Wojciech Szweda

Yersiniosis - a zoonotic foodborne disease of relevance to public health.

INTRODUCTION Y. enterocolitica is the causative agent of yersiniosis - a foodborne zoonosis with substantial importance to public health. Y. enterocolitica is widespread in the environment and animal populations, posing a potential source of infection to humans. OBJECTIVE Presentation of yersiniosis as a zoonotic foodborne disease of relevance to public health. State of knowledge. Swine play an important role as a reservoir of Y. enterocolitica and insufficiently thermally processed pork is the main source of infection to humans. The correlation between strains isolated from pigs and from clinical cases of human yersiniosis has been sufficiently proven. Yersiniosis usually appears with gastrointestinal disturbances in children, whereas in adults it manifests in a pseudo-appendix form. The extra-enteric form of yersiniosis is rare. Classical bacteriological methods used for classifying Y. enterocolitica as pathogenic does not take into account the new aspects of the pathogenesis of yersiniosis. The examples are biotype 1A strains, commonly regarded as non-pathogenic, although they are increasingly often isolated from clinical cases of yersiniosis. Molecular methods seem much more effective and accurate in the diagnostic. New diagnostic tools such as real-time PCR, allows not only qualitative examination, but also quantitative evaluation of genes expression level, or single nucleotide polymorphism detection. CONCLUSIONS Yersiniosis is an important food-borne zoonosis with wide range of clinical symptoms. Considering the fact that pork is the main source of infection for humans, public information campaigns seems to be an important element of the preventive measures against Y. enterocolitica infections.

Presence of ail and ystB genes in Yersinia enterocolitica biotype 1A isolates from game animals in Poland

The pathogenicity of Yersinia enterocolitica is associated with the presence of plasmid and chromosomal virulence genes. Strains belonging to biotype 1A do not possess pYV plasmids, often harbour the ystB gene and usually lack the ail gene, which is the main virulence marker for Y. enterocolitica. The simultaneous presence of ail and ystB is uncommon. In this study, 21/218 (9.6%) biotype 1A Y. enterocolitica isolates from rectal swabs of wild boar (Sus scrofa; n = 18), red deer (Cervus elaphus; n = 2) and roe deer (Capreolus capreolus; n = 1) in Poland harboured both ail and ystB genes.

Detection and characterisation of Yersinia enterocolitica strains in cold-stored carcasses of large game animals in Poland

Yersinia enterocolitica is an important foodborne pathogen. The aim of the present study was to identify the bioserotypes and virulence markers of Y.enterocolitica strains isolated from three different anatomical regions of cold-stored carcasses of large game animals intended for human consumption. Y.enterocolitica strains were found in 12/20 (60%) of the roe deer carcasses examined, 7/16 (43.8%) of red deer carcasses and 11/20 (55%) of wild boar carcasses. Of the 52 Y.enterocolitica strains, 19 were isolated from the perineum, followed by 17 strains from the peritoneum of the longissimus dorsi muscle and 16 from the tonsils. Only one strain was isolated from warm culture. Bioserotype 1A/NI was the most commonly found and was detected in 29/52 isolates. All isolates contained amplicons corresponding to ystB gene fragments. The relatively high degree of carcass contamination with Y.enterocolitica is of concern due to the growing popularity of game meat with consumers.

A study of single nucleotide polymorphism in the ystB gene of Yersinia enterocolitica strains isolated from various wild animal species

INTRODUCTION AND OBJECTIVE Y. enterocolitica is the causative agent of yersiniosis. The objective of the article was a study of single nucleotide polymorphism in the ystB gene of Y. enterocolitica strains isolated from various wild animal species. MATERIALS AND METHOD High-resolution melting (HRM) analysis was applied to identify single nucleotide polymorphism (SNP) of ystB gene fragments of 88 Y. enterocolitica biotype 1A strains isolated from wild boar, roe deer, red deer and wild ducks. RESULTS HRM analysis revealed 14 different melting profiles - 4 of them were defined as regular genotypes (G1, G2, G3, G4), whereas 10 as variations. 24 of the examined Y. enterocolitica strains were classified as G1, 18 strains as a G2, 21 strains as a G3, and 15 strains as a G4. Nucleotide sequences classified as G1 revealed 100% similarity with the Y. enterocolitica D88145.1 sequence (NCBI). Analysis of G2 revealed one point mutation - transition T111A. One mutation was also found in G3, but SNP was placed in a different gene region - transition G193A. Two SNPs - transitions G92C and T111A - were identified in G4. Direct sequencing of 10 variations revealed 5 new variants of the ystB nucleotide sequence: V1 - transition G129A (3 strains); V2 - transitions T111A and G193A (2 strains); V3 - transitions C118T and G193A (1 strain); V4 - transitions C141A and G193A (2 strains); and V5 characterized by 19 SNPs: G83A, T93A, A109G, G114T, C116T, A123G, T134C, T142G, T144C, A150C, G162A, T165G, T170G, T174A, T177G, G178A, A179G, A184G and G193A (2 strains). The predominant genotype in isolates from wild ducks was G1; in red deer G2; in wild boar G3; in roe deer G1 and G4. CONCLUSIONS The proposed HRM method could be used to analyze Y. enterocolitica biotype 1A strains isolated from different sources, including humans.

The prevalence of Yersinia enterocolitica in game animals in Poland

Natural reservoirs of Yersinia (Y.) enterocolitica comprise different animal species, but little is known about the role of wild animals in the epidemiology of yersiniosis. The aim of the study was to evaluate the prevalence of Y. enterocolitica among game animals in Poland. The bio-serotypes and the pathogenicity markers of the analyzed isolates were determined. The experimental material comprised rectal swabs from 857 free-living animals hunter-harvested over a period of 2 years (2013–2014) in hunting districts across Poland. The isolates from bacteriological studies were confirmed by PCR and bio-serotyped based on the results of biochemical and agglutination tests. In the group of the 218 analyzed isolates of Y. enterocolitica, 133 were derived from wild boars, 70 from red deer, 11 from roe deer and 4 from fallow deer, and they accounted for 61.0%, 32.1%, 5.1% and 1.8% of all isolates, respectively. Bio-serotyping assays revealed that 91.7% of the examined isolates belonged to biotype 1A (200/218). The remaining 18 isolates belonged to bio-serotypes 1B/NI (3/218, 1.4%), 1B/O:8 (1/218, 0.5%), 2/NI (6/218, 2.8%), 2/O:27 (1/218, 0.5%), 2/O:3 (1/218, 0.5%), 2/O:9 (2/218, 0.9%), 3/NI (2/218, 0.9%), 4/O:3 (1/218, 0.5%) and 4/O:9 (1/218, 0.5%). The ail gene, a suggestive virulence gene for Y. enterocolitica, has been found in 30 isolates from 20 wild boars, in 6 isolates from red deer, and in 1 isolate from roe deer. Our study demonstrated that Y. enterocolitica is frequently isolated from game animals in Poland, which poses a risk of spreading these infectious agents to other animal species and humans.

Characterisation of ail-positive Yersinia enterocolitica of different biotypes using HRMA

Yersiniosis is one of the four most frequent foodborne zoonotic diseases in Europe, and Yersinia enterocolitica is the primary agent in human infections. The ail gene is an important chromosomal virulence marker of Y. enterocolitica which encodes Ail, a 17-kDa outer membrane protein that promotes attachment and invasion. In the present study, ail-positive Y. enterocolitica strains of different biotypes were examined using high resolution melting analysis (HRMA) and DNA sequencing. Genotype data relating to Y. enterocolitica strains isolated from different sources and belonging to different biotypes were compared. Applied method allowed efficient distinguishing of three genotypes and phylogenetic groups: 1A - included non-pathogenic Y. enterocolitica strains; 1B - consisted of highly pathogenic Y. enterocolitica strains and 2/4 - involved weakly pathogenic Y. enterocolitica strains. Amplicon genotyping based on HRMA supports rapid identification of ail SNPs correlated with biotype of examined Y. enterocolitica strains.

The Most Important Virulence Markers of Yersinia enterocolitica and Their Role during Infection

Yersinia enterocolitica is the causative agent of yersiniosis, a zoonotic disease of growing epidemiological importance with significant consequences for public health. This pathogenic species has been intensively studied for many years. Six biotypes (1A, 1B, 2, 3, 4, 5) and more than 70 serotypes of Y. enterocolitica have been identified to date. The biotypes of Y. enterocolitica are divided according to their pathogenic properties: the non-pathogenic biotype 1A, weakly pathogenic biotypes 2–5, and the highly pathogenic biotype 1B. Due to the complex pathogenesis of yersiniosis, further research is needed to expand our knowledge of the molecular mechanisms involved in the infection process and the clinical course of the disease. Many factors, both plasmid and chromosomal, significantly influence these processes. The aim of this study was to present the most important virulence markers of Y. enterocolitica and their role during infection.

The use of the HRM method for identifying possible mutations in the ymoA gene region and evaluating their influence on the enterotoxic properties of Y. enterocolitica strains

BackgroundThe yst gene that encodes the production of Yst enterotoxins is one of the most important and reliable virulence markers. Its ability to produce Yst has been demonstrated in pathogenic strains isolated from clinical cases of yersiniosis with diarrhea. However, not all yst positive strains produce enterotoxins. According to some authors, Yst production can be restored in a silent strain by ymoA mutation. In this study, the HRM method was applied to identify ymoA single nucleotide polymorphism with the aim of evaluating their influence on the enterotoxic properties of Y. enterocolitica strains.ResultsTwo genotypes (A and G) of the examined nucleotide sequence and some variations were detected in the HRM analysis. A phylogenetic analysis of 10 genotype A nucleotide sequences revealed 100% similarity with the Yersinia enterocolitica subsp. enterocolitica 8081 genome NCBI Acc. No. AM286415. An analysis of 10 genotype G nucleotide sequences and 3 variations sequences revealed two point mutations in the examined region: transition A3387326G and insertion A in position 3387368. However, no mutations were observed in the coding region of any of the examined ymoA gene fragments. Genotype G was identified in nearly all Y. enterocolitica strains isolated from pigs. Only 4 nucleotide sequences were similar to AM286415 and did not feature point mutations. In case of human Y. enterocolitica strains 31 were classified as belonging to genotype A, the remaining 59 belonged to genotype G and were characterized by the presence of point mutations.ConclusionsNo correlations were observed between enterotoxic properties and the presence of mutations in the ymoA gene region of Y. enterocolitica strains isolated from both humans and pigs.

Experimental immunology Relationship between Yersinia enterocolitica antibody level and bacterial shedding after challenge in previously immunized pigs

AsymptomaticcarriersofYersiniaenterocoliticaareoftenfoundinpigswhicharethemainreservoir and source of human infection. Carrying of that bacteria is usually connected with a long-term shedding and microbiological contamination of the environment. Thus, the aim of this study was to determine the effectofimmunizationwitha suspensionofselectedstrainsofY.enterocoliticaonthedurationofpathogen sheddinginexperimentallyinfectedpigs. Fifteenpigsweredividedintothreegroups,twoexperimentalandonecontrol.Immunizingsuspension wasadministeredsubcutaneouslyindosesof2 ml(groupI)and5 ml(groupII)withthedensityof2.7◊10 9 CFU/mlofformaldehydeinactivatedcellssuspendedinphosphate-bufferedsalineintwoinjectionswithin an interval of two weeks. Pigs were experimentally infected with a 10 ml dose of a pathogenic strain of Y.enterocoliticaO:3withthedensityof2.7◊10 9CFU/mltwoweeksafterimmunization. Subcutaneous immunization stimulated higher antibody levels in group II, which was administered ahigherinoculantdoseof5 mlsuspensionwiththedensityof2.7◊10 9CFU/ml.Inthisgroup,shedding was not reported in two out of five pigs, and the period of pathogen excretion was shorter in comparison withgroupI andthecontrolgroup.ExperimentalimmunizationagainstinfectionscausedbyY.enteroco liticadidnotpreventpathogenshedding,butmerelylimitedtheintensityanddurationofbacterialexcre tion.