Abstract:The aim of the present study was to estimate the genetic diversity of the cestode Echinococcus multilocularis Leuckart, 1863 in Poland based on sequence analysis of the mitochondrial genes of worms isolated from red foxes, Vulpes vulpes (Linnaeus). Overall, 83 adults of E. multilocularis from the same number of foxes in different parts of Poland were used for analysis. Sequences of the three mitochondrial genes, cytochrome b (cob), NADH dehydrogenase subunit 2 (nad2) and cytochrome c oxidase subunit 1 (cox1), were analysed. Seventy-four individual biological samples were successfully sequenced. Combined sequence analysis of these three genes exhibited fifteen Polish haplotypes (EmPL1-EmPL15). Most isolates (n = 29; 39%) were classified to the EmPL1 haplotype, which occurred mainly in the east, north and centre of Poland. Haplotype EmPL4 (n = 14; 19%) and other haplotypes appeared predominantly in the south and west area. Fourteen haplotypes were grouped in the European clade. One Polish haplotype (EmPL9) (n = 7, 10%) was assigned to the Asian clade with haplotypes from Japan and Kazakhstan. This haplotype was found only in northeast Poland and this is the westernmost report of haplotype of E. multilocularis belonging to the Asian clade in Europe. The investigation demonstrated that populations of E. multilocularis in Poland (and probably also in eastern Europe) included not only different European haplotypes but also those of the Asian origin.

Faecal samples from 162 wild animals were collected from 32 distinct sites of Łęczyńsko-Włodawskie Lakeland (eastern Poland). The presence of Giardia duodenalis (Stiles, 1902) was assessed by a Direct Fluorescence Assay (DFA) and by Polymerase Chain Reaction (PCR) and sequencing of a fragment of the beta-giardin gene. DFA showed the presence of cysts of G. duodenalis in 12 of 162 faecal samples (7%), namely in four wild boars (15%), four foxes (19%), two roe deer (4%), and two wolves (29%). PCR identified 34 of the 162 (21%) samples as positive, including 11 wild boars (41%), five red deer (18%), 11 roe deer (23%), four moose (17%), two wolves (29%) and a single sample from the European badger. Thus, PCR detected a significantly higher number of infection than DFA (P = 0.0005). However, 14 of 34 PCR products could not be sequenced because of their insufficient amount; the low number of cysts, poor conservation of the faeces or presence of PCR inhibitors may have contributed to weak DNA amplification. Sequence analysis of the remaining 20 products showed the presence of assemblage B in wild boars, red deer and roe deer, whereas samples from wolves were identified as assemblage D. This is the first detection of assemblage B in wild boars and deer. As assemblage B has zoonotic potential, wild animals from eastern Poland may act as reservoirs of cysts of G. duodenalis infectious for humans.

Protozoa of the genus Sarcocystis (phylum Apicomplexa, family Sarcocystidae) is one of the most common parasites affecting animals. Interspecies diagnostic of Sarcocystis genus was based on electron microscopy for many years. Because of absence of visible differences between species with reachable magnifications, light microscopy is useless. In many cases serological diagnostic method have lack of sensitivity. A variety of molecular methods have been developed and used to detect and identify Sarcocystis spp. and to assess the genetic diversity among this protozoan from different population/hosts. Nowadays, molecular diagnostic is the common, time/cost effective method used all over the world to interspecies differentiation.

Toxoplasma gondii (Nicolle et Manceaux, 1908) is an obligatory intracellular protozoan parasite prevalent in animals and humans worldwide having medical and veterinary importance on account of causing abortion or congenital disease in intermediate hosts, including man. Since T. gondii has already been identified in the milk of goats, Capra aegagrus hircus (Linnaeus), the possibility of acquiring infection by ingesting unpasteurised goat milk should be taken into consideration. Thus, the aim of the present study was to determine the presence of T. gondii DNA in goat milk. First, 73 goats (females) from 36 farms located in Poland were examined serologically by direct agglutination test (DAT) to estimate the T. gondii serological status. Milk samples from 60 selected lactating females were examined for the presence of T. gondii DNA by Real time PCR and nested PCR (B1 gene). To estimate the clonal type of detected T. gondii, multiplex PCR was performed using 6 markers. In DAT, positive results were found in 70% of 73 goats. Among examined 60 milk samples, 65% were positive in Real time PCR and 43% in nested PCR. It is noteworthy that 11 samples positive in PCR were collected from seronegative goats. The multilocus PCR analysis mostly revealed the occurrence of genotype III, which is relatively rare in Europe. The recorded high prevalence of anti-Toxoplasma antibodies in tested goats (70%), associated with a high prevalence of T. gondii DNA in goat milk samples (65%), indicates a potential risk of the parasite transmission through goat milk ingestion.

Faecal samples from 297 farm animals were collected from 18 households in distinct sites of the Łęczyńsko-Włodawskie Lake District of eastern Poland. They included samples from 86 cattle (Bos taurus), 84 pigs (Sus scrofa f. domestica), 81 sheep (Ovis aries), 10 horses (Equus caballus), and 36 dogs (Canis lupus familiaris). The samples were examined for the presence of Giardia intestinalis by the Direct Fluorescence Assay (DFA) and semi-nested PCR. All amplicons were sequenced on both strands. By DFA, cysts of Giardia spp. were detected in 66 of 297 faecal samples (22.2%). Positive specimens for Giardia spp. were derived from 29.8% of examined pigs, 21.0% of sheep, 18.6% of cattle, 10% of horses, and 19.4% of dogs. Based on the detection of the β-giardin gene by PCR, 39 (13.1%) of the 297 examined samples were recognized as positive. Detection of the presence of Giardia cysts by DFA test was overall significantly higher compared to PCR (p=0.0045). By PCR, Giardia was found in 28.1% of sheep, 11.6% of cattle, 10% of horses, 9.5% of pigs and 5.6% of dogs. Partial β-giardin gene sequences were obtained for 73.7% of the PCR positive samples. From sequenced samples derived from the studied animals, Giardia were identified as assemblage A (8 samples), B (1 sample) and E (18 samples). As assemblages A and B may be zoonotic, the farm animals living in eastern Poland could be regarded as a potential source of Giardia infection for humans.

The purpose of this study was to assess the possible influence of beavers on the contamination of lake water with zoonotic parasites Giardia duodenalis and Cryptosporidium spp., with respect to the risk to human health. A total of 79 water samples were taken around the habitats of beavers from 14 localities situated in the recreational Masurian Lake District (north-eastern Poland). Water was sampled in the spring and autumn seasons, at different distances from beavers' lodges (0-2, 10, 30, and 50 m). The samples were examined for the presence of (oo)cysts of zoonotic protozoa Giardia duodenalis and Cryptosporidium spp. by direct fluorescence assay (DFA) and by nested and real time PCR. By DFA, the presence of Giardia cysts was found in 36 samples (45.6%) and the presence of Cryptosporidium oocysts in 26 samples (32.9%). Numbers of Giardia cysts, Cryptosporidium oocysts, and summarised (oo)cysts of both parasites showed a significant variation depending on locality. The numbers of Giardia cysts significantly decreased with the distance from beavers' lodges while the numbers of Cryptosporidium oocysts did not show such dependence. The amount of Giardia cysts in samples collected in spring was approximately 3 times higher than in autumn. Conversely, a larger number of Cryptosporidium oocysts were detected in samples collected in autumn than in spring. By PCR, Giardia DNA was found in 38 samples (48.1%) whereas DNA of Cryptosporidium was found in only 7 samples (8.9%). Eleven Giardia isolates were subjected to phylogenetic analysis by restriction fragment length polymorphism PCR or sequencing which evidenced their belonging to zoonotic assemblages: A (3 isolates) and B (8 isolates). In conclusion, water in the vicinity of beavers' lodges in the tested region was markedly contaminated with (oo)cysts of Giardia duodenalis and Cryptosporidium spp., which confirms the potential role of beavers as a reservoir of these parasites and indicates a need for implementation of appropriate preventive measures to protect tourists' health.

Giardia intestinalis is a widespread parasitic protozoa which has great significance as a public health threat. Molecular diagnostics of stool sample can be unreliable because of the presence of inhibitors of enzymatic reactions. The aim of this study was to determine the effectiveness of selected pre-treatment methods of fecal samples for further PCR-based diagnostics of G. intestinalis, and the effect of each component of pre-treatment solutions on PCR reactions. Seven stool concentration techniques were compared. The results showed that the most efficient concentration method for stool sample preparation for detection of G. intestinalis by PCR is centrifugal flotation with Percoll (with saturated NaNO 3 as the flotation solution). This method is relatively inexpensive, less labor-intensive, and suitable for epidemiological monitoring and clinical investigations.

Expression of CD4, CD8, and CD25 surface markers on T lymphocytes and levels of IFNγ, IL-10, and TNF-α in colostrum and milk were determined in sows vaccinated against Trueperella pyogenes in the final stage of pregnancy. The autovaccine, prepared from Trueperella pyogenes, administered twice to pregnant sows six and three weeks before the anticipated delivery significantly increased the percentages of TCD4+, TCD8+, and TCD25+ as well as levels of IFNγ, TNF-α, and IL-10 in colostrum and milk. The enhanced immune potential of colostrum effectively protected the piglets against T. pyogenes infections during weaning and thus reduced the economic losses on the particular farm concerned, where T. pyogenes infections occur endemically. Knowledge of the profile of cellular and humoral immune response in colostrum and milk of vaccinated sows will enable the design of a T. pyogenes infection prophylactic programme for suckling pigs and weaners, which are most susceptible to infections.