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.

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.

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.

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.

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.

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 influence of experimental Yersinia enterocolitica infection on the pregnancy course in sows--preliminary studies. III. Histopathological lesions.

The aim of the study was to evaluate the anatomo- and histopathological lesions in internal organs of sows and their stillborn piglets after experimental Y. enterocolitica infection in different phases of pregnancy. Twelve pregnant sows were divided into 4 groups, infected per os on 33 (n = 3), 54 (n = 3) and 89 (n = 3) day of pregnancy with the pathogenic Y. enterocolitica strain isolated from the aborted swine fetus, and uninfected control group. Histopathological examinations of internal organs and intestine samples of stillborn piglets, slaughtered sows and samples of placentas were performed. Anatomo- and histopathological lesions were the most intense in the group of sows infected in the final phase of pregnancy, where the highest number of stillborn piglets was also found. Lesions of internal organs in stillborn piglets suggested a severe generalized bacterial infection. Although the analysis of experimental Y. enterocolitica infection of pregnant sows revealed that the most intense clinical, anatomopathological and histopathological abnormalities were recorded in the group of animals infected in the final phase of pregnancy. Infection in the first phase of pregnancy could have had an influence on the formation of the granulomatous inflammation. Differences in anatomopathological lesions between infected and control animals suggest that the period of pregnancy in which the infection appears could have had an influence on the course of yersiniosis in pigs.

Partial sequence analysis of the L1 gene of bovine papillomavirus type 1 detected by PCR with MY09/MY11 primers in equine sarcoids in Poland.

BPV-1 is now recognized as a main etiological agent of equine sarcoids. The etiopathogenesis of the equine sarcoids is equivocal and is not yet fully understood. The aim of the present study was to analyse a partial sequence of the L1 gene of BPV associated with equine sarcoids in Polish horses. After clinical diagnosis, 40 skin lesions obtained from 29 horses were collected. The amplicons of a fragment of BPV L1 DNA were detected using PCR with MY09/MY11 primers in 31 specimens. All of them were recognized as BPV-1. Phylogenetic analysis has allowed the amplicons of partial L1 gene to be divided into 3 phylogenetic groups (A, B, C) and one separate isolate (20c). Sequence variants from phylogenetic groups B, C and isolate 20c represented new genetic variants of BPV-1 L1. Sequence variants from groups B and C were submitted to GenBank NCBI.

Effectiveness of coupled administration of Bioimmuno immunomodulator and Respisure One vaccine in the prophylaxis of mycoplasmal pneumonia of swine.

The study was aimed at determining the protective value of the coupled application of immunomodulation and vaccination in the prophylaxis of mycoplasmal pneumonia of swine (MPS). It was conducted on a pig farm, on 28 piglets with body weights of 7-8 kg in the peri-weaning period, divided into four groups of 7 animals each. In the experiment, the following were used: Bioimmuno preparation (Institute of Inland Fisheries, Olsztyn) elaborated based on methisoprinol and Saccharomyces cerevisiae, to be administered with feed, as well as an inactivated vaccine against MPS - Respisure One (Pfizer). The first two groups of piglets were administered Bioimmuno with a feed mixture at a dose of 1 kg/50 kg feed for 48 hours prior to the immunization of one of the groups with Respisure One on day 28 of life. The third group of piglets was only vaccinated, whereas the fourth group served as control. Clinical observations were conducted that covered the monitoring of side reactions (local and constitutional), an evaluation of the incidence and character of symptoms manifested by the respiratory system and other health status disorders as well as the determination of the number and percentage of medical interventions and death cases of animals. Animal husbandry examinations and post-slaughter analyses involved the determination of the mean body weight at slaughter, mean daily body weight gains, mean daily feed utilization, and mean feed conversion ratio per 1 kg b.w. gain and an index of quantitative lesions in the lungs. The study did not demonstrate any local side reactions at an injection site of the Respisure One vaccine nor any constitutional side reactions linked with the administration of either preparation. In turn, the clinical and animal husbandry examinations showed a positive effect of administering the Bioimmuno immunomodulator on the effectiveness of immunization against MPS with the Respisure One vaccine and on the improvement of the health status of pigs, which was manifested by a reduced incidence of infections in the piglets with respiratory symptoms, increased b.w. gains, improved feed utilization and shortened fattening period. Due to the diminished intensification of respiratory symptoms and pathological lesions in lungs and the improved fattening indices, the coupled administration of the Bioimmuno and Respisure One biopreparations to piglets may constitute an important element of MPS prophylaxis in pig herds.

The influence of experimental Yersinia enterocolitica infection on the pregnancy course in sows--preliminary studies. I. Bacteriological examination.

The aim of the study was to determine the influence of Yersinia enterocolitica (Y. enterocolitica) experimental infection on the carrying and shedding states of the microorganism by pregnant sows and on the bacteria occurrence in tissues of the infected animals and aborted or stillborn piglets. Twelve pregnant sows were divided into 4 groups and infected per os on 33--group I (n = 3), 54--group II (n = 3) and 89--group III (n = 3) day of pregnancy with the Y. enterocolitica strain isolated from the palatine tonsil of aborted swine fetus. The control group (n = 3) remained uninfected. Rectal, oral and vaginal swabs from sows, placentas and the specimens of tissues from stillborn piglets were collected for bacteriological examination. Eight weeks after delivery, the sows were slaughtered and samples of internal organs were subjected to bacteriological examination. Pregnancy in all groups of sows took a normal course, and no cases of abortion were observed. Y. enterocolitica was isolated from oral, rectal and vaginal swabs of all infected sows. The number of stillborn piglets in the litters was highest in group III, where two macerated fetuses with putrefactive lesions were found. The bacteria were isolated from tissues of stillborn piglets in groups I and III, and only from placenta in group II. In rectal swabs of piglets in all groups, Y. enterocolitica was not isolated. The results of experimental infection of pregnant sows with Y. enterocolitica revealed that in animals infected in the last part of pregnancy, the microorganisms were isolated most frequently from vaginal, rectal and oral swabs of sows as well as from internal organ tissues of stillborn piglets.

Isolation of Yersinia enterocolitica from aborted fetuses and sows in pig farms with reproductive disturbances.

The purpose of the study was to determine the occurrence of Yersinia enterocolitica in tissues of aborted fetuses, placentas, vaginal and rectal swabs of aborting sows from pig farms where reproductive disturbances were found and to determine and analyze the biotype and serotype affinity of the strains isolated. Altogether 97 fetuses aborted in various stages of pregnancy, 25 placentas and swabs from 231 sows were taken. All sows originated from farms where reproductive disorders appeared. In general, 1069 samples were collected. Two enrichment methods were used in this study; fast enrichment techniques in ITC broth, then plating onto CIN agar (ITC/CIN), and cold enrichment in phosphate buffered saline followed by plating onto CIN agar (PBS/CIN). From all samples examined, 96 Y. enterocolitica strains were isolated including 57 (59.4%) from rectal swabs of sows, followed by 6 (6.3%) from vaginal swabs and 2 (2.1%) from placentas. The bacteria were isolated from tissues of 18 out of 97 aborted fetuses. A total of 60 strains were selected for further examination--29 strains originated from aborting sows and 31 from aborted fetuses. Among strains examined 54 isolates (90%) belonged to the biotype 1A of Y. enterocolitica and to the different serotypes O:3, O:5, O:6, O:7/13, O:8 and NT (not typable). Only 6 strains belonged to serotype O:3, biotype 4 Y. enterocolitica. Our study has revealed the possibility of Y enterocolitica isolation from internal organs of aborted swine fetuses and sows from farms with reproductive disturbances. The results suggest the connection between fetal death, pregnancy course disorders and Y. enterocolitica infection.

The influence of experimental Yersinia enterocolitica infection on the pregnancy course in sows--preliminary studies. II. Antibodies, C-reactive proteins and haptoglobin as an immunological response.

The aim of the study was to determine the time of emergence and level of Y. enterocolitica antibodies in pregnant sows challenged orally with Y. enterocolitica in particular trimesters of pregnancy (groups I, II and III, respectively) and also the assignation of its influence on the CRP and Hp concentration in sera of pigs. Levels of antibodies measured by tube agglutination test increased slowly from 2 weeks post infection (wpi) and positive results were obtained not in all animals. In ELISA, in 2 weeks in all groups of infected animals high levels of antibodies against Y. enterocolitica were formed and lasted up to the end of the experiment. In newborn piglets in all groups, a significant decrease in antibody levels 6 weeks after birth was observed in both agglutination and ELISA tests. Concentrations of CRP as Hp in all groups of infected animals increased in 1 week post infection. Statistically significant differences (P < or = 0.05) between CRP levels in groups I and II (46-fold and 44-fold) as well as III (29-fold) were revealed. In case of Hp, statistically significant differences between groups of animals in the first week post infection were not observed. Our findings indicate that Y. enterocolitica infection evoked strong and long-lasting immunological reaction in the form of specific antibodies production in all inoculated animals. The significant increase in CRP and moderate increase in Hp concentrations in the sera of pregnant sows also occurred. However, relationships between colostrums antibody levels in piglets' sera and phase of pregnancy when the Y. enterocolitica infection happened in sows were not observed.

Analysis of the presence of ail, ystA and ystB genes in Yersinia enterocolitica strains isolated from aborting sows and aborted fetuses.

Forty-five Yersinia enterocolitica strains isolated from aborted fetuses and placentas and from vaginal and rectal swabs of aborting sows were subjected to serotyping, biochemical typing and polymerase chain reaction multiplex analyses to detect the presence of the ail, yst A and ystB genes. The isolates were recovered from the internal organs (tonsil, lung, liver, spleen, kidney, mesentheric lymph nodes, small intestine and rectal intestine) of 18 (18.6%) of 97 aborted fetuses examined, two (8%) of 25 aborted placentas and 27 (15.8%) of 172 examined aborting sows. Serotyping of Y. enterocolitica revealed that only six (13.3%) of the examined isolates belonged to serotype O:3, with a considerable number of isolates (31.1%) having serotype O:5, while biochemical studies showed that as many as 40 of the 45 strains belonged to biotype 1A. As expected, the Y. enterocolitica strains of bioserotype 4/O:3 contained ail and ystA genes, while strains of biotype 1A contained only the ystB gene.

Evaluation of specific humoral immune response in pigs vaccinated intradermally with deleted Aujeszky's disease vaccine and challenged with virulent strain of Herpesvirus suis type 1.

A comparison of intradermal (ID) versus intramuscular (IM) routes of pig vaccination with deleted Aujeszky's disease (AD) vaccine on the formation of specific postvaccinal and postchallenge humoral immune response was performed. The studies were carried out on 21 eight week-old piglets, divided into three groups--two experimental and one control of 7 piglets each. Animals of first two groups were vaccinated twice in 12 and 16 week of age with deleted, live attenuated AD vaccine Porcilis Begonia (Intervet). Group I was vaccinated with a dose of 2.0 ml (10(6.0) TCID50)) intramuscularly (IM) into neck muscles, and group II received 0.2 ml (10(5.0) TCID50) intradermally (ID) in neck area using needleless apparatus SERENA model SD 1-2 (Emplast, Italy). In group K (control) 2.0 ml PBS IM was used. Seventy days after the first vaccination all pigs were intranasally infected with a dose of 10(5.5) TCID50 of virulent Northern Ireland Aujeszky-3 (NIA-3) strain of Herpesvirus suis type 1 (SHV-1) by instilling 0.5 ml of virus suspension into each nostril. Specific humoral immune response was evaluated using seroneutralization (SN) test and gE-ELISA-Pseudorabies virus gpI Antibody Test Kit (Herd Chek Anti-PRV gpI), IDEXX Lab Inc (USA). It was found that challenge caused anamnestic reaction in both groups of vaccinated pigs, but postchallenge immune response was stronger in ID-vaccinated group--on 14 day post infection (dpi) SN antibody level was considerably higher than in IM-vaccinated group. The obtained results suggest that secondary immunological response after challenge is decidedly more effective in the range of evaluated parameters in animals vaccinated by ID route, which can be linked to, perhaps underestimated yet and seldom utilized, skin immunity mechanisms in specific prophylaxis of infectious diseases. Advantages and disadvantages of SN test and ELISA are also discussed.

Modulative influence of lysozyme dimer on defence mechanisms in the carp (Cyprinus carpio) and European sheatfish (Silurus glanis) after suppression induced by herbicide Roundup.

Immunomodulation is a commonly used method of prophylaxis in humans and animals. Lysozyme dimer (KLP-602) was used at a dose of 50 ug/kg b.w. in order to correct the immunosuppression caused by the action of herbicide glyphosate (Roundup- Monsanto), which was used in a single bath for 10 minutes in a concentration of 100 mg/l of water. The investigations were carried out on 2 species of fish: the carp (Cyprinus carpio L.) and european catfish (Silurus glanis L.). Herbicide glyphosate caused a decrease in metabolic and phagocytic activity (RBA and PKA) and in proliferative response stimulated by Con A and LPS in carp and european catfish. The immunosuppression sustained for about 2 weeks. The results obtained indicate the possibility of correction of immunosuppression applying lysozyme dimmer (KLP-602) after use of which, the level of the studied indexes increased.

[Is Aujeszky's disease a zoonosis?]. / Czy choroba Aujeszkyego jest zoonoza?

Several cases of human illness with symptoms suggestive of Aujeszky's Disease (AD) at an outbreak of the disease in cattle and swine were evaluated during this study. Additionally serological studies of people in the group at high risk for illness caused by Aujeszky's Disease Virus (ADV) were carried out. In a herd of 180 bulls sudden clinical disease occurred with symptoms of muscular tremors, salivation, sweating and severe pruritus of the head. The clinical disease lasted two to eight hours and finished in death. In 7 days 10 bulls died and 60 were either killed or culled. A biological test for AD was positive. ADV was identified in the brain of a dead bull. Serological studies for the bulls were negative, however 92.9% on the premises farm were asymptomatically infected. On third to the fifth day of disease in the cattle clinical signs appeared in six of seven workers which had direct contact with diseased cattle. A pruritus of the palms, which spread onto the lower and upper arms, shoulders and back lasted several days.