Phylogenetic analysis of the trypanosomatid parasite Lotmaria passim in honey bees (Apis mellifera) in Poland.

Introduction: Lotmaria passim (L. passim) is a single-celled flagellate which colonises the bee gastrointestinal tract and is highly prevalent in honey bees. This parasite is associated with colony losses. Honey bee (Apis mellifera) colonies were sampled from five apiaries in the north-eastern part of Poland for the phylogenetic analysis of L. passim. Material and Methods: Each apiary consisted of approximately 60 bee colonies, of which 20 were randomly selected. Samples of 60 differently aged worker bees were collected from each colony and pooled. A total of 100 bee colonies from five apiaries were examined. Protozoa of the Trypanosomatidae family were identified by PCR. L. passim was detected in 47 (47%) of the samples. The 18S ribosomal (r) RNA amplicons of L. passim were sequenced by a commercial service. Their sequences were analysed with BLASTN and noted to be compatible with the GenBank sequences of this region of the organism's genome. A sequence analysis was performed using the BioEdit Sequence Alignment Editor and Clustal W software. Results: The amplicon sequences of L. passim were 100% homologous with the sequences deposited in GenBank under accession numbers KM066243.1., KJ684964.1 and KM980181.1. Conclusion: This is the first study to perform a phylogenetic analysis of L. passim in Polish honey bees. The analysis demonstrated high levels of genetic similarity between isolates of L. passim colonising apiaries in the north-eastern region of Poland.

Isolation, characterization and antimicrobial resistance of Yersinia enterocolitica from Polish cattle and their carcasses.

BACKGROUND: Yersinia enterocolitica is a heterogeneous bacterial species that has been divided into six biotypes and more than 70 serotypes. Each year, the European Food Safety Authority classifies yersiniosis caused by Y. enterocolitica as one of the most important zoonotic diseases. The prevalence of Y. enterocolitica in cattle has not been thoroughly analyzed in Poland, and beef and bovine carcasses contaminated with antimicrobial resistant Y. enterocolitica pose a health risk for both, farm workers and consumers. Therefore, the aim of this study was to evaluate the prevalence of Y. enterocolitica in cattle and to determine the antimicrobial susceptibility of the isolated strains. RESULTS: A total of 1020 samples were analyzed, including 660 rectal swabs collected from live cattle and 360 swabs from cold-stored beef carcasses. The results of this study indicate that Y. enterocolitica was isolated from three of the 15 examined cattle herds and the prevalence within these herds ranged from 0% to nearly 32%. Y. enterocolitica was isolated from 14.7% of the examined heifers, 7.4% of calves and 5.5% of adult cows. More than 65% of the strains were isolated from cold enrichment. The strains isolated from live cattle tested positive for the ystB gene, while ail and ystA genes were not found. Most of the isolated strains belonged to bioserotype 1A/NT. The majority of the isolated strains were resistant to ampicillin, cefalexin and amoxicillin with clavulanic acid, however these are expected phenotypes for Y. enterocolitica. CONCLUSIONS: The results of this study indicate that Y. enterocolitica is present in cattle herds in Poland. The strains isolated from live cattle were ystB-positive, most of them belonged to bioserotype 1A/NT. The prevalence of Y. enterocolitica strains was generally low in cold-stored beef carcasses.

Phylogenetic analysis of Starmerella apis in honey bees (Apis mellifera).

Honey bees are among the most effective pollinators that promote plant reproduction. Bees are highly active in the pollen collection season, which can lead to the transmission of selected pathogens between colonies. The clade Starmerella comprises yeasts that are isolated mainly from bees and their environment. When visiting plants, bees can come into contact with Starmerella spp. The aim of this study was to determine the prevalence and phylogenetic position of S. apis in bee colonies. Bee colonies were collected from nine apiaries in three regions. Ten colonies were sampled randomly from each apiary, and pooled samples were collected from the central part of the hive in each colony. A total of 90 (100%) bee colonies from nine apiaries were examined. Starmerella apis was detected in 31 (34.44%) samples, but related species were not identified. The 18S rRNA amplicon sequences of S. apis were compatible with the GenBank sequences of Starmerella spp. from India, Japan, Syria, Thailand, and the USA. The amplicon sequences of S. apis were also 99.06% homologous with the sequences deposited in GenBank under accession numbers JX515988 and NG067631.This is the first study to perform a phylogenetic analysis of S. apis in Polish honey bees.

Assessment of the Role of Free-Living and Farmed Fallow Deer (Dama dama) as A Potential Source of Human Infection with Multiple-Drug-Resistant Strains of Yersinia enterocolitica and Yersinia pseudotuberculosis.

Yersinia enterocolitica and Y. pseudotuberculosis are Gram-negative, facultative anaerobic bacteria that cause yersiniosis-one of the most important zoonotic diseases of the digestive tract. The aim of this study was to determine the prevalence of potentially human-pathogenic Y. enterocolitica and Y. pseudotuberculosis strains in free-living and farmed fallow deer, and to evaluate their sensitivity to chemotherapeutics. A total of 372 rectal swabs were analyzed, including 262 from free-living and 110 from farmed fallow deer. Due to the psychrophilic properties of Yersinia, two samples were collected from each animal. Seven Y. enterocolitica strains were isolated from free-living fallow deer, while two strains were isolated from farmed fallow deer. Yersinia pseudotuberculosis strains were not identified. All isolated Y. enterocolitica strains were ystB-positive, and phylogenetic analysis based on the nucleotide sequences of this gene revealed the presence of two phylogenetic groups. Yersinia enterocolitica strains isolated from fallow deer belonged to biotype 1A, and serotyping analysis demonstrated that the vast majority did not agglutinate with any diagnostic sera. All strains were multiple drug resistant and were not sensitive to at least four of the tested chemotherapeutics (amoxicillin with clavulanic acid, ampicillin, cefalexin, and streptomycin). One Y. enterocolitica strain isolated from a free-living animal was resistant to nine out of the 13 analyzed chemotherapeutics and was intermediately sensitive to the four remaining chemotherapeutics. The highest sensitivity was noted in case of ciprofloxacin (five strains) and trimethoprim-sulfamethoxazole (three strains). Only one strain isolated from a free-living animal was sensitive to three out of the 13 examined antibiotics, whereas the remaining strains were sensitive to only one drug or were not sensitive to any of the chemotherapeutics used. The results of this study indicate that multiple drug-resistant Y. enterocolitica strains can be carried by free-living and farmed fallow deer. This observation gives serious cause for concern because the meat of fallow deer and other ruminants is often consumed semi-raw (steak) or raw (tartar steak).

Comparison of Yersinia enterocolitica DNA Isolation from Swabs Without Pre-Enrichment on Selective Media With Isolation Preceded by Warm and Cold Enrichment.

Bacteriological methods for the identification of Yersinia enterocolitica are laborious and time-consuming. The aim of this study was to compare Y. enterocolitica DNA isolation from swabs without pre-enrichment on selective media with isolation preceded by warm and cold enrichment. The material for the study consisted of 150 rectal swabs taken from 50 clinically healthy fattening pigs. Forty-one Y. enterocolitica strains were isolated after warm enrichment and 43 after cold enrichment. The presence of ail, ystA, and yadA gene fragments was detected in all isolates. DNA isolation from swabs without pre-enrichment supported the detection of Y. enterocolitica in only nine samples. This study demonstrates that Y. enterocolitica DNA can be isolated from swabs without pre-enrichment on selective media, but this method is less sensitive than the approach where DNA isolation is preceded by warm or cold enrichment.

Evaluation of the Correlation between the mRNA Expression Levels of ystA and ymoA Genes in Y. enterocolitica Strains with Different Enterotoxic Properties.

Yersinia enterocolitica is one of the main causative agents of human diarrhea. Pigs are a reservoir and the most common source of infection for humans. The aim of this study was to analyze the expression of ystA and ymoA genes in Y. enterocolitica strains with different enterotoxic properties, isolated from humans and pigs. The experiment involved two groups of Y. enterocolitica strains producing and not producing enterotoxin YstA, which were isolated from humans and pigs. All strains were ystA- and ymoA-positive. The expression of ystA and ymoA genes was analyzed by quantitative real-time PCR (qPCR). The relative expression level of the ystA gene was significantly higher than the expression level of the ymoA gene in Y. enterocolitica strains isolated from humans with clinical symptoms of yersiniosis. In other strains, a significant decrease in ystA gene transcription was observed, and the relative expression level of the ymoA gene was significantly higher than the expression level of the ystA gene. Statistically significant differences were not observed in either group of strains isolated from pigs. The results of our study revealed a correlation between mRNA expression levels of ystA and ymoA genes in Y. enterocolitica strains isolated from humans.

The Prevalence of Yersinia enterocolitica and Yersinia pseudotuberculosis in Small Wild Rodents in Poland.

Rodents are a large group of mammals that can be carriers of zoonotic pathogens such as Yersinia strains that cause yersiniosis. The prevalence of Yersinia enterocolitica and Yersinia pseudotuberculosis was determined in 214 small wild rodents from south-eastern Poland. Samples were analyzed by precultivation and PCR. Nine (4.2%) Y. enterocolitica and one (0.5%) Y. pseudotuberculosis isolates were received. Most of them (n = 5) were obtained from the common vole (Microtus arvalis). All Y. enterocolitica strains were classified as biotype (BT) 1A. A PCR analysis of virulence markers revealed that all Y. enterocolitica isolates contained the ystB gene and five isolates harbored a rare genetic combination of ail/ystB. Three of the four ail/ystB-positive isolates belonged to serotype O:5.27. The Y. pseudotuberculosis inv-positive isolate was classified as BT 1. A genetic analysis of Y. enterocolitica harboring the ystB gene revealed 100% similarity between the analyzed sequences and the sequences from diarrhea patients in India and the United Kingdom as well as high similarity with the sequences from different species of wild animals from Poland. The Y. pseudotuberculosis inv sequence was 100% identical to the sequence isolated from fully virulent clinical strain from France and Australia. The results of our study suggest that small wild rodents, especially voles and yellow-necked mice, may act as carriers of Yersinia strains. The high similarity of the tested gene sequences between our isolates and the isolates from other free-living animals indicates that small wild rodents can play a role in the epidemiology of yersiniosis and can shed Yersinia spp. into the environment.

Bioserotypes, Virulence Markers, and Antimicrobial Susceptibility of Yersinia enterocolitica Strains Isolated from Free-Living Birds.

The risk of meat contamination with Yersinia enterocolitica poses a threat to consumers and persons who come into contact with bird carcasses. The occurrence of Y. enterocolitica in the vast majority of migratory game species, the capercaillie, and the black grouse has never been studied in Poland, Europe, or in the world. The material for the study consisted of cloacal swabs obtained from 143 Eurasian coots, 50 mallards, 30 pochards, 27 greylag geese, 22 white-fronted geese, 22 bean geese, 20 green-winged teals, and 10 tufted ducks, as well as fecal swabs obtained from 105 capercaillie and 18 black grouse. Bacteriological examinations of 894 samples taken from 447 birds led to the isolation of 20 strains with the biochemical features characteristic of the genus Yersinia. All 20 strains were molecularly examined, and the genes characteristic of Y. enterocolitica were detected in 8 strains. The isolated strains harbored amplicons whose size corresponded to ystB gene fragments. Four strains belonged to bioserotype 1A/NI, one strain was identified as bioserotype 1B/O:9, and one as 1A/O:9. The prevalence of Y. enterocolitica was determined at 1.4% in green-winged teals, at 5.0% in Eurasian coots, and at 4.8% in capercaillie. All strains were resistant to amoxicillin with clavulanic acid, ampicillin, and cefalexin. The strains isolated from migratory birds were also resistant to kanamycin and streptomycin, and they were characterized by resistance or intermediate resistance to cefotaxime, ceftazidime, chloramphenicol, gentamycin, and tetracycline, to which the strains isolated from the capercaillie were susceptible. Yersinia enterocolitica was not detected in the remaining bird species. The presence of Y. enterocolitica in green-winged teals, Eurasian coots, and capercaillie indicates that these birds could be carriers, potential reservoirs, and sources of infection for humans. They can also be regarded as reliable bioindicators of Y. enterocolitica in their respective habitats.

Shiga toxin-producing Escherichia coli isolates from red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Poland.

Shiga toxin-producing Escherichia (E.) coli (STEC) pathogens are responsible for the outbreaks of serious diseases in humans, including haemolytic uraemic syndrome (HUS), bloody diarrhoea (BD) and diarrhoea (D), and they pose a significant public health concern. Wild ruminants are an important environmental reservoir of foodborne pathogens that can cause serious illnesses in humans and contaminate fresh products. There is a general scarcity of published data about wildlife as a reservoir of foodborne pathogens in Poland, which is why the potential epidemiological risk associated with red deer, roe deer and fallow deer as reservoirs of STEC/AE-STEC strains was evaluated in this study. The aim of the study was to investigate the prevalence of STEC strains in red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) populations in north-eastern Poland, and to evaluate the potential health risk associated with wild ruminants carrying STEC/AE-STEC strains. We examined 252 rectal swabs obtained from 134 roe deer (Capreolus capreolus), 97 red deer (Cervus elaphus) and 21 fallow deer (Dama dama) in north-eastern Poland. The samples were enriched in modified buffered peptone water. Polymerase chain reaction (PCR) assays were conducted to determine the virulence profile of stx1, stx2 and eae or aggR genes, to identify the subtypes of stx1 and stx2 genes, and to perform O and H serotyping. E. coli O157:H7 isolates were detected in the rectal swabs collected from 1/134 roe deer (0.75%) and 4/97 red deer (4.1%), and they were not detected in fallow deer (Dama dama). The remaining E. coli serogroups, namely O26, O103, O111 and O145 that belong to the "top five" non-O157 serogroups, were detected in 15/134 roe deer (11.19%), 18/97 red deer (18.56%) and 2/21 fallow deer (9.52%). STEC/AE-STEC strains were detected in 33 roe deer isolates (24.63%), 21 red deer isolates (21.65%) and 2 fallow deer isolates (9.52%). According to the most recent FAO/WHO report, stx2a and eae genes are the primary virulence traits associated with HUS, and these genes were identified in one roe deer isolate and one red deer isolate. Stx2 was the predominant stx gene, and it was detected in 78.79% of roe deer and in 71.43% of red deer isolates. The results of this study confirmed that red deer and roe deer in north-eastern Poland are carriers of STEC/AE-STEC strains that are potentially pathogenic for humans. This is the first report documenting the virulence of STEC/AE-STEC strains from wild ruminants in Poland.

Phenotypic and genotypic presence of the Yersinia virulence plasmid do not affect the production of enterotoxin YstA by Yersinia enterocolitica strains.

The aim of the study was to determine whether the presence of the Yersinia virulence plasmid could affect the production of enterotoxin YstA by Y. enterocolitica strains isolated from pigs which are the main source of infection for humans. The phenotypic features characteristic for the Yersinia virulence plasmid were detected on CRMOX agar in 8 out of 12 strains producing enterotoxin YstA, in 5 out of 12 doubtful strains, and in 11 out of 12 strains not producing YstA. Autoagglutination ability was detected in all 12 Y. enterocolitica strains that were positive in the suckling mice bioassay, in 11 doubtful strains and 10 negative strains. CRMOX+ colonies were generally ystA, myfA, virF and yadA positive, while CRMOX- colonies were only ystA and myfA positive. The amplicons of yadA were not detected in 2 (8.3%) out of 24 CRMOX+ and virF positive strains. The results of this study indicate that the presence of pYV does not affect the enterotoxin-producing ability of 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 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.

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.

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.

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.

Ultrasonographic fetometry formulas of inner chorionic cavity diameter and biparietal diameter for medium-sized dogs can be used in giant breeds.

The aim of this study was to evaluate the suitability of the ultrasonographic fetometry method, involving inner chorionic cavity diameter (ICC) and biparietal diameter (BP) measurements, to predict the parturition date in giant breed dogs. Overall, 30 ICC and 24 BP measurements were taken on 24 giant breed bitches. The measured values were substituted into Luvoni and Grioni (2000) formulas for medium-sized bitches because formulas with ICC and BP to dogs with a body mass greater than 40 kg have not been defined. The accuracy of the parturition date predictions proved the method to be highly useful in the observed group of dogs. Prediction accuracy in the giants ranged between 54.16% (± 1 day, using BP) and 90% (± 2 days, using ICC), depending on the parameter measured and precision levels used. Numerically, the results obtained using ICC were better; however, no statistically significant differences between ICC and BP accuracy were found when comparing the effectiveness of the parturition date predictions. Regression lines based on the own fetometric measurements were highly convergent with the lines defined by Luvoni and Grioni (2000) formulas for medium-sized bitches. This outcome suggests a similar gestational development of fetuses in giant dogs and the possible use of Luvoni and Grioni (2000) formulas for medium-sized dogs with breeds weighing greater than 40 kg.

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.

BACKGROUND: The 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. RESULTS: Two 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. CONCLUSIONS: No 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.

Bioserotypes and virulence markers of Yersinia enterocolitica strains isolated from mallards (Anas platyrhynchos) and Pheasants (Phasianus colchicus).

Yersinia enterocolitica is the causative agent of yersiniosis in different animal species and in humans. Food contaminated with Y. enterocolitica is the main source of infection for humans, and swine plays a major role in the transmission of the disease. There are a limited number of reports of the prevalence of Y. enterocolitica in wild animals and birds. This study characterized virulence markers associated with Y. enterocolitica isolates recovered from mallards and pheasants. Y. enterocolitica strains were isolated from 5 (11.11%) of 45 mallards originating from a cold culture (peptone, sorbitol, and bile salts medium) belonging to biotype 1A. Serotyping showed that three of these five serotypes represented serotype O:8, one belonged to serotype O:5, and one did not agglutinate with any of the sera and was classified as nonidentified. Molecular analysis for virulence markers detected the ystB gene, which encodes an enterotoxin, in five isolates. Y. enterocolitica was not detected in any of the 16 examined pheasants.