MINIREVIEW

Molecular epidemiology of Yersinia enterocolitica infections Maria Fredriksson-Ahomaa1, Andreas Stolle1 & Hannu Korkeala2 1

Institute of Hygiene and Technology of Food of Animal Origin, Ludwig-Maximilian University, Munich, Germany; and 2Department of Food and Environmental Hygiene, University of Helsinki, Finland

Correspondence: Maria FredrikssonAhomaa, Institute of Hygiene and Technology of Food of Animal Origin, Schoenleutnerstrasse 8, D-85764 Oberschleissheim, Germany. Tel.: 149 89 2180 78533; fax: 149 89 2180 78502; e-mail: [email protected] Received 6 February 2006; revised 8 March 2006; accepted 8 March 2006. First published online 16 May 2006. doi:10.1111/j.1574-695X.2006.00095.x Editor: Willem van Leeuwen Keywords Yersinia enterocolitica ; molecular typing; epidemiology.

Abstract Yersinia enterocolitica is an important food-borne pathogen that can cause yersiniosis in humans and animals. The epidemiology of Y. enterocolitica infections is complex and remains poorly understood. Most cases of yersiniosis occur sporadically without an apparent source. The main sources of human infection are assumed to be pork and pork products, as pigs are a major reservoir of pathogenic Y. enterocolitica. However, no clear evidence shows that such a transmission route exists. Using PCR, the detection rate of pathogenic Y. enterocolitica in raw pork products is high, which reinforces the assumption that these products are a transmission link between pigs and humans. Several different DNA-based methods have been used to characterize Y. enterocolitica strains. However, the high genetic similarity between strains and the predominating genotypes within the bio- and serotype have limited the benefit of these methods in epidemiological studies. Similar DNA patterns have been obtained among human and pig strains of pathogenic Y. enterocolitica, corroborating the view that pigs are an important source of human yersiniosis. Indistinguishable genotypes have also been found between human strains and dog, cat, sheep and wild rodent strains, indicating that these animals are other possible infection sources for humans.

Introduction Yersinia enterocolitica is an important zoonotic pathogen that can cause yersiniosis in humans and animals. The predominant symptom in humans, particularly in young children, is diarrhoea (Bottone, 1999). Secondary immunologically induced sequelae, such as reactive arthritis, are not uncommon, especially in HLA-B27-positive individuals. Most cases of yersiniosis occur sporadically without an apparent source (Bottone, 1997, 1999). Yersinia enterocolitica is thought to be a significant food-borne pathogen, even though pathogenic strains have seldom been isolated from foods. In studying raw pork, higher detection rates have been obtained by PCR than by culture methods (Fredriksson-Ahomaa & Korkeala, 2003a). In some case-control studies, an increased risk of yersiniosis has been demonstrated when raw or undercooked pork has been consumed (Tauxe et al., 1987; Ostroff et al., 1994). The pig is the only reservoir from which pathogenic Y. enterocolitica strains have frequently been isolated (Bottone, 1999). Nevertheless, the epidemiology of Y. enterocolitica infections is FEMS Immunol Med Microbiol 47 (2006) 315–329

complex and remains poorly understood. To identify reservoirs of infection, transmission vehicles and associations between clinical cases, several DNA-based methods have been used in molecular typing of Y. enterocolitica (Iteman et al., 1996; Wojciech et al., 2004; Fearnley et al., 2005; Virdi & Sachdeva, 2005).

Yersinia enterocolitica infection Yersinia enterocolitica infections have been observed on all continents but appear to be most common in Europe (Bottone, 1999). Food-borne outbreaks are rare and most infections are sporadic. The prevalence of Y. enterocolitica O:3/O:9-specific antibodies in healthy blood donors has been shown by immunoblotting to be relatively high in Finland (31%) and in Germany (43%) (M¨aki-Ikola et al., 1997). This may indicate a high rate of subclinical yersiniosis in the healthy population. Yersinia enterocolitica can cause gastrointestinal symptoms ranging from mild self-limiting diarrhoea to acute mesenteric lymphadenitis, which can lead to appendicitis (Bottone, 1997). The clinical manifestations 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

316

of the infection depend to some extent on the age and physical state of the patient, the presence of any underlying medical conditions and the bioserotype of the organism. Gastroenteritis, caused by Y. enterocolitica, is the most frequent form of yersiniosis, typically affecting infants and young children. In older children and young adults, acute yersiniosis can be present as a pseudoappendicular syndrome, which is frequently confused with appendicitis. Sometimes long-term sequelae, including reactive arthritis, erythema nodosum, uveitis, glomerulonephritis and myocarditis, will occur. Postinfection manifestations are mainly seen in young adults. Sepsis is a rare complication of Y. enterocolitica infection, except in patients who have a predisposing underlying disease or are in an iron-overloaded state. Sepsis can also occur during blood transfusion (Bottone, 1999). Yersinia enterocolitica infection is typically initiated by ingestion of contaminated food or water. Yersinia enterocolitica initially travels to the small intestine and invades the intestinal barrier via M cells, a specialized group of follicleassociated epithelial cells that function in antigen uptake (Bottone, 1999; Pujol & Bliska, 2005). Adherence to and invasion of epithelial layers require at least two chromosomal genes, inv (invasion) and ail (attachment invasion locus). After invasion of the intestinal epithelium, the bacteria replicate within lymphoid follicles known as Peyer’s patches. The presence of an approximately 70 kb virulence plasmid, termed pYV (plasmid for Yersinia virulence) enables Y. enterocolitica to survive and multiply in lymphoid tissues. The bacteria can spread from the lymphoid follicles to mesenteric lymph nodes, resulting in mesenteric lymphadenitis. Yersinia enterocolitica strains belonging to certain few bioserotypes can cause human disease. Most strains associated with yersiniosis belong to the following bioserotypes: 1B/O:8; 2/O:5,27; 2/O:9; 3/O:3; 4/O:3. These bioserotypes have been shown to have different geographical distributions. Strains largely responsible for human yersiniosis in Europe, Japan, Canada and the USA belong to bioserotype 4/O:3 (Bottone, 1999). Strains of five biotypes (1B, 2, 3, 4 and 5) can carry the pYV, which is required for full expression of virulence, and several chromosomally encoded virulence determinants. Strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and are considered to be nonpathogenic. However, growing clinical, epidemiological and experimental evidence suggests that some biotype 1A strains are virulent and can cause gastrointestinal disease (Tennant et al., 2003). Several studies have been conducted to investigate the distribution of different virulence genes (ail, inv, yst, yadA, virF and yopT) among Y. enterocolitica strains by PCR (Thoerner et al., 2003; Falcao et al., 2004; Lee et al., 2004; G¨urtler et al., 2005). A correlation between biotypes and the presence of plasmid 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

M. Fredriksson-Ahomaa et al.

and chromosomal virulence genes has been found. However, plasmid-borne genes (yadA, virF and yopT) have been detected with variable efficiency owing to heterogeneity within the bacterial population for the presence of the virulence plasmid.

Molecular detection of Y. enterocolitica in natural samples Considerable difficulties are associated with isolating Y. enterocolitica from clinical, food and environmental samples. Conventional culture-dependent methods have several limitations, such as long incubation steps taking up to 4 weeks, lack of identification between species, and lack of discrimination between pathogenic and nonpathogenic strains (Cocolin & Comi, 2005). In addition, the ability of bacteria, including Y. enterocolitica, to persist in samples in a viable but nonculturable state can be a problem (Alexandrino et al., 2004). Thus, investigations have been undertaken to develop rapid and reliable methods for detecting pathogenic Y. enterocolitica in natural samples. In the field of molecular diagnostics, PCR is the accepted method for detecting nucleic acids in a variety of samples. PCR is the most commonly used nucleic acid amplification technique for the diagnosis of infectious diseases, surpassing the probe and signal amplification methods. Using PCR, pathogenic Y. enterocolitica can be detected in samples rapidly and with high specificity and sensitivity (Fredriksson-Ahomaa & Korkeala, 2003). Several PCR assays have been developed to detect pYVpositive Y. enterocolitica in clinical, food and environmental samples (Fredriksson-Ahomaa & Korkeala, 2003a). Many of these samples use primers targeting the yadA or virF gene located on the pYV. Because of possible plasmid loss, PCR methods targeting chromosomal virulence genes have also been created for natural samples. The ail, inv and yst genes, located in the chromosome of pathogenic Y. enterocolitica strains, are the most frequently used chromosomal targets. In addition, some PCR assays using the Yersinia-specific region of the 16S rRNA gene have been designed to detect Yersinia spp., especially in blood samples (Table 1). Real-time PCR is a powerful advancement of the basic PCR technique. Since real-time PCR thermal cyclers became commercially available in the late 1990s, the technology has been recognized as an outstanding tool in molecular diagnostics. Compared with conventional PCR, real-time PCR facilitates automation, computerization and quantification of nucleic acids. A significant improvement introduced by real-time PCR is the increased speed with which it can produce results. This is largely due to reduced cycle times, removal of separate post-PCR detection procedures and use of sensitive fluorescence detection equipment, allowing FEMS Immunol Med Microbiol 47 (2006) 315–329

317

Molecular epidemiology of Y. enterocolitica

Table 1. Real-time PCR assays developed for detection of Yersinia enterocolitica in clinical, food and environmental samples Sample

Gene region

Detection system

Reference

Blood

16S rRNA 16S rRNA ail 16S rRNA yadA ail yst 16S rRNA 16S rRNA, yadA 16S rRNA, yadA

Taqman SYBRGreen Taqman SYBRGreen SYBRGreen Taqman Taqman SYBRGreen SYBRGreen SYBRGreen

Sen (2000); Sen & Asher (2001) Wolffs et al. (2004) Jourdan et al. (2000) Wolffs et al. (2004) Fukushima et al. (2003) Boyapalle et al. (2001); Jourdan et al. (2000) Vishnubhatla et al. (2000); Wu et al. (2004) Wolffs et al. (2004) Wolffs et al. (2005) Wolffs et al. (2005)

Faeces

Food

Water

earlier amplicon detection. This helps to increase throughput and reduce carry-over contamination. At present, the most popular real-time PCR assays are based on ‘Taqman’ and ‘SYBRGreen’ approaches. The Taqman system is a 5 0 -nuclease assay that utilizes specific hybridization of a dual-labelled Taqman probe to the PCR product. The SYBRGreen system is based on the binding of the fluorescent SYBRGreen dye to the PCR product. Although both assays are potentially rapid and sensitive, their principles of detection and optimization are different, as is the resulting price per assay. Recently, several real-time PCR assays for qualitative detection of Y. enterocolitica in natural samples have been developed (Table 1). Boyapalle et al. (2001) reported that the Taqman assay was 1000– 10 000 times more sensitive than the culture method or traditional PCR assay when pig samples were studied. Sensitive methods are particularly necessary to detect pathogenic Y. enterocolitica in asymptomatic carriers, when studying, for example, possible animal reservoirs for this pathogen. Rapid and sensitive methods are also needed to detect small numbers of Y. enterocolitica organisms in blood units used for transfusion.

Molecular identification of Y. enterocolitica strains Identification systems for Y. enterocolitica based on biochemical reactions do not guarantee reliable identification at species level. Members of nonpathogenic Yersinia species can easily be misidentified as Y. enterocolitica. To identify presumptively typed Y. enterocolitica, Neubauer et al. (2000b) described a PCR method targeting the 16S rRNA gene combined with sequencing. Based on different DNA– DNA hybridization values and 16S rRNA gene sequences, Neubauer et al. (2000a) demonstrated that Y. enterocolitica should be divided into two subspecies: Y. enterocolitica ssp. enterocolitica and Y. enterocolitica ssp. palearctica. Yersinia enterocolitica ssp. enterocolitica consists of strains of biotype 1B, and Y. enterocolitica ssp. palearctica contains the remaining strains. FEMS Immunol Med Microbiol 47 (2006) 315–329

Plasmid-encoded targets, like virF and yadA genes, and chromosomally encoded targets, like ail, inv, yst and rfb genes, have also been used in PCR to identify Y. enterocolitica strains (Fredriksson-Ahomaa & Korkeala, 2003a; Bhaduri et al., 2005; Thisted Lambertz & Danielsson-Tham, 2005). Thisted Lambertz & Danielsson-Tham (2005) designed a multiplex PCR targeting virF, ail, yst and rfbC genes for identification of pathogenic Y. enterocolitica isolates. Using these four primer pairs, it was possible to differentiate Y. enterocolitica serotype O:3 from other pathogenic serotypes as well as from Yersinia pseudotuberculosis in the same reaction. Jacobsen et al. (2005) have recently developed a real-time PCR assay based on the perosamine synthetase (per) gene to detect Y. enterocolitica O:9. This serotype and Brucella spp. cross-react in serological tests due to identical O-antigens in their lipopolysaccharides.

Molecular subtyping of Y. enterocolitica strains Outbreaks of infectious disease often result from exposure to a common source of the aetiological agent. Generally, the aetiological agent causing an outbreak of bacterial infection is derived from a single cell whose progeny are genetically identical or closely related to the source organism. In epidemiological studies, subtyping is important in recognizing outbreaks of infection, in detecting the cross-transmission of pathogens and in determining the source of the infection. Subtyping has been accomplished by a number of different approaches. All organisms within a species must be typeable by the method used. Moreover, the subtyping methods should have high reproducibility and good discriminatory power. Subtyping of Y. enterocolitica strains has previously largely relied on phenotypic characteristics such as biochemical properties (biotyping), O and H antigens (serotyping), antimicrobial susceptibility (antibiogram typing) and bacteriophage lysis patterns (phage typing). However, because of their low discriminatory power, these techniques have partly been replaced by DNA-based molecular methods. 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

318

M. Fredriksson-Ahomaa et al.

Genotyping of Y. enterocolitica has made great strides in the last decades, and several different DNA-based methods have been used to characterize Y. enterocolitica strains (Table 2). Ideally, the typing methods should have high typeability, reproducibility and discriminatory power. They should also be inexpensive, generally available and easy to use. Finally, the results should be easy to interpret. However, the high similarity between strains and the predominating genotypes within the bioserotype have limited the benefit of these methods in epidemiological studies.

Restriction endonuclease analysis of plasmid and restriction endonuclease analysis of chromosome Plasmid analysis, the first bacterial typing tool, has been used to differentiate bacterial strains, but pathogenic strains of Y. enterocolitica contain only one virulence plasmid (pYV) of about 70 kb. Thus, for subtyping of pYV-positive Y. enterocolitica strains, the isolated plasmid has been cut with different frequent-cutting restriction enzymes (Table 3). Restriction endonuclease analysis of the plasmid (REAP) has been applied in several studies to compare pYV-positive Y. enterocolitica strains. This method has the advantage of being rapid and easy to perform but cannot be used for plasmid-negative strains. In most studies, the profiles were serotype-specific (Nesbakken et al., 1987; Kapperud et al., 1990; Kwaga & Iversen, 1993; Iteman et al., 1996). Serotype O:8 strains display the highest degree of REAP polymorphism, whereas strains of serotypes O:3 and O:9 display low diversity. Fukushima et al. (1993, 1997) showed a close correlation between geographical distribution and REAP patterns among strains of serotype O:5,27. However, because of the low degree of polymorphism within serotypes O:3 and O:9, the use of REAP for epidemiological tracing of these serotypes did not yield much information. Chromosomal DNA restriction analysis was the first of the chromosomal DNA-based typing schemes. A major limitation of this technique is the difficulty in interpreting complex profiles, which consist of hundreds of bands that

may be unresolved and overlapping. Kapperud et al. (1990) used restriction endonuclease analysis of chromosome (REAC) to study polymorphism in restriction fragment patterns among Y. enterocolitica strains belonging to different bioserotypes. A total of 22 distinct REAC patterns were distinguished among the 72 Yersinia strains examined, and each pattern was bioserotype-specific. Some DNA profiles also varied between strains belonging to the same bioserotype. REAC proved to have the greatest discriminatory power within bioserotype 1B/O:8. Compared with bioserotype 1B/O:8, bioserotypes 3/O:3, 4/O:3, 2/O:5,27 and 2/O:9 were homogeneous. Most (12/13) of the strains of bioserotype 4/O:3 isolated in Norway could not be distinguished from each other by REAC, showing an overall genetic homogeneity.

REAC and Southern blotting To avoid problems associated with complex REAC patterns, probes that hybridize to specific DNA sequences are used. The separated DNA fragments are transferred from the agarose gel to a membrane by Southern blotting. The membrane-bound nucleic acid is then hybridized to one or more labelled probes. Ribotyping refers to the use of nucleic acid probes to detect polymorphisms associated with the ribosomal operons. Automated ribotyping is very useful in epidemiological surveys as the first step owing to its ability to analyse a large number of bacterial isolates in a very short time and with minimal human effort. Ribotyping has been applied to characterize Y. enterocolitica isolates in several studies, but interstudy comparisons are difficult because of the use of different restriction enzymes (Table 3). However, these studies demonstrate a good correlation between ribotypes and bioserotypes. Ribotyping has also allowed, to some extent, the subtyping of strains within a given bioserotype. Polymorphisms appear to be higher for nonpathogenic strains of biotype 1A found in the environment than for pathogenic strains of bioserotypes 4/O:3 and 2/O:9, which are adapted to an animal host. This technique has limited potential for outbreak investigations because of the

Table 2. Methods for molecular typing of Yersinia enterocolitica isolates Typing method

Typeability

Reproducibility

Discriminatory power

Use

Interpretation

REAP REAC Ribotyping PFGE PCR AFLP DNA sequencing

Variable Excellent Excellent Excellent Excellent Excellent Excellent

Good Moderate Excellent Excellent Moderate Good Excellent

Poor Moderate Variable Good Variable Good Good

Easy Easy Moderate Moderate Easy Moderate Difficult

Easy Difficult Easy Easy Moderate Moderate Moderate

Modified from Virdi et al. (2005). REAP, restriction endonuclease analysis of plasmid; REAC, restriction endonuclease analysis of chromosome; PFGE, pulsed-field gel electrophoresis; AFLP, amplified fragment length polymorphism.

2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

FEMS Immunol Med Microbiol 47 (2006) 315–329

319

Molecular epidemiology of Y. enterocolitica

Table 3. Restriction enzymes used for typing of Yersinia enterocolitica strains Typing method

Enzymes used

Reference

Restriction endonuclease analysis of plasmid

EcoRI EcoRI, BamHI

Iwata et al. (2005) Nesbakken et al. (1987); Kapperud et al. (1990); Fukushima et al. (1993), (1997); Iteman et al. (1996); Hayashidani et al. (2003) Kwaga & Iversen (1993) Pulkkinen et al. (1986) Kapperud et al. (1990); Fukushima et al. (1993)

Restriction endonuclease analysis of chromosome Southern hybridization Ribotyping

YeO:3RS IS Yen2w Pulsed-field gel electrophoresis

EcoRI, BamHI, HindIII EcoRI, BamHI, HindIII, XbaI HaeIII

EcoRI NciI PvuII AvaI, NciI EcoRI, EcoRV HindIII, BglI EcoRI, EcoRV, NciI HindIII, BglI, SalI, NciI, EcoRI NciI, BglI EcoRI NotI

XbaI NotI, BlnI NotI, XbaI

Amplified fragment length polymorphism

XbaI, SpeI NotI, ApaI, XhoI NotI, XbaI, SpeI NotI, XbaI, SpeI, SfiI BamHI, BspDI

Hayashidani et al. (2003); Iwata et al. (2005) Blumberg et al. (1991) Burnens et al. (1996) Andersen & Saunders (1990) Fukushima et al. (1998) Lobato et al. (1998) Iteman et al. (1996) Mendoza et al. (1996) Hallanvuo et al. (2002) Golubov et al. (2005) Saken et al. (1994); Pilon et al. (2000); Hayashidani et al. (2003); Korte et al. (2004); Iwata et al. (2005); Thisted Lamberzt & Danielsson-Tham, 2005 Favier et al. (2005) Jones et al. (2003) Buchrieser et al. (1994); Asplund et al. (1998); Fredriksson-Ahomaa et al. (2000a,b) Filetici et al. (2000) Fredriksson-Ahomaa et al., (1999, 2001a,b, 2003, 2004) Najdenski et al. (1994) Shayegani et al. (1995) Fearnley et al. (2005)

YeO:3RS, probe for Y. enterocolitica O:3 repeated sequence. w

IS Yen2, probe for Y. enterocolitica insertion sequence element.

low degree of polymorphism within the most common pathogenic bioserotypes. However, it could be used to trace the global dissemination of Y. enterocolitica clones. Blumberg et al. (1991) studied 53 Y. enterocolitica O:3 strains and distinguished four different ribopatterns. All but two of the strains belonged to ribotype patterns I and II. Ribotype I strains corresponded to phage type 9b, and ribotype II strains to phage type 8. Phage type 9b is known as the ‘Canadian phage type’ because it was initially thought to be present only in Canada. It was later identified in the USA, and now this clone appears to be widely disseminated. Import of a large number of Canadian pigs into the USA may explain how strains of ribotype pattern I were introduced. Phage type 8, originally known as ‘European and Japanese phage type’, also appears to be widely disseminated. This type was identified almost 10 years after the first documentation of phage 9b. Although many fewer pigs have been imported to the USA from Europe than from Canada, this practice may have led to the introduction of ribotype pattern II strains. Andersen & Saunders (1990) have also found some evidence of geographical variation in the FEMS Immunol Med Microbiol 47 (2006) 315–329

distribution of ribotypes among the strains of bioserotype 4/O:3; Canadian strains were found to be different from Danish strains. Fukushima et al. (1998) studied 103 strains of serotype O:9 with REAP and ribotyping. They demonstrated a close correlation between genotypes (ribotypes and REAP profiles) and geographical distribution of strains of serotype O:9. Ribotype 1 has been widely detected in isolates from humans, animals and food worldwide, ribotype 2 in West European countries and the USA, ribotype 3 in Russia and ribotype 4 in Japan. Hallanvuo et al. (2002) used a probe containing the O-antigen-coding region, which is present in multiple copies in the genome of Y. enterocolitica. This repeated sequence, termed YeO:3RS, was found only in strains of the European pathogenic serotypes (O:1, O:2, O:3, O:5,27 and O:9) among the genus Yersinia. Using the YeO:3RS probe and the NciI and BglI restriction enzymes, all European pathogenic Y. enterocolitica isolates of bioserotype 4/O:3, 2/ O:5,27 and 2/O:9 could efficiently be detected and subtyped when 112 Y. enterocolitica isolates from humans, animals and food were studied. Within bioserotype 4/O:3, YeO:3RS 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

320

typing using NciI and BglI restriction enzymes was as discriminatory as pulsed-field gel electrophoresis (PFGE) typing using NotI and XbaI enzymes; the discrimination indices of the YeO:3RS and PFGE methods were 0.73 and 0.69, respectively. When these methods were combined, the two predominant PFGE types could be divided into six subtypes, thus increasing the discriminatory power to 0.85. Golubov et al. (2005) investigated a new Y. enterocolitica insertion sequence element, IS Yen2, which is present in multiple copies in Y. enterocolitica O:3 and O:9. The novel IS element is restricted to strains of biotypes 2–5 and is absent in the strains of biotype 1B, but also in the strains of nonpathogenic biotype 1A. Using the IS Yen2 probe and EcoRI restriction enzyme, all strains of biotypes 2–5 could be differentiated from strains of biotypes 1A and 1B. Furthermore, the number of IS Yen2 copies varied among the Y. enterocolitica O:3 strains, indicating that this method can serve as an additional tool for Y. enterocolitica differentiation and epidemiological studies.

M. Fredriksson-Ahomaa et al.

isolates (Table 3). However, one drawback is that thus far no optimal enzyme and gel electrophoresis conditions have been found; all enzymes used in subtyping Y. enterocolitica isolates produce a large number of closely spaced restriction fragments, which can make analysis of patterns difficult. The global homogeneity of the pulsotypes among strains of bioserotype 4/O:3 has been shown to be high (Najdenski et al., 1994; Saken et al., 1994; Iteman et al., 1996; Asplund et al., 1998). However, using NotI, ApaI and XhoI enzymes, this group can efficiently be divided into several genotypes with a discrimination index of 0.93 (Fredriksson-Ahomaa et al., 1999). Diversity of different genotypes of Y. enterocolitica 4/O:3 strains recovered from pig tonsils in southern Germany and Finland has been studied using these three restriction enzymes. All genotypes found in German strains differed from Finnish strains, indicating that genotypes of Y. enterocolitica 4/O:3 strains have a different geographical distribution (Fredriksson-Ahomaa et al., 2003).

PCR PFGE PFGE is a variation of agarose gel electrophoresis that permits analysis of large-sized DNA fragments of the whole bacterial genome. PFGE used with various rare-cutting restriction enzymes is considered the gold standard in subtyping of bacteria. PFGE provides a highly reproducible restriction profile that typically shows distinct, well-resolved fragments representing the entire bacterial DNA. The method has proved to be highly discriminatory and superior to other available techniques (Lukinmaa et al., 2004). With the aid of computerized gel scanning and analysis software, creating data banks of PFGE patterns is possible, which enables the development of reference databases to which any new strain can be compared in order to define its relationship to other strains. One of the factors that has limited the use of PFGE is the time involved in completing the analysis. Although the procedural steps are straightforward, the time needed to complete the procedure can be 2–3 days. This can reduce the laboratory’s ability to analyse large numbers of samples. Several studies have been conducted to characterize Y. enterocolitica with PFGE (Table 3). Iteman et al. (1996) compared PFGE with ribotyping and REAP, and found PFGE to be the most suitable technique for epidemiological tracing of Y. enterocolitica. PFGE allows subtyping of strains belonging to the same bioserotype (Buchrieser et al., 1994; Najdenski et al., 1994; Saken et al., 1994). Najdenski et al. (1994, 1995) showed that the pulsotype is more closely associated with the biotype than with the serotype. They also demonstrated that the degree of genomic instability is low among Y. enterocolitica strains. NotI enzyme is the most frequently used enzyme for PFGE typing of Y. enterocolitica 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

Randomly amplified polymorphic DNA (RAPD) assay, also called arbitrary primed PCR, is a variation of the PCR technique employing a single short (typically 10 bp) primer that is not targeted to amplify any specific bacterial sequence. RAPD is a very simple and quick method, but its reproducibility can be low and standardization of the technique is difficult. However, a stable interlaboratory RAPD protocol may be obtained by careful optimization of technical procedures (Blixt et al., 2003). Some studies have been conducted to characterize Y. enterocolitica isolates with RAPD (Rasmussen et al., 1994; Odinot et al., 1995; Leal et al., 1999). This method allows discrimination between Y. enterocolitica isolates belonging to different bioserotypes and also, in some cases, between isolates belonging to the same bioserotype (Odinot et al., 1995; Leal et al., 1999). With RAPD, subtypes of pathogenic Y. enterocolitica strains correlated well with the geographical origin. Blixt et al. (2003) divided all 27 Y. enterocolitica O:3 strains into two subclusters, representing the geographical origin of the strains. One of the subclusters contained 21 Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while the other subcluster comprised six Danish and English O:3 strains together with O:9 and O:5,27 strains. Repetitive element PCR (rep-PCR) uses primers corresponding to interspersed repetitive DNA elements present in various locations within the bacterial genome to generate specific genomic fingerprints. Rep-PCR with primers based on repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) sequences has been successfully used to differentiate Y. enterocolitica strains (Sachdeva & Virdi, 2004; Wojciech et al., 2004). Sachdeva & Virdi (2004) characterized 81 Y. enterocolitica strains of FEMS Immunol Med Microbiol 47 (2006) 315–329

321

Molecular epidemiology of Y. enterocolitica

biotype 1A isolated from India, Germany, France and the USA. Although both REP and ERIC fingerprinting gave comparable results, ERIC primers discriminated the strains better than REP primers; ERIC and REP primers produced 23 and 19 genotypes, respectively. The rep-PCR genotyping showed that strains having different serotypes produced identical rep-profiles, indicating a limited genetic diversity among strains of biotype 1A. Wojciech et al. (2004) analysed 35 Y. enterocolitica strains belonging to different bioserotypes. The strains were isolated from humans, pigs and foxes in Poland. They also found that different serotypes produced identical rep-profiles. However, they demonstrated that strains belonging to the same serotype can represent different rep-profiles. REP-PCR proved to be more discriminatory than ERIC-PCR. PCR-ribotyping (ITC-profiling) is a modification of conventional ribotyping, in which the PCR is used to amplify the spacer region between the 16S and 23S rRNA genes. This spacer region has shown a significant length and sequence polymorphism across species lines, in contrast to rRNA genes, which are highly conserved. To analyse the intergenic space, the relevant DNA is amplified by PCR with defined primers. The PCR product can further be digested with a restriction enzyme, and the resulting fragments resolved electrophoretically. Internal transcribed spacer (ITS)-profiling can also be used to subtype Y. enterocolitica strains (Wojciech et al., 2004). Its discriminatory power was shown to be similar to ERIC-PCR but lower than REP-PCR when 35 Y. enterocolitica strains of different bioserotypes were studied.

Amplified fragment length polymorphism (AFLP) Amplified fragment length polymorphism (AFLP) is a recently adopted PCR-based typing method. The AFLP approach is based on selective PCR amplification of restriction fragments from total digest of genomic DNA. The technique involves four steps: restriction of DNA, ligation of oligonucleotide adapters, selective amplification of sets of restriction fragments and gel analysis of the amplified products. Selective amplification is achieved by the use of primers that extend beyond the restriction site into the restriction fragments, amplifying only the subset of fragments in which the primer extension matches the nucleotide flanking the restriction sites. Conventionally, AFLP is performed using a combination of a 6-base-specific and a 4base-specific restriction enzyme. The primers are labelled with a fluorescent label on the 5 0 end such that the amplified DNA fragments are labelled in the PCR reaction. The number of fragments that can be analysed simultaneously is dependent on the resolution of the detection system. Typically, 50–100 restriction fragments are amplified and FEMS Immunol Med Microbiol 47 (2006) 315–329

detected on denaturing polyacrylamide gels. This protocol yields AFLP fragments that are 100–500 bp long. Gel electrophoresis of the fragments is carried out on a DNA sequencing machine, and interpretation of the results is performed using gene scan analysis software. The AFLP technique provides a very powerful means of DNA fingerprinting for DNA of bacterial origin. Fearnley et al. (2005) have developed an AFLP method to genotype Y. enterocolitica. The combination of restriction enzymes BamHI (four base specific) and BspDI (two base specific) resulted in bands ranging from 35 to 500 bp. In total, 70 strains belonging to different bioserotypes isolated from humans, pigs, sheep and cattle from the UK were studied. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A included biotypes 2–5 and cluster B biotypes 1A and 1B. Within these two clusters, subclusters were formed, largely on the basis of serotype. AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating a high discriminatory power of the technique for Y. enterocolitica. Strains of bioserotype 4/O:3 were the most clonal, with 93.2% identity found among the strains tested. Strains of bioserotype 3/O:5,27 and 3/O:9 had 92.2% and 91.8% identity, respectively.

Sequencing Full-genome sequences have given us new opportunities to study bacteria and diseases. So far, the genome of more than 160 bacteria, including food-borne bacteria, has been partly or completely sequenced (ftp://ftp.ncbi.nih.gov/genbank/ genomes/bacteria). Sequencing allows the cataloguing of all genetic variables, providing knowledge about bacterial pathogenicity and helping us to understand the origin and spread of microbial disease better. Based on the sequencing of specific housekeeping genes, a multilocus sequence typing (MLST) method has been developed for strain typing. MLST is a relatively new approach that has been used to determine the genetic relatedness among strains of various bacterial species (Kotetishvili et al., 2005). Genetic variation is studied by nucleotide sequencing of chromosomal genes encoding enzymes and proteins of several functional types. DNA sequences of multiple housekeeping genes reveal the existence of clonal grouping even within bacterial species, such as Neisseria meningitis, Streptococcus pneumoniae and Helicobacter pylori, which have a high level of recombination (Achtman et al., 1999). Using five housekeeping genes and a gene involved in lipopolysaccharide biosynthesis, Achtman et al. (1999) showed that some sequence polymorphisms occurred among strains between bioserotypes and within the same bioserotype when 13 Y. enterocolitica strains were 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

322

studied. However, Y. enterocolitica proved to be relatively uniform, especially the bioserotypes of 2/O:5, 2/O:9 and 4/ O:3. Kotetishvili et al. (2005) identified two groups of Y. enterocolitica, one containing biotypes 1A and 2, and the other composed of biotype 1B, when four housekeeping genes were sequenced. Short nucleotide sequences repeated in tandem, called tandem repeats or short sequence repeats, constituting microsatellite elements, are very frequent in eukaryotic organisms as well as in bacteria. In some cases, these bacterial repetitive loci are polymorphic owing to differences in the number of repeated units that they contain. These are known as variable number tandem repeats (VNTRs). This method has been used to characterize bacterial species that are genetically homogeneous and difficult to type with other methods. The VNTR method is easy to use, fast and reproducible (Lukinmaa et al., 2004). The banding patterns can also be easily analysed. Preliminary results from de Benito et al. (2004) suggest that the analysis of VNTR in the orf528 locus may provide a new method to discriminate Y. enterocolitica 4/O:3 isolates found to be identical with other epidemiological tools.

Epidemiology of Y. enterocolitica Indirect evidence suggests that food, particularly pork, is an important link between the pig reservoir and human infections. In case-control studies, a correlation has been demonstrated between the consumption of raw or undercooked pork and the prevalence of yersiniosis (Tauxe et al., 1987; Ostroff et al., 1994). To identify reservoirs of infections, transmission vehicles and associations between clinical cases, several DNA-based methods have been used to subtype Y. enterocolitica strains (Table 2). However, the high genetic similarity between Y. enterocolitica strains and the predominating genotypes among the strains have limited the benefit of these methods in epidemiological studies. Thus, many factors related to the epidemiology of Y. enterocolitica, such as sources and transmission routes of yersiniosis, remain obscure.

Reservoirs Animals have long been suspected of being reservoirs for Y. enterocolitica and, hence, sources of human infections (Bottone, 1997). Numerous studies have been carried out to isolate Y. enterocolitica strains from a variety of animals. However, most of the strains isolated from animal sources differ both biochemically and serologically from strains isolated from humans with yersiniosis. Y. enterocolitica strains that belong to bioserotypes associated with human disease have frequently been isolated from tonsils and faecal samples of slaughtered pigs. In several countries, Y. enterocolitica of bioserotype 4/O:3 has been shown to be the 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

M. Fredriksson-Ahomaa et al.

predominant bioserotype in asymptomatic pigs. Occasionally, pathogenic Y. enterocolitica strains, mostly of bioserotype 4/O:3, have been isolated from dogs and cats (Fredriksson-Ahomaa et al., 2001b). Dogs excrete this organism in faeces for several weeks after infection. Thus, pets may be one source of human infections because of their close contact with people, especially young children. Y. enterocolitica strains of biotypes 2 and 3 and serotypes O:5,27 and O:9 have sporadically been isolated from slaughter pigs, cows, sheep and goats; however, the reservoir of these bioserotypes is not clearly established (Fukushima et al., 1993; Wojciech et al., 2004; Fearnley et al., 2005). Wild rodents and pigs have been shown to be reservoirs for Y. enterocolitica O:8 strains in Japan (Hayashidani et al., 2003). Strains of very rare bioserotypes, such as bioserotype 5/O:2,3, have been isolated from sheep, hares and goats, and bioserotype 3/O:1,2a,3 from chinchillas. The PCR method has been applied in only a few studies investigating the prevalence of pathogenic Y. enterocolitica in pigs (Fredriksson-Ahomaa et al., 2000a; Korte et al., 2004; Bhaduri et al., 2005). The detection rate is clearly higher with PCR, especially when using real-time PCR (Boyapalle et al., 2001; Bhaduri et al., 2005). The prevalence of ailpositive Y. enterocolitica in mesenteric lymph nodes of pigs was surprisingly high (40%) when the Taqman assay was used (Boyapalle et al., 2001) (Table 4). Korte et al. (2005) determined the prevalence of yadApositive Y. enterocolitica in pig tonsils in Finland using a conventional PCR method. The detection rate in fattening pigs (56%) was high compared with sows (14%), indicating that fattening pigs are an important reservoir of pathogenic Y. enterocolitica. The authors further characterized the Y. enterocolitica 4/O:3 strains isolated from sows and fattening pigs using PFGE. A total of nine and 34 NotI profiles were obtained for 26 sow strains and 122 fattening pig strains, respectively. Only three sow strains differed genotypically from pig strains, suggesting that Y. enterocolitica-positive sows could be an important reservoir in pig houses. Sows can transmit pathogenic Yersinia directly to weaning pigs or indirectly to other pigs via a contaminated environment. Similar genotypes were found in 1995 and 1999, demonstrating that the genomes of Y. enterocolitica 4/O:3 strains are very stable. Pilon et al. (2000) studied the distribution of different genotypes of pathogenic Y. enterocolitica in pig herds in eastern Canada. Most (143/153) of the isolates belonged to serotype O:3, which is the predominant serotype in eastern provinces. The genotypic profiles obtained with PFGE using NotI enzyme were similar within a given farm over all visits. This indicates that in each herd certain strains are present, which persist on the farm for a long time. All environmental isolates, except one, has a NotI profile identical to that of an isolate recovered in pig faeces from the same farm. This suggests that the environment represents a FEMS Immunol Med Microbiol 47 (2006) 315–329

323

Molecular epidemiology of Y. enterocolitica

Table 4. Detection of pathogenic Yersinia enterocolitica in natural samples with PCR and culture methods Sample Clinical Pig tonsils

Pig faeces

Mesenteric lymph nodes Submaxillary lymph nodes Foodw Pig tongues Pig offalz Chitterlings Ground pork Ground beef Minced pork Pork‰ Chicken Fish Lettuce Tofu Vegetables Environment Water Slaughterhouse

No. of samples

No. of culture-positive samples (%)

No. of PCR-positive samples (%)

185 252 24 255 24 2793 257 24 24

48 0 15 0 3 114 0 1 1

58 90 18 80 3 345 103 2 3

(31) (36) (75) (31) (13) (12) (40) (8) (13)

Fredriksson-Ahomaa et al. (2000a) Boyapalle et al. (2001) Nesbakken et al. (2002) Boyapalle et al. (2001) Nesbakken et al. (2002) Bhaduri et al. (2005) Boyapalle et al. (2001) Nesbakken et al. (2002)

15 99 110 350 350 100 100 255 300 91 43 200 101 50 27

7 79 38 8 0 32 23 4 6 6 0 0 0 0 1

(67) (83) (70) (79) (38) (47) (31) (25) (17) (10)

Vishnubhatla et al. (2001) Fredriksson-Ahomaa & Korkeala (2003b)

(32) (23) (2) (2) (7)

(4)

10 82 77 278 133 47 31 63 50 9 0 0 3 6 4

(3) (12) (15)

Vishnubhatla et al. (2001) Cocolin & Comi (2005)

105 89

1 5

(1) (6)

11 12

(10) (13)

Sandery et al. (1996) Fredriksson-Ahomaa et al. (2000b)

(26) (63) (13) (4) (4) (4) (47) (80) (35) (2)

Reference

Boyapalle et al. (2001) Vishnubhatla et al. (2001) Fredriksson-Ahomaa & Korkeala (2003b) Johannessen et al. (2000) Thisted Lambertz & Danielsson-Tham (2005) Fredriksson-Ahomaa & Korkeala (2003b)

Pathogenicity of isolates confirmed. w

All meat samples are raw. Liver, heart, kidney. ‰ Except pig tongues and offal. z

source of contamination of pigs by Y. enterocolitica. However, because the prevalence of pathogenic Y. enterocolitica in the environment was clearly lower than in pigs, the pigs probably are the main source of pathogenic isolates on the farms. Several studies using different typing methods have been conducted to compare human strains with animal, mostly pig, strains. Most of the reports support the hypothesis that pigs are the main source of human Y. enterocolitica infections (Andersen & Saunders, 1990; Kapperud et al., 1990; Lee et al., 1990; Fukushima et al., 1993; FredrikssonAhomaa et al., 2001a; Wojciech et al., 2004; Fearnley et al., 2005). Studies of REAC patterns have shown that strains of serotypes O:3 and O:9, which are generally implicated in human disease in Europe, had patterns identical to those of strains isolated from pigs (Kapperud et al., 1990). Andersen and Saunders (1990) detected no differences in the ribotypes of Y. enterocolitica strains from pigs and human patients in Denmark. Similarly, Lee et al. (1990) found identical riboFEMS Immunol Med Microbiol 47 (2006) 315–329

type patterns of Y. enterocolitica O:3 in chitterlings and human specimens from the Atlanta outbreak. With three PCR-based techniques (ITC-profiling, ERIC-PCR and REPPCR), Wojciech et al. (2004) demonstrated that many of the human Y. enterocolitica strains of bioserotypes 4/O:3 and 2/O:9 share common genotypes with pig strains. However, the Y. enterocolitica strains recovered from humans and pigs exhibited limited heterogeneity, and two prevailing genotypes were observed. Fredriksson et al. (2001a) compared 212 Y. enterocolitica 4/O:3 strains isolated from humans with sporadic yersiniosis with 334 Y. enterocolitica 4/O:3 strains isolated from porcine origin using PFGE with NotI, ApaI and XhoI enzymes. They found 80% (170/212) of human strains to be indistinguishable from 247 strains of porcine origin in Finland. Two common genotypes obtained among human strains were also found among dog and cat strains, indicating that pet animals are also a possible source of human infection. Investigation of the relationship between strain and host AFLP profiles showed that pigs and sheep 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

324

may be a potential source of human Y. enterocolitica 4/O:3 and 3/O:9 infections in Great Britain (Fearnley et al., 2005). Fukushima et al. (1993) examined 123 strains of serotype O:5,27 using REAP and obtained three distinct restriction patterns. All human strains, most of the pig strains and some of the dog strains showed similar REAP patterns, suggesting the possibility that pigs and dogs are sources of serotype O:5,27 infections in humans in Japan. Using PFGE, ribotyping and REAP, wild rodents and pigs have been shown to be reservoirs for Y. enterocolitica O:8 strains in Japan (Hayashidani et al., 2003). Indistinguishable genotypes were found among strains isolated from humans, wild rodents and pigs, indicating that wild rodents and pigs are possible infection sources for human Y. enterocolitica O:8 infections. Iwata et al. (2005) recently reported the first fatal cases of Y. enterocolitica O:8 infection in monkeys. Using PFGE, ribotyping and REAP, they demonstrated that the genotypes of almost all outbreak isolates were undistinguishable from each other, indicating a common infection source. Furthermore, the strains from black rats had the same genotype as the strains from monkeys, which further supports the hypothesis that wild rodents are the infection source and an important reservoir for serotype O:8. Hosaka et al. (1997) characterized a Y. enterocolitica O:8 strain isolated from a human with septicaemia using PFGE with XbaI enzyme and compared this strain with an earlier Y. enterocolitica O:8 strain isolated from humans with gastroenteritis. The XbaI patterns between the strains were very similar. No specific food could be determined as the source of infection for the septicaemia patient. The patient had not been exposed to small rodents, which are considered to be carriers of this pathogen in Japan. Furthermore, the patient had not travelled to the area, where serotype O:8 had been sporadically isolated. The authors therefore suggested that the bacterium persists latently in healthy humans throughout Japan; however, the true infection source remains obscure.

Contamination of food and environment Food has often been suggested to be the main source of Y. enterocolitica infection, although pathogenic isolates have seldom been recovered from food samples. Raw pork products have been widely investigated because of the association between Y. enterocolitica and pigs. However, the isolation rates of pathogenic bioserotypes of Y. enterocolitica have been low in raw pork, except for in edible pig offal, with the most common type isolated being bioserotype 4/O:3. The low isolation rates of pathogenic Y. enterocolitica in food samples may be due to limited sensitivity of culture methods (Fredriksson-Ahomaa & Korkeala, 2003a). The occurrence of pathogenic Y. enterocolitica in some foods has been estimated by both culture and PCR methods (Table 4). In 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

M. Fredriksson-Ahomaa et al.

all of these studies, the prevalence was higher by PCR than by culturing. Prevalence of yadA-positive Y. enterocolitica in food has been studied in Finland using the PCR method (Fredriksson-Ahomaa & Korkeala, 2003b). The highest detection rate was obtained from pig offal, including pig tongues (83%), livers (73%), hearts (71%) and kidneys (67%). The detection rate was clearly higher in minced meat with the PCR method than with the culture method (Table 4). Using PCR, Thisted Lambertz et al. (2005) detected ail-positive Y. enterocolitica in 10% (9/91) of raw pork samples (loin, fillet, chop, ham and minced meat) and in one of 27 ready-to-eat pork products. Surprisingly, Vishnuhatla et al. (2001) found a high occurrence of yst-positive Y. enterocolitica in ground beef. Pathogenic strains have only sporadically been isolated from beef. In the same study, yst-positive Y. enterocolitica was also detected in tofu by real-time PCR. These PCR results indicate that the true rate of contamination of pathogenic Y. enterocolitica in pork and other processed meats and foods is underestimated using culture methods. No pathogenic Y. enterocolitica has been detected in fish and chicken samples in Finland; however, three (3%) lettuce samples were positive with PCR (Table 4). In Korea, Lee et al. (2004) isolated one ail-positive Y. enterocolitica strain of bioserotype 3/O:3 from 673 ready-to-eat vegetables, which supports the PCR results and shows that vegetables can be a source of human infection. Furthermore, Sakai et al. (2005) reported a food-borne outbreak of Y. enterocolitica O:8 in Japan where the same PFGE pattern was obtained from all patient and salad isolates. Recently, Y. enterocolitica 2/O:9 has been isolated from chicken eggshell surfaces in Argentina (Favier et al., 2005). Using PFGE, XbaI patterns revealed a genomic heterogeneity among the strains, which suggests different contamination sources. Contamination of the egg surface might have occurred from contact with other Y. enterocolitica-contaminated animal products, such as pork, during collection on farms or during transportation or handling in retail shops. In a case-control study, untreated drinking water has been reported to be a risk factor for sporadic Y. enterocolitica infections in Norway (Ostroff et al., 1994). Drinking water has been relatively widely investigated and revealed to be a significant reservoir for nonpathogenic Y. enterocolitica. However, Sandery et al. (1996) detected pathogenic Y. enterocolitica in 10% of environmental water by PCR. Falcao et al. (2004) recently tested 67 Y. enterocolitica strains isolated in Brazil from untreated water for the presence of virulence genes by PCR. They found that all 38 strains of serotype O:5,27 possessed inv, ail and yst genes, suggesting that water may be responsible for human infection with Y. enterocolitica. In Japan, Y. enterocolitica O:8 strains have been isolated from stream water (Hayashidani et al., 2003; Iwata et al., 2005). Indistinguishable genotypes using PFGE, FEMS Immunol Med Microbiol 47 (2006) 315–329

325

Molecular epidemiology of Y. enterocolitica

ribotyping and REAP were found among human and stream water strains, indicating that stream water is a possible infection source for human Y. enterocolitica O:8 infections. The combination of ribopatterns obtained with five different restriction enzymes allowed differentiation of Y. enterocolitica O:3 strains, including 48 human strains and 24 food strains from Spain, into 11 genotypes (Mendoza et al., 1996). Raw pig minced meat and raw sausages were identified as possible sources of human yersiniosis because all Y. enterocolitica O:3 strains from the 24 food samples were grouped into the three most frequent genotypes together with human strains. Thisted Lambertz and DanielssonTham (2005) characterized Y. enterocolitica 4/O:3 strains recovered from patients and pork products in Sweden with PFGE using the NotI enzyme. Ten NotI profiles were revealed for 44 human strains and four profiles for six pork strains. These four pork profiles were also present among isolates recovered from the yersiniosis patients. Furthermore, Fredriksson-Ahomaa et al. (2001a) recovered indistinguishable genotypes among 140 human and 66 retailed pork strains of Y. enterocolitica 4/O:3 using NotI, ApaI and XhoI enzymes, which further reinforces the assumption that Y. enterocolitica infections are of food origin. Pathogenic Y. enterocolitica has frequently been detected on pig carcasses and especially on pig offal in slaughterhouses in Finland (Fredriksson-Ahomaa & Korkeala, 2003b). The major contamination source of edible offal may be the tonsils, which are frequently Yersinia-positive. The tonsils are removed along with the pluck set (tongue, oesophagus, trachea, lungs, heart, diaphragm, liver and kidneys) and then hung on a hook or placed on a conveyer belt. During this process the spread of pathogenic Yersinia from the tonsils to the pluck set is unavoidable. The most common PFGE types found in pig tonsils in Finland (Fredriksson-Ahomaa et al, 2000a) were also found on pig carcasses and offal (ear, livers, hearts and kidneys) in the slaughterhouse (Fredriksson-Ahomaa et al., 2000b), supporting the hypothesis that tonsils contaminate the offal. In another study by Fredriksson-Ahomaa et al. (2001a), a major contamination source of human Y. enterocolitica 4/O:3 infections was revealed to be edible pig offal: 151 (71%) of the human strains were indistinguishable from the strains isolated from tongues, livers, kidneys and hearts. The same genotypes have been found at the retail level on edible pig offal and pork. These results reveal that contaminated pig offal is an important vehicle in transmission of Y. enterocolitica bioserotype 4/O:3 from the slaughterhouse to humans. yadA-positive Y. enterocolitica has been detected by PCR in a variety of environmental sources in Finnish slaughterhouses (Fredriksson-Ahomaa et al., 2000b). The PCR method yielded more positive samples than did the traditional culture methods (Table 4), indicating that virulent Yersinia may occur at higher frequencies in the slaughterhouse FEMS Immunol Med Microbiol 47 (2006) 315–329

environment than previously assumed. Pathogenic Y. enterocolitica was detected on the brisket saw, the hook from which the pluck set hangs, the knife used for evisceration, the aprons used by trimming workers, the computer keyboard used in the meat inspection area and the handle of the coffee maker used by slaughterhouse workers. PCR-positive samples were also obtained from the floor in the eviscerating and weighing areas and from the table in the meat-cutting area, revealing that the pig slaughterhouse environment is contaminated with pathogenic Y. enterocolitica. The Y. enterocolitica 4/O:3 isolates recovered from the brisket saw, hook and knife showed the same PFGE pattern that had dominated among carcass and offal isolates, indicating that tools and machines may spread Yersinia in the slaughterhouse. Some of the PFGE types found on the computer were also observed on livers and kidneys, which reinforces the assumption that meat inspectors spread pathogenic Y. enterocolitica from offal with their hands. Distribution of genotypes of Y. enterocolitica 4/O:3 strains in butcher shops in Munich has been studied with PFGE using NotI, ApaI and XhoI enzymes (Fredriksson-Ahomaa et al., 2004). Twelve genotypes were obtained among 33 isolates from 14 pork and two environmental samples, demonstrating that several different strains were distributed in butcher shops. The genotypes differed among butcher shops, possibly because raw material was purchased from different sources. In most shops, more than one genotype was found, indicating that the raw material was contaminated with different strains. These results show that pathogenic Y. enterocolitica can easily be transmitted from slaughterhouses via contaminated raw material to the retail level.

Possible transmission routes The most common transmission route of pathogenic Y. enterocolitica is thought to be faecal–oral via contaminated food. Direct person-to-person contact has not been demonstrated, but Lee et al. (1990) reported Y. enterocolitica O:3 infections in infants who were probably exposed to infection by their carers. This may happen when basic hygiene and hand-washing habits are inadequate. Indirect person-toperson transmission has apparently occurred in several instances by transfusion of contaminated blood products (Bottone, 1999). One transmission link may be direct contact with pigs, a common risk for pig farmers and slaughterhouse workers. However, transmission of pathogenic Y. enterocolitica from pigs to humans has not yet been proven. The main sources of human infection are assumed to be pork and pork products. Pathogenic Y. enterocolitica can be transmitted from slaughterhouses to meat processing plants and then to retail level via contaminated pig carcasses and 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

326

offal (Fredriksson-Ahomaa et al., 2001a, 2004). Contaminated pork and offal are important transmission vehicles from retail shops to humans (Fredriksson-Ahomaa et al., 2001a). Cross-contamination of offal and pork will occur directly or indirectly via equipment, air and food handlers in slaughterhouses (Fredriksson-Ahomaa et al., 2000b), retail shops (Fredriksson-Ahomaa et al., 2004) and residential kitchens. The detection rate of pathogenic Y. enterocolitica in raw pork products has been shown to be high using PCR. However, consumption of raw pork would play only a limited role in the development of yersiniosis as this is not a common habit in most developed countries. Nevertheless, in Germany, raw minced pork with pepper and onion is a delicacy that can be purchased in ready-to-eat form from butcher shops. Transmission probably more often occurs via cooked pork and other food products that have been undercooked or improperly handled. Pet animals have also been suspected as being sources of human yersiniosis because of their close contact with humans, especially young children. However, transmission from pets to humans has not yet been proven. Pathogenic Y. enterocolitica may be transmitted to humans indirectly from pork and offal via dogs and cats (Fredriksson-Ahomaa et al., 2001b). Transmission of Y. enterocolitica 4/O:3 to pets via contaminated pork has been studied using PFGE with NotI, ApaI and XhoI enzymes. A total of 132 isolates, of which 16 were from cat and dog faeces and 116 from raw pork samples, were studied in Finland. The predominant genotype recovered from pig heart, liver, kidney, tongue and ear samples was also found in the cat, whose diet consisted mostly of raw pig hearts and kidneys. The dog, which was fed raw minced pork, excreted the same genotype found in the minced meat. These results show that raw pork should not be given to pets because pathogenic isolates can easily be transmitted from highly contaminated raw pork to pets. Dogs and cats may be an important transmission link of pathogenic Y. enterocolitica between pigs and young children.

Concluding remarks Yersinia enterocolitica is an important zoonotic pathogen that can cause yersiniosis in humans and animals. Pigs are assumed to be the main source of human yersiniosis, even though a definite connection between pathogenic Y. enterocolitica strains isolated from pigs and human infections has not been established. A close genetic relationship between pig and human strains of Y. enterocolitica has been demonstrated by several DNA-based methods. However, the high similarity between strains and the predominating genotypes within the bioserotype have limited the benefit of these methods in epidemiological studies. PFGE typing is highly effective in molecular epidemiological studies of Y. enterocolitica and is superior to other methods in discriminating 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

M. Fredriksson-Ahomaa et al.

between Y. enterocolitica isolates of the same bioserotype. The AFLP technique is a recently adopted typing method that utilizes fingerprinting technique to subtype Y. enterocolitica strains. This method could provide a means of discriminating Y. enterocolitica strains found to be identical with other epidemiological tools. There are considerable difficulties associated with isolating Y. enterocolitica from clinical, food and environmental samples. Conventional culture-dependent methods have several limitations, such as low sensitivity, long incubation time, lack of identification between species, and lack of discrimination between pathogenic and nonpathogenic strains. Using PCR, pathogenic Y. enterocolitica can be detected in natural samples rapidly and with high specificity and sensitivity. Recently, several real-time PCR assays for qualitative detection of Y. enterocolitica in clinical, food and environmental samples have been developed. However, to date, the PCR method has been used in only a few studies. Prevalence of pathogenic Y. enterocolitica in pigs has been determined by PCR in some countries; however, epidemiological data about other possible animal reservoirs and from many countries are still missing. Food has often been suggested to be the main source of yersiniosis, although pathogenic strains have seldom been isolated from food samples. Raw pork products have been widely investigated because of the association between Y. enterocolitica and pigs. However, the isolation rates of pathogenic Y. enterocolitica have been low, which may be due to the limited sensitivity of the culture methods. The prevalence of pathogenic Y. enterocolitica in pork products has been shown to be higher with PCR than with culturing, indicating that the true contamination rate of pathogenic Y. enterocolitica in pork has been underestimated. Occasionally, pathogenic Y. enterocolitica has been detected by PCR in vegetables and environmental water; thus, vegetables and untreated water are also potential sources of human yersiniosis. To identify other possible transmission vehicles, different food items should be studied more extensively by PCR. Using genotyping, only a few animal reservoirs of Y. enterocolitica infections have been identified. The primary source of pathogenic Y. enterocolitica is fattening pigs. A close genetic relationship between pig and human strains of Y. enterocolitica has been demonstrated by several DNAbased methods. Human pathogenic Y. enterocolitica strains share common genotypes with dog strains, indicating that dogs are a possible source of human yersiniosis. In Great Britain, sheep are suspected of being a potential reservoir of human yersiniosis. Similar AFLP patterns between human and sheep strains reinforce this assumption. Wild rodents have been shown to be an important reservoir of Y. enterocolitica O:8 strains in Japan. Indistinguishable genotypes have been found among strains isolated from humans and wild rodents. FEMS Immunol Med Microbiol 47 (2006) 315–329

327

Molecular epidemiology of Y. enterocolitica

Tonsils of fattening pigs are an important contamination source in slaughterhouses. Yersinia-positive tonsils will easily contaminate the carcass, the offal and the environment during the slaughtering process. Using PFGE, Yersiniacontaminated pork and edible pig offal have proven to be important transmission vehicles of pathogenic Y. enterocolitica from the slaughterhouse to the retail level and further to humans. Indirect transmission of pathogenic Y. enterocolitica from pets to humans may occur via contaminated pork and offal. Indistinguishable genotypes have been found among strains isolated from humans and environmental water, indicating that untreated water is a possible infection source for human yersiniosis. However, many factors related to the epidemiology of Y. enterocolitica, such as sources and transmission routes, remain obscure because of the low sensitivity of culture methods and the predominating genotypes among Y. enterocolitica strains.

References Achtman M, Zurth K, Morelli G, Torrea G, Guiyoule A & Carniel E (1999) Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc Natl Acad Sci USA 96: 14043–14048. Alexandrino M, Grohmann E & Szewzyk U (2004) Optimization of PCR-based methods for rapid detection of Campylobacter jejuni, Campylobacter coli and Yersinia enterocolitica serovar O:3 in waste water samples. Water Res 38: 1340–1346. Andersen JK & Saunders NA (1990) Epidemiological typing of Yersinia enterocolitica by analysis of restriction fragment length polymorphisms with a cloned ribosomal RNA gene. J Med Microbiol 32: 179–187. Asplund K, Johansson T & Siitonen A (1998) Evaluation of pulsed-field gel electrophoresis of genomic restriction fragments in the discrimination of Yersinia enterocolitica O:3. Epidemiol Infect 121: 579–586. Bhaduri S, Wesley IV & Bush EJ (2005) Prevalence of pathogenic Yersinia enteorcolitica strains in pigs in the United States. Appl Environ Microbiol 71: 7117–71121. Blixt Y, Knutsson R, Borch E & R˚adstr¨om P (2003) Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria. Int J Food Microbiol 83: 15–26. Blumberg HM, Kiehlbauch JA & Wachsmuth IK (1991) Molecular epidemiology of Yersinia enterocolitica O:3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes. J Clin Microbiol 29: 2368–2374. Bottone EJ (1997) Yersinia enterocolitica: the charisma continues. Clin Microbiol Rev 10: 257–276. Bottone EJ (1999) Yersinia enterocolitica: overview and epidemiologic correlates. Microb Infect 1: 323–333. Boyapalle S, Wesley IV, Hurd HS & Reddy PG (2001) Comparison of culture, multiplex, and 5 0 nuclease polymerase chain

FEMS Immunol Med Microbiol 47 (2006) 315–329

reaction assay for the rapid detection of Yersinia enterocolitica in swine and pork products. J Food Prot 64: 1352–1361. Buchrieser C, Weagant SD & Kaspar CW (1994) Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes. Appl Environ Microbiol 60: 4371–4379. Burnens AP, Frey A & Nicolet J (1996) Association between clinical presentation, biogroups and virulence attributes of Yersinia enterocolitica strains in human diarrhoeal disease. Epidemiol Infect 116: 27–34. Cocolin L & Comi G (2005) Use of culture-independent molecular method to study the ecology of Yersinia spp. in food. Int J Food Microbiol 105: 71–82. de Benito I, Cano ME, Ag¨uero J & Lobo JMG (2004) A polymorphic tandem repeat potentially useful for typing in the chromosome of Yersinia enterocolitica. Microbiology 150: 199–204. Falcao JP, Brocchi M, Proenca-Modena JL, Acrani GO, Correa EF & Falcao DP (2004) Virulence characteristics and epidemiology of Yersinia enterocolitica and yersiniae other than Y. pseudotuberculosis and Y. pestis isolated from water and sewage. J Appl Microbiol 96: 1230–1235. Favier GI, Escudero ME & de Guzman MS (2005) Genotypic and phenotypic characterization of Yersinia enterocolitica isolated from the surface of chicken eggshells obtained in Argentina. J Food Prot 68: 1812–1815. Fearnley C, On SLW, Kokotovic B, Manning G, Cheasty T & Newell DG (2005) Application of fluorescent amplified length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica. Appl Environ Microbiol 71: 4960–4965. Filetici E, Anastasio MP, Pourshaban M & Fantasia M (2000) Genotypic and phenotypic characterization of Yersinia spp. isolates from food and man. Food Microbiol 17: 261–267. Fredriksson-Ahomaa M & Korkeala H (2003a) Low occurrence of pathogenic Yersinia enterocolitica in clinical, food and environmental samples: a methodological problem. Clin Microbiol Rev 16: 220–229. Fredriksson-Ahomaa M & Korkeala H (2003b) Molecular epidemiology of Yersinia enterocolitica 4/O:3. Adv Exp Med Biol 529: 295–369. Fredriksson-Ahomaa M, Autio T & Korkeala H (1999) Efficient subtyping of Yersinia enterocolitica bioserotype 4/O:3 with pulsed-field gel electrophoresis. Lett Appl Microbiol 29: 308–312. Fredriksson-Ahomaa M, Bj¨orkroth J, Hielm S & Korkeala H (2000a) Prevalence and characterization of pathogenic Yersinia enterocolitica in pig tonsils from different slaughterhouses. Food Microbiol 17: 93–101. Fredriksson-Ahomaa M, Korte T & Korkeala H (2000b) Contamination of carcasses, offals and the environment with yadA-positive Yersinia enterocolitica in a pig slaughterhouse. J Food Prot 63: 31–35. Fredriksson-Ahomaa M, Hallanvuo S, Korte T, Siitonen A & Korkeala H (2001a) Correspondence of genotypes of sporadic

2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

328

Yersinia enterocolitica 4/O:3 strains from human and porcine origin. Epidemiol Infect 127: 37–47. Fredriksson-Ahomaa M, Korte T & Korkeal H (2001b) Transmission of Yersinia enterocolitica 4/O:3 to pets via contaminated pork. Lett Appl Microbiol 32: 375–378. Fredriksson-Ahomaa M, Niskanen T, Bucher M, Korte T, Stolle A & Korkeala H (2003) Different Yersinia enterocolitica 4/O:3 genotypes found in pig tonsils in Southern Germany and Finland. System Appl Microbiol 26: 132–137. Fredriksson-Ahomaa M, Koch U, Klemm C, Bucher M & Stolle A (2004) Different genotypes of Y. enterocolitica 4/O:3 strains widely distributed in butcher shops in Munich area. Int J Food Microbiol 95: 89–94. Fukushima H, Gomyoda M, Aleksic S & Tsubokura M (1993) Differentiation of Yersinia enterocolitica serotype O:5,27 strains by phenotypic and molecular techniques. J Clin Microbiol 31: 1672–1674. Fukushima H, Hoshina K, Itowa H & Gomyoda M (1997) Introduction into Japan of pathogenic Yersinia through imported pork, beef and fowl. Int J Food Microbiol 35: 205–212. Fukushima H, Gomyoda M & Aleksic S (1998) Genetic variation of Yersinia enterocolitica serotype O:9 strains detected in samples from western and eastern countries. Zentbl Bakteriol 288: 167–174. Fukushima H, Tsunomori Y & Seki R (2003) Duplex real-time SYBR Green PCR assays for detection of 17 species of food- or waterborne pathogens in stools. J Clin Microbiol 41: 5134–5146. Golubov A, Gierczynski R, Heesemann J & Rakin A (2005) A novel insertion sequence element, IS Yen2, as an epidemiological marker for weekly pathogenic bioserotypes of Yersinia enterocolitica. Int J Med Microbiol 295: 213–226. G¨urtler M, Alter T, Kasimir S, Linnebur M & Fehlhaber K (2005) Prevalence of Yersinia enterocolitica in fattening pigs. J Food Prot 115: 640–644. Hallanvuo S, Skurnik M, Asplund K & Siitonen A (2002) Detection of a novel repeated sequence useful for epidemiological typing of pathogenic Yersinia enterocolitica. Int J Med Microbiol 292: 215–225. Hayashidani H, Ishiyama Y, Okatani TA, et al . (2003) Molecular genetic typing of Yersinia enterocolitica O:8 isolated in Japan. Adv Exp Med Biol 529: 363–365. Hosaka S, Uchiyama M, Ishikawa M, Akahoshi T, Kondo H, Shimauchi C, Sasahara T & Inoue M (1997) Y. enterocolitica serotype O:8 septicemia in an otherwise healthy adult: analysis of chromosomal DNA pattern by pulsed-field gel electrophoresis. J Clin Microbiol 35: 3346–3347. Iteman I, Guiyoule A & Carniel E (1996) Comparison of three molecular methods for typing and subtyping pathogenic Yersinia enterocolitica strains. J Med Microbiol 45: 48–56. Iwata T, Une Y, Okatani TA, Kaneko S, Namai S, Yoshida S, Horisaka T, Horikita T, Nakadai A & Hayashidani H (2005) Yersinia enterocolitica serovar O:8 infection in breeding monkeys in Japani. Microbiol Immunol 49: 1–7.

2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c

M. Fredriksson-Ahomaa et al.

Jacobsen NR, Bogdanovich T, Skurnik M, L¨ubeck PS, Ahrens P & Hoorfar J (2005) A real-time PCR assay for the specific identification of serotype O:9 of Yersinia enterocolitica. J Microbiol Meth 63: 151–156. Johannessen GS, Kapperud G & Kruse H (2000) Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method. Int J Food Microbiol 54: 75–80. Jones TF, Buckingham SC, Bopp CA, Ribot E & Schaffner W (2003) From pig to pacifier: chitterling-associated yersiniosis outbreak among black infants. Emerg Infect Dis 9: 1007–1009. Jourdan AD, Johnson SCJ & Wesley IV (2000) Development of a fluorogenic 5 0 nuclease PCR assay for detection of the ail gene of pathogenic Yersinia enterocolitica. Appl Environ Microbiol 66: 3750–3755. Kapperud G, Nesbakken T, Aleksic S & Mollaret HH (1990) Comparison of restriction endonuclease analysis and phenotypic typing methods for differentiation of Yersinia enterocolitica isolates. J Clin Microbiol 28: 1125–1131. Korte T, Fredriksson-Ahomaa M, Niskanen T & Korkeala H (2004) Low prevalence of yadA-positive Yersinia enterocolitica in sows. Foodborne Pathog Dis 1: 45–52. Kotetishvili M, Kreger A, Wauters G, Morris JG Jr, Sulakvelidze A & Stine OC (2005) Multilocus sequence typing for studying genetic relationship among Yersinia species. J Clin Microbiol 43: 2674–2684. Kwaga J & Iversen JO (1993) Plasmids and outer membrane proteins of Yersinia enterocolitica and related species of swine origin. Vet Microbiol 36: 205–214. Leal TCA, Leal NC & de Almeida AMP (1999) RAPD-PCR typing of Yersinia enterocolitica O:3 serotype strains isolated from pigs and humans. Gen Mol Biol 22: 315–319. Lee LA, Gerber AR, Lonsway MS, Smith JD, Carter GP, Nancy DP, Parrish CM, Sikes RK, Finton RJ & Tauxe RV (1990) Yersinia enterocolitica O: infections in infants and children, associated with the household preparation of chitterlings. N Engl J Med 14: 984–987. Lee TS, Lee SW, Seok WS, Yoo MY, Yoon JW, Park BK, Moon KD & Oh DH (2004) Prevalence, antibiotic susceptibility, and virulence factors of Yersinia enterocolitica and related species from ready-to-eat vegetables available in Korea. J Food Prot 67: 1123–1127. Lobato MJ, Landeras E, Gonz´ales-Hevia MA & Mendoza MC (1998) Genetic heterogeneity of clinical strains of Yersinia enterocolitica traced by ribotyping and relationships between ribotypes, serotypes, and biotypes. J Clin Microbiol 36: 3297–3302. Lukinmaa S, Nakari UM, Eklund M & Siitonen A (2004) Application of molecular genetic methods in diagnostics and epidemiology of foodborne bacterial pathogens. APMIS 112: 908–927. M¨aki-Ikola O, Heesemann J, Toivanen A & Granfors K (1997) High frequency of Yersinia antibodies in healthy populations in Finland and Germany. Rheumatol 16: 227–229.

FEMS Immunol Med Microbiol 47 (2006) 315–329

329

Molecular epidemiology of Y. enterocolitica

Mendoza MC, Alzugaray R, Landeras E & Gonz´alez-Hevia MA (1996) Discriminatory power and application of ribotyping of Yersinia enterocolitica O:3 in an epidemiological study. Eur J Clin Microbiol Infect Dis 15: 220–226. Najdenski H, Iteman I & Carniel E (1994) Efficient subtyping of pathogenic Yersinia enterocolitica strains by pulsed-field gel electrophoresis. J Clin Microbiol 32: 2913–2920. Najdenski H, Iteman I & Carniel E (1995) The genome of Yersinia enterocolitica is the most stable of the three pathogenic species. Contrib Microbiol Immunol 13: 281–284. Nesbakken T, Kapperud G, Srum H & Dommarsnes K (1987) Structural variability of 40–50 Mdal virulence plasmids from Yersinia enterocolitica. Acta Path Microbiol Immunol Scand Sect B 95: 167–173. Nesbakken T, Eckner K, Hidal HK & Rtterud OJ (2002) Occurrence of Yersinia enterocolitica and Campylobacter spp. in slaughter pigs and consequences for meat inspection, slaughtering and dressing procedures. Int J Food Microbiol 80: 231–240. Neubauer H, Aleksic S, Hensel A, Finke EJ & Meyer H (2000a) Yersinia enterocolitica 16S rRNA gene types belong to the same genospieces but form three homology groups. Int J Med Microbiol 290: 61–64. Neubauer H, Hensel A, Aleksic S & Mayer H (2000b) Identification of Yersinia enterocolitica within the genus Yersinia. System Appl Microbiol 23: 58–62. Odinot PT, Meis JFGM, van den Hurk PJJC, HoogkampKorstanje JAAA & Melchers WJG (1995) PCR-based characterization of Yersinia enterocolitica: comparison with biotyping and serotyping. Epidemiol Infect 115: 269–277. Ostroff SM, Kapperud G, Huteagner LC, Nesbakken T, Bean NH, Lassen J & Tauxe RV (1994) Sources of sporadic Yersinia enterocolitica infections in Norway: a prospective case-control study. Epidemiol Infect 112: 133–141. Pilon J, Higgins R & Quessy S (2000) Epidemiological study of Yersinia enterocolitica in swine herds in Qu´ebec. Can J Vet 41: 383–387. Pujol C & Bliska JB (2005) Turning Yersinia pathogenesis outside in: subversion of macrophage function by intracellular yersiniae. Clin Microbiol 114: 216–226. Pulkkinen L, Granberg I, Granfors K & Toivanen A (1986) Restriction map of the virulence plasmid in Yersinia enterocolitica O:3. Plasmid 16: 225–227. Rasmussen HN, Olsen JE & Rasmussen OF (1994) RAPD analysis of Yersinia enterocolitica. Lett Appl Microbiol 19: 359–362. Sachdeva P & Virdi JS (2004) Repetitive elements sequence (REP/ ERIC)-PCR based genotyping of clinical and environmental strains of Yersinia enterocolitica biotype 1A reveal existence of limited number of clonal groups. FEMS Microbiol Lett 240: 193–201. Sakai T, Nakayama A, Hashida M, Yamamoto Y, Takebe H & Imai S (2005) Outbreak of food poisoning by Yersinia enterocolitica serotype O:8 in Nara prefecture: the first case report in Japan. Jpn J Infect Dis 58: 257–258.

FEMS Immunol Med Microbiol 47 (2006) 315–329

Saken E, Roggenkamp A, Aleksic S & Heesemann J (1994) Characterisation of pathogenic Yersinia enterocolitica serogroups by pulsed-field gel electrophoresis of genomic NotI restriction fragments. J Med Microbiol 41: 329–338. Sandery M, Stinear T & Kaucner C (1996) Detection of pathogenic Yersinia enterocolitica in environmental water by PCR. J Appl Bacteriol 80: 327–332. Sen K (2000) Rapid identification of Yersinia enterocolitica in blood by the 5 0 nuclease PCR assay. J Clin Microbiol 38: 1953–58. Sen K & Asher DV (2001) Multiplex PCR for detection of Enterobacteriaceae in blood. Transfusion 41: 1356–1364. Shayegani M, Maupin PS & Waring A (1995) Prevalence and molecular typing of two pathogenic serogroups of Yersinia enterocolitica in New York State. Contrib Microbiol Immunol 13: 33–38. Tauxe RV, Vandepitte J, Wauters G, Martin SM, Goossens V, De Moll P, van Noyen R & Theirs G (1987) Yersinia enterocolitica infections and pork: the missing link. Lancet i: 1129–1132. Tennant SM, Grant TH & Robins-Brown GRM (2003) Pathogenicity of Yersinia enterocolitica biotype 1A. FEMS Immun Med Microbiol 38: 127–137. Thisted Lambertz S & Danielsson-Tham ML (2005) Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis. Appl Environ Microbiol 71: 3674–3681. Thoerner P, Bin Kingombe CI, B¨ogli-Stuber K, Bissig-Choisat B, Wassenaar TM, Frey J & Jemmi T (2003) PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution. Appl Environ Microbiol 69: 1810–1816. Virdi J & Sachdeva P (2005) Molecular heterogeneity in Yersinia enterocolitica and ‘Y. enterocolitica-like’ species – Implications for epidemiology, typing and taxonomy. FEMS Immunol Med Microbiol 45: 1–10. Vishnubhatla A, Fung DYC, Oberst RD, Hays MP, Nagaraja TG & Flood SJA (2000) Rapid 5 0 nuclease (Taqman) assay for detection of virulent strains of Yersinia enterocolitica. Appl Environ Microbiol 66: 4131–4135. Wojciech L, Staroniewicz Z, Jakubczak A & Ugorski M (2004) Typing of Yersinia enterocolitica isolates by ITS profiling, REPand ERIC-PCR. J Vet Med B 51: 238–144. Wolffs P, Knutsson R, Norling B & R˚adstr¨om P (2004) Rapid quantification of Yersinia enterocolitica in porksamples by a novel sample preparation method, flotation, prior to real-time PCR. J Clin Microbiol 42: 1042–1047. Wolffs P, Norling B & R˚adstr¨om P (2005) Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells. J Microbiol Method 60: 315–323. Wu VCH, Fung DY & Oberst RD (2004) Evaluation of a 5 0 nuclease (Taqman) assay with athin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples. J Food Prot 67: 271–277.

2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


c