Prevalence of pathogenic Yersinia enterocolitica in pigs slaughtered at a Swiss abattoir

https://doi.org/10.1016/j.ijfoodmicro.2007.07.050Get rights and content

Abstract

Human yersiniosis is the third most common enteric disease after campylobacteriosis and salmonellosis in many European countries. However, epidemiological data on the prevalence of pathogenic Yersinia enterocolitica in animals and humans is insufficient. Pigs are assumed to be the main reservoir of pathogenic Y. enterocolitica because pig is so far the only animal species from which pathogenic strains have frequently been isolated. This work was conducted to study the frequency of ail-positive Y. enterocolitica in pigs slaughtered at a Swiss abattoir. In total, 212 pig tonsils were screened by real-time PCR and culture methods. The prevalence rate of ail-positive Y. enterocolitica in pigs at slaughter was 88% and 34% with PCR and culture methods, respectively. The 148 ail-positive isolates from the 72 culture-positive tonsils were bio-and serotyped. The most common bioserotype was 4/O:3 found in 96% (69/72) of the culture-positive samples. However, pig was also shown to be a reservoir for ail-positive Y. enterocolitica belonging to bioserotypes 2/O:5,27 and 2/O:9, which were detected in 8% (6/72) and 1% (1/72) of the culture-positive samples, respectively. Using PFGE with NotI, only a limited number of different patterns was found. In all, 6 genotypes were obtained when 86 isolates of bioserotype 4/O:3 from 69 samples were characterised and two genotypes (N1 and N4) dominated. The biotype 4 differs clearly from biotype 2 with PFGE. Antimicrobial resistance testing of 77 ail-positive Y. enterocolitica isolates from 72 samples studied with disc-diffusion revealed that all strains were sensitive to cefotaxime, chloramphenicol, ciprofloxacin and tetracycline, which are antimicrobial agents used for treatment of human disease. The isolates of bioserotype 2/O:5,27 differed from the isolates of bioserotypes 2/O:9 and 4/O:3 in resistance to ampicillin and amoxicillin/clavulanic acid.

Introduction

Yersinia enterocolitica is a foodborne pathogen that can cause acute gastroenteritis and mesenteric lymphadenitis mimicking appendicitis (Bottone, 1997). Human yersiniosis is the third most common enteric disease after campylobacteriosis and salmonellosis in many European countries (EFSA, 2006). However, epidemiological data on the prevalence of pathogenic Y. enterocolitica in animals in EU-member states are missing as the reporting of this pathogen in animals is not mandatory in most countries. Pigs and mainly their tonsils are assumed to be the main reservoir of pathogenic Y. enterocolitica because pig is so far the only animal species from which pathogenic strains have frequently been isolated. Furthermore, high similarity between pig and human strains has been demonstrated with several DNA-based methods (Fredriksson-Ahomaa et al., 2006a, Kuehni-Boghenbor et al., 2006).

There are some difficulties associated with isolating Y. enterocolitica from naturally contaminated samples. The culture methods have several limitations, such as low sensitivity and lack of discrimination between pathogenic and non-pathogenic strains. However, it is important to assess the pathogenicity of Y. enterocolitica isolates, since the majority of the isolates recovered from asymptomatic carriers, food and environmental samples are non-pathogenic having no clinical importance (Fredriksson-Ahomaa and Korkeala, 2003). One of the chromosomal encoded genes required for the virulence of Y. enterocolitica is the ail-gene, which is only present in biotypes associated with human disease (Miller et al., 1990). Using PCR, ail-positive Y. enterocolitica can be detected in natural samples with high sensitivity and specificity (Fredriksson-Ahomaa et al., 2007).

However, in order to get epidemiological information, Y. enterocolitica isolates are needed. Thus, at least one culture method has to be used in parallel to PCR method. Selective enrichment is needed, especially when food samples are studied. However, no single procedure is currently available which will recover all bioserotypes. The most commonly used selective broth is the irgasan-ticarcillin-potassium chlorate (ITC) broth, which has been useful in recovery of bioserorype 4/O:3 (Fredriksson-Ahomaa and Korkeala, 2003).

Bio-and serotyping have been extensively used to characterise Y. enterocolitica isolates. The biotyping scheme proposed by Wauters, Kandolo and Janssens (1987) has been universally adopted. Pathogenic isolates belonging to biotypes 1B, 2-5 can be differentiated from non-pathogenic isolates belonging to biotype 1A with pyrazinamidase test, which is included in this biotyping scheme. Serotypes like O:3, O:5 and O:8 associated with human disease can also be found in non-pathogenic Y. enterocolitica strains and even in various Yersinia species. Thus, an accurate biochemical characterisation is needed before or after serological typing for correct assessment of the relevance of the strains especially from food and the environment, since related species and biotype 1A strains are widely distributed in these samples. In addition, subtyping of Y. enterocolitica strains belonging to the same bioserotype is sometimes necessary, for example to study animal reservoirs or to recognise outbreaks. Pulse-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) techniques have shown to be effective in molecular epidemiological studies of Y. enterocolitica (Fearnley et al., 2005, Fredriksson-Ahomaa et al., 2006a, Kuehni-Boghenbor et al., 2006).

Nation-specific data on the prevalence of pathogenic Y. enterocolitica using different detection methods is needed to get an overall epidemiological view. So far, only very few data is available from Switzerland. Thus, the aims of this study were i) to describe the prevalence of ail-positive Y. enterocolitica in tonsils of pigs at slaughter using real-time PCR and culture methods and ii) to further pheno-and genotype the isolates to get more epidemiological information on the current situation in Switzerland.

Section snippets

Sampling and sample preparation

Tonsils of 212 finishing pigs were sampled from a slaughterhouse in Switzerland during February and March 2006. Seventeen to 35 samples were collected from two different herds on 8 different days (Table 2). The 212 tonsil samples represent 16 farms that are located in the central and north-eastern region of Switzerland, which is the region with the highest density of pig farms. The tonsils were cut out from unsplit heads with sterilised instruments at the slaughterline and put into sterile

Results and discussion

The prevalence of ail-positive Y. enterocolitica in tonsils of slaughter pigs in the sampled herds was shown to be high using real-time PCR. The prevalence rates were 88% and 85% using SYBRGreen and probe approaches, respectively (Table 1). The PCR method has been applied in only a few studies investigating the prevalence of pathogenic Y. enterocolitica in pigs (Fredriksson-Ahomaa et al., 2000, Boyapalle et al., 2001, Korte et al., 2004, Bhaduri et al., 2005). In these studies, the detection

Conclusions

The prevalence of ail-positive Y. enterocolitica was high in tonsils of slaughter pigs using real-time PCR. Strains of bioserotype 4/O:3 but also of bioserotypes 2/O:5,27 and 2/O:9 could be isolated. Serotypes O:5,27 and O:9 belonging to biotype 2 were distinguished from each other using PCR serotyping, PFGE typing and resistance pattern. All pathogenic Y. enterocolitica isolates showed a high degree of susceptibility to antimicrobial agents used to treat human infections.

Acknowledgements

We thank A.M. Zychowska from the slaughterhouse of Zurich for her help in collecting the tonsil samples.

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