The occurrence and molecular identification of Thelazia spp. in European bison (Bison bonasus) in the Bieszczady Mountains.

Infection with Thelazia nematodes results in eye disease in wild and domestic animals. The aim of the present study was to describe the occurrence of Thelazia nematodes in European bison, and to subject the isolated parasites to molecular identification and phylogenetical analysis. The eyeballs of 18 European bison from the Bieszczady Mountains, culled due to dysfunctional vision, were collected for study. The conjunctival sacs, tear ducts, corneal surface and nictitating membrane were rinsed with a saline solution. Any obtained nematodes were isolated under a stereoscopic microscope, and then identified as T. gulosa or T. skrjabini by molecular analysis of partial cox1 sequences. The prevalence of infection with Thelazia spp. was found to be 61%, with a 95% confidence interval (CI 95%) of 39-80%. Thelazia skrjabini was isolated from 56% (CI 95% 34-75%) of examined animals; T. gulosa was significantly less common (p = 0.038) with the prevalence of infection reaching 22% (CI 95% 9-45%). Three European bison were cross-infected with both T. gulosa and T. skrjabini. Phylogenetic analysis found the obtained sequences to be similar to those of Thelazia species from domestic ungulates in Europe. Infection intensity ranged from 1 to 16 nematodes per individual (median of three nematodes), and was significantly higher in females (6 nematodes) than in males (1 nematode; p = 0.019). A tendency for seasonal occurrence of nematodes in European bison was also observed. Our study provides further information regarding the patterns of Thelazia transmission in European bison in Poland.

Sarcocystis cruzi infection in free-living European bison (Bison bonasus bonasus L.) from the Bialowieza Forest, Poland - A molecular analysis based on the cox1 gene.

European bison are susceptible to a range of pathogens which may influence their health, and hence, to ensure their protection, it is essential to provide effective monitoring of potential exposure. This study presents the first molecular confirmation of Sarcocystis cruzi infection in European bison based on PCR amplification of the cytochrome c oxidase subunit I (cox1) gene. A sample of heart tissue taken from one fifteen-year-old European bison cow was examined by light microscopy for the presence of heart sarcocysts. The genomic DNA isolated from any identified sarcocysts was subjected to PCR to amplify cox1 gene sequences, and the obtained amplicons were sequenced by Sanger dideoxy sequencing. Two partial cox1 sequences were obtained; they were identified as S. cruzi and deposited in the GenBank™ database under the accession numbers MW490605 and MW490606. BLAST analysis found them to demonstrate the closest similarity to S. levinei (MH255771-MH255779 and KU247874-KU247884), sharing an identity of 93.14-93.8 %. This is the first report to identify sarcocysts isolated from heart tissue of infected European bison living in the Bialowieza forest to species level using cox1 analysis. Our findings confirm that the European bison is a natural intermediate host for S. cruzi. As such, coordinators of future conservation programmes should consider the impact of these diseases on reintroduced animals.

Use of meat juice from racoons (Procyon lotor) collected from Central Europe for immunological detection of Trichinella spp.

The raccoon (Procyon lotor) is a species native to North America, but which is now spreading throughout Europe. Raccoons have been found to host various Trichinella species. The aim of the present study was to evaluate the effectiveness of using immunological testing of meat juice for determining the occurrence of Trichinella in raccoons. The studies were carried out on 139 animals from three European countries: the Czech Republic, Germany and Poland. Seven meat juice samples were found to be positive for antibodies to Trichinella by ELISA, and another seven were unclear. The ELISA results were confirmed by immunoblot: anti-Trichinella antibodies were identified in 9.35 % of the examined animals. Slight agreement (κ = 0.13) was found between the digestion method and the combined ELISA and immunoblot approach. From the results of our study, we concluded that meat juice may be used as a simple and convenient sample for detection of anti-Trichinella in racoons.

Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes.

The Trichinella genus poses an interesting puzzle for researchers, having diverged very early in the evolution of the nematodes. The Trichinella spiralis proteome is a cosmopolitan and well-studied model of Trichinella; however, Trichinella britovi also circulates in the sylvatic environment and both species infect humans, resulting in the development of trichinellosis. Few experiments have examined the proteins belonging to the T. britovi proteome. The aim of the present study was to compare the protein expression profiles of crude extracts of T. spiralis and T. britovi muscle larvae using a highly-sensitive two-dimensional differential in-gel electrophoresis (2D DIGE) technique coupled with 2DE immunoblotting. Selected immunoreactive protein spots were then identified by liquid chromatography coupled with mass spectrometry analysis (LC-MS/MS), and their function in Trichinella and the host-parasite interaction was determined by gene ontology analysis. Spots common to both T. spiralis and T. britovi, spots with different expressions between the two and spots specific to each species were labelled with different cyanine dyes. In total, 196 protein spots were found in both proteomes; of these 165 were common, 23 expressed exclusively in T. spiralis and 8 in T. britovi. A comparative analysis of volume ratio values with Melanie software showed that among the common spots, nine demonstrated higher expression in T. spiralis, and 17 in T. britovi. LC-MS/MS analysis of 11 selected spots identified 41 proteins with potential antigenic characteristics: 26 were specific for T. spiralis, six for T. britovi, and eight were found in both proteomes. Gene Ontology analysis showed that the identified T. spiralis proteins possess hydrolytic endopeptidase, endonuclease and transferase activities. Similarly, most of the T. britovi proteins possess catalytic activities, such as lyase, hydrolase, isomerase and peptidase activity. The applied 2D DIGE technique visualized Trichinella spp. protein spots with different molecular weights or isoelectric point values, as well as those with different expression levels. The identified immunoreactive proteins participate in multiple processes associated with host muscle cell invasion and larval adaptation to the host environment. Their reactivity with the host immune system makes them possible candidates for the development of a novel trichinellosis diagnostic test or vaccine against helminthiasis caused by T. spiralis or T. britovi.

Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Trichinella britovi infection and muscle distribution in free-living martens (Martes spp.) from the Gleboki Bród Forest District, Poland.

Trichinella nematodes occur in many carnivorous and omnivorous animal species in the sylvatic cycle. Due to their widespread occurrence throughout Poland and diet, free-living Mustelids can act as a potential reservoir for nematodes of the genus Trichinella and play a role in their circulation. The study was designed to determine the presence and predilection sites for Trichinella nematodes in martens (Martes spp.) from the Gleboki Bród Forest District, Poland. Trichinella britovi larvae were detected by molecular methods in 17.54% examined martens (prevalence: 41.67% among pine martens and 13.88% among Martes spp.). The intensity of infection varied from 0.17 to 37.29 larvae per gram (LPG) (mean 5.43; median 3.4). The highest larval burdens were detected in the tongue in pine martens (Martes martes) and the diaphragm in Martes spp., respectively; the lowest levels were found in the masseter in pine martens and the tongue in Martes spp. No statistically significant difference in the intensity of infection was observed between males and females in either group. Our findings indicate that T. britovi is present in martens from the Gleboki Bród Forest District, and the predilection sites for the nematode may differ between males and females. However, due to the low number of examined animals, further studies are necessary to confirm whether they are an important element in the maintenance of Trichinella nematodes in the examined area.

The Nematodes Thelazia gulosa Railiet and Henry, 1910 and Thelazia skrjabini Erschov, 1928 as a Cause of Blindness in European Bison (Bison bonasus) in Poland.

PURPOSE: The nematodes of the genus Thelazia are the cause of eye diseases of wild and domestic ruminants throughout the world. The aim of the study was to describe clinical cases of thelasiosis in European bison (Bison bonasus) in Poland, and provide morphometrical features of Thelazia gulosa Railiet and Henry, 1910 and Thelazia skrjabini Erschov, 1928 regarded as potentially useful for species differentiation METHODS: The conjunctival sacs, tear ducts, the surface of the cornea and nicitating membrane collected from bison were rinsed with saline solution. Any nematodes isolated from the sediment were subjected to morphometric analysis. RESULTS: Thirteen of the 16 examined European bison were infected with Thelazia nematodes, belonging to the species T. gulosa and T. skrjabini. The intensity of infection ranged from one to six (mean intensity 5), and four to 29 (mean intensity 14) nematodes T. skrjabini and T. gulosa respectively. Congestion of conjunctival sac, keratitis and corneal opacity, corneal ulceration and perforation as well as purulent eyeball inflammation were observed in infected animals. CONCLUSIONS: Thelazia gulosa and T. skrjabini can be identified by morphometrical features. As thelasiosis might be a serious threat for protected population of European bison, further studies are needed of the epidemiology and pathology of this emerging parasitosis in Poland.

The Seroprevalence of Toxoplasma gondii in Wild Boars from Three Voivodeships in Poland, MAT Analyses.

PURPOSE: The European wild boar (Sus scrofa) is a popular game animal species. Its meat, however, can represent a reservoir of dangerous foodborne diseases and can play an important role in the transmission of many pathogens, including Toxoplasma gondii, in humans and animals worldwide. The aim of the present study was to determine the presence of antibodies to T. gondii in the serum of hunted wild boars in Poland. METHODS: Using the commercial direct agglutination test, 398 serum samples collected during the hunting season 2009/2010 were tested for the presence of T. gondii antibodies, and the titre of 40 was considered indicative of T. gondii infection in analysed samples. RESULTS: It was found that nationwide, 37.7% were seropositive to T. gondii, although seroprevalence varied from 11.6 to 50% depending on the Voivodeship. Significant differences were observed between the Great Poland and Lubusz Voivodeships and between Great Poland and Warmian-Masurian. CONCLUSION: Serological test indicated widespread exposure to T. gondii by wild boar; therefore, consumption of raw or undercooked game meat of infected animals can carry a significant risk of T. gondii infection.

Molecular identification of sarcocysts from tissue of fallow deer (Dama dama) farmed in the open pasture system based on ssu rRNA gene.

PURPOSE: Sarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals, including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed in an open pasture system. METHODS: Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using their DNA as templates. RESULTS: Analysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata were detected in oseophagus. CONCLUSIONS: These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more molecular studies are needed.

Ashworthius sidemi in cattle and wild ruminants in Poland - the current state of play.

Ashworthius sidemi, a blood-sucking abomasal nematode, has been identified in various wild ruminants, including deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) and moose (Alces alces). Although it has been observed throughout Poland, most sightings have been in the eastern part of the country. However, more recently, A. sidemi has been confirmed in the Ruszów Forest District (Lower Silesian Wilderness). It is now possible to test the faeces of cattle for the DNA of the third-stage infectious larvae (L3) of A. sidemi. The present paper describes such a molecular study of 120 faecal samples collected from cattle grazed in the Ruszów Forest District and Biebrza Marshland, where A. sidemi had previously been detected in wildlife. In this study, no A. sidemi DNA was identified in any of the examined samples.

Seroprevalence of Trichinella spp. infection in bank voles (Myodes glareolus) - A long term study.

Rodents play an important role as reservoir hosts of zoonotic diseases. As a component of our long-term programme of monitoring parasitic infections in bank vole populations in three ecologically similar sites in NE Poland, we screened blood samples for signs of a serological response to the presence of Trichinella spp. The overall seroprevalence of Trichinella spp. was 1.52%, but prevalence was largely concentrated in one of our three study sites and confined to the oldest individuals in the study. Seroprevalence of Trichinella spp. did not differ between the sexes. Although a local prevalence of 1.52% may seem low, when this is extrapolated to the national population of bank voles in peak years, perhaps numbering hundreds of millions of animals, the number of infected bank voles on a country wide scale is likely to be huge. Our results suggest that bank voles may be reservoirs of Trichinella spp. However, on the basis of our results we consider their importance as epidemiologically significant hosts for Trichinella spp. to be moderate and their role in this context to require further investigation.

The occurrence and muscle distribution of Trichinella britovi in raccoon dogs (Nyctereutes procyonoides) in wildlife in the Gleboki Bród Forest District, Poland.

The raccoon dog (Nyctereutes procyonoides) is an introduced, invasive species in Europe. Literature data show that raccoon dogs act as a reservoir of many dangerous parasites, including nematodes of the genus Trichinella. The aims of the study were to determine the prevalence of Trichinella spp. infection in raccoon dogs collected from the Gleboki Bród Forest District between 2013 and 2016, and to evaluate their distribution in the muscle tissue of the host. The larvae of Trichinella spp. were detected in 45 raccoon dogs (39.82%), and all of them were identified as T. britovi. No mixed infection was observed. The intensity of infection ranged from 0.02 to 622.92 larvae per gram (LPG), and the highest mean was observed in the tongue and lower forelimb in both examined sexes. The raccoon dog may play a significant role as a reservoir of T. britovi in the wildlife in the examined area.

Survey of Toxoplasma gondii and Neospora caninum in raccoons (Procyon lotor) from the Czech Republic, Germany and Poland.

The studies were carried on raccoons from Poland, Germany and the Czech Republic. Tissue samples from raccoon hearts, lungs and brains were used for molecular examination while meat juice was collected for immunological tests. Antibodies against T. gondii were detected in six out of 44 raccoons (13.6%), while T. gondii DNA was found in 18 (40.9%). Antibodies against N. caninum were found in seven raccoons (15.9%) but no parasite DNA was observed in any sample. DNA of T. gondii was observed in raccoons of both sexes (in 42.3% of females and 38.9% of males) from all three countries. The proportion of raccoons that tested positive for DNA of T. gondii was higher in the Czech Republic (47.1%) than in Germany (33.3%), however the difference was non-significant (p = 0.7032). It seems that the raccoons appear to have been exposed to both T. gondii and N. caninum, but only T. gondii infection was confirmed. The role of raccoons as reservoir, and as possibly contributing to spread of these parasites merits further studies.

Trichinella britovi muscle larvae and adult worms: stage-specific and common antigens detected by two-dimensional gel electrophoresis-based immunoblotting.

BACKGROUND: Trichinella britovi is the second most common species of Trichinella that may affect human health. As an early diagnosis of trichinellosis is crucial for effective treatment, it is important to identify sensitive, specific and common antigens of adult T. britovi worms and muscle larvae. The present study was undertaken to uncover the stage-specific and common proteins of T. britovi that may be used in specific diagnostics. METHODS: Somatic extracts obtained from two developmental stages, muscle larvae (ML) and adult worms (Ad), were separated using two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. The positively-visualized protein spots specific for each stage were identified through liquid chromatography-tandem mass spectrometry (LC-LC/MS). RESULTS: A total of 272 spots were detected in the proteome of T. britovi adult worms (Ad) and 261 in the muscle larvae (ML). The somatic extracts from Ad and ML were specifically recognized by T. britovi-infected swine sera at 10 days post infection (dpi) and 60 dpi, with a total of 70 prominent protein spots. According to immunoblotting patterns and LC-MS/MS results, the immunogenic spots recognized by different pig T. britovi-infected sera were divided into three groups for the two developmental stages: adult stage-specific proteins, muscle larvae stage-specific proteins, and proteins common to both stages. Forty-five Ad proteins (29 Ad-specific and 16 common) and thirteen ML proteins (nine ML-specific and four common) cross-reacted with sera at 10 dpi. Many of the proteins identified in Ad (myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin) are involved in structural and motor activity. Among the most abundant proteins identified in ML were 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. Heat-shock protein, intermediate filament protein ifa-1 and intermediate filament protein B were identified in both proteomes. CONCLUSIONS: To our knowledge, this study represents the first immunoproteomic identification of the antigenic proteins of adult worms and muscle larvae of T. britovi. Our results provide a valuable basis for the development of diagnostic methods. The identification of common components for the two developmental stages of T. britovi may be useful in the preparation of parasitic antigens in recombinant forms for diagnostic use.

Comparison of sensitivity of two primer sets for the detection of Toxoplasma gondii DNA in wildlife.

Toxoplasma gondii, a coccidian parasite known to infect almost all warm-blooded animals, is the cause of one of the most common zoonotic parasitic diseases. The aim of the study is to determine whether the 529 bp fragment or the TGR1E gene is more useful target for PCR identification of T. gondii, for common use. The brains of 221 carnivores and omnivores collected between 2013 and 2015 from north-eastern Poland were examined for the presence of this parasite. The DNA was extracted and then amplified using specific primers. Positive results were obtained in 24% of brain samples using the TGR1E target and 19% using the 529 bp sequence. The results demonstrate that both TGR1E and 529 bp repeat element are suitable for detecting T. gondii DNA in wildlife animals, and the combination of two methods is necessary to obtain reliable results.

Seroprevalence of Toxoplasma gondii and Neospora caninum infection in sheep, goats, and fallow deer farmed on the same area.

Toxoplasma gondii and Neospora caninum are coccidian parasites with a global distribution that cause reproductive failure and production losses in livestock. The seroprevalence of both parasite species in ruminants and Cervidae has been investigated worldwide and found to vary greatly. Studies carried out on mixed flocks with 3 ruminant species (sheep, goats, and fallow deer) living under the same conditions are excellent models for identifying any differences in the rate of infection with the 2 parasites between the animal species. Additionally, the species used in the present study differ in their feeding categories: grazers, browsers, and intermediate feeders. The aim of the study is to identify any variation in the prevalence of the 2 parasites in mixed flocks and to identify any possible relationships with food choice. The seroprevalence against T. gondii and N. caninum in 167 captive fallow deer, 64 sheep, and 39 goats were detected using commercially available ELISA. The seroprevalence for T. gondii achieved 10% in fallow deer, 21% in goats, and 47% in sheep. The seroprevalence for N. caninum achieved 13% in sheep and fallow deer and 21% in goats. Overall, 53% of the sheep, 33% of the goats, and 22% of the fallow deer were seropositive for both infections. Coinfection of T. gondii and N. caninum was detected in 6% of sheep, 8% of goats, and 2% of fallow deer. Statistical analyses of the seroprevalence levels observed between 2 parasites for each animal species revealed that only the results obtained for sheep were significant (P < 0.01). Additionally, the differences in the seroprevalence levels for T. gondii between sheep and goats and between sheep and fallow deer were statistically significant (P < 0.01). The results of the N. caninum seroprevalence levels observed among animal species were not significant. Although the variations in susceptibility to T. gondii and N. caninum infections demonstrated by the examined animals may affect the differences in seropositivity, these appear to be related to the feeding habits of the animal species. Therefore, the risk of infection by agents found close to the ground, such as coccidian oocysts, varies. Sheep as grazers are at a greater risk of infection by T. gondii than goats and fallow deer.

First detection of Trichinella pseudospiralis infection in raccoon (Procyon lotor) in Central Europe.

The raccoon (Procyon lotor) is a North American carnivore introduced to Europe in the 20th Century. Raccoons are believed to be the potential hosts of many parasites, or to be involved in their transmission to other animals. Nematodes of the genus Trichinella can infect many carnivorous and omnivorous animals worldwide. The aim of the present study was to determine the occurrence of Trichinella spp. infection in raccoons in Central Europe. Muscle samples were collected from various regions of Poland, the Czech Republic and Germany during the years 2012-2016. The larvae of Trichinella spp. were detected in 11 raccoons, and these were identified as T. spiralis and T. pseudospiralis by multiplex PCR (89.9% and 9.1%, respectively). No mixed infection was observed. This is the first report describing the occurrence of T. spiralis and T. pseudospiralis in P. lotor in Central Europe. Our findings also show that the raccoon population acts as a reservoir of Trichinella pseudospiralis.

First Toxoplasma gondii isolate from an aborted foetus of European bison (Bison bonasus bonasus L.).

The study was performed on a male European bison (Bison bonasus bonasus L.) foetus spontaneously aborted at the fourth or fifth month of pregnancy in the Bialowieza Forest. Serum samples from the foetus and mother revealed the presence of antibodies against T. gondii (S/P% = 88% and 75%, respectively). Mobile extracellular tachyzoites were first observed in a Vero cell culture, 110 days following inoculation of brain homogenate. PCR amplification with TGR1E1 and TGR1E2 primers confirmed the presence of T. gondii DNA, which was classified as Type I by PCR-RFLP genotyping. The sequences of 18S ribosomal RNA (18S rRNA) and 5.8S ribosomal RNA (5.8S rRNA) genes; internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2), obtained from T. gondii isolate, have been deposited in GenBank (accession number KX459518.1). This is the first in vitro isolation and molecular identification of T. gondii from an aborted European bison foetus. The origin of this protozoan isolate indicates that the species is a significant threat to the European bison conservation program implemented in the Bialowieza Forest.

The Occurrence of Trichinella spp. in Red Foxes (Vulpes vulpes) in Different Regions of Poland: Current Data.

Trichinellosis is one of the most widespread parasitic zoonoses. Trichinella Owen, 1835 nematodes are found in pigs, horses, and humans in the domestic cycle, and in many carnivores and omnivores in the sylvatic cycle, such as wild boars, red foxes, raccoon dogs, and wolves. Carnivores are known to be involved in the circulation of Trichinella nematodes and they act as a reservoir in the sylvatic environment. The aim of this study was to determine the occurrence of Trichinella spp. infection in red foxes in Poland. Samples were collected from 2010 to 2015 in different regions of the country and then tested for Trichinella nematodes using HCl-pepsin digestion. Trichinella larvae were found in 10.02% of examined samples (145/1447). The larvae were identified as T. spiralis (11.03%), T. britovi (71.72%), and T. pseudospiralis (0.69%). No mixed infection was observed. The prevalence of infection varied between years and different voivodeships of the country. Our findings confirm that red foxes are involved in the maintenance of Trichinella spp. in the sylvatic cycle in Poland.

The usefulness of direct agglutination test, enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Toxoplasma gondii in wild animals.

The aim of the study was to compare the usefulness of two antibody-based methods, the direct agglutination test (DAT) and enzyme linked immuosorbent assay (ELISA), with that of the polymerase chain reaction (PCR) for detecting anti-Toxoplasma gondii in samples derived from naturally-infected wild animals. Antibodies against T. gondii were detected in meat juice samples collected from 129 free- living carnivores and omnivores. T. gondii seroprevalence was confirmed in 73,6% of examined samples when DAT and ELISA were used separately, but in only 88,4% samples when both immunological tests were used in parallel. PCR results confirmed the presence of DNA of the parasite in 24 of all the 129 samples. Sixteen samples were classified as positive when all three tests were used. A moderate degree of agreement was found between DAT and ELISA (κ=0.55). However, no agreement was found between the molecular and serological tests: κ=-1.75 for DAT versus PCR; κ=-1.67 ELISA versus PCR. By using both serological tests, antibodies against T. gondii were found in 77.5% of red foxes, 12.5% of badgers, 40% of martens and 8.3% of raccoon dogs. Antibodies against the parasite were detected also in one mink, but not in the sample derived from a polecat. T.gondii DNA was found in the brain tissue of 20 red foxes, three badgers and one raccoon dog. Our studies confirm that ELISA and DAT are suitable and reliable techniques for T. gondii antibody detection in meat juice from wild animals when serum samples are unavailable. Positive results obtained by immunological tests do not always reflect that the host was infected by T. gondii. They indicate only a contact with parasite. PCR should be used to confirm te presence of DNA from T. gondii.