Genetic evaluation of bovine papillomavirus types detected in equine sarcoids in Poland.

BACKGROUND: Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe. OBJECTIVE: The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers. RESULTS: The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomavirus 4 isolate 09 asi UK (Acc. No. MF384289) and 99% nucleotide identity with BPV-1 isolate EqSarc1 (Acc. No. JX678969). A comparison with BPV-1 isolate EqSarc1 revealed one silent mutation in C5827T which did not change the aminoacid codon. The remaining 6 isolates (28.6%) shared 100% nucleotide identity with the BPV-1 (Acc. No. X02346) "wild type" isolate, and 1 isolate (4.8%) demonstrated 99% nucleotide identity with BPV-2 (Acc. No. M20219). CONCLUSIONS: Variants of BPV-1 isolate EqSarc1 (Acc. No. JX678969) constitute the most prevalent type of BPV-1 in Polish horses.

Pathogenic potential to humans of Shiga toxin-producing Escherichia coli isolated from wild boars in Poland.

The aim of the study was to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) in the wild boar population of north-eastern Poland, and to evaluate the potential health risk associated with wild boars carrying STEC/AE-STEC strains. In Poland, the African Swine Fever (ASF) virus has been a growing problem in domestic pigs and wild boars, one of the main reservoirs of the virus, because of this hunters, veterinary practitioners and foresters thus face a greater risk of coming into contact with animals. Rectal swabs samples were obtained from 152 wild boars hunter-harvested in the 2017/2018 season (autumn-winter) in north-eastern Poland. The samples were enrichment in modified buffered peptone water. Polymerase chain reaction (PCR) assays were conducted to determine the virulence profile of stx1, stx2 and eae and aggR genes, identify subtypes of stx1 and stx2 genes, and perform O and H serotyping. STEC/AE-STEC virulence genes were detected in 43 isolates (28.29%): STEC in 17 isolates (11.18%) and AE-STEC in 26 isolates (17.11%), respectively. None of the tested isolates carried the aggR gene. The most dangerous AE-STEC virulence profile associated with HUS was found in 2 isolates (1.32%): stx1NS/stx2a/d/eae serotype ONT:H7 and stx2a/eae serotype O146:H7. Six of the 152 tested samples belonged to serogroup O157 (3.95%), including one AE-STEC isolate with virulence profile stx2g/eae and five EPEC isolates. The results of this study suggest that wild boars in north-eastern Poland can carry STEC/AE-STEC strains that are potentially pathogenic for humans. This is the first report documenting the virulence of STEC and AE-STEC isolates from wild boars in Poland.

Sequence variants of Yersinia enterocolitica ystB gene detected in wild animals in Poland.

The purpose of the study was to analyze a part of the nucleotide sequences of ystB gene Y. enterocolitica strains isolated from wild animals. The material for the study consists of 30 Y. enterocolitica biotype 1A strains obtained from different wild animal species and belonging to different genotypes. Phylogenetic analysis of ystB nucleotide sequences belonging to four regular genotypes G1, G2, G3, G4 and to five groups of variations V1, V2, V3, V4, V5 revealed significant differences of Y. enterocolitica strains isolated from wild animals. The most phylogenetically distant were strains belonging to V5.

Detection of Bordetella avium by TaqMan real-time PCR in tracheal swabs from wildlife birds.

Bordetella avium, the causing agent of bordetellosis, a highly contagious infection of the respiratory tract in young poultry, causes significant losses in poultry farming throughout the world. Wildlife birds can be a reservoir of various pathogens that infect farm animals. For this reason the studies were conducted to estimate the prevalence of Bordetella avium in wildlife birds in Poland. Tracheal swab samples were collected from 650 birds representing 27 species. The bacterial DNA was isolated directly from the swabs and screened for Bordetella avium by TaqMan real-time PCR. The assay specificity was evaluated by testing DNA isolated from 8 other bacteria that can be present in avian respiratory tract, and there was no amplification from non-Bordetella avium agents. Test sensitivity was determined by preparing standard tenfold serial dilutions of DNA isolated from positive control. The assay revealed to be sensitive, with detection limit of approximately 4.07x10^2 copies of Bordetella avium DNA. The genetic material of Bordetella avium was found in 54.54% of common pheasants, in 9.09% of Eurasian coots, in 3.22% of black-headed gulls and in 2.77% of mallard ducks. The results of this study point to low prevalence of Bordetella avium infections in wildlife birds. The results also show that described molecular assay proved to be suitable for the rapid diagnosis of bordetellosis in the routine diagnostic laboratory.

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.

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

AIMS: 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 Drweca River in Poland. METHODS AND RESULTS: Thirty-nine water samples were collected. Strains were isolated, identified with the use of the API(®) 20E test kit (Biomerieux, Marcy l'Etoile, France) at 37°C, serotyped and subjected to a molecular analysis. Multiplex PCR was carried out to amplify three virulence genes: ail, ystA and ystB. Fragments of ail and ystA genes were not identified in the genetic material of the analysed strains. The ystB gene was identified in four strains. Yersinia enterocolitica strains of biotype 1A, which contain the ystB gene, may cause gastrointestinal problems. CONCLUSIONS: In our study, Y. enterocolitica strains of biotype 1A/ystB with serotypes 0 : 3, 0 : 5 and 0 : 8 were identified in samples collected from the Drweca River which flows through the areas protected by Natura 2000, one of the largest networks of nature conservation areas in the European Union. The presence of Y. enterocolitica in the Drweca River indicates that the analysed bacteria colonize natural water bodies. SIGNIFICANCE AND IMPACT OF THE STUDY: Most research focuses on food or sewage as a source of Y. enterocolitica infections. Little is known about the occurrence of this pathogen in natural waters. Our results show that natural waters are also a potential threat to human and animal health.

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.

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.

The first pathogenic Yersinia enterocolitica bioserotype 4/O:3 strain isolated from a hunted wild boar (Sus scrofa) in Poland.

The objective of this study was to identify the bioserotypes and virulence markers of Yersinia enterocolitica strains isolated from wild boars in Poland. Bacteriological examination of 302 rectal swabs from 151 wild boars resulted in the isolation of 40 Y. enterocolitica strains. The majority of the examined strains (n = 30), belonged to bioserotype 1A/NI. The presence of individual Y. enterocolitica strains belonging to bioserotypes 1B/NI (3), 1A/O:8 (2), 1A/O:27 (2), 2/NI (1), 2/O:9 (1) and 4/O:3 (1) was also demonstrated. Amplicons corresponding to ail and ystA genes were observed only in one Y. enterocolitica strain--bioserotype 4/O:3. The ail and ystB gene amplicons were noted in 11 Y. enterocolitica biotype 1A strains, although single amplicons of ystB gene were found in 28 of the tested samples. In four out of eight cases when two Y. enterocolitica strains were isolated from the same animal, the strains differed in biotype, serotype or virulence markers. The European population of wild boars continues to grow and spread to new areas, therefore, wild boars harbouring potentially pathogenic Y. enterocolitica 4/O:3 strains pose a challenge to public health.

Quantitative PCR high-resolution melting (qPCR-hRM) curve analysis, a new approach to rapid detection and differentiation of bovine papillomavirus detected in equine sarcoids.

The aim of the study was to evaluate a novel diagnostic scheme which combines quantitative PCR and High-Resolution Melting (qPCR-HRM) curve analysis for rapid differentiation based on E5 partial CDS of bovine papillomavirus type 1 or 2 (BPV-1 or BPV-2), and to perform a phylogenetic analysis of the complete CDS of the E5 gene of BPV detected in equine sarcoids. Samples of 38 skin lesions obtained from 27 horses were collected for molecular examinations. All lesions were clinically diagnosed as sarcoids, but results of histopathological examinations did not always corroborate the clinical diagnosis. Although all the samples were positive for the presence of BPV DNA, after qPCR-HRM analysis 6 (16%) specimens were recognized as BPV-1 "wild", 24 (63%) as BPV-1 "European" and 8 (21%) as a "variant" of BPV E5 ORF partial CDS. Phylogenetic analysis based on nucleotide sequences of E2 ORF partial CDS and E5 ORF complete CDS was conducted on 7 specimens, whose sequences were published in GenBank and recognized as: 2PL (Accession Number --Acc. No. KC684939)--"variant" BPV-1, 7aPL (Acc. No. KC684940)--"European" BPV-1, 10PL (Acc. No. KC693480)--"variant" BPV-1, 16PL (Acc. No. KC693484)--"variant" BPV-2, 17PL (Acc. No. KC693481)--"variant" BPV-1, 20aPL (Acc. No. KC693482)--"European" BPV-1 and 20cPL (Acc. No. KC693483)--"wild" BPV-1. Amino acid (aa) sequences of E5 ORF complete CDS were also analyzed. The E5 variant of aa sequences found in isolate 10PL (protein identification--ID: AGM 20700) is a novel variant of E5 ORF complete CDS of BPV-1 detected in equine sarcoid in Poland.

Bioserotypes and virulence markers of Y. enterocolitica strains isolated from roe deer (Capreolus capreolus) and red deer (Cervus elaphus).

Free-living animals are an important environmental reservoir of pathogens dangerous for other animal species and humans. One of those is Yersinia (Y.) enterocolitica, the causative agent of yersiniosis--foodborne, enzootic disease, significant for public health. The purpose of the study was to identify bioserotypes and virulence markers of Y enterocolitica strains isolated from roe deer (Capreolus capreolus) and red deer (Cervus elaphus) obtained during the 2010/2011 hunting season in north-eastern Poland. From among 48 rectal swabs obtained from 24 roe deer, two strains of Y enterocolitica from one animal were isolated. Although both belonged to biotype 1A they were identified as different serotypes. The strain obtained from cold culture (PSB) belonged to serotype 0:5, while the strain isolated from warm culture (ITC) was regarded as nonidentified (NI), what may suggest mixed infection in that animal. The presence of ystB gene, coding for YstB enterotoxin, directly related to Y enterocolitica pathogenicity was detected in both strains using triplex PCR. The effect of the examination of 32 swabs obtained from 16 red deer was the isolation of two Y enterocolitica strains from two different animals. Both belonged to biotype 1A with NI serotype, but were originated from different types of culture. They gave positive results in case of products of a size corresponding to the ystB gene. No amplicons corresponding to ail and ystA genes were found. Roe deer and red deer may carry and shed Y. enterocolitica, what seems to be important in aspect of an environmental reservoir of this pathogen. The Y enterocolitica strains isolated from wild ruminants had the amplicons of the ystB gene, what suggest they can be potential source of Y enterocolitica infection for humans.

Antimicrobial resistance in bacteria isolated from pigeons in Poland.

The present study investigated the drug-resistance to the selected antibiotics in Escherichia coli, Salmonella typhimurium and beta-haemolytic coagulase-positive staphylococci isolated from pigeons bred in Poland. In the case of E. coli, tetracyclines and amoxicillin were least effective. In the staphylococci, the highest resistance was detected for oxytetracycline and quinolones and 5% were resistant to methicillin. The lowest drug-resistance was reported for Salmonella typhimurium.

Distribution of the ymoA and ystA genes and enterotoxins yst production by Yersinia enterocolitica strains isolated from humans and pigs.

Yersinia (Y.) enterocolitica is the third etiological agent of human diarrhea in terms of the number of confirmed clinical cases. One of the important virulence markers is the yst gene which encodes the production of enterotoxins Yst (Yersinia stable toxins). However, not all strains with yst genes produce enterotoxins, what seems to be caused by the ymoA gene encoding the production of the YmoA protein inhibiting the expression of various genes. The purpose of our study was to evaluate the distribution of the ymoA and ystA genes and Yst production by Y. enterocolitica isolated from humans and pigs. All the studied strains obtained from pigs had the ystA gene which indicates that they belong to the group of strains commonly regarded as pathogenic, but the ability to produce YstA was detected in only 14 out of 96 examined strains. The fragments of ystA gene were also detected in all Y. enterocolitica strains isolated from human cases of diarrhea. Amplification of a fragment of the ymoA gene was detected in all the studied strains, both from humans and pigs, based on the presence of a 330 bp band. Thus no correlation was identified between the occurrence of the ymoA and ystA genes and the production of a specific type of enterotoxin.

A comparison of the effectiveness of the microscopic method and the multiplex PCR method in identifying and discriminating the species of Nosema spp. spores in worker bees (Apis mellifera) from winter hive debris.

The objective of this study was to compare the effectiveness of the multiplex PCR method and traditional light microscopy in identifying and discriminating the species of Nosema spp. spores in worker bees from winter hive debris in the Province of Warmia and Mazury (NE Poland). A total of 1000 beesdead after from the bottom of the hive from bee colonies were analyzed. Spores were identified with the use of a light microscope (400-600x magnification). Spores were assigned to species by the multiplex PCR method. The microscopic evaluation revealed the presence of Nosema spp. spores in 803 samples (80.3%). Nosema ceranae spores were observed in 353 positive samples (43.96%), Nosema apis spores were found in 300 samples (37.35%), while 150 samples (19.67%) showed signs of a mixed infection. A multiplex PCR analysis revealed that 806 samples were infested with Nosema spp., of which 206 were affected only by Nosema ceranae, 600 showed signs of mixed invasion, while no samples were infected solely by Nosema apis parasites. In two cases, the presence of spores detected under a light microscope was not confirmed by the PCR analysis. The results of the study indicate that Nosema ceranae is the predominant parasitic species found in post-winter worker bees from the bottom of the hive in the region of Warmia and Mazury.

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).