A. Szczerba-Turek

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.

Yersinia enterocolitica strains isolated from beavers (Castor fiber)

Pseudocloacal swabs and palatine tonsils from beavers have been examined for the Yersinia enterocolitica presence. Thirty-six samples from 24 beavers were collected and subjected to bacteriological examinations including sero- and biotypisation. Amplicons confirmed by PCR as Y. enterocolitica were sequenced. Positive samples originated from 4 out of the 24 beavers (16.7 %) and all the strains belonged to biotype 1A. The study suggested that Y. enterocolitica could be isolated from beavers, which may therefore be treated as a reservoir, a significant factor of water contamination and a vector of the Y. enterocolitica.

Monitoring of Yersinia enterocolitica strains from free-living animals using different methods.

The aim of the study was to monitor Y. enterocolitica strains from free-living animals captured during 2011-2014 hunting seasons in Poland using warm (ITC) and cold (PSB) enrichment and molecular examination. Over 1600 samples have been cultured. After ITC/PSB enrichment 237 strains presenting features characteristic for Y. enterocolitica were isolated. Molecular examination using multiplex PCR revealed 140 isolates from PSB and 78 from ITC. The concentration of pathogenic Yersinia in asymptomatic carriers is low and the PCR detection should be preceded by bacteriological examination.

Characterization of Yersinia enterocolitica strains potentially virulent for humans and animals in river water.

The aim of this study was to isolate and identify potentially pathogenic strains of Yersinia enterocolitica in water samples collected from the upstream section of the Drwęca River in Poland.

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.

Phylogenetic analysis of bovine papillomavirus E5 detected in equine sarcoids in Poland

The aim of the study was to analyse a part of the sequence of the E5 gene of bovine papillomaviruses (BPV) associated with equine sarcoids in Polish horses. Samples of 40 skin lesions obtained from 29 horses were collected for molecular examination. The PCR amplicons of BPV DNA were detected in 38 specimens. After phylogenetic analysis 37 specimens were recognized as BPV-1 and one as BPV-2. Phylogenetic analysis has allowed the classification of the amplicons into two phylogenetic groups (A1,) and four separate isolates (2, 10, 16, 17).

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 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.