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Meat of horses may be infested with nematodes of the genus

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 Małopolskie, Świętokrzyskie, and Dolnoślaskie 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.

Background Many parasites (protozoa and helminths) can be transmitted through food and lead to infections with high morbidity, as well as disease outbreaks. Although the importance of foodborne parasites (FBP) is recognised by many sectors of the food industry, standardized analytical methods and validation procedures for testing food for FBP are lacking. Scope and approach:Current methods for detection of FBP, and their validation, are critically reviewed, focusing on priority FBP in Europe: the helminths Echinococcus multilocularis, Echinococcus granulosus, Taenia saginata, Trichinella spp., and Anisakidae, and the protozoa Toxoplasma gondii, Cryptosporidium spp., and Giardia duodenalis. Key findings and conclusions:Standard methods exist for detection of T. saginata in beef, and Trichinella spp. in meat (and are mandatory at meat inspection in Europe), Anisakidae in fish, and Cryptosporidium spp. and G. duodenalis in leafy green vegetables and berry fruits. For other FBP or foods, methods used in sample surveys have been described, but validation data are generally absent; limits of detection are not provided, ring trials have rarely been performed, and for most FBP quality control materials, proficiency schemes, and reference standards are lacking. The use of surrogate particles or organisms for method development or validation purposes needs to be carefully considered. Documented procedures for validation, such as ISO17468 and ISO16140-2:2016 that were established for bacteria, are mostly inappropriate for FBP. The development and application of standardized and validated detection methods would enhance understanding of the foodborne route of transmission, improve risk assessments, and help identify and verify critical control points.

Background This is the first report of the finding of Trichinella spiralis in beaver meat (Castor fiber) in Poland and Europe. In Poland, the beaver is a strictly protected animal species, except the few regions where high population density leads to economic losses. In these areas, the reduction culling of the animals was introduced. This uncommon hunting game animal is consumed and treated as a delicacy by hunters. However, currently, there is a lack of knowledge on possible risk factors for humans associated with the consumption of beaver meat. This paper presents the result of the study on the occurrence of nematodes of the genus Trichinella in beavers. Methods In total, 69 beavers were examined for the presence of Trichinella spp. The 50g samples were taken from each animal and digested separately, according to a procedure based on the EU reference method. The larva DNA was examined by PCR and sequencing. Results One of the 69 examined beavers was infected. Only one Trichinella larva was detected by the digestion method. The result of PCR confirms the presence of T. spiralis in beaver meat. Conclusions This case further confirms the ability of these typical herbivores to be infected with Trichinella spp. This is the second confirmed case of Trichinella spp. infection in beavers in Europe and the first of T. spiralis., Graphical abstract Image 1, Highlights • First report of the presence of Trichinella spiralis larva in beaver meat. • Detection of Trichinella spiralis by digestion method and confirmed by PCR and sequencing. • New parasitic risks factor for humans associated with consumption of beaver meat.

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).

Trichinella spiralis and Trichinella britovi are species of nematodes which are responsible for the majority of Trichinella infections in the world and the most prevalent in Poland. The most abundant species – T. spiralis, is considered to be more genetically homogeneous in Europe than T. britovi. The aim of the present study was to determine the genetic variability in T. spiralis and T. britovi populations based on nuclear 5S rDNA intergenic spacer region (5S rDNA) and cytochrome c oxidase 1 (COX1) gene sequences. For the study, 55 isolates of T. spiralis and 50 isolates of T. britovi isolated from wild boars, pigs, brown rat and a red fox were analyzed. Based on the analysis of both genes, the genetic variability within populations of T. spiralis and T. britovi differed. In T. spiralis, two single nucleotide polymorphisms (SNPs) were observed in the 612 bp 5S rDNA gene fragment, and one SNP was detected in the 700 bp COX1 gene fragment. In T. britovi, 17 single nucleotide variations (SNVs) were detected in the 5S rDNA gene fragment (among them 16 SNPs), while COX1 sequence analysis revealed the occurrence of 20 SNVs between the sequences tested (among them 19 SNPs). For the majority of T. spiralis isolates the investigated larvae presented uniform haplotypes. In contrast, most of the isolates of T. britovi consisted of larvae of different haplotypes. Geographical analysis showed that each region exhibited different haplotype composition and richness. Warmińsko-Mazurskie and Zachodniopomorskie regions were the richest in haplotypes (15 and 16 haplotypes, respectively). We used heatmaps showing a characteristic pattern for each region graphically. This may allow to differentiate regions based on the occurrence of particular haplotypes. Furthermore, a PCA analysis on the SNP level yielded biplots that show that certain haplotypes/genotypes are associated with (clusters of) regions., Graphical abstract Image 1, Highlights • The genetic variability within the Polish T. britovi population is high. • 5S rDNA and COX1 variability in T. spiralis is low compared to T. britovi. • T. britovi variability may correlate to geographical origin. • Heatmap and PCA analysis could be helpful analytical tool for epidemiological investigations.

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.

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.

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. Graphical abstract

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 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.

Serodiagnosis of human anisakidosis is presently hampered by the current lack of standardised serological assays that allow sensitive and specific detection of Anisakidae-specific antibodies in human patients. In the present study, we comparatively evaluated the diagnostic value (by IgG-ELISA) of excretory-secretory antigens (ESAgs) of Anisakis simplex, Pseudoterranova decipiens and Contracaecum osculatum, representing the most frequently found genera responsible for human infection. In addition, we tested also a mix of the three ES preparations (Mix-ESAgs) as well as two recombinant allergens of A. simplex, rAni s 1 and rAni s 7. ES antigen from C. osculatum yielded the best diagnostic performance in IgG-ELISA-based serodiagnosis of the Spanish anisakidosis patients investigated in this study (relative serodiagnostic sensitivity 100%; specificity 89%) as compared to A. simplex ES-antigen (93% versus 57%) and P. decipiens (67% versus 93%) or a mix of the three ES antigens (100% versus 44%), respectively. Cross-reactions of C. osculatum ES antigen with serum-antibodies from patients suffering from other helminth infections were rare and were exclusively found with few sera from toxocariasis, ascariasis, and filariasis patients. The two recombinant allergens rAni s 1 and rAni s 7 did not prove sufficiently sensitive and specific in order to justify a further evaluation of these antigens regarding their suitability in IgG-ELISA-based serodiagnosis of human anisakidosis. In conclusion, the C. osculatum-ESAg-ELISA remains as key candidate to be further assessed for the serodiagnosis of symptomatic anisakidosis in different endemic regions.

In 2012, WHO/FAO ranked 24 foodborne parasites (FBP) using multicriteria decision analysis (MCDA) to provide risk assessors with a basis for prioritising control of highly ranked FBP on the global level. One conclusion was that ranking may differ substantially per region. In Europe, the same methodology was used to rank FBP of relevance for Europe. Of the 24 FBP, the top-five prioritised FBP were identified for Europe as Echinococcus multilocularis, Toxoplasma gondii, Trichinella spiralis, E. granulosus, and Cryptosporidium spp., all of which are zoonotic. The objective of the present study was to provide an overview of surveillance and reporting systems in Europe for these top five prioritised FBP in the human and animal populations, to identify gaps, and give recommendations for improvement. Information on the surveillance systems was collected from 35 European countries and analysed according to the five different regions. For most FBP, human surveillance is passive in most countries and regions in Europe and notification differs between countries and regions. Adequate surveillance programmes for these FBP are lacking, except for T. spiralis, which is notifiable in 34 countries with active surveillance in susceptible animals under EU directive. Although human and animal surveillance data are available for the five prioritised FBP, we identified a lack of consistency in surveillance and reporting requirements between national experts and European bodies. Recommendations for improved surveillance systems are discussed.

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.

The third-stage larvae (L3) of Anisakis simplex are the most important source of hidden allergens in seafood products. However, there exist no commercial methods for detecting Anisakis proteins in food. Furthermore, only a few methods have been validated for the detection of A. simplex in thermally processed food. The aims of our study are (i) the development and validation of high-sensitivity chemiluminescent (CL) immunoassays for the detection of A. simplex proteins in processed seafood, (ii) and A. simplex antigen detection in common seafood products from Polish markets. We developed and validated CL sandwich ELISA (S-ELISA) and CL competitive ELISA (C-ELISA) methods for A. simplex proteins detection in food, with respective detection limits of 0.5 and 5 ng/mL. The usefulness of the assays for detecting A. simplex proteins in highly processed food was evaluated by examination of autoclaved canned fish spiked with A. simplex larvae (1&ndash, 8 larvae/200 g). Commercial real-time PCR was unable to detect A. simplex in autoclaved samples at all levels of enrichment with Anisakis larvae. CL-S-ELISA was used to test various types of seafood products from Polish markets. Among all tested products (n = 259), 28% were positive. A. simplex antigens were found mostly (n = 39) in smoked fish products: mackerel, herring, cod, and hake. Other positive samples were found in marinated herrings, canned cod livers, canned mackerels, and surimi sticks. In tuna, Atlantic argentine, anchovy, sardine, sprat, and squid products, A. simplex antigens were not detected. This study provides novel effective tools for the detection of A. simplex proteins in processed food and highlights the potential allergic hazards for Anisakis-sensitized Polish consumers of seafood.

Anisakis simplex third-stage larvae are the main source of hidden allergens in marine fish products. Some Anisakis allergens are thermostable and, even highly processed, could cause hypersensitivity reactions. However, Anisakis proteome has not been studied under autoclaving conditions of 121 &deg, C for 60 min, which is an important process in the food industry. The aim of the study was the identification and characterization of allergens, potential allergens, and other proteins of heat-treated A. simplex larvae. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify 470 proteins, including allergens&mdash, Ani s 1, Ani s 2, Ani s 3, Ani s 4, Ani s 5&mdash, and 13 potential allergens that were mainly homologs of Anisakis spp., Ascaris spp., and Acari allergens. Ani s 2, Ani s 3, Ani s 5, and three possible allergens were found among the top 25 most abundant proteins. The computational analysis allowed us to detect allergen epitopes, assign protein families, and domains as well as to annotate the localization of proteins. The predicted 3D models of proteins revealed similarities between potential allergens and homologous allergens. Despite the partial degradation of heated A. simplex antigens, their immunoreactivity with anti-A. simplex IgG antibodies was confirmed using a Western blot. In conclusion, identified epitopes of allergenic peptides highlighted that the occurrence of Anisakis proteins in thermally processed fish products could be a potential allergic hazard. Further studies are necessary to confirm the IgE immunoreactivity and thermostability of identified proteins.

The aim of the study was to analyze the distribution of the main groups of parasitic helminths within the small intestine of the red fox on the example of animals coming from eastern Poland. Two hundred and sixteen red foxes shot in eastern Poland were used in the investigation. Before examination, each small intestine was divided into three equal parts: anterior (A), middle (M), and posterior (P). Each part was examined separately with the sedimentation and counting technique. Six different types of intestinal parasites were detected: Alaria alata (78.7%), Mesocestoides spp. (78.2%), hookworms (72.7%), Taenia spp. (53.2%), Toxocara/Toxascaris (43.1%), and Echinococcus multilocularis (18.5%). Alaria alata was most often found in A and in the only-A variant. Taenia spp. and Toxocara/Toxascaris occurred often in A and were the second (after A. alata) parasites in terms of frequency occurring in the only-A variant. Mesocestoides spp. was most commonly located in M. Parasites with predilection sites located mainly in M and P were E. multilocularis and hookworms. In all parasite species, the variant covering the entire intestine (A + M + P) was found in samples with a higher intensity compared to variants limited to one or two fragments. Our investigation, as one of the few of its type, conducted a comprehensive analysis of the distribution of intestinal helminths in the small intestine of the red fox. It showed significant differences in the distribution of parasitic helminths in the small intestine of the red fox. Determining typical predilection sites for parasites in the intestine can be helpful in creating effective diagnostic methods.

Cephalopods, an appreciated seafood product, are common hosts of marine cestodes. The aim of this work is to report visible alive plerocercoids in longfin inshore squid (Doryteuthis pealeii), a cephalopod species commercialized as fresh and whole in Italy. Seventy D. pealeii from the Northwest Atlantic (FAO area 21) were collected and visually inspected. In total, 18 plerocercoid larvae were found in the viscera of 10 host specimens (P: 14.3% 95% CI 7.1&ndash, 24.7, MI: 1.8, MA: 0.26, range 1&ndash, 4) and molecularly analyzed targeting the variable D2 region of the large subunit (LSU) rRNA gene and the cytochrome c oxidase subunit I (COI) gene. The molecular characterization allowed to identify all the plerocercoids as Clistobothrium sp., a cestode of the Phyllobothriidae family with Lamnidae sharks as definitive hosts, and cephalopods as second intermediate hosts. These findings represent the first molecular record of Clistobothrium sp. in D. pealeii, thus contributing to elucidate its poorly known life cycle. Even if not affecting consumer&rsquo, s health, these visible parasites may represent a reason for disgust for consumers. Therefore, the results suggest that Food Business Operators should also check for the presence of these visible parasites during inspection and underline the importance of a correct consumers&rsquo, education.

Introduction Health, religious, and commercial aspects justify the need for meat species identification. The lack of officially approved methods prompts the undertaking of research on validation of isoelectric focusing of proteins (IEF) for official purposes. Material and Methods Samples were prepared from pigs (Sus scrofa ferus domestica), cattle (Bos taurus), and poultry (Gallus gallus domesticus). Meat mixtures were made by blending 50%, 25%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.2% meat of other species. Samples were examined on ultrathin polyacrylamide gels with pH 3–9 gradient. Results The results of the study confirmed the stable and reproducible pattern of meat protein bands. The detection limit of raw meat admixtures from pigs, cattle, and poultry mostly ranged from 2% down to 0.2% (0.2% for poultry). However, the IEF method can be used to detect the addition of pig meat to bovine meat in an amount higher than 3%. At the significant mixture level (i.e at least 5% addition of meat of another species) IEF proves itself with 100% specificity, sensitivity, and accuracy. Conclusion The achieved detection limits provide a basis for recommending the IEF method for routine tests in laboratories detecting the species origin of meat.

The aim of our study was the collection of seroprevalence data for Toxoplasma gondii, Coxiella burnetii, Trichinella spp., and Francisella tularensis from hunters in Lublin Province. The antibodies against T. gondii and C. burnetii were recorded in 38.5% and 16.2% of the sera, respectively. 4.05% of the sera were seropositive for both T. gondii and C. burnetii. None of the sera tested reacted positively with F. tulariensis or Trichinella spp. Seroprevalence of T. gondii and C. burnetii is common among the hunters from Lublin Province. It seems reasonable to undertake similar research among hunters from other regions of eastern Poland.

Anisakis simplex is a zoonotic nematode which can cause human anisakiasis. Furthermore, A. simplex allergens, even of dead larvae, can cause allergic reactions, including anaphylaxis. Due to the frequent occurrence in fish muscles and pathogenicity, A. simplex is a serious danger for consumers of fish products. Therefore, it is necessary to examine fish and fish products for the presence of these parasites before placing them on the market. The purpose of this paper is the review of the methods for A. simplex detection in fish and fishery products. These methods differ according to their effectiveness and type of the target analyte. They also have different suitability for examination of matrices with different properties. Moreover, this paper presents legislation associated with A. simplex detection. .

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.

Introduction The highest risk of trichinellosis for human is considered in eating meat products containing live larvae, mostly from wild boars or pigs. Spreading of Trichinella spp. may occur in various ways, one of which is transmission by vectors. The rat is considered to be the most common vector for Trichinella parasite. The population of rats living on pig farms can play an important role in maintaining or spreading the parasite to other animals. Objective The aim of presented survey was to investigate the occurrence of Trichinella spp. in rats on farms with pigs infected with this parasite. Material and methods From pig farms selected for study, the muscles of collected rats were investigated by magnetic stirrer digestion method to assess occurrence of Trichinella in the rat population. Isolated Trichinella parasites were identified under stereomicroscope and multiplex PCR were performed for species identification. Results Rats infected with Trichinella spp. were discovered on three of five investigated pig farms. The mean extent of invasion in rats from the studied farms was 23.33%. The calculated medium intensity of invasion was 4.09 lpg (larvae per gram) (SD 5.41). All larvae of Trichinella discovered from rats were identified as T.spiralis. Conclusions The results obtained indicate that in farms with a high prevalence of Trichinella invasion in pigs there are very likely to be found rats infected by this nematode. This suggests possibility to maintain the invasion in herd and spread into neighborhood farms.

The highest risk of trichinellosis for human is considered in eating meat products containing live larvae, mostly from wild boars or pigs. Spreading ofiTrichinella/ispp. may occur in various ways, one of which is transmission by vectors. The rat is considered to be the most common vector foriTrichinella/iparasite. The population of rats living on pig farms can play an important role in maintaining or spreading the parasite to other animals.The aim of presented survey was to investigate the occurrence ofiTrichinella/ispp. in rats on farms with pigs infected with this parasite.From pig farms selected for study, the muscles of collected rats were investigated by magnetic stirrer digestion method to assess occurrence ofiTrichinella/iin the rat population. IsolatediTrichinella/iparasites were identified under stereomicroscope and multiplex PCR were performed for species identification.Rats infected withiTrichinella/ispp. were discovered on three of five investigated pig farms. The mean extent of invasion in rats from the studied farms was 23.33%. The calculated medium intensity of invasion was 4.09 lpg (larvae per gram) (SD 5.41). All larvae ofiTrichinella/idiscovered from rats were identified asiT.spiralis/i.The results obtained indicate that in farms with a high prevalence ofiTrichinella/iinvasion in pigs there are very likely to be found rats infected by this nematode. This suggests possibility to maintain the invasion in herd and spread into neighborhood farms.

Introduction: The aim of the study was to determine the prevalence of intestinal helminths in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in the Augustów Primeval Forest (north-eastern Poland), with particular regard to zoonotic parasites. Material and Methods: Intestines from 53 raccoon dogs and 66 red foxes were examined with the use of sedimentation and counting technique (SCT). Samples of faeces from 51 red foxes and 50 raccoon dogs were examined with the use of flotation method. Results: Parasitic helminths were found by SCT in 98.5% of red foxes and 96.2% of raccoon dogs. Both species were infected with: Alaria alata (93.9% and 94.3%, respectively), hookworms (68.2% and 83.0%), Apophallus spp. (7.6% and 15.1%), Mesocestoides spp. (57.6% and 24.5%), Taenia spp. (40.9% and 1.9%), and Toxocara/Toxascaris nematodes (33.3% 15.1%). Echinococcus multilocularis was detected only in red foxes (6.1%), but trematodes Echinostomatidae and nematodes Molineus spp. only in raccoon dogs (18.9% and 41.5%, respectively). Additionally, Capillaria spp. eggs were detected by flotation method in 78.4% of foxes and 20.0% of raccoon dogs. Conclusion: The study showed a very high percentage of red foxes and raccoon dogs infected with intestinal helminths in the Augustów Primeval Forest. Moreover, dangerous zoonotic parasites also were found, which should be taken into consideration in the assessment of infection risk for humans in this region.

Introduction: The paper describes identification of Trichinella species isolated from wild boars (Sus scrofa) in the most popular hunting region of the West Pomeranian Province of Poland. Material and Methods: The Trichinella larvae were identified by digestion method. For species identification of the larvae, multiplex PCR was used according to the European Reference Laboratory for Parasites Multiplex PCR protocol. The results were confirmed by molecular amplification of 5S rDNA gene and sequence analysis. Results: Prevalence of 0.54% Trichinella–positive wild boars in the West Pomeranian Province was recorded. Examination of the larvae showed the occurrence of T. spiralis in 79 %, T. britovi in 16.5 %, mixed infection with T. spiralis/T. britovi in 3.5%, and T. pseudospiralis in 1.0% of the boars. Conclusion: This is the first record of wild boar infected with non-encapsulated larvae of T. pseudospiralis in Poland. The species is very difficult to determine, especially using trichinoscopic method. The discovery of the larvae in the animals which may be intended for human consumption confirms that digestion technique should be the only method used for the inspection of meat, especially that from wild boars..

The aim of the study was to detect Trichinella larvae in muscle tissue samples of red foxes (Vulpes vulpes) living in Podkarpackie Voivodeship and to determine their species composition. Muscle tissue samples had been collected from a total number of 139 red foxes culled during 2011-2012 in Podkarpackie Voivodeship and studied for the presence of Trichinella. Ten-gram muscle tissue samples were collected from each animal, combined into a fifty-gram pooled sample and then tested for the presence of Trichinella by the reference method for pooled sample digestion with a magnetic stirrer. Whenever a pooled sample gave a positive result, the suspected animals were resampled, and each ten-gram muscle tissue sample was tested separately. Trichinella larvae were identified to species by Multiplex PCR. The studies revealed the presence of one Trichinella larva in a pooled sample composed of five individual samples from foxes culled in the following locations: Leszczowate in Bieszczadzki District (one sample), Szczutków in Lubaczowski District (two samples), Podemszczyzna in Lubaczowski District (one sample) and Strzyżów in Strzyżowski District (one sample). No Trichinella larvae were detected during the test carried out on individual samples that composed the pooled sample. Numerous Trichinella larvae were found in samples from 4 foxes coming from Berezka and Glinka in Leski District, Dybawka in Przemyski District, and Golcowa in Brzozowski District. All Trichinella larvae isolated from the foxes belonged to the species T. britovi. The study confirmed the role of foxes as an important vector of Trichinella larvae circulating in the habitat of humans and animals. T. britovi is a species occurring most frequently in the fox population.

Anisakis simplex, Pseudoterranova decipiens, and Contracaecum osculatum third-stage larvae (L3) are fish-borne nematodes that can cause human anisakidosis. Although A. simplex is a known source of allergens, knowledge about the allergic potential of P. decipiens and C. osculatum is limited. Therefore, we performed comparative proteomic profiling of A. simplex, P. decipiens, and C. osculatum L3 larvae using liquid chromatography-tandem mass spectrometry. In total, 645, 397, and 261 proteins were detected in A. simplex, P. decipiens, and C. osculatum L3 larvae, respectively. Western blot analysis confirmed the cross-reactivity of anti-A. simplex immunoglobulin (Ig)G antibodies with protein extracts from P. decipiens and C. osculatum L3 larvae. The identified proteins of the Anisakidae proteomes were characterized by label-free quantification and functional analysis, and proteins involved in many essential biological mechanisms, such as parasite survival, were identified. In the proteome of A. simplex 14, the following allergens were identified: Ani s 1, Ani s 2 (2 isomers), Ani s 3 (2 isomers), Ani s 4, Ani s 8, Ani s 9, Ani s 10, Ani s 11-like, Ani s 13, Ani s fructose 1,6-bisphosphatase, Ani s phosphatidylethanolamine-binding protein (PEPB), and Thu a 3.0101. The following 8 allergens were detected in P. decipiens: Ani s 2, Ani s 3 (2 isomers), Ani s 5, Ani s 8, Ani s 9, Ani s PEPB, and Ani s troponin. In C. osculatum 4, the following allergens were identified: Ani s 2, Ani s 5, Ani s 13, and Asc l 3. Furthermore, 28 probable allergens were predicted in A. simplex and P. decipiens, whereas in C. osculatum, 25 possible allergens were identified. Among the putative allergens, heat shock proteins were most frequently detected, followed by paramyosin, peptidyl-prolyl cis-trans isomerase, enolase, and tropomyosin. We provide a new proteomic data set that could be beneficial for the discovery of biomarkers or drug target candidates. Furthermore, our findings showed that in addition to A. simplex, P. decipiens and C. osculatum should also be considered as potential sources of allergens that could lead to IgE-mediated hypersensitivity.

Tritrichomonas foetus is described as a pathogen of cattle and cats and also exhibits commensalism with pigs. In order to estimate the prevalence and determine the risk factors for parasite infection, specimens from animal hosts (cat, pigs, and cattle) from Poland were investigated. To our best knowledge, this is the first such study to examine samples from wild boars (Sus scrofa) for the presence of T. foetus. Data were collected from 117 cats, 172 pigs, 236 wild boars, and 180 cattle. The sensitivity of T. foetus identification was increased by using two molecular assays: PCR and loop-mediated isothermal amplification (LAMP). The prevalence of feline tritrichomonosis was 20.51%, and statistically significant differences were obtained between groups of animals regarding age, breed, number of cats, diarrhea, and place of living. Positive PCR and LAMP results for T. foetus were estimated for 16.28% of pigs, and the obtained data were significantly correlated with age. Conversely, no significant differences were observed concerning the farm size factor. In our survey, no cases of bovine tritrichomonosis were found, which is consistent with the data from the other countries of the European Union. Similarly, all wild boar samples were also T. foetus-negative according to LAMP and PCR.

Background The red fox (Vulpes vulpes) is widely distributed in the world; in central Europe, it is the most numerous wild species of the family Canidae. It can play the role of a definitive host for many intestinal parasites, including zoonotic helminths. Poland, with its geographical location (central Europe), is an interesting area for parasitological investigations of this species. The aim of this study was to evaluate and compare the prevalence of intestinal helminths in red foxes in different regions of Poland. Methods Intestines of 473 red foxes from four different regions were examined using the sedimentation and counting technique (SCT). In addition, 344 samples of faeces were examined using flotation. Results Overall, intestinal helminths were found in 98.9% of red foxes. The average prevalence of detected parasites was as follows: Mesocestoides spp. (84.1%); hookworms (67.9%); Alaria alata (61.5%); Toxocara/Toxascaris (49.5%); Taenia spp. (42.5%); Echinococcus multilocularis (25.6%); and Trichuris vulpis (2.3%). The prevalence of the majority of parasite species was similar in each region. Significant differences between regions were observed in the case of E. multilocularis: a low prevalence in the south-western and northern regions (0% and 0.9%, respectively) and a high prevalence in the south-east and northeast (39.3% and 42.7%, respectively). In the case of A. alata, important differences were found between northern (96.5% and 93.7% in northern and northeast regions, respectively) and southern regions (15.2% and 24.7% for south-western and south-east regions, respectively). The percentage of positive samples obtained with coproscopic examination (except for Trichuris) was significantly lower than that obtained with SCT. Analysis of the prevalence estimated in individual regions with the use of both methods (flotation and SCT) showed a high correlation for all parasite species (except for Mesocestoides spp.). The flotation method also allowed us to detect the eggs of the lung nematode Eucoleus aerophilus (syn. Capillaria aerophila) (76.2% of positive foxes). Conclusions This study showed a very high percentage of red foxes infected with intestinal helminths in different parts of Poland. Depending on the location, some differences were observed regarding the prevalence of dangerous zoonotic parasites, which should be considered in the assessment of infection risk for humans.

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.

The objective of the present study was to evaluate the contamination of sewage sludge produced by municipal waste treatment plants in Poland by viable eggs of intestinal parasites of the genera Ascaris, Toxocara and Trichuris (ATT). Ninety-two municipal, mechanical-biological sewage treatment plants located within Poland were selected. These plants belonged to types of agglomerations: group 0 (large), group 1 (medium), group 2 (smaller) and group 3 (small). Samples were collected at the final stage of sewage treatment after the addition of flocculent to sludge, followed by dehydration. The samples were examined by a method adjusted to examine sewage sludge dehydrated using polyelectrolytes. The viability of the isolated eggs was evaluated based on incubation in a moist chamber. Live eggs of intestinal nematodes were found in 99% of samples. Most samples were contaminated by the eggs of Ascaris spp. (95%) and Toxocara spp. (96%). However, Trichuris spp. eggs were detected in 60% of samples. The mean number of eggs in 1 kg of dry mass (eggs/kg d.m.) was 5600 for Ascaris, 3700 for Toxocara and 1100 for Trichuris. The highest number of ATT eggs was detected in samples from sewage treatment plants located in south-eastern and central Poland. The highest number of ATT eggs was found in sewage sludge produced in large sewage treatment plants (agglomeration Groups 0 and 1), with mean values of 15,000 and 8900 eggs/kg d.m. The present study is the first parasitological investigation conducted on a large number of samples (92 samples) taken from various types of municipal sewage treatment plants located throughout Poland (16 regions) after the common introduction of polyelectrolytes during sewage sludge dehydration. The results of this study indicate that sludge produced in municipal sewage treatment plants is highly contaminated with parasite eggs.