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.

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.

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 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 occurrence of nematodes of the genus Trichinella in wolves (Canis lupus) from the Bieszczady Mountains and Augustowska Forest in Poland.

The aim of the present study was to investigate the prevalence of Trichinella infection in wolves (Canis lupus) in two regions in Poland. Muscle samples were collected from 21 wolves between 1999 and 2015 and processed by artificial digestion. In two cases, the muscle larvae (ML) were obtained and stored in alcohol. ML were detected in 12 wolves and genotyped by multiplex PCR. Trichinella britovi was confirmed in 12 wolves (54.5%). The larval burdens in infected animals ranged from 0.009 to 27 larvae per gram. The high prevalence of Trichinella infection in wolves might suggest that this predator is a significant reservoir of Trichinella species in the sylvatic cycle in Poland.

Wild boars meat as a potential source of human trichinellosis in Poland: current data.

Trichinellosis is an epidemiological problem with a global distribution. In Poland a substantial increase of the wild boar population has been observed since 2010, together with an increased incidence of trichinellosis after ingestion of raw or undercooked wild boar products containing Trichinella spp. larvae. However, the actual number of human cases remains particularly difficult to determine. The aim of the present study was to determine the current prevalence and spread of these parasites within wild boars. The diaphragm pillars and tongue from 833 wild boars were collected from 2010 to 2014, as well as one wild boar meat sausage known to be a source of infection. The samples were tested for Trichinella spp. using pepsin digestion. Recovered larvae were identified at species level by multiplex polymerase chain reaction (multiplex PCR). The overall prevalence in all examined samples was found to be 2.0% (17/833). Recovered larvae were identified as T. spiralis and T. britovi (9/18 and 5/18, respectively). T. spiralis larvae were isolated from the sausage. Mixed infection was confirmed only once. Three isolates were not identified. The results of our study confirm that the wild boar plays a key role in the maintenance of Trichinella nematodes through the sylvatic cycle.

Proteomic analysis of potential immunoreactive proteins from muscle larvae and adult worms of Trichinella spiralis in experimentally infected pigs.

The present study was undertaken to identify potentially immunoreactive proteins of the muscle larvae (ML) and adult stage (Ad) of the nematode Trichinella spiralis Owen, 1835. To identify immunoreactive proteins that are specifically recognised by anti-Trichinella antibodies, ML and Ad crude extracts and their excretory-secretory (E-S) products were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with serum samples from pigs experimentally infected with T. spiralis. A total of 18 bands were selected for final identification by liquid chromatography-tandem mass spectrometry. To further understand the functions of the proteins identified in this study, gene ontology terms were applied. Results showed that the specific antibodies against T. spiralis reacted with protein bands matching heat shock proteins, aminopeptidase, enolase, isocitrate dehydrogenase NADP-dependent, tropomyosin, P49 antigen, serine proteinase, secreted 5'-nucleotidase, antigen targeted by protective antibodies, 53 kDa E-S antigen, putative trypsin and paramyosin. Three proteins common for both adult stage and muscle larvae, including heat shock proteins, enolase and 5'-nucleotidase, might play important role during T. spiralis infection. These proteins are presumably presented to the host immune system and may induce humoral immune response. Thus, these proteins may be potential antigens for early diagnosis and the development of a vaccine against the parasite.

The first identification of a blood-sucking abomasal nematode Ashworthius sidemi in cattle (Bos taurus) using simple polymerase chain reaction (PCR).

A simple polymerase chain reaction (PCR) test was used to identify Ashworthius sidemi, a blood-sucking gastrointestinal nematode that commonly infects bison, red and roe deer, and moose in Poland. The present study uses this technique to confirm the possibility of transmission of A. sidemi infection from wildlife to domestic animals, such as cattle and sheep, grazing on the same natural pastures. A 406 bp fragment of genomic A. sidemi DNA was actually detected in DNA isolated from larval cultures derived from feces from cattle. A. sidemi DNA has been detected in cattle which represent a new host for this parasite. This is the first evidence of A. sidemi in cattle. The results reveal that a PCR test based on DNA from L3 larvae can be used for in vivo detection of A. sidemi invasions in breeding animals. In conclusion, the transfer of A. sidemi infection from wildlife to the farm animals sharing the same pastures appears possible.

The parasitic fauna of the European bison (Bison bonasus) (Linnaeus, 1758) and their impact on the conservation. Part 1. The summarising list of parasites noted.

During the current century, 88 species of parasites have been recorded in Bison bonasus. These are 22 species of protozoa (Trypanosoma wrublewskii, T. theileri, Giardia sp., Sarcocystis cruzi, S. hirsuta, S. hominis, S. fusiformis, Neospora caninum, Toxoplasma gondii, Cryptosporidium sp., Eimeria cylindrica, E. subspherica, E. bovis, E. zuernii, E. canadensis, E. ellipsoidalis, E. alabamensis, E. bukidnonensis, E. auburnensis, E. pellita, E. brasiliensis, Babesia divergens), 4 trematodes species (Dicrocoelium dendriticum, Fasciola hepatica, Parafasciolopsis fasciolaemorpha, Paramphistomum cervi), 4 cestodes species (Taenia hydatigena larvae, Moniezia benedeni, M. expansa, Moniezia sp.), 43 nematodes species (Bunostomum trigonocephalum, B. phlebotomum, Chabertia ovina, Oesophagostomum radiatum, O. venulosum, Dictyocaulus filaria, D.viviparus, Nematodirella alcidis, Nematodirus europaeus, N. helvetianus, N. roscidus, N. filicollis, N. spathiger, Cooperia oncophora, C. pectinata, C. punctata, C. surnabada, Haemonchus contortus, Mazamastrongylus dagestanicus, Ostertagia lyrata, O. ostertagi, O. antipini, O. leptospicularis, O. kolchida, O. circumcincta, O. trifurcata, Spiculopteragia boehmi, S. mathevossiani, S. asymmetrica, Trichostrongylus axei, T. askivali, T. capricola, T. vitrinus, Ashworthius sidemi, Onchocerca lienalis, O. gutturosa, Setaria labiatopapillosa, Gongylonema pulchrum, Thelazia gulosa, T. skrjabini, T. rhodesi, Aonchotheca bilobata, Trichuris ovis), 7 mites (Demodex bisonianus, D. bovis, Demodex sp., Chorioptes bovis, Psoroptes equi, P. ovis, Sarcoptes scabiei), 4 Ixodidae ticks (Ixodes ricinus, I. persulcatus, I. hexagonus, Dermacentor reticulatus), 1 Mallophaga species (Bisonicola sedecimdecembrii), 1 Anoplura (Haematopinus eurysternus), and 2 Hippoboscidae flies (Lipoptena cervi, Melophagus ovinus). There are few monoxenous parasites, many typical for cattle and many newly acquired from Cervidae.

The parasitic fauna of the European bison (Bison bonasus) (Linnaeus, 1758) and their impact on the conservation. Part 2. The structure and changes over time.

During the last century the recorded parasite fauna of Bison bonasus includes 88 species. These are 22 species of protozoa, 4 trematode species, 4 cestode species, 43 nematode species, 7 mites, 4 Ixodidae ticks, 1 Mallophaga species, 1 Anoplura, and 2 Hippoboscidae flies. There are few monoxenous parasites, the majority of parasites are typical for other Bovidae and Cervidae species and many are newly acquired from Cervidae. This is an evident increased trend in the parasite species richness, in both the prevalence and intensity of infections, which is associated with the bison population size, host status (captive breeding or free-ranging) and the possibility of contact with other ruminant species. In light of the changes to parasite species richness during the last decades, special emphasis shall be given to new parasite species reported in European bison, their pathogenicity and potential implications for conservation.

The usefulness of DNA derived from third stage larvae in the detection of Ashworthius sidemi infection in European bison, by a simple polymerase chain reaction.

BACKGROUND: Ashworthius sidemi, a blood-sucking nematode, is a primary parasite of Asiatic cervides, primarily sika deer (Cervus nippon). As A. sidemi infections are common in bison, red and roe deer, and gastrointestinal nematodes are often exchanged between animals, it is possible that other farm animals such as cows and sheep that may use the same pastures can be infected. Hence, histopathological changes observed in the walls of the abomasa and duodena of infected wildlife caused by a strong parasite presence may become an important health problem also for farm animals. METHODS: In the present study, a simple PCR test for the detection of A. sidemi infection in European bison based on DNA from third stage infective larvae (L3) has been optimized. RESULTS: The species-specific primers generated a 406 bp fragment, and A. sidemi DNA could be detected at concentrations of 0.1 pg/µl. The specificity of PCR was confirmed by the use of the genomic DNA of adult Ostertagia ostertagi, Haemonchus contortus, Cooperia oncophora as negative controls. CONCLUSION: It is possible to detect A. sidemi infection in European bison using DNA from L3. If this nematode infection is transmitted to cows this method may be effective to diagnose invasion in breeding animals in vivo.

Detection of antibodies to Neospora caninum in moose (Alces alces): the first report in Europe.

Neospora caninum Dubey, Carpenter, Speer, Topper et Uggla, 1988 is a protozoan parasite originally reported as a major cause of bovine abortions worldwide. It is documented that the parasite is widely spread among non-carnivorous cervids. The purpose of this study was to investigate the seroprevalence of N. caninum in moose (Alces alces Linnaeus). Blood samples collected in 2010 and 2012 in the northeastern Poland were tested for antibodies to N. caninum by agglutination test (NAT), a commercial competitive screening enzyme-linked immunosorbent assay (cELISA) and enzyme-linked immunoassay (ELISA). Sera that gave a positive result were further investigated by western blot (WB) analysis to verify the presence of antibodies. Antibodies to N. caninum were detected in one of seven moose. The antibody titer was confirmed by NAT (1 : 1 280), cELISA (I = 91%) and ELISA (OD = 0.736). The main immunodominant antigens detected by WB were 120, 70, 55, 35 and 16 kDa proteins. This is the first evidence of N. caninum seropositivity in moose living in a natural environment in Europe.

The first report of Toxoplasma gondii antibodies in free-living European bison (Bison bonasus bonasus Linnaeus).

Toxoplasma gondii Nicolle et Manceaux, 1908 is an apicomplexan parasite with a worldwide distribution. It is of great medical and veterinary importance because it may cause abortion or congenital disease in its intermediate hosts, including man. The European bison, the largest herbivorous animal in Europe, is a species that has been saved from extinction. Twenty-four of 95 examined sera of the European bison (Bison bonasus bonasus) from the Bialowieza Forest, Poland collected from 2008 to 2011 were found to be positive for the presence of T. gondii-specific IgG antibodies using a direct agglutination test, with the antibody titre in positive animals ranging from 40 to 18000. Statistically significant differences were observed only between years of sample collection. This is the first report on T. gondii in lowland European bison living in the natural environment.

First report of Trichinella pseudospiralis in Poland, in red foxes (Vulpes vulpes).

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585-1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96-95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.

Recognition of antigens of three different stages of the Trichinella spiralis by antibodies from pigs infected with T. spiralis.

Infective muscle larvae (ML), adults (Ad) and new born larvae (NBL) of Trichinella spiralis express many immunogenic proteins which can elicit a host protective response, and may be useful in the diagnosis of Trichinella infected humans and animals. The present study was carried out to identify T. spiralis antigens recognized by antibodies from pigs infected with T. spiralis. To that end, the crude extracts of ML, Ad, NBL and ML excretory-secretory (E-S) and Ad E-S proteins were analyzed by sodium dodecyl sulfate polycrystalline gel electrophoresis (SDS-PAGE). To identify antigens of T. spiralis that are recognized by host antibodies, crude extracts and E-S proteins were subjected to immunoblot with antisera derived from pigs experimentally infected with 200 or 20,000 T. spiralis ML. Searching for T. spiralis antigens with diagnostic potential, immunoblots showed that all T. spiralis antisera, regardless of the infective dose, recognized common proteins in each examined life stage with molecular weights around 20-27 kDa, 41 kDa and 197-105 kDa. Interestingly, all the common proteins were detected by T. spiralis sera throughout the infection, from 5 days post infection (dpi) to 60 dpi. These results extend our knowledge of specific antigenic components of T. spiralis. The finding of common components among all T. spiralis life stages may be useful in the preparation of parasite antigens for diagnostic use, as these antigens are relevant regardless of infection phase.

Molecular identification of Trichinella britovi in martens (Martes martes) and badgers (Meles meles); new host records in Poland.

Trichinella larvae were detected in a marten (Martes martes) and a badger (Meles meles) in Poland. The animals were found dead following car accidents. All examined animals derived from the Mazurian Lake district, north-east Poland, near the village Kosewo Górne where Trichinella infection were earlier confirmed in wildlife; red foxes and wild boars. The muscle samples were examined by artificial pepsin-HCl digestion method. The parasites were identified as Trichinella britovi by multiplex polymerase chain reaction method. Larvae were found in two out of three martens and one out of seven examined badgers. This is the first report of the identification of Trichinella britovi larvae from martens and badgers in Poland.

Multilocus genotyping of Giardia duodenalis isolates from red deer (Cervus elaphus) and roe deer (Capreolus capreolus) from Poland.

A total of 181 faecal samples were collected from wild cervids in two regions of Poland. Giardia cysts were detected in one faecal specimen from red deer and in two samples from roe deer. Fragments of the beta-giardin (bg) triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes were successfully amplified from the Giardia isolate obtained from red deer, whereas only amplicons of bg and gdh were obtained from Giardia isolates derived from two roe deer. The result of genotyping and phylogenetic analysis showed that the G. duodenalis isolate from red deer belonged to sub-assemblage AIII, which has never been identified in humans, whereas isolates from roe deer clustered within zoonotic sub-assemblage AI. Further studies are necessary to explain which Giardia assemblages and/or sub-assemblages occur in wild cervids in various regions of the world. Moreover, the impact of Giardia infection on the health of wild cervids should also be elucidated.

Comparative analysis of excretory-secretory antigens of Trichinella spiralis and Trichinella britovi muscle larvae by two-dimensional difference gel electrophoresis and immunoblotting.

BACKGROUND: Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi. RESULTS: According to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase. CONCLUSIONS: Both 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host.

Detection of specific antibodies anti-Neospora caninum in the fallow deer (Dama dama).

Sera from 335 farmed fallow deer (Dama dama) at the breeding station in Kosewo Górne in the Mazurian Lake District, North-East Poland, were investigated for the presence of antibodies against Neospora caninum. The distribution of age groups was as follow: >4 years - 154 animals; 2 years - 76 animals; 1 year - 105 animals. Ten sera with the optical density exceeding 0.159 absorbance units (i.e., cut-off value) in ELISA test were also analyzed by Western blot. Western blot analysis revealed seroreactivity against immunodominant N. caninum antigens of 37, 25, and 16kDa; however, in some sera additional bands were also visible. This is the first screening studies for antibodies against N. caninum in farmed fallow deer in Poland, in the region where neosporosis was confirmed in cattle and in farmed and free-ranging European red deer (Cervus elaphus).