Occurrence of Alaria alata in wild boars (Sus scrofa) in Poland and detection of genetic variability between isolates.

Alaria alata is a trematode included among several emerging zoonotic parasites. The mesocercarial larval stage of A. alata named Distomum musculorum suis (DMS) may potentially be infective for humans. In the past, DMS was often observed in wild boar meat during the official Trichinella inspection by artificial digestion before a more specific and effective detection method, the A. alata mesocercariae migration technique (AMT), was introduced. In the present study, the AMT method was used to screen 3589 tissue samples collected from wild boars hunted in Poland during the 2015-2019 period. The survey mainly focused on the southern part of Poland with the majority of samples coming from Malopolskie, Swietokrzyskie, and Dolnoslaskie provinces; samples from ten additional provinces were also included. The total prevalence was 4.2% with mean abundance of 4.7 DMS. Occurrence was dependent upon environmental conditions (i.e., wetland habitats and water reservoirs) rather than on sex of the host or season in which they were hunted. The recovered trematodes were identified as Alaria spp. according to their morphological features. Molecular analysis of 18S rDNA and COI genes confirmed the species identification to be A. alata and documented genetic variability among the isolates.

Species identification of Trichinella originated from various host and different geographical location by MALDI-TOF.

The foodborne zoonotic nematode Trichinella spp. can cause human trichinellosis when raw or undercooked contaminated meat is ingested. To date, twelve Trichinella species/genotypes have been described. According to EU regulation any Trichinella larvae detected during mandatory routine examinations need to be identified at a species level by a competent laboratory. Currently, Trichinella species identification is performed using molecular biology tools such as multiplex PCR, PCR-sequencing or PCR-RFLP. These techniques require high level of skills for good interpretation of the results. Due to its rapidness and ease of use a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol was previously developed for the identification of Trichinella species. Using this method, spectra from different Trichinella species and strains were acquired allowing to generate new Main Spectra (MSP). Finally a new MSP database from Trichinella spp. Samples of different countries (France, Germany and Poland), including field samples, was generated. Comparing the different main spectra, Trichinella worms were identified at the species level and differences in the genetic diversities within the different species are discussed. In conclusion, using the previously described method on field samples is a reliable, rapid, easy-to-use and cheap tool for Trichinella species identification. The new Trichinella database could be incremented with new samples. It constitutes a tool, which could be used as an alternative method to replace the actual molecular methods for Trichinella species identification.

Detection and Molecular Characteristics of Toxoplasma gondii DNA in Retail Raw Meat Products in Poland.

Raw and undercooked meat are regarded as important sources of Toxoplasma gondii infection of people in Europe; however, data concerning this issue in Poland are still insufficient. The aim of this study was to determine the prevalence of T. gondii DNA isolated from raw meat products retailed in Poland. The molecular characteristics of detected DNA were also performed. Samples of cured bacon, raw or smoked sausages, ham, and minced meat were examined for the presence of T. gondii DNA. Samples were digested by pepsin solution, followed by the DNA isolation. Nested and real-time polymerase chain reaction (PCR) was performed based on the amplification of 35-fold-repetitive B1 fragment gene of T. gondii. For selected B1-positive samples, multiplex PCR was performed using SAG1, SAG2 (5'-SAG2 and 3'-SAG2), altSAG2, SAG3, GRA6, BTUB, C29-2, and L358 genetic markers. Amplicons were sequenced and analyzed with NCBI database. Among 3223 examined samples, 175 (5.4%) were PCR positive. The highest percentages of positive results were found for samples originating from south-east regions of Poland-Podkarpackie (17.9%), Malopolskie (12.6%), and Lubelskie (10.8%) (p < 0.001). The percentages of positive results for particular types of meat products-sausages, smoked meat products, ham, and minced meat-ranged from 4.5% to 5.8% and the differences between them were not significant (p > 0.05). Sequence analysis of selected B1-positive samples demonstrated mostly the alleles of clonal type III (49.0%), and less-type II (17.3%), and type I (10.2%) based on nine used genetic markers. The combinations of types I/II or II/III or I/III alleles at different loci were also found in 23.5% of cases. Detection of T. gondii DNA in raw meat products may indicate the potential health threat for consumers in Poland; however, for complete risk assessment of T. gondii infection, the additional studies, including detection of live parasite, are needed.

Negative effect of flocculant (cationic acrylamide) on detectability of the nematode eggs in sewage sludge.

The use of sewage sludge in agriculture brings the risk of microbiological and parasitological contamination of soil, ground and surface water, as well as cultivated plants. Therefore prior the application to the soil, sewage sludge must be examined, among others, for the presence of live eggs of intestinal parasites. However, the efficiency of commonly used for this purpose parasitological methods is not satisfactory. This is probably due to the presence of flocculants in the sediments used in the dehydration process. The objective of the study was analysis of the effect of flocculant (cationic acrylamide) on the possibilities of isolation of parasite eggs from dehydrated sewage sludge. For this purpose 10 samples of sewage sludge were prepared: 5 containing flocculant and 5 without flocculant. Samples were tested by flotation method according to Quinn. From sewage sludge free of flocculant, 67.8 eggs were isolated, on average, whereas from sludge containing flocculant - only 2.8 eggs. The experiments confirmed that the isolation of eggs from sewage sludge containing flocculant (cationic acrylamide) is much more difficult than from sludge free from this substance and therefore the simple parasitological methods should not be used to examine the dehydrated sewage sludge.

First case of Trichinella nativa infection in wild boar in Central Europe-molecular characterization of the parasite.

The examination of wild boars gained in Poland shows for the first time occurrence of Trichinella nativa, freeze-resistant species of Trichinella in this host from the central Europe region. This finding is not only one of several cases of T. nativa invasion in wild boars all over the world but also one of the very few cases of T. nativa detected so far beyond the known boundary of occurrence of this species. The molecular characterization of discovered larvae based on analysis of partial genes: 5s rDNA-ISR and CO1 confirm the findings. Moreover, the analyzed DNA sequences of both genes present new haplotypes of T. nativa in comparison to that described previously.

Seroprevalence of Toxoplasma gondii infection in goats from the south-west region of Poland and the detection of T. gondii DNA in goat milk.

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.

Genetic diversity of Echinococcus multilocularis in red foxes in Poland: the first report of a haplotype of probable Asian origin.

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.

Characterisation of a new, highly effective method for detecting nematode eggs (Ascaris spp., Toxocara spp., Trichuris spp.) in sewage sludge containing flocculants.

Because traditional methods used for sewage sludge parasitological examinations have low sensitivity, a new, highly effective method (own method - OM) was devised. The principle of this method is to eliminate the flocculent effect on the structure of sewage sludge by mechanically damaging floccules in the presence of surfactants and to increase the effectiveness of egg isolation processes in large volumes of liquids. The objective of this study was to estimate the effectiveness of the OM in detecting nematode eggs in sewage sludge samples containing flocculants. In the first stage, the effectiveness of the OM was compared to 4 other methods routinely used in parasitological examinations of dehydrated sewage sludge. Next, method standardisation was performed using sewage sludge samples supplemented with eggs from 3 parasite species (Ascaris suum, Toxocara canis and Trichuris vulpis). The study demonstrated that OM efficiency was 6-65 times greater than other methods, depending on the method and type of detected eggs. Limit of detection (LOD) calculations for the OM were performed on samples supplemented with a known number of parasite eggs resulting in 10, 5 and 3 eggs/50 g of sample for A. suum, T. vulpis and T. canis eggs, respectively. The limits of quantification (LOQ) of the OM were established as 200 eggs/50 g of sample for A. suum and T. vulpis eggs and 50 eggs/50 g of sample for T. canis eggs. The rectilinear regression functions, which determined the relationship between the number of eggs detected in OM measurements and the number of eggs contained in the samples, were characterised by high and statistically significant coefficients of determination (r2). The slopes of the trend lines were 0.3188, 0.3821 and 0.3276, and the intercepts were -11.223, -9.0261 and -23.15 for A. suum, T. canis and T. vulpis eggs, respectively. Method sensitivity, calculated as the slope coefficient of the regression function and expressed as a percentage, ranged from 32% to 38% depending on egg type. The study confirmed that the OM may be applied to quantify parasite eggs in dehydrated sewage sludge containing polyelectrolytes.

The prevalence of Echinococcus multilocularis in red foxes in Poland--current results (2009-2013).

The aim of the study was to determine the prevalence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in Poland. Overall, 1,546 intestinal samples from 15 of the 16 provinces in Poland were examined by the sedimentation and counting technique (SCT). The mean prevalence of E. multilocularis in Poland was 16.5% and was found in 14 of the 15 examined provinces. The mean intensity of infection was 2,807 tapeworms per intestine. Distinct differences in prevalence were observed between regions. In some provinces of eastern and southern Poland, the level of prevalence was 50.0% (Warminsko-Mazurskie), 47.2% (Podkarpackie), 30.4% (Podlaskie) and 28.6% (Malopolskie), while in other provinces (west and south-west), only a few percent was found: 2.0% (Dolnoslaskie), 2.5% (Wielkopolskie) and 0.0% (in Opolskie). The border between areas with higher and lower prevalence seems to coincide with a north-south line running through the middle of Poland, with prevalence from 17.5 to 50.0% in the eastern half and from 0.0 to 11.8% in the western half. The dynamic situation observed in the prevalence of this tapeworm indicated the necessity of continuing to monitor the situation concerning E. multilocularis in red foxes in Poland.

Scheme of Effective Epidemiological Investigations in Trichinella Outbreaks on Pig Farms.

Trichinellosis is a parasitic, zoonotic disease caused by larvae of the genus Trichinella. Infection occurs via the consumption of raw or undercooked meat containing this parasite. Symptoms of the disease manifest as intestinal disorders, followed by facial swelling, fever, muscle pain and other symptoms, eventually leading to neurological and cardiac complications and even death. In Europe, trichinellosis is most often associated with the consumption of meat from wild boars, pigs and horses. In recent years, wild boars that are hunted illegally and not tested for Trichinella spp. have been the most common cause of trichinellosis in humans; however, there have also been cases where infected pigs have been the source of infection. When trichinellosis is suspected in humans, epidemiological measures are taken to identify the source. Similarly, an epidemiological investigation should be initiated whenever Trichinella spp. has been detected in pigs. However, commonly used actions do not provide sufficient data to determine the source of infection for pigs and to prevent further transmission. Therefore, in this article, we propose a scheme for effective epidemiological investigations into Trichinella outbreaks on pig farms that can help trace the transmission mechanisms of the parasite and that takes into account currently available testing tools. The proposed pathway can be easily adopted for epidemiological investigations in routine veterinary inspection work.

Occurrence of Eucoleus aerophilus in wild and domestic animals: a systematic review and meta-analysis.

BACKGROUND: Eucoleus aerophilus (syn. Capillaria aerophila) is a nematode with a worldwide geographical distribution. It causes a disease called lung capillariosis by affecting the respiratory tract of wild and domestic animals, and has also occasionally been described in humans. Despite steady increases in knowledge of the morphology of this neglected parasite, many aspects are still poorly understood. Epidemiological data regarding, for example, geographic distribution, range of hosts, clinical relevance and the actual zoonotic potential of this nematode are scarce and incomplete. METHODS: This article is a systematic review based on the screening of three databases (PubMed, Web of Science and Science Direct) to identify eligible studies published from 1973 to the end of 2022. RESULTS: From a total of 606 studies describing the occurrence of E. aerophilus, 141 articles from 38 countries worldwide were included in this meta-analysis, all of which presented results obtained mainly with flotation and necropsy. Due to the occurrence of E. aerophilus in many different species and different matrices (lungs and faeces), we decided to conduct the meta-analysis separately for each species with a given matrix. This systematic review confirmed the status of the Red fox as the main reservoir and main transmitter of E. aerophilus (average prevalence of 43% in faeces and 49% in lungs) and provided evidence of a higher prevalence of E. aerophilus in wild animals in comparison to domestic animals, such as dogs (3% in faeces) and cats (2% in faeces and 8% in lungs). Previous studies have investigated many host-related factors (age, sex, environmental/living conditions) in relation to the prevalence of E. aerophilus, but they show wide variations and no simple relationship has been demonstrates. Furthermore, mixed infections with other pulmonary nematodes, such as Crenosoma vulpis and/or Angiostrongylus vasorum, are reported very frequently, which greatly complicates the diagnosis. CONCLUSIONS: This systematic review focused on identifying data gaps and promoting future research directions in this area. To the best of our knowledge, this is the first systematic review that evaluates and summarizes existing knowledge on the occurrence and prevalence of E. aerophilus in wild and domestic animals originating from different geographical locations worldwide.

Comparison of the effectiveness of various parasitological methods in detecting nematode eggs in different types of soil.

INTRODUCTION AND OBJECTIVE: Natural fertilizers, sewage sludge, digestates, as well as organic fertilizers produced on their basis, can become a source of parasitological contamination of cultivated land. High concentration of invasive forms of parasites in the soil may pose a threat to human and animal health. Therefore, it is necessary to control the hygienic condition of fertilizers and fertilized soils with particular emphasis on parasites. The aim of the study was to compare the effectiveness of methods commonly used for parasitological examination of soil with own methods which were used to develop the standards. MATERIAL AND METHODS: The study was carried out using samples of sandy soil (SS), horticultural mix soil (HS) and peat-based substrate (PS). Each sample was spiked with 100 dyed Ascaris suum eggs and examined with the use of 6 methods: Vasilkova, Dada, Quinn, and 3 methods according to the Polish Standards (PN-19000, PN- 19005, PN-19006). For each variant, 8 repetitions were made. RESULTS: The largest number of A. suum eggs were found with PN-19006 (mean number of detected eggs was 21.25, 46.50, 23.00 for HS, SS, PS, respectively. Slightly lower results were obtained using PN-19005 - the mean number eggs was 21.25, 36.00, 16.75, respectively. On the other hand, the mean number of A. suum eggs found with the Dada method was about 2-3 times lower than with the PN-19006 - 15.75, 22.50, 6.50 for HS, SS, PS soil, respectively. Other methods were much less effective. CONCLUSIONS: PN-19006 method turned out to be the most effective in detecting A. suum eggs. This method can be used for parasitological examination of soils and can be the basis for developing a system of methods dedicated to testing different types of soils for the presence of nematode eggs.

The Occurrence of Freshwater Fish-Borne Zoonotic Helminths in Italy and Neighbouring Countries: A Systematic Review.

In recent years, the consumption of fish products has surged in European countries, being an essential part of a healthy diet. Despite representing a small part of EU production, freshwater fisheries hold considerable significance for lake-dwelling populations and tourists seeking traditional dishes. This increased fish consumption has brought to light potential health risks associated with fish-borne zoonotic helminths (FBZHs), now acknowledged as global food-borne parasites. Fish-borne zoonotic helminths belong to various taxonomic groups, including nematodes (Anisakidae), trematodes (Opisthorchiidae and Heterophyidae), and cestodes (Diphyllobothriidae). More than 50 species of FBZH are known to cause human infections, derived from eating raw or undercooked aquatic foods containing viable parasites. Despite increased attention, FBZHs remain relatively neglected compared to other food-borne pathogens due to factors like chronic disease progression and under-diagnosis. This systematic review concentrates on the prevalence of six freshwater FBZHs (Clinostomum complanatum, Contracaecum rudolphii, Dibothriocephalus latus, Eustrongylides excisus, Opisthorchis felineus, and Pseudamphistomum truncatum) in Italy and neighbouring countries. The study explores the expansion of these parasites, analysing their biological and epidemiological aspects, and the factors that influence their proliferation, such as the increased cormorant population and the lake eutrophication phenomena. In summary, this research highlights the necessity for further research, the development of spatial databases, and the establishment of a unified European policy to effectively manage these multifaceted health concerns. It strongly advocates adopting a One-Health approach to address the growing incidence of parasitic zoonoses within the context of food safety in EU countries.

Echinococcus multilocularis genetic diversity based on isolates from pigs confirmed the characteristic haplotype distribution and the presence of the Asian-like haplotype in Central Europe.

Introduction: The aim of the study was to determine the genetic diversity of Echinococcus multilocularis in pigs in highly endemic areas in Poland, as well as to attempt to confirm the occurrence and geographical distribution of haplotypes characteristic for these areas, which were previously described on the basis of examination of adult tapeworms isolated from foxes. Material and Methods: Twenty samples of E. multilocularis larval forms were obtained from pigs' livers in four provinces of Poland. Genetic analyses were conducted on sequences of two mitochondrial genes: cox1 and nad2. Results: Seven haplotypes were found for the cox1 gene (OQ874673-OQ874679) and four haplotypes for nad2 (OQ884981-OQ884984). They corresponded to the haplotypes described earlier in foxes in Poland (some of them differing only in one nucleotide). The analysis showed the presence of the Asian-like haplotype in both the cox1 and nad2 genes. The remaining haplotypes were grouped in the European clade. The geographical distribution of haplotypes identified in the pig samples was noticed to bear a similarity to the distribution of haplotypes previously isolated from foxes in the same regions. Conclusion: The characteristic geographical distribution of E. multilocularis haplotypes in Central Europe (including the presence of the Asian-like haplotype) previously described in the population of definitive hosts (foxes) has now been confirmed by the analysis of samples from non-specific intermediate hosts (pigs).

Gelatin medium for preserving of Trichinella spp. for quality control in laboratories - estimation of larvae viability.

INTRODUCTION AND OBJECTIVE: Official food control laboratories ensure food safety using reliable, validated methods. Council Regulations (EC) No. 853/2004, 854/2004 and 882/2004 of the European Parliament established hygiene rules the production of food of animal origin, together with requirements for official controls. This leads to detailed requirements for Trichinella control set out in Commission Implementing Regulation (EU) 2015/1375 of 10 August 2015. These regulations require the laboratory to participate in proficiency testing (PT) to confirm their competence and improve the quality of testing, and require the PT Organizer to use methods for the preparation and preservation of parasite larvae in order to evaluate and improve detection. Traditional methods of preparing such larvae expose them to rapid degradation, making it necessary to simultaneously isolate the larvae and place them in meatballs to ensure quality. MATERIAL AND METHODS: We developed a technique for preserving of Trichinella spp. for quality control such as PT sample preparation. The procedure protects larvae against toxic oxygen activity and bacterial destruction via a gelatin barrier. To estimate the viability of larvae preserved by this method, gelatin capsules with 10 larvae of T. spiralis in each were stored (4-8 °C) during 45 days of an experiment. Samples were tested at 2 day intervals (3 samples each day of testing). RESULTS: In total, 75 samples were tested. Larvae remained alive up to 3 weeks. The number of living larvae diminished after 27 days through day 43, after which no living larvae were observed. CONCLUSIONS: The gelatin medium procedure facilitated easy, high-throughput sample preparation and supported 100% recovery for 3 weeks. The method allows fast, efficient and accurate PT sample preparation.

Molecular confirmation of the systemic toxoplasmosis in cat.

INTRODUCTION AND OBJECTIVE: Systemic toxoplasmosis with tissue-spread parasites occurring in intermediate hosts may also occur in immunocompromised cats (e.g., infected with FLV or FIV). To the best of our knowledge, no reports have been published on the detection and genotyping of T. gondii DNA in cats with extraintestinal toxoplasmosis in Poland. The article describes the case of the sudden death of 3 out of 4 cats in a cattery, and the detection and molecular characterization of T. gondii DNA detected in the tissues of one of the dead cats. MATERIAL AND METHODS: Samples of brain, lungs, heart, and liver of the cat that died suddenly were examined for the presence of T. gondii DNA (B1 gene) by nested PCR and real-time PCR. DNA positive samples were also genotyped at 12 genetic markers using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) and multilocus sequence typing (MLST). RESULTS: A total of 9 out of the 20 DNA samples were successfully amplified with nested and/or Real-time PCR. DNA from 3 out of 5 types of tested samples were genotyped (brain, heart and muscle). Mn-PCR-RFLP and MLST results revealed type II (and II/III at SAG1) alleles at almost all loci, except a clonal type I allele at the APICO locus. This profile corresponds to the ToxoDB#3 genotype, commonly identified amongst cats in Central Europe. CONCLUSIONS: To the best of our knowledge, this is the first study describing the genetic characteristics of T. gondii population determined in a cat in Poland. These data confirm the importance of this host as a reservoir for this pathogen, and demonstrate the genotypic variation of this parasite. Veterinarians should take into account that cats may develop disseminated toxoplasmosis, and that it is a systemic disease which may lead to the death of the cat, and to transmission of the pathogen to other domestic animals and to humans.

Trich-tracker - a practical tool to trace Trichinella spiralis transmission based on rapid, cost-effective sampling of genome-wide genetic variation.

Molecular epidemiology using traditional sequencing has been notoriously difficult in inbred parasites due to a lack of genetic variation available for discriminating among parasites. Next generation sequencing techniques offer a solution to this problem by increasing the number of loci that can be sequenced. Here, we introduce Trich-tracker, a tool that makes efficient use of diagnostic variation distributed throughout the genome of Trichinella spiralis to more rapidly, and conclusively, resolve connections and distinctions among focal outbreaks of T. spiralis. In particular, we rapidly characterised genetic variation among a sample of parasites from Polish farms and wildlife, sampling genomic variation using double digest restriction site-associated DNA sequencing (ddRADseq). Approximately 400,000 bases of sequence were generated from each sample and shown to be distributed across the genome with single nucleotide polymorphisms occurring at a frequency of approximately one base in 10,000. Both phylogenetic and Bayesian clustering analyses indicated that ddRADseq genotypes formed distinct clusters for specific outbreaks and were quite distinct from wild boar samples. Two of the investigated outbreaks were more similar to each other than to other outbreak samples, suggesting a link between these outbreaks. Hence, the Trich-tracker procedure identified informative genomic variation which afforded unprecedented epidemiological resolution. Trich-tracker is very flexible tool, quickly and inexpensively mining genomes of even highly inbred populations of T. spiralis to support outbreak investigations. The simplicity of the entire procedure, and time and cost effectiveness of Trich-tracker support its practical application in ongoing Trichinella outbreaks. The discriminating power of this tool is tunable and scalable, allowing application in a variety of epidemiological contexts, and is easily adapted to other parasite systems.

Grass Snakes (Natrix natrix) as a Reservoir of Alaria alata and Other Parasites.

The aim of the study was to investigate the occurrence of Alaria alata (Goeze, 1782) in fifty-one grass snakes (Natrix natrix) collected in Gostyninsko-Wloclawski Landscape Park. Each snake was tested for the presence of A. alata mesocercariae using the AMT and MSM methods. 18S ribosomal RNA (18S rRNA), cytochrome C oxidase subunit I (COI) and 28S ribosomal RNA (28S rRNA) genes were amplified by PCR and sequenced for the purpose of species identification. Fifty grass snakes were infected with helminths. The molecular characterization of trematodes allowed us to identify A. alata in 30 snakes (58.8%), Conodiplostomum spathula (Dubois, 1937) in 16 snakes (31.3%), Strigea falconis (Szidat, 1928) in 12 snakes (23.5%), and Neodiplostomum attenuatum (Linstow, 1906) in 2 snakes (3.9%), while, in 4 snakes (7.8%), the trematodes species could not be identified. Based on the analysis of 18S and COI sequences, Crenosoma vulpis (Dujardin, 1845) was identified in four snakes (7.8%), while nematodes collected from three snakes remained unidentified. The tapeworm sample was identified as Ophiotaenia. The obtained results indicate that grass snakes are an excellent vector of A. alata and may be a potential source of infection for mammals, e.g., wild boars and foxes, which results in an increased risk of alariosis for consumers of raw or undercooked game meat.

Validation of the Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples Based on Proficiency Tests Results.

Trichinellosis is a zoonotic disease caused by the nematodes of the genus Trichinella. Infection takes place through the consumption of infected meat containing live larvae. The only way to prevent the disease is to break its epizootic chain. To ensure effective control of Trichinella spp., a range of preventive and control measures have been undertaken. These efforts have been focused on controlling Trichinella in domestic pigs, the main source of the disease. Artificial digestion is also the reference point for other methods for Trichinella risk control. Descriptive data validation of the digestion assay was presented in 1998 based on results published by scientific laboratories. Herein, we supplement those data by characterizing the method's performance in inter-laboratory comparisons. The source of data was the results of Proficiency Testing conducted in 2015-2019. Samples were contaminated by 0, 1, 3, and 5 larvae. In total, 7580 samples were examined by the laboratories. Based on Proficiency Testing results, the main parameters characterizing the method performance in field conditions were established as follows: specificity, 97.3%; sensitivity, 86.5%; accuracy, 89.2%; uncertainty, 0.3; limit of detection (LOD), 1 larva; and limit of quantification (LOQ), 3 larvae.

Validation Parameters of the Magnetic Stirrer Method for Pooled Sample Digestion for Trichinella spp. in Horse Meat Based on Proficiency Tests Results.

Meat of horses may be infested with nematodes of the genus Trichinella spp. and can cause serious disease in humans. Rules for the carcasses sampling of species susceptible to Trichinella spp. infection and examination are laid down in Commission Regulation 1375/2015, where the magnetic stirrer method for pooled-sample digestion is recommended (Commission Regulation 1478/2020). All personnel involved in the examination should be properly trained and participate in quality control programs. Proficiency tests (PTs) play a key role in the quality verification process. This paper presents the results of PTs organized for 68 Polish laboratories in 2014-2019. Results were assessed qualitatively at three levels of sample contamination (0, 3, 5 larvae) and quantitatively at one level (5 larvae). The laboratories have achieved the average correct qualitative results 100%, 96.2% and 96.8% for the samples contaminated with 0, 3 and 5 larvae, respectively. In the quantitative evaluation, an average 94.1% of the reported results were correct. The data from PTs enabled us to define, for the first time, validation parameters of the digestion method for the horse meat matrix in a large-scale experiment including: specificity (100%), sensitivity (95.6%), accuracy (97.1%), the limit of detection (LOD) (1.14 ≈ 1) and the limit of quantification (LOQ) (3.42 ≈ 3).