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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Studies on Neospora caninum DNA detection in the oocytes and embryos collected from infected cows.

Neosporosis is a major cause of abortion in cattle over the world. One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is embryo transfer (ET) from infected dams into uninfected recipients. Oocytes and embryos taken from seropositive cows were examined for the presence of Neospora caninum DNA. A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams. The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams.