important reservoir of Y. enterocolitica biotype 4/O:3 carrying virulence genes. 1. Introduction Yersinia enterocolitica is an important zoonotic foodborne pathogen that can cause acute diarrhea, terminal ileitis, and mesenteric lymphadenitis in humans and animals [1, 2]. The isolates largely responsible for human yersiniosis in Europe, Japan, Canada, and the USA belong to biotype 4/O:3 [1]. The epidemiology of the disease is not completely known. The pig is considered the only reservoir from which pathogenic Y. enterocolitica isolates, such as biotype 4/O:3, have been frequently isolated [1]. The prevalence of this biotype in pig slaughterhouses has been reported to be 56% in Finland [3] and 60% in southern Germany [4]. Serotype O:3 is predominant among the isolates recovered from slaughter pigs in the USA [5]. Martínez et al. [6] reported that fattening pigs seem to be an important reservoir of pathogenic Y. enterocolitica in Belgium, Italy, and Spain. The virulence of the pathogenic biotype 4/O:3 is attributed to the presence of chromosomal and extrachromosomal genes. The plasmid for Yersinia virulence (pYV) encodes the adhesin A (YadA), the Yersinia outer proteins (Yops) from type III secretion system, and the transcriptional regulator gene (virF) [7, 8]. The chromosomal virulence genes include inv (invasin),
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