Aleksandra Kornacka

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 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.

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

Abstract 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.

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.

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.

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.

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.

Seroprevalence of Toxoplasma gondii and Neospora caninum infection in sheep, goats, and fallow deer farmed on the same area1

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.