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.

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).

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.

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.

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.

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.

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).

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.

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.

Trichinella Outbreaks on Pig Farms in Poland in 2012-2020.

Trichinella nematodes continue to circulate in various hosts both in the domestic and sylvatic cycles. In the majority of countries in Europe, wild boars have been noticed as a primary source of Trichinella spp. infections in humans. However, in some regions, the meat of pigs containing Trichinella spp. larvae can still be a cause of trichinellosis. Therefore, in the present study, we aimed to determine and present actual data on the occurrence of Trichinella spp. on pig farms (Sus scrofa f. domestica) in Poland. In this study, over 194 million pigs, slaughtered for commercial and personal purposes between 2012 and 2020, were tested with a digestion method according to the official rules for Trichinella control. Positive results were noticed in 172 pigs which gives an overall prevalence of 0.000088%. On seven farms, rats (Rattus norvegicus) infected with Trichinella spp. were also discovered. The species identification showed pigs were infected with Trichinella spiralis on 26 farms, and on four farms pigs with Trichinella britovi infections were found. Therefore, it is important to constantly monitor pigs for the presence of these parasites, especially in view of the growing interest in organic meat originated from ecological farms.

Results of Proficiency Testing for Trichinella in Poland, 2015-2019.

Trichinellosis is a zoonotic meat-borne disease caused by the nematodes of the genus Trichinella. Meat containing live Trichinella larvae is a source of infection. The examination of meat for Trichinella was introduced in 1869, but the digestion method for this did not appear in Poland until the late 1970s. Nowadays, the meat of all food animals susceptible to Trichinella spp. is examined in the frame of official post mortem control with the digestion method. The majority of laboratories in Poland meet the requirements of the ISO/IEC 17025 Standard (352 field laboratories). Laboratory personnel participate in quality control programs. This paper presents the results of proficiency tests (PTs) organized within 2015-2019 in Poland. Over this period, the laboratories examined 7580 samples (contamination levels: zero, one, three, and five larvae). Each laboratory was provided with a set of samples (one negative and three positive). Over 95% of the samples were considered correct in qualitative assessments, though the results of the quantitative evaluations were slightly lower, with 89% of samples being considered correct. Based on a sample evaluation, 88% of laboratories passed the PT comparison. A slight decrease was observed in the examination of samples spiked with five larvae, and great progress was achieved in samples containing three larvae. Low levels of sample contamination are sought after in laboratories but may make evaluations difficult. For this reason, we must consider increasing the number of larvae added to the samples in the next PTs.

Infection, genetics, and evolution of Trichinella: Historical insights and applications to molecular epidemiology.

Genetic variation in pathogen populations provides the means to answer questions in disease ecology and transmission, illuminating interactions between genetic traits, environmental exposures, and disease. Such studies elucidate the phylogeny, evolution, transmission and pathogenesis of viruses, bacteria and parasites. Here, we review how such studies have fostered understanding of the biology and epidemiology of zoonotic nematode parasites in the genus Trichinella spp., which impose considerable economic and health burdens by infecting wildlife, livestock, and people. To use such data to define ongoing chains of local transmission and source traceback, researchers first must understand the extent and distribution of genetic variation resident in regional parasite populations. Thus, genetic variability illuminates a population's past as well as its present. Here we review how such data have helped define population dynamics of Trichinella spp. in wild and domesticated hosts, creating opportunities to harness genetic variation in the quest to prevent, track, and contain future outbreaks.

The First Record of Echinococcus ortleppi (G5) Tapeworms in Grey Wolf (Canis lupus).

The aim of this study is to confirm the presence and molecular identification of Echinococcus tapeworms in wolves from south-eastern Poland. An investigation was carried out on the intestines of 13 wolves from south-eastern Poland. The small intestines were divided into three equal segments. Each segment was separately examined using the sedimentation and counting technique (SCT). The detected Echinococcus tapeworms were isolated and identified by PCRs and sequencing (nad1 and cox1 genes). Additionally, DNA isolated from the feces of wolves positive for Echinococcus tapeworms was examined with two diagnostic PCRs. The intestines of one wolf were positive for E. granulosus s.l. when assessed by SCT; the intestine was from a six-year-old male wolf killed in a communication accident. We detected 61 adult tapeworms: 42 in the anterior, 14 in the middle, and 5 in the posterior parts of the small intestine. The PCRs conducted for cox1 and nad1 produced specific products. A sequence comparison with the GenBank database showed similarity to the deposited E. ortleppi (G5) sequences. An analysis of the available phylogenetic sequences showed very little variation within the species of E. ortleppi (G5), and identity ranged from 99.10% to 100.00% in the case of cox1 and from 99.04% to 100.00% in the case of nad1. One of the two diagnostic PCRs used and performed on the feces of Echinococcus-positive animals showed product specific for E. granulosus. This study showed the presence of adult E. ortleppi tapeworms in wolves for the first time.

Alaria alata in Terms of Risks to Consumers' Health.

Alaria alata flukes are cosmopolitan parasites. In Europe, the definitive hosts are red foxes (Vulpes vulpes), wolves (Canis lupus), and raccoon dogs (Nyctereutes procyonoides), as well as animals that belong to the Felidae family. Intermediate hosts, such as snails and frogs, are the sources of infection for definitive hosts. The developmental stages of A. alata mesocercariae may occur in paratenic hosts, including many species of mammals, birds, and reptiles, as well as in wild boars (Sus scrofa), which are important from the zoonotic point of view. Because there are no regulations concerning the detection of A. alata in meat, this fluke is usually detected during official obligatory Trichinella spp. inspections. However, a method dedicated to A. alata detection was developed. The growing popularity of game and organic meat has led to an increased risk of food-associated parasitic infections, including alariosis, which is caused by the mesocercarial stage of A. alata. The aim of this article is to highlight the problem of A. alata as an emerging parasite, especially in the terms of the increasing market for game and organic meats that have been processed with traditional methods, often without proper heat treatment.

Molecular Confirmation of Massive Taenia pisiformis Cysticercosis in One Rabbit in Poland.

The aim of this study was to provide molecular characterization, together with phylogenetic analysis, of Taenia pisiformis cysts isolated from rabbit. On the basis of morphological features and molecular analysis, the cysticerci were identified as T.pisiformis metacestodes. PCR was performed with three different protocols to obtain partial sequences of 12S ribosomal RNA (12S rRNA), NADH dehydrogenase subunit 1 (nad1), and cytochrome oxidase subunit 1 (cox1) of Taenia spp. The products from the PCRs were sequenced. Interpretation of the sequencing results of the obtained amplicons, by comparing them with the GenBank database, proved that the causative agent, in this case, was T. pisiformis. The phylogenetic analysis of the received sequences identified a new haplotype. The received data can be used to supplement the species description. To our knowledge, this is the first molecular confirmation of T. pisiformis metacestodes infection in the rabbit, in Poland.

Genetic evidence substantiates transmission of Trichinella spiralis from one swine farm to another.

BACKGROUND: Trichinella spiralis ranks seventh in the risk posed by foodborne parasites. It causes most human cases of trichinellosis and is the most frequent cause of Trichinella outbreaks on pig farms and in wild boar, worldwide. Veterinary inspectors seek the source of outbreaks in hopes of limiting the spread. Established molecular tools are inadequate for distinguishing among potential T. spiralis infection sources because genetic variability in these zoonotic pathogens is limited in Europe. Microsatellite markers proved successful in tracing an outbreak of T. britovi, a related parasite harboring much more genetic variation. Here, we successfully employed microsatellite markers to determine the genetic structure of T. spiralis isolates from two pig outbreaks, discovering notable uniformity among parasites within each farm and discovering an epidemiological link between these two outbreaks. METHODS: The individual larvae from five isolates of T. spiralis from two pig farms and from ten wild boars were genotyped using nine microsatellite markers to examine their genetic structure. RESULTS: Notably uniform parasite populations constituted each farm outbreak, and the parasites from the first and second outbreaks resembled each other to a notable degree, indicating an epidemiological link between them. Wild boar harbored more genetically variable larval cohorts, distinguishing them from parasites isolated from domestic pigs. CONCLUSIONS: Microsatellite markers succeeded in distinguishing isolates of the highly homogeneous T. spiralis, aiding efforts to track transmission. Each outbreak was composed of a homogenous group of parasites, suggesting a point source of contamination.

Divergence at mitochondrial and ribosomal loci indicates the split between Asian and European populations of Trichinella spiralis occurred prior to swine domestication.

Available evidence suggests that Trichinella spiralis first originated in Asia and subsequently spread to the rest of the world. Notably limited genetic diversity in European T. spiralis isolates indicates that the parasite went through a dramatic genetic bottleneck at some point in its history. Did this genetic bottleneck result from the transport of a limited number of T. spiralis infected pigs from Asian centers of domestication, or was the parasite resident in Europe far earlier than the domestication of pigs there? In order to explore this hypothesis, we generated complete mitochondrial genomes and ribosomal DNAs from seventeen European T. spiralis isolates, six North American isolates and seven Asian isolates using next generation sequencing. A total of 13,858 base pairs of mitochondrial DNA and 7431 nucleotides of the nuclear ribosomal DNA sequence from each isolate were aligned and subjected to phylogenetic analysis using T. nelsoni as an outgroup. We confirmed that North American and European isolates were tightly clustered within a single "western clade" and all Chinese T. spiralis isolates were placed within a well-supported sister clade. These results indicate that European T. spiralis did not directly descend from extant Chinese parasite populations. Furthermore, the amount of nucleotide divergence between the two clades suggests that they diverged before pigs were domesticated. Over evolutionary time periods, Chinese and European T. spiralis were likely maintained as separate populations. The data presented here indicates the genetic bottleneck observed in European T. spiralis did not result from a small number of founders introduced with Chinese pigs in the recent past, but derives from an earlier bottleneck in host populations associated with the end of the last glacial maximum.

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.