Influence of a nanoscale coating on plucking fingers and stainless steel on attachment and detachment of Salmonella Enteritidis, Escherichia coli and Campylobacter jejuni.

Gastroenteritis caused by Campylobacter represents the most common reported foodborne bacterial illness worldwide, followed by salmonellosis. Both diseases are often caused by the consumption of contaminated, insufficiently heated poultry meat. This can result from contamination of the meat during the slaughtering processes. Food contact surfaces like stainless steel or plucking fingers contribute significantly to cross-contamination of poultry carcasses. Modification of these surfaces could lead to a reduction of the bacterial burden, as already proven by successful application in various food industry sectors, such as packaging.In this study, nanoscale silica-coated and uncoated stainless-steel surfaces and plucking fingers were compared on a pilot scale regarding attachment and detachment of Campylobacter jejuni, Salmonella Enteritidis and Escherichia coli.The bacteria did not adhere less to the coated plucking fingers or stainless-steel sections than to the uncoated ones. The coating also did not lead to a significant difference in detachment of Campylobacter jejuni, Salmonella Enteritidis and Escherichia coli from the investigated surfaces compared to the uncoated ones.Our study did not reveal any differences between the coated and uncoated surfaces with regard to the investigated bacteria. In order to achieve a better adaptation of the coating to slaughterhouse conditions, future studies should focus on its further development based on the investigation of specific coating parameters.

Impact of nanoscale silicon dioxide coating of stainless-steel surfaces on Listeria monocytogenes.

High resistance to environmental factors as well as the ability to form biofilms allow Listeria monocytogenes to persist for a long time in difficult-to-reach places in food-producing plants. L. monocytogenes enters final products from contaminated surfaces in different areas of plants and poses a health risk to consumer. Modified surfaces are already used in the food industry to prevent cross-contamination. In this study, stainless-steel surfaces were coated with nanoscale silicon dioxide and the effects on attachment, bacterial growth and detachment of L. monocytogenes were evaluated. Attachment was considered for three different ways of application to simulate different scenarios of contamination. Bacterial growth of L. monocytogenes on the surface was recorded over a period of up to 8 h. Detachment was tested after cleaning inoculated stainless-steel surfaces with heated distilled water or detergent. Coating stainless-steel surfaces with nanoscale silica tends to reduce adherence and increased detachment and does not influence the bacterial growth of L. monocytogenes. Further modifications of the coating are necessary for a targeted use in the reduction of L. monocytogenes in food-processing plants.

Direct detection and quantification of Toxoplasma gondii in meat samples from feral raccoons (Procyon lotor) in Germany by magnetic-capture real-time PCR.

Because the number of wild raccoons in Germany is increasing constantly, it appears to be economic reasonable to use their meat as food. For this purpose, it is essential to generate data regarding the pathogen load of the meat to be consumed and handled. It is known that raccoons, particularly in Germany, show a high seroprevalence of Toxoplasma gondii. Because serological data only indicates contact of a host to a parasite additional direct detection is needed to prove presence of parasitic stages in particular tissues. Therefore, a total of 150 samples from raccoons with known serostatus were tested and quantified using magnetic-capture real-time PCR for Toxoplasma gondii. As it represents potentially consumption-relevant parts of raccoons, meat from forelimb and hindlimb was examined. Samples were stratified into three groups based on the animals' serostatus (each 50 negative, low positive, and high positive). All samples from seronegative animals were found negative by MC-PCR as well. In a total of 56 meat samples from 100 seropositive animals, T. gondii DNA was detected. Statistically significant more samples were positive by MC-PCR in the high positive than in the low positive serostatus group (38/50 vs. 18/50, p < 0.0001). Furthermore, samples from the former group were also found to have statistically significant higher DNA equivalent values compared to samples from the low positive serostatus group (p < 0.0001). These results suggest that meat from seropositive raccoons may contain considerable numbers of T. gondii presenting a potential public health risk for humans whilst handling and consumption.

First detection of Trichinella spiralis in raccoon (Procyon lotor) in Germany.

Trichinella spp. are foodborne parasites that can cause severe and potentially fatal disease in humans. Infections occur through consumption of meat containing the infectious stage (L1). In Germany the domestic cycle has been eradicated. In wild animals sporadic occurrence is observed in species such as wild boar, red foxes and raccoon dogs. The omnivore raccoon which is an invasive species in Europe is known as a potential host but has not been studied intensely regarding this parasite in Germany until now, thus resulting in a lack of knowledge about its role in the sylvatic cycle. Raccoons from the urban area of Leipzig were investigated for several pathogens including Trichinella spp. in a cooperative project. Muscle samples of 88 individuals were examined using the artificial digestion method (ADM). One animal was found positive, which is the first detection of this parasite in a raccoon in Germany.

Toxoplasma gondii in raccoons (Procyon lotor) in Germany: a serosurvey based on meat juice.

Toxoplasma gondii seroprevalence was determined in meat juice samples of 820 free-living raccoons from Germany. The animals were collected between December 2017 and April 2021. Using a commercial enzyme linked immunosorbent assay (ELISA), the overall seroprevalence was found to be 48.5%. Statistical analysis revealed significant seroprevalence differences between seasons, sex, and weight of analysed raccoons. The prevalence in late winter/spring (57.7%) was significantly higher than in autumn (38.4%) (p < 0.0003). Male raccoons (50.5%) were more often seropositive than females (41.0%) (p = 0.028). Increasing animal weight had a significant impact on the relative probability of a positive serostatus (odds ratio: 1.783, p < 0.0001). Furthermore, we found regional differences in seroprevalence, but there was no statistically significant difference resulting from animal age, degree of habitat urbanization and hunting year. Meat juice is a suitable medium for serological surveys for T. gondii in meat producing animals, as sampling is even possible after slaughter or during meat inspection when blood is no longer available. The observed high seroprevalence indicates that T. gondii infection is widespread among the German raccoon population providing a potentially relevant source of T. gondii transmission to humans upon consumption or handling of animal products.

Distribution of Alaria spp. mesocercariae in waterfrogs.

The distribution of Alaria-spp.-mesocercariae within the host is relevant for the examination via Alaria spp. mesocercariae migration technique (AMT) regarding predilection sites and may indicate an interaction between parasite and host. Naturally Alaria-exposed frogs of Pelophylax species (n = 13) were examined for systemic distribution and localization-specific parasite density of Alaria spp. mesocercariae. The frogs were necropsied and their body was divided into the following localizations: inner organs, head, torso, forelimbs, and hind limbs. The localizations were analyzed individually and in toto using Alaria spp. mesocercariae migration technique. Our results showed neither statistical differences concerning the number of mesocercariae in the different localizations nor in respect of the rate of positive localizations. Therefore, an accumulation in a particular predilection site seems unlikely. Further research on a representative sample is necessary before final conclusions can be drawn.

Occurrence of selected zoonotic food-borne parasites and first molecular identification of Alaria alata in wild boars (Sus scrofa) in Italy.

Wild boar is a source of human infections with zoonotic pathogens, including food-borne parasites. With the aim of a characterization of the human exposure risk, a survey on wild boars intended for human consumption was planned, selecting three pathogens, Toxoplasma gondii, Alaria alata, and Trichinella spp., as markers of meat infection. Diaphragm muscle samples from 100 wild boars hunted in Piedmont region (Northern Italy) in two hunting seasons (2015-2016) were collected. Concerning T. gondii, a combined approach of antibody detection and molecular techniques with genotyping was performed. For the detection of A. alata and Trichinella spp., the larva migration technique and the magnetic stirrer method were employed, respectively; in addition, molecular confirmation of the morphological identification of the recovered specimen was performed. Anti-T. gondii antibodies were found in meat juice samples (43.3%) and T. gondii DNA (type II) was detected in three animals (7.1%) out of 42 seropositive examined. In none of the sampled wild boars (0%), Trichinella spp. larvae were found, whereas one animal (1%) scored positive to A. alata mesocercariae. The molecular diagnosis proved the morphological identification of the trematode. This is the first finding of A. alata in Italian wild boar population. The present study confirmed the role of wild boars as a source of parasitic zoonotic diseases and thus the risk derived for humans posed by the consumption of game meat. Considering the zoonotic implications, the results underline the importance of monitoring and surveillance of zoonotic parasites in Italian wild boar populations.

Mastitis vaccination using a commercial polyvalent vaccine or a herd-specific Staphylococcus aureus vaccine. Results of a controlled field trial on a dairy farm.

UNLABELLED: Objective of this study was the improvement of selected parameters of udder health by mastitis vaccination in a dairy herd with elevated bulk milk somatic cell counts and Staphylococcus (S.) aureus as predominant mastitis causing pathogen. MATERIAL AND METHODS: On a dairy farm, pregnant heifers (status group [SG] 1; n = 181) as well as cows stratified for their udder health state (classification based on results of cytobacteriological investigations of quarter milk samples obtained before dry cow therapy [MS0]) (SG 2-4; n = 416) were randomly assigned to one of the following vaccination groups (VG): Startvac® (VG SV), Bestvac® Rind Mastitis (containing herd-specific S. aureus-strains; VG BV) and the unvaccinated control (VG Co, placebo), respectively. The collected data (5 [MS5] and 52 [MS52] days in milk [DIM]: quarter milk somatic cell count [QSCC] and bacteriological investigation of quarter milk samples; dairy herd improvement test [DHIT] days 1-10: milk yield and individual cow somatic cell count; until 305 DIM: clinical mastitis cases) were compared between the VG within their SG. RESULTS: S. aureus prevalences were significantly lower in VG SV (p < 0.001) and VG BV (p = 0.006) within SG 3 and in VG SV (p = 0.008) within SG 4, respectively, in comparison to VG Co. Milk yields (DHIT days [p = 0.042] and 305-day milk yield [p = 0.040]) were significantly less in VG SV within SG 4 compared to VG Co. Significant different changes over time in comparison to VG Co indicating a vaccine effect during lactation were only observed for QSCC within SG 4 for VG BV (p = 0.017; increase towards MS52) and for S. aureus prevalence within SG 3 for VG BV (p < 0.001; opposing trends from MS0 towards MS52). All other interactions of time and VG under investigation were not significant in any of the SG. Furthermore, there were no descriptive differences in the incidence of clinical mastitis and duration of a necessary mastitis therapy, respectively, between the VG within their SG. CONCLUSION: In this field study, the application of two different mastitis vaccines was not an appropriate tool to improve the considered parameters of udder health sustainably.

Effect of temperature on the survival of Alaria alata mesocercariae.

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.

Effects of in vitro conditions on the survival of Alaria alata mesocercariae.

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 (<1 min). Finally, our studies have revealed that it required 120 min to reliably inactivate all A. alata mesocercariae within HCl-pepsin digestion solution with a pH of 1.5-2.0 at 37°C. Consequently, the results showed that 3.0% is the minimum concentration of NaCl in meat products recommended for human consumption because at lower NaCl concentration the parasite survived for a substantial period of time. Finally, the common concentrations of ethanol used for the disinfection of surfaces in household and/or laboratory, are sufficient for the inactivation of A. alata mesocercariae.

Tenacity of Alaria alata mesocercariae in homemade German meat products.

A renewed interest in the pathogenic potential of Alaria alata mesocercariae emerged over the last 10years 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 24h 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 mesocercariae in raccoons (Procyon lotor) in Germany.

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 Müritz 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.

First interlaboratory test for the detection of Alaria spp. mesocercariae in meat samples using the Alaria spp. mesocercariae migration technique (AMT).

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.

Campylobacter spp. - prevalence on pig livers and antimicrobial susceptibility.

The objective of the study was to determine the prevalence of Campylobacter spp. on surfaces of slaughtered pig livers. Multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter coli isolates. Additionally, C. coli and Campylobacter jejuni isolates were tested for antimicrobial susceptibility by the broth dilution method. The minimal inhibitory concentrations were determined for erythromycin, gentamicin, ampicillin, ampicillin/sulbactam, nalidixic acid, ciprofloxacin, tetracycline and trimethoprim/sulphamethoxazole. Samples were taken during the slaughtering process in a slaughterhouse in Lower Saxony, Germany. Altogether, 10% of 1500 surfaces of pig livers from 50 fattening herds was found to be Campylobacter positive, with C. coli as the predominant species (76%) followed by C. jejuni (21%). Resistance to erythromycin and tetracycline was higher in C. jejuni compared to C. coli, whereas C. coli were more resistant to quinolone compared to C. jejuni. Fluoroquinolone resistance is usually associated with cross-resistance to quinolone, but in the presented investigation C. coli as well as C. jejuni showed a higher resistance to ciprofloxacin (28.6% and 20.0%, respectively) than to nalidixic acid (9.5% and 0%, respectively). A high genetic diversity of the C. coli isolates was demonstrated by MLST. Differences in STs and antimicrobial resistance pattern indicate that the Campylobacter strains originated from the pig itself and not from the slaughterhouse. A comparison of the STs with those reported in the C. jejuni/coli PubMLST database showed an overlap of porcine and human isolates, indicating that C. coli isolates from pigs should be considered as potential sources of human infection.

Alaria alata in wild boars (Sus scrofa, Linnaeus, 1758) in the eastern parts of Germany.

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.

Development of a PCR approach for differentiation of Alaria spp. mesocercariae.

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.

Carry-over of thermophilic Campylobacter spp. between sequential and adjacent poultry flocks.

Nineteen flocks of four poultry species were monitored at a veterinary field station to investigate the distribution and spread of Campylobacter genotypes between sequential and adjacent flocks. Caecal and liver samples were obtained at frequent intervals from birds of all flocks and examined for Campylobacter. Amplified fragment length polymorphism (AFLP) analysis was performed to genotype Campylobacter isolates. Of the 1643 caecal and liver samples investigated, 452 (27.5%) caecal samples and 11 (0.7%) liver samples contained Campylobacter. Of the caecal isolates 76.3% were identified as Campylobacter jejuni and 23.7% were identified as Campylobacter coli. Poultry flocks were largely colonized by more than one AFLP type and an intense exchange of Campylobacter genotypes between different poultry flocks occurred. These findings indicate that multiple genotypes can constitute the Campylobacter population within single poultry flocks, hinting to different sources of exposure and/or genetic drifts within the Campylobacter population. Nevertheless, in most flocks single Campylobacter genotypes predominated. Some strains superseded others resulting in colonization by successive Campylobacter genotypes during the observation period. In conclusion, the data demonstrate that the large genetic diversity of Campylobacter must be considered in epidemiological evaluations and microbial risk assessments of Campylobacter in poultry.

A novel detection method for Alaria alata mesocercariae in meat.

Distomum musculorum suis (DMS), the mesocercarial stage of the trematode Alaria alata, can cause severe damages within their hosts, and since several reports about cases of human larval alariosis have been published, it became apparent that infected game animals and in particular wild boars are a potential source of infection for both humans and animals. A final statement concerning the health risks for consumers could not be given due to the lack of information about both the prevalence of DMS and the suitability of Trichinella inspection methods to detect this parasite in wild boar meat. Our studies concentrate on (1) the verification of suitability of the official digestion methods for Trichinella spp. for DMS detection in wild boars, (2) development, optimization, and validation of methods, and (3) the distribution of the parasites within their paratenic hosts. A total of 868 individual samples/digests from 48 wild boars were analyzed by the reference method for Trichinella detection in meat samples according to regulation (EC) No. 2075/2005. In addition to the official protocol, a method modification with Pankreatin(c) and bile acid was applied for analysis of adipose tissue samples (n = 89). On the basis of our results, a new detection method based on a larvae migration technique was developed and used for detection of DMS in 574 single samples. Furthermore, the distribution patterns of DMS in wild boars in a total of 1377 single sample migrations/digestions from 35 positive animals were analyzed by application of all three methods. The official digestion method for Trichinella spp. in wild boars meat is inapplicable for the detection of A. alata mesocercariae as it shows shortcomings in both digestion and sampling. A direct comparison between the newly developed A. alata mesocercariae migration technique and the official digestion method for Trichinella spp. based on 574 single samples from 18 animals clearly shows that the sensitivity to detect A. alata developmental stages in tissues of wild boars of the new method is nearly 60% higher compared with the magnetic stirrer method for pooled sample digestion as laid down in regulation (EC) No. 2075/2005. Among other advantages, this method offers a simple, highly applicable, fast, and cost effective way to detect DMS in wild boars which is already applicable in routine veterinary inspection.

Biology of Alaria spp. and human exposition risk to Alaria mesocercariae-a review.

Recent incidental background findings of Alaria alata mesocercariae ["Distomum muscularis suis," Duncker, 1896] in meat of wild boars during official Trichinella inspection initiated a re-assessment of the potential human health risk as posed by this parasite. The present review of the literature on Alaria biology shows that the human exposition risk should no longer be accepted to be negligible, as it demonstrates a general lack of knowledge in relevant areas of Alaria biology confounding any risk analysis. Sound risk assessment needs future studies which should concentrate on the most pressing questions of (1) the optimization and/or development of methods for reliable Alaria mesocercariae detection, (2) the distribution of the mesocercariae within their paratenic hosts, i.e., identification of potential predilection sites, particularly in wild boars, and (3) their prevalence in sylvatic populations of animals with respect to their introduction into the human food chain. Further, the degree and possibly also the species specificity of Alaria mesocercariae tenacity within the paratenic hosts and respective meat as pertaining to food technological treatments need to be elucidated. While these questions remain unanswered, it is an incontrovertible fact that Alaria mesocercariae have a potentially high human pathogenicity by both occupational and alimentary exposition.