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Distomum musculorum suis (DMS), the mesocercariae of Alaria alata, is typically found accidently during examination of wild boar meat for Trichinella spp. The aim of the study was to compare DMS detection methods. Briefly, 232 wild boar meat samples were tested by mesocercariae migration technique (AMT) as a reference method; of these, 104 were found to be positive. Selected positive samples were tested again with the three other methods: compressorium method (Compressor), digestion with magnetic stirrer (Digestion) and by modified digestion with Pancreatin® bile and pancreatic enzymes (D + P). The results were analyzed by logistic regression, the non-parametric Kruskal-Wallis test and the Mann-Whitney U test. Of the 43 samples found positive by the AMT, 20 were found positive by Digestion and 25 by D + P. The Compressor identified DMS in seven of the 19 tested samples. The Digestion and D + P methods gave similar intensities (P = 0.506), i.e. 1.4 and 1.3 DMS respectively, but the AMT detected seven times higher number of parasites. The probability of detection of DMS in the meat sample by the Digestion or by D + P was higher than 0.5 when at least seven (Digestion) or five (D + P) DMS were present in the sample (AMT). The Compressor was the least sensitive method: at least 14 DMS must be present in the meat sample for detection. AMT should be considered the most accurate method of DMS detection.

Currently, an increase in cases of infection of wild and domestic carnivores with the trematode Alaria alata (Trematoda, Strigeidida) is currently recorded in the Central Black-Earth Zone. The trematode A. alata has a complex life cycle involving intermediate, insert and reservoir hosts. We obtained new data on the realization of the life cycle of A. alata in the natural environment of the Central Black-Earth Zone. The moor frog (Rana arvalis), green toad (Bufotes viridis) and spade-footed toad (Pelobates fuscus) are recorded as insert hosts. Infection with mesocercarias A. alata of the moor frog is 20% (the prevalence) and 23 specimens (infection intensity). The moor frog is the most numerous species among amphibians and is characterized by high in-touch capabilities with carnivorous mammals. The moor frog is the main insert host in the circulation of A. alata in natural biocenoses. We have identified the localization of the Alaria mesocercarias in the moor frog, namely, the pericardium and subcutaneous tissue of the intermaxillary space. We determined the distribution of mesocercarias in moor frogs of this year's brood: 71% in the head; 25.8% in the hind legs; 22.6% in the trunk; and 3.2% in the front legs. The green toad and spade-footed toad were also classified as insert hosts of A. alata (prevalence of infection was less than 10%; and infection intensity was 4.0 specimens). Mesocercarias were found in frog larvae (24.6%; 2.6 sp.).

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.

Various species of mammals, including humans and wild boars, can serve as paratenic hosts of Alaria alata mesocercariae – Distomum musculorum suis (DMS). Cases of DMS can be reliably detected by the recent introduction of the A. alata mesocercariae migration technique (AMT). The aim of this study is to present current data on the occurrence of DMS in wild boars in north-eastern Poland, and to compare the findings with those obtained in other European countries. A. alata was identified in 98 of 221 (44.3%) tissue samples of wild boar taken from five provinces in north-eastern Poland during the hunting seasons 2015–2016 and 2016–2017 based on AMT analysis. Positive results were found in all studied regions, but the percentage of infected individuals ranged from 26.3% in the Pomorskie province to 65.5% in the Warmińsko-Mazurskie province. The mean number of larvae exceeded seven larvae per 30 g sample for three provinces: Pomorskie, Mazowieckie and Lubelskie. In turn, lower values were found in the Warmińsko-Mazurskie province (3.3 larvae per 30 g), and the lowest in the Kujawsko-Pomorskie province (1.8 larvae per 30 g). The occurrence and intensity of A. alata infestation in wild boars was found to depend on the environment in which they live. Neither the sex or the age of the wild boar appeared to influence the occurrence nor the intensity of infestation., Graphical abstract Image 1, Highlights • High percentage (44.3) of Alaria alata in samples of wild boars in Poland. • High prevalence of A. alata infestation in wild boars compared to other reports. • Sex does not affect the risk or intensity of infestation by A. alata in wild boars. • Age does not affect the risk or intensity of A. alata infestation in wild boars. • A. alata poses a potential risk for wild boar consumers in Poland.

An archaeoparasitological analysis of the soil samples from Nadym Gorodok site of Western Siberia has been carried out in this study. The archaeological site was dated as the 13 to 18th century, being characterized as permafrost region ensuring good preservation of ancient parasite eggs. Parasite eggs as Opisthorchis felineus, Alaria alata, and Diphyllobothrium sp. were found in the archaeological soil samples, which made clear about the detailed aspects of Nadym Gorodok people’s life. We found the Diphyllobothrium sp. eggs throughout the 14 to 18th century specimens, allowing us to presume that raw or undercooked fish might have been commonly used for the foods of Nadym Gorodok inhabitants and their dogs for at least the past 400 years. Our study on Nadym Gorodok specimens also demonstrate that there might have been migratory interactions and strong economic ties between the people and society in Western Siberia, based on archaeoparasitological results of Opisthorchis felineus in Western Siberia.

Alaria (A.) alata mesocercariae (AM) have increasingly appeared as incidental findings during the mandatory inspection of wild boars for Trichinella in many European countries. An Alaria spp.-specific PCR is available for the identification of AM, however, it is time- and cost-intensive. Therefore, we propose a rapid and cost-efficient MALDI-TOF assay for the identification of AM in wild boar meat that can be applied in routine diagnostics. In this study, a fast and methodologically simple protocol for the protein extraction of AM from different host species in different countries was established, and an AM-specific reference spectra database was created as part of the ongoing development of an existing Trichinella spp. database. A formic acid protein extraction was performed after pooling 10 AM from the same host individual. In total, 61 main spectra profiles (MSPs) from different host individuals were stored in an AM-specific MSP library. The cluster analysis of these 61 MSPs indicated a possible variation within the A. alata species with a tentative association with the geographical origin of the host, but not the host species. This MALDI-TOF assay allows for a fast verification of the AM isolates, which is the next step in the development of a universal database for the identification of several parasites isolated from meat.

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.

Simple Summary There are many parasites that may be transmitted to humans via food, and meat is a major source of such infections. Trichinella spp. is one of the most important meat-transmitted parasites, while Alaria spp. may be considered an emerging pathogen, albeit to date rarely reported in humans. Raw and undercooked wild boar meat has been proven as a major source of human infection by both parasites. In the present study, an investigation of the presence of these parasites in wild boar meat was conducted for the first time in Greece. Classical parasitological methods and molecular techniques were implemented for the examination of samples collected from 128 hunted wild boars, and none of them were found positive for Trichinella spp. or Alaria spp. For the detection of Alaria spp., a novel molecular method was developed, offering a powerful complementary diagnostic tool that may be useful for the epizootiological surveillance of the parasite. The epizootiology/epidemiology, clinical implications, and importance of monitoring of these parasitic infections are briefly discussed. Abstract Foodborne parasitic diseases represent a major threat to public health. Trichinellosis, caused by the nematode parasite Trichinella spp., is one of the most important foodborne diseases, while alariosis, caused by the trematode parasite Alaria spp., is less common in humans, and rare cases have been reported only in the USA and Canada. Both parasites can infect humans via the consumption of raw or undercooked wild boar meat. In order to investigate the prevalence of these parasites in wild boar meat in Greece, samples from the diaphragm pillars and the region of the mandibular angle from 128 wild boars, hunted in Greece, were collected. The samples were examined by classical parasitological (compression, artificial digestion, and Alaria spp. migration) and by molecular (real-time PCR) methods. For Trichinella spp. an existent real-time PCR detecting all species likely to be present in Greece was applied, while for Alaria spp. a real-time PCR was developed, employing an LNA TaqMan probe targeting the large subunit ribosomal RNA gene. All examined wild boar samples from Greece resulted negative for Trichinella and Alaria species, indicating a low prevalence of infection in the examined population. The novel real-time PCR for Alaria spp. has 81.5% amplification efficiency and is able to detect 0.12 larvae per 50 g of tissue and could be utilized as a complementary to AMT diagnostic tool in surveillance.

The neozoan species raccoon dog (Nyctereutes procyonoides) and raccoon (Procyon lotor) are widespread in Europe and potential vectors of many diseases that can threaten human and domestic animal health. Facing a further spread of these species, it is important to know about (i) pathogens imported and/or (ii) pathogens acquired in the new habitat. Thus, we investigated the parasite fauna of wild raccoon dogs and raccoons from Austria, at the edge of their new distribution range. The eight examined raccoons were nearly free of pathogens including Baylisascaris procyonis, and thus assumed to have a low epidemiological impact, so far. Out of ten raccoon dog specimens, we found one from western Austria to be infected with Echinococcus multilocularis and another three from the eastern wetland regions to harbour adults of Alaria alata. Furthermore, we detected Babesia cf. microti in five of eight raccoon dogs all over Austria but none of our samples were tested positive for Trichinella spp. Nevertheless, the raccoon dog seems to be a relevant host, at least for the zoonotic pathogens E. multilocularis and A. alata, and we suggest to further monitor the raccoon dogs parasite fauna.

Alaria alata trematode is widely distributed throughout Europe and has a complex life cycle in which wild boar serve as a reservoir host. The primary aims of the present study are to establish the occurrence of A. alata mesocercariae in naturally infected wild boar in Latvia and to assess the risk for humans to acquire A. alata infection via consumption of wild boar meat. By summarizing long-term data using the Trichinella inspection method from 2014 to 2019, the overall A. alata prevalence was 8.3%, of which significantly higher A. alata prevalence was observed during the summer seasons. Additionally, 43.9% (n = 485) of wild boar were found to be infected with A. alata using Alaria mesocercariae migration technique. The present study indicates that the probability for humans to acquire A. alata mesocercariae is possible, yet improbable and varies from 0.2% to 2.2%. Most likely, it depends on both frequency of A. alata presence in wild boar population and of a method of preparing wild boar meat for consumption that will allow for the parasite inactivation.

The paper summarises information on 11 species of trematodes parasitic in 9 species of wild carnivorans of Ukraine. The largest number of trematode species (9) was found in the red fox (Vulpes vulpes). Alaria alata (Diplostomidae) appeared to be the most common trematode parasite in the studied group; it was found in 4 host species from 9 administrative regions and Crimea.

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 trematode Alaria alata has a complex life cycle in which carnivore mammals may become infected by feeding on mesocercariae-infected, second intermediate hosts or paratenic hosts. Afterwards, young flukes migrate through various organs of the definitive host, including the diaphragm and the lungs, before reaching the small intestine. The aim of the present study was to establish the prevalence and infection intensity of A. alata in carnivore mammals, the host age and related occurrence of different A. alata development stages in particular animals in Latvia. Overall, samples from 992 animals (539 red foxes, 411 raccoon dogs and 42 grey wolves) were examined using sedimentation and counting techniques to observe the presence of A. alata metacercariae and adult trematodes in each particular host. The present study, based on data collected over five hunting seasons (2010/2011–2014/2015), is a comprehensive study on the circulation of A. alata in wild canids in Latvia. A. alata was present in all species examined and the overall prevalence reached 86.2%. A significant positive correlation was observed between the burden of A. alata metacercariae and adult trematodes in raccoon dogs (rs = 0.27, p = 0.001) and red foxes (rs = 0.17, p = 0.0002). A significantly higher (p = 0.003) A. alata metacercariae mean intensity was observed in juvenile raccoon dogs compared to the one observed in adult raccoon dogs and red foxes from both age groups. The presence of both A. alata development stages in a particular animal concurrently and at different intensity levels may indicate primary infection and re-cured infection of the host.

Alaria alata (Diplostomidae, Trematoda), a potentially zoonotic pathogen, is the only Alaria species in Europe. In recent years, increasing reports of mesocercariae of A. alata in wild boars have been recorded in European countries; however there have been no described cases in domestic pigs over the past decades. Out of 272 diaphragm samples from wild boars (Sus scrofa) and domestic pigs (Sus scrofa), from Serbia, included in the present investigations, mesocercariae of A. alata were found in six (3%) samples from wild boars and in two (2.77%) samples from domestic pigs. Due to the lack of sensitivity of artificial digestion with a magnetic stirrer which was applied in the study, the true prevalence is estimated to be considerably higher in the area of investigation. Confirming the infection in domestic pigs and a wide distribution in wild boars in the area of investigation, the present paper should influence the growing awareness of alariosis as a potential human disease. The meat from free-range domestic pigs and game meat as a potential source of alariosis should always be adequately thermally treated before consumption.

In the last 15 years, the mesocercariae of Alaria alata have frequently been reported in the wild boar during routine Trichinella inspections made compulsory for the trade of venison meat in Europe. If these studies have focused primarily on mesocercariae isolated from meat, few works have been done so far to understand the circulation of the parasite in natural conditions especially in the intermediate hosts. This study focuses on the second intermediate hosts of this parasite assessing the suitability of two amphibian groups-brown frogs and water frogs sensu lato-for mesocercarial infection on an area where A. alata has already been identified in water snails and wild boars. During this study, both groups showed to be suitable for mesocercarial infection, with high prevalence and parasite burdens. Prevalence was higher in the brown frog group (56.9 versus 11.54 % for water frogs) which would indicate that it is a preferential group for infection on the study area, though reasons for this remain to be investigated. No significant difference among prevalences was observed between tadpoles and frogs. This study, the first focusing on A. alata in these amphibians in Europe, provides further information on circulation of this parasite in natura.

Recent findings of Alaria alata mesocercariae in wild boars and other animals in Europe reinforced the concern about the public health risk posed by this parasite especially if the game meat is insufficiently heated during preparation. Cooking and freezing are effective methods for the inactivation of parasites in meat whereas refrigeration is considered as an essential part of the Good Hygiene Practice. Additionally, microwave dielectric heating may represent an equally effective tool for parasite inactivation. Therefore, isolated vital mesocercariae were examined with respect to their resilience against heating, refrigeration, freezing, and microwave heating. A. alata mesocercariae stored in Ringer's solution do not survive heating temperatures that exceed 60.0 °C. Similarly, exposure to microwave heating ensured an inactivation of all parasite developmental stages after 90 s of treatment. In contrast, the parasites' tolerance towards cold is far higher as the mesocercariae survived refrigeration temperatures (4.0 ± 2 °C) in Ringer's solution for up to 13 days. An effective inactivation by cold is therefore only guaranteed if the infested game meat is frozen to a core temperature of -13.7 °C for a minimum of 2 h at least. Game meat should be handled with the same or even higher caution than meat of husbandry animals since wild animals may be infected with parasites or other zoonotic agents that are not common in livestock. It is therefore of crucial importance that appropriate temperature time protocols are used for the reliable inactivation of these zoonotic agents.

The presence of the mesocercarial stage of Alaria alata (Goeze, 1792) in wild boar meat represents a potential risk for human, but little is known about the circulation of mesocercaria in wild boar populations. Routine Trichinella inspection, mandatorily performed in wild boar in France, also allowed detecting mesocercaria. We analyzed the results of this detection in the carcasses of 27,582 wild boars hunted in 2007-2011, in 502 hunting areas of the Rhine valley. Prevalence was globally low (0.6%), but 12% of the hunting areas were affected. These were clustered in lowlands of the Rhine valley, and prevalence strongly decreased with increasing elevation. In the lowlands, prevalence doubled between 2007 and 2011. This time trend and the geographic aggregation of positive wild boars suggest risk management measures based on targeted surveillance, control and prevention.

Objective: To analyze the prevalence of Alaria sp. mesocercariae in slaughter pigs in the Mekong Delta in Vietnam. Methods: From December 2012 to March 2013, 621 carcasses of slaughter pigs originating from nine Vietnamese provinces (69 carcasses per province) bordering the Mekong Delta were tested for the presence of Alaria alata mesocercariae. From each carcass, 30 g tissue (a mix of peritoneal fat and cheek tissue) were examined using the Alaria-migration-technique. Animals originated from traditional farming with free access of pigs to water/wetlands. Fattening pigs (5.5-7.5 months of age, 84% males) constituted 97.1% of the sample set, whereas sows>26 months accounted for 2.9%. Results: All samples tested were negative for motile mesocercariae. Conclusions: Results indicate that the actual prevalence of this parasite will not exceed 5% (α=0.05) in pigs from the study area. Further studies could elucidate if this is due to (unapproved) antimollusc and antiparasitic treatment in the study area or if the parasite is absent in the wildlife at all.

From March 2012 to February 2013, 221 wild boar carcasses obtained from regular hunts in the Czech Republic were tested for Alaria alata mesocercariae using the Alaria-migration-technique. Most samples originated from South Moravia (173), and all 15 positive samples were found in this region, in particular in Tvrdonice (3/10) and Lanžhot (12/28), close to the site of the first description of this parasitic stage in wild boars in the territory of former ČSSR. These hunting grounds are located between the rivers March and Thaya, and rich in floodplains. Among the 38 carcasses tested in this area, higher carcass weights were associated with higher frequency of positive carcasses (P0.05). Overall frequency of positive carcasses was 15/221 (6.8%). In positive samples (adipose and glandular tissue and muscle), the median number of mesocercariae was 14.3 per 100 g (range 3-69).

Emerging and re-emerging zoonotic diseases affect both public and animal health and require the development and contemporary implementation of suitable detection methods. A growing number of findings of the mesocercarial stage of the digenean trematode Alaria alata in game inhabiting wetlands have necessitated the development of a specific detection method. With the Alaria spp. mesocercariae migration technique (AMT), a specific and sensitive detection method is now available. To make the method accessible to the official controls, method validation is necessary. In this context, interlaboratory tests (IT) are a key factor to demonstrate both (1) the suitability of the respective method and (2) the reference materials. In the first IT performed on this issue, 15 laboratories from nine German federal states took part. Every lab received two negative and four positive standard samples each as well as a standardized examination device for AMT, and a standard operating procedure. All participating laboratories showed very good results in terms of qualitative analysis: 96.7 % of the samples were assessed correctly positive or negative. An analysis of the qualitative performance shows that 263 (58.4 %) of 450 mesocercariae that were inserted in the meatballs were identified by the participants, and 5 (33.3 %) out of 15 labs were able to count at least 70 % of the Alaria spp. mesocercariae. A direct comparison with the results of the German Trichinella IT, which were conducted since 2004, shows that the overall sensitivity of the AMT is even higher than that registered for the reference method for Trichinella detection (e.g. 93 % in 2010). Also, in terms of quantitative analysis, AMT stands up to the comparison with the results from the German Trichinella IT. The refinement of the implementation protocol of this innovative, easy-to-use and cost-effective method harbours great potential for further optimization and successful implementation in the official controls.

The digenean trematode Alaria alata, an intestinal parasite of wild canids is widely distributed in Europe. The recent finding of the mesocercarial life cycle stage in the paratenic wild boar host suggests that it may potentially infect humans Mohl et al. (Parasitol Res 105:1-15, 2009). Over 500 foxes were examined during a wildlife survey for zoonotic diseases in 2009 and 2010. The prevalence of A. alata ranged from 21% to 26% in 2009 and 2010, and the intensity of infection varied, with the majority of foxes having between one and ten trematodes, but a small number of animals had parasitic burdens greater than 500. The location of foxes was geo-referenced and mapped using a geographic information system. The results of the spatial analysis suggest that A. alata may have a limited distribution being confined mainly to areas of pasture especially in the central plain and north Munster. Hot spot analysis indicated a clustering and that the level of parasitism was greatest in foxes from those areas where the prevalence of infection was highest.

The trematode Alaria alata is a cosmopolite parasite found in red foxes (Vulpes vulpes), the main definitive host in Europe. In contrast only few data are reported in wild boars (Sus scrofa), a paratenic host. The aim of this paper is to describe the importance and distribution of Alaria alata mesocercariae in wild boars, information is given by findings of these larvae during Trichinella mandatory meat inspection on wild boars’ carcasses aimed for human consumption. More than a hundred cases of mesocercariae positive animals are found every year in the East of France. First investigations on the parasite’s resistance to deep-freezing in meat are presented in this work.

2 ABSTRACT:- RUAS, J.L.; MULLER, G.; FARIAS, N.A.R.; GALLINA, T.; LUCAS, A.S.; PAPPEN, F.G.; SINKOC, A.L.; BRUM, J. G.W. (Helminths of Pampas fox Pseudalopex gymnocercus (Fischer, 1814) and of Crab-eating fox Cerdocyon thous (Linnaeus, 1766) in the Southern of the State of Rio Grande do Sul, Brazil). Helmintos do Cachorro do campo Pseudalopex gymnocercus (Fischer, 1814) e do Cachorro do mato Cerdocyon thous (Linnaeus, 1766) no sul do estado do Rio Grande do Sul, Brasil. Revista Brasileira de Parasitologia Veteri- naria, v. 17, n. 2, p. 87-92, 2008. Laboratorio Regional de Diagnostico, Faculdade de Veterinaria, Universidade Federal de Pelotas, Rio Grande do Sul, Brasil. Caixa Posta 354, CEP.: 96.010-900. E-mail: ruas@ ufpel.edu.br Forty wild canids were captured by live trap at Municipalities of Pedro Osorio and Pelotas in Southern of the State of Rio Grande do Sul and they were transported to the Parasitology Laboratory at the Universidade Federal de Pelotas. After they were posted, segments of intestinal, respiratory and urinary tracts and liver were separated and examined. Animal skulls were used for taxonomic identification. Of forty wild animals trapped, 22 (55%) were Pseudalopex gymnocercus and 22 (55%) Cerdocyon thous. The most prevalent nematodes were: Ancylostoma caninum (45.4 in P. gymnocercus and 22.2% in C. thous), Molineus felineus (9.9 in P. gymnocercus and 5.6% in C. thous), Strongyloides sp. (22.7 in P. gymnocercus and 16.7% in C. thous), Trichuris sp. (13.6 in P. gymnocercus and 11.1% in C. thous), and Capillaria hepatica (13.6 in P. gymnocercus and 5.5 % in C. thous). The trematodes observed were: Alaria alata (36.4 in P. gymnocercus and 50.0% in C. thous), and Asthemia heterolecithodes in 5.6% C. thous. Cestodes were identified as Spirometra sp. (61.1 % in C. thous and 54.5 in P. gymnocercus), Diphyllobothriidae, (81.8 in P. gymnocercus and 77.8 % in C. thous) and an Acantocephala of the genus Centrorhynchus was also observed in 5.6% of C. thous only. These results indicated the helminths fauna in wild canids from the studied area.

To the Editor. Alariosis is a reemerging zoonotic disease caused by infection with larval stages of trematodes of the genus Alaria. The trematodes are found in wildlife that inhabit wetlands, and these animals may serve as possible reservoirs for these organisms that cause human infection (1). The main sources for human infection are suids and frogs (1). In humans, the clinical features of alariosis caused by infections with the North American species of Alaria vary from mild and asymptomatic to moderate with respiratory or cutaneous signs (2) or neuroretinitis (3), to severe-to-lethal anaphylactic shock caused by larva migrans (4,5). The genus Alaria has 7 species; only A. alata is found naturally in Europe (6), a species which has not thus far been shown to be responsible for human infections. A. alata infection is common in its typical definitive host (red fox, Vulpes vulpes) and in certain paratenic hosts (wild boar, Sus scrofa) (1). However, the role of other paratenic hosts is poorly known. Among these, mustelids are reported to harbor mesocercariae of A. alata trematodes (7). The pathogenic effect of A. alata infection has been poorly studied, because most lesions described were in humans infected with other species of Alaria. Except for 2 experimental studies that described gross lesions produced by A. alata trematodes (6,8), to our knowledge, no data have been published concerning lesions produced by natural infection in nonhuman hosts. Our report provides a detailed description of the lesions, shown by microscopy, which suggests the pathogenic mechanisms. One adult female European mink (Mustela lutreola) was found dead during standard surveillance operations in which box traps were used; this trapping was part of biodiversity and ecology studies in the central part of the Danube delta in Romania (45°08′N, 29°19′E) in March 2010. The corpse was deep-frozen and analyzed after 3 months in the laboratory. During necropsy, multiple, well-defined, whitish nodules were observed in most muscular and subcutaneous tissues (Figure, panel A), with no evident preferential localization. We collected samples from these tissues for artificial digestion (9,10) and histologic examination, using the routine paraffin-embedding protocol and the following staining methods: hematoxylin-eosin, Masson trichrome, and Gordon and Sweet. Figure Lesions produced by mesocercariae of Alaria alata in European mink. A) Mesocercariae in the muscle and subcutaneous tissue produce whitish, round or slightly oval, well-defined nodules. B) Free mesocercarium after artificial digestion, showing the characteristics ... Artificial digestion released parasites (6 larvae/5 gm tissue) with typical larval trematode structures (Figure, panel B). By microscopy, we observed that morphologic features of these larvae were consistent with A. alata mesocercariae (6). Histopathologic examination confirmed the presence of parasitic forms in muscle sections (Figure, panel C). The mesocercariae were located in the connective fibrous tissue of the perimysium or between the muscle fibers. The typical structure of muscle fibers was altered around the larvae, with inflammatory cell reactions, represented mainly by lymphocytes, macrophages, and plasma cells (Figure, panel D). In other areas, the inflammatory reaction around the parasite was minimal or absent (Figure, panel E). In certain histologic sections, the damaged muscular tissue was replaced by granulation tissue in various stages of development (Figure, panel F). The maturity of the granulation tissue differed substantially, depending on the muscular areas examined. Some lesions were found in adult connective tissue, formed by mature collagen scar fibers (type I collagen) and few inflammatory cells, whereas other lesions had reticulin fibers (type III collagen) with numerous inflammatory cells. The lesions of the subcutaneous connective tissue consisted of an inflammatory reaction (panniculitis). The inflammation was characterized by a low number of mononuclear leukocytes and fibrinous exudate and fibroplasia. The polyphasic nature of muscle and subcutaneous lesions produced by A. alata infection in its paratenic host appears to be caused by mesocercarial migration. This view is sustained by the presence of mononuclear cells that it infiltrates and by the appearance of the granulomatous tissue in various stages of maturation, which leads to muscle and subcutaneous fibroplasia. The reparatory nature of the lesions suggests that the inflammation is probably the result of direct tissue damage rather than an immune reaction targeted toward the parasitic antigens. This assumption could explain the local absence of inflammatory reaction around the parasites. The lack of inflammation was previously observed also with A. americana infection of humans (4). The structure of all mesocercariae observed by microscopy suggested that they were alive and active before the mink carcass was frozen. Because no mesocercariae were surrounded by adult connective tissue or by granulomatous inflammation, together with the multiple presences of migratory routes, the continuous mobility of the parasites through the host’s tissues was strongly suggested. Although data on the pathologic changes caused by Alaria spp. in general, and A. alata parasites in particular, are scarce, the migration pattern and the lesions seem to be dependent on the particular parasite and host species. The reparatory nature of the lesions suggests that the inflammation is the result of direct tissue damage rather than an immune reaction targeted toward the parasitic antigens.

Im Lebensmittel-Untersuchungslabor wurden im Jahr 2001 insgesamt 210 Fleischproben von Wildschweinen auf das Vorhandensein des ParasitenTrichinella spiralis mit negativem Ergebnis untersucht. Als Zufallsbefund wurden in drei Proben (entspricht 1,80 %) Mesozerkarien des SaugwurmsAlaria alata festgestellt. Entsprechend der in der kroatischen Republik gultigen gesetzlichen Regelung das Fleisch der betroffenen Tiere als genussuntauglich deklariert und unschAdlich beseitigt.

The aim of this study was to determine the effect of different concentrations of table salt (NaCl) and ethanol (v/v) solutions on the viability of Alaria alata mesocercariae. Furthermore, the survival of A. alata mesocercariae during simulated human gastric digestion was evaluated. For this purpose, A. alata mesocercariae migration technique (AMT) was used for the isolation of the parasite from high-positive A. alata mesocercariae meat from wild boar, raccoon, raccoon dog, and badger meat. In total, we have studied the behavior of 582 larvae under different conditions (NaCl, ethanol, and artificial gastric juice) in three independent in vitro experiments. The larvae survived at a NaCl concentration of up to 2.0 % until day 21 with a median survival time of 11 days. At 3.0 % NaCl concentration, the larvae lost their vitality after less than 24 h. In addition, it was found that ethanol concentrations from 8.0 to 70.0 % were effective at reducing survival of A. alata mesocercariae within a short period of time (

Intestinal washes, faecal flotations and serological examinations for antibodies to Toxoplasma gondii and Neospora caninum were used to assess the prevalence of parasites in carcases of foxes killed on roads or shot in the Dublin area and surrounding counties. The ascarids Uncinaria stenocephala and Toxocara canis were prevalent, as was the trematode Alaria alata. Taenia species, eggs of Capillaria species and sporocysts of Sarcocystis species were also found. Only one fox out of 70 examined was seropositive for N. caninum, whereas 24 of 51 were seropositive for T. gondii.

A renewed interest in the pathogenic potential of Alaria alata mesocercariae emerged over the last 10 years as a result of increased findings of this parasite in feral pigs during official examination for Trichinella spp. Cases of food associated human alariosis in North America suggest that a risk associated with the consumption of traditional raw cured products from infected wild boar meat cannot be neglected because the commonly applied preservation techniques may not necessarily kill the mesocercariae. In addition, changes in consumer behavior and new preparation methods for game meat (e.g. pink roasting and grilling) may increase the risk for food-associated parasitic infections. Thus, there is a strong need for the evaluation of the tenacity of A. alata mesocercariae against different physical and chemical influences as pertaining to common preservation and preparation techniques. Against this backdrop the aim of our work was a sound analysis of the survivability of A. alata mesocercariae during curing, fermentation, cold smoking and drying in raw cured meat products. Eighty three samples of traditional German meat products were prepared from naturally infected game meat and partly spiked with additional vital mesocercariae to achieve an adequate dose of infection. The resultant products were examined chronologically for dead and viable A. alata mesocercariae with the Alaria mesocercariae migration technique. After 24 h of production, vital A. alata mesocercariae were still found in raw type sausages but no vital parasites were detected in the final products. Based on these results a possible risk for the consumer for an infection with A. alata mesocercariae through the consumption of contaminated raw cured products can be largely ruled out if the respective food technological procedures are carried out properly. However, a risk for the consumer cannot be excluded in cases of very early consumption of these products.

Alaria alata is a trematode of carnivores from Europe. The mesocercarial stage was recently identified in wild boar meat from Europe. Previous histopathologic studies showed the presence of unidentified parasitic cysts within the tongues of raccoons from northern Germany. For identification of the parasite species, tissue samples of 105 raccoons originating from a National Park in northern Germany and from Berlin metropolitan area were collected. Histological examination of cryotome sections of frozen as well as paraffin-embedded tongues were used to identify parasite cysts. These were located in the connective and adipose tissue and in close proximity to small arterioles, suggesting a hematogenous spread of the parasite. Often, cysts were surrounded with mild infiltration by inflammatory cells. Additionally, mesocercariae were isolated from defrosted tongue samples of 11 raccoons. Molecular-biology assays confirmed the parasite species as A. alata. Except for one positive raccoon from Berlin City, all other positive raccoons originated from the sylvan Muritz National park, indicating an abundance of intermediate hosts in this area. Our results show that raccoons can act as paratenic hosts for A. alata and extend the broad host range of this parasite to a species introduced into Germany.

The trematode Alaria alata, an intestinal parasite of different carnivore species is widely distributed throughout Europe. The mesocercarial stages of Alaria spp. may infect almost all vertebrate species, including humans, and, in particular, omnivorous scavengers such as wild boars serve as paratenic hosts for the parasite. The introduction of the A. alata mesocercariae migration technique (AMT) opened the way to a reliable detection of Alaria spp. mesocercariae in different body tissues of their paratenic hosts. For the first time, it was possible to detect vital A. alata mesocercariae from two Bulgarian wild boars by means of this new method. In addition, polymerase chain reaction (PCR) examination of the respective parasitic DNA allowed the unequivocal species identification of the parasites as A. alata. Isolation and molecular biological identification of the parasite's developmental stages make significant contributions to completion of data on both the distribution of Alaria spp. in stocks of European game and the relationship between different Eurasian Alaria spp. isolates.

Graphical abstract, Highlights • Raccoon dogs recently invaded Denmark. • Raccoon dogs and foxes harboured several helminths, some are zoonotic. • A markedly different helminth fauna was evident between the two hosts. • A fox was infected with Echinococcus multilocularis, no Trichinella spp. were detected. • Wildlife surveillance is a foundation for the protection against communicable parasites., Invasive species negatively influence the biodiversity of the ecosystems they invade and may introduce pathogens to native species. Raccoon dogs have very successfully invaded Europe, including, recently, Denmark. This study included analyses of gastrointestinal helminths and Trichinella spp. from 99 raccoon dogs and 384 native red foxes collected from October 2009 to March 2012. The sedimentation and counting method used revealed that raccoon dogs and foxes harboured 9 and 13 different helminth species, respectively, of which several known to be zoonotic. Significantly more nematode and cestode species were found in foxes while raccoon dogs had more trematode species. Rodent transmitted parasites were more prevalent in foxes, while amphibian transmitted parasites were more prevalent in raccoon dogs. One fox was infected with Echinococcus multilocularis (0.3%), while no Trichinella spp. were detected in raccoon dogs or foxes. The trematode Brachylaima tokudai was detected for the first time in Denmark in five of 384 foxes (1.3%). Prevalences of Pygidiopsis summa (3.0% and 3.4%) and Cryptocotyle spp. (15.2% and 15.4%) were comparable in raccoon dogs and foxes, respectively. Four helminth species were more prevalent in foxes than in raccoon dogs: Toxocara canis (60.9% and 13.1%); Uncinaria stenocephala (84.1% and 48.5%); Mesocestoides spp. (42.7% and 23.2%); and Taenia spp. (30.7% and 2.0%), respectively. Three helminth species were more prevalent in raccoon dogs than in foxes: Dipylidium caninum (5.1% and 0.3%); Mesorchis denticulatus (38.4% and 4.2%); and Alaria alata (69.7% and 34.4%), respectively. T. canis was more abundant in foxes while A. alata was more abundant in raccoon dogs. The intestinal distribution of a number of helminth species was comparable between hosts, but highly variable between parasite species. Inherent biological factors and host invasion of new areas might have shaped these marked differences in helminth fauna between the invasive raccoon dog and the native red fox.

Over the last decade, incidental findings of Alaria alata in stocks of German wild boar during the official Trichinella inspection have been increased. As early as 2006, the German Federal Institute for Risk Assessment pointed out the possible health risk to the consumer posed by this trematode. However, at that time, reliable data concerning the prevalence of the parasite in German wild boars and feral pigs were lacking especially because no appropriate detection method was available. The development of the A. alata mesocercariae technique (Riehn et al., Parasitol Res 107(1):213–220, 2010) now makes it possible to close the remaining gaps in knowledge in this field. Over a 2-year period, 286 retained samples of fresh meat from wild boars originating from different hunting areas in Brandenburg and Saxony-Anhalt, which were tested negative for A. alata during the official Trichinella inspection in the competent veterinary inspection offices, were reexamined with the A. alata mesocercariae migration technique (AMT). In 33 out of 286 retained meat samples (11.5 %) with a preliminary negative report, the trematode was demonstrated during the follow-up examination using AMT. This result especially in connection with the highly heterogeneous distribution of the parasite within the hosts' body (Riehn et al., Parasitol Res 107(1):213–220, 2010; Moehl et al., Parasitol Res 105(1):1–15, 2009) shows clearly that a high number of unreported cases of alariosis in wild boars have to be assumed.

A steadily increasing number of incidental findings of Alaria alata mesocercariae in meat of wild boars during official Trichinella inspection necessitates the development of a specific detection method for this parasite. A reliable verification of infested paratenic hosts seems to be one of the key factors for both understanding the biology of Alaria spp. and determining a sound risk assessment within the meaning of the consumer’s health protection. Consequently, our studies concentrate on (1) the verification of suitability of the official digestion methods for Trichinella spp. for Alaria alata mesocercariae detection in wild boars, (2) development, optimisation and validation of methods, and, (3) the distribution of the parasites within their paratenic hosts.

To date classification and differentiation of Alaria spp. are based largely on external characteristics and comparative morphology of adult flukes. The accurate differentiation between various Alaria spp. mesocercariae is indeed difficult because there are only few data on morphological and morphometrical features of the parasite’s developmental stages. We established a conventional polymerase chain reaction (PCR) for a molecular-based diagnosis of Alaria alata mesocercariae that can aid in their identification. Twenty Alaria spp. mesocercariae specimens were collected from three different wild boars originating from different areas of eastern Germany. DNA from the prepared isolates was extracted, and a primer pair was selected to amplify a 303-bp region of the A. alata genome. The DNA preparations extracted from the field samples as well as A. alata positive controls were successfully amplified and yielded a single sharp band of the expected size. In all samples, molecular identification was consistent with morphological identification. With our new PCR assay, we present the first approach for identification and characterization of A. alata mesocercariae specimens using molecular methods. This practicable and reproducible protocol can be used for both diagnostic and epidemiological purposes.

Echinococcus multilocularis was found in 1 of 75 foxes collected from the Morris area, 30 mi south of Winnipeg. Toxocara canis was present in 71, Taenia crassiceps in 7, and Alaria alata in 3 of the foxes examined.