Schlüsselwörter
(Englisch)
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Yersiniosis, zoonosis, molecular detection, genotyping, surveillance
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Kurzbeschreibung
(Englisch)
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Yersinia enterocolitica and Y. pseudotuberculosis comprise of both pathogenic and non-pathogenic strains. Both species can be foodborne pathogens, mainly transmitted through pork, milk or water. The differentiation between pathogenic and non-pathogenic strains is classically based on phenotypic tests; these are frequently ambigious to read with unreliable outcome, since they depend on expression of (plasmid-encoded) virulence genes, which can be lost during culture.
A multi-target genotyping method was developed, to specifically detect the presence of virulence genes, either plasmid- or chromosomal encoded, in Yersinia spp. (Project 1.00.03). The developed PCR test clearly showed that it is possible to characterize Yersinia isolates by means of genotype with reliable outcome. The known pathogenic biotypes 1B, 2, 3 and 4 were clearly grouped and are over 90%s homogeneous in virulence gene content. This suggests that the population genetics of Yersinia spp. Is relatively clonal, as is also indicated by multilocus sequence typing of the related Y. pestis and Y. pseudotuberculosis (Achtman et al., 1999). However, biotype 1A, traditionally considered non-pathogenic, was frequently recovered from patients, and the genotype of such apparent pathogenic strains was identical to that of presumably apathogenic 1A strains. Thus, genotypic characteristic of biotype 1A Y. enterocolotica does not discriminate between pathogenic and non-pathogenic strains. Since phenotypic assays do not have the desired discriminatory power either, the development of a more sensitive genotyping technique for biotype 1A isolates is required.
The aim of the present proposal is to adapt the developed genotypic test for direct use on food- and animal samples. Thus, a rapid PCR test would not only determine the presence of the species, but also determine the genetical pathotype of Yersinia spp. Furthermore, the genetic fingerprint method AFLP will be established for Yersiniae, to determine the genetic clusters within and between serotypes and biotypes. AFLP is a multilocus PCR-based genotyping technique and analyses, in contrast to classical PCR-RFLP, 50 to 100 loci of the genome. AFLP has been shown to a highly sensitive, reliable, and reproducible genotyping method for bacteria with a complex genetic population, such as Campylobacter (Duim et al, 2000). The third aim of this project is to establish a fast PCR RFLP assay to differentiate pathogenic serotype 1A strains from apathogenic strains.
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Projektziele
(Englisch)
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The following objectives are defined:
Objective 1:
To adapt the genotyping test for Yersinia spp. for direct analysis of pork and pork products.
The developed multiplex PCR test will be adapted to be applied directly on different food-stuffs for which Yersinia contamination is common. The outcome of the test will be valida-ted by microbiological culture procedure followed by biochemical speciation.
Objective 2:
To adapt AFLP for genotyping of Yersinia spp. and to produce AFLP finger-prints of representative strains from the BVET strain collection.
The technique of AFLP will be adapted for Yersinia spp, and the optimal restriction nucleo-tides and additional specific nucleotides (which together make up the recognition sites for the PCR primers used in AFLP) will be established by a systematic empiric approach.
Objective 3:
To develop a PCR RFLP test to differentiate pathogenic from apathogenic biotype 1A strains.
First, the presence of polymorphisms within gene YstB of Y. enterocolitica will be esta-blished by DNA sequence analysis. If present, these polymorphisms will be used to deve-lop a fast PCR RFLP screening test to differentiate pathogenic and apathogenic Y. entero-colitica strains within serotype 1A. Otherwise, a band will be selected from the AFLP pat-terns of biotype 1A strains that can be used as target locus for a PCR RFLP test. If both approaches fail, differences reported in ribotype will be used to identify a locus suitable for a PCR RFLP test. In either case, once a PCR RFLP is developed, this will be adapted to be used on food products, with proper validation.
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Abstract
(Englisch)
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We applied the whole genome fingerprinting method amplified fragment length polymorphism (AFLP) to 266 Y.enterocolitica strains of human and porcine origin and to reference strains. The biotypes 1A, 1B, 2, 3 and 4 clustered among themselves and could be clearly distinguished from each other. Y.enterocolitica biotype 2, 3 and 4 are more closely related to each other (83 % similarity) than to biotypes 1A and 1B. Biotype 1A showed the most heterogeneous pattern of all. Among the strains of biotype 1A we found 3 major clusters with strains human and porcine origin. As the strains of human origin were all isolated from patients suffering from diarrhea while porcine strains were generally not related to disease, our findings suggest that Y.enterocolitica biotype 1A contains both pathogenic and non-pathogenic strains, or that biotype 1A strains are of moderate virulence which cause disease only in particular susceptible individuals. AFLP showed to be a valuable genetic method to type Y.enterocolitica and other Yersinia spp.
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Publikationen / Ergebnisse
(Deutsch)
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Kühni, Kathrin, Tierärztin von Langnau i.E. BE (2004): Genotyping of Human and Porcine Yersinia enterocolitica Strains from Switzerland by Amplified Fragment Length Polymorphism. Dissertation Institut für Veterinärbakteriologie der Universität Bern, Switzerland.
Zugehörige Dokumente
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Publikationen / Ergebnisse
(Englisch)
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Kühni, Kathrin, et al. (2006) Genotyping of Human and Porcine Yersinia enterocolitica Strains from Switzerland by Amplified Fragment Length Polymorphism Analysis. Applied and Environmental Microbiology, Vol. 72, No. 6, p.4061-4066.
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