Detection of Spirocerca lupi and an unknown Trichinella-like nematode in raccoon (Procyon lotor).

The raccoon Procyon lotor (Carnivora: Procyonidae) is an invasive species of growing importance for the introduction of alien pathogens or as additional hosts for autochthonous pathogens in Europe, including zoonotic parasites. As the population is steadily increasing and outcompeting the red fox (Vulpes vulpes) in Germany, the consumption of raccoon meat raises concerns about pathogens they may transmit. Therefore the presence of Trichinella larvae was here investigated in muscle samples (n = 904) of raccoons from northern Germany. No Trichinella larvae were found, thus confirming the general low occurrence of this parasite in Germany. However, Spirocerca lupi (n = 12) and an unidentified Trichinella-like nematode (n = 1) were accidently detected in the examined samples. The first is not a zoonotic parasite but has a high veterinary relevance as it can cause severe diseases in dogs. It is the first documented autochthonous infection of this nematode in Germany. The larvae of an unidentified Trichinella-like nematode were found in high abundance in all examined muscles of one raccoon, though they could not be identified to species level. Histological investigation revealed intramuscular cystic structures. This is the largest study investigating muscular parasites of raccoons in Europe so far, which suggests that this invasive animal species is infected by S. lupi and by a yet unknown Trichinella -like parasite.

Impact of climate change on foodborne infections and intoxications.

Background: Temperature, precipitation, and humidity are important factors that can influence the spread, reproduction, and survival of pathogens. Climate change affects these factors, resulting in higher air and water temperatures, increased precipitation, or water scarcity. Climate change may thus have an increasing impact on many infectious diseases. Methods: The present review considers those foodborne pathogens and toxins in animal and plant foods that are most relevant in Germany, on the basis of a selective literature review: the bacterial pathogens of the genera Salmonella, Campylobacter and Vibrio, parasites of the genera Cryptosporidium and Giardia, and marine biotoxins. Results: As climate change continues to progress, all infections and intoxications discussed here can be expected to increase in Germany. Conclusions: The expected increase in foodborne infections and intoxications presents a growing public health risk in Germany.

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.

Reprint of: Survival of Trichinella spiralis in cured meat products.

Processing of meat is one possible approach to control meat-borne parasites. Processing methods such as freezing, cooking and irradiation are recommended for the control of Trichinella in pork, horse or game meat if specific technical conditions are fulfilled. Curing is a widely used preservation process influencing product characteristics such as shelf life, food safety, and taste. As curing methods are characterized by high parameter variability and predictions about inactivation of parasitic stages in raw meat products are difficult, curing and smoking are not recommended for Trichinella control. The objective of this study was to investigate the survival of T. spiralis in cured raw sausages taking into account water activity (aw-value), pH value, temperature, and time. For this purpose, four different types of sausage (Knackwurst, vacuum packed Knackwurst, short ripened salami, long ripened salami) were produced using T. spiralis infested batter. After production, the sausages were stored at product specific conditions for up to 35 days. During storage, pH value and aw-value of the sausages were monitored over time. Further, sausages of each type were digested using the magnetic stirrer method and the viability of the isolated larvae was assessed using a previously published larval motility test as a proxy for viability and infectivity of Trichinella larvae. In this context, we also introduce a three-level rated infectivity score (RIS) with a clear categorization scheme allowing the assessment of the infectivity of larvae. Based on the RIS, larvae isolated from the salamis were regarded as potentially infective until day 2 (short ripened salami) or day 3 (long ripened salami) post ripening, whereas in Knackwurst, potentially infective larvae were still found by day 8 post ripening. In contrast potentially infective larvae were detected in vacuum-packed Knackwurst until day 24 post ripening. Finally, using the RIS approach, data from previously published studies were collected and subjected to a correlation analysis to identify matrix factors linked to short Trichinella inactivation times.

Comparison of Direct and Indirect Toxoplasma gondii Detection and Genotyping in Game: Relationship and Challenges.

The importance of game as a source of Toxoplasma gondii (T. gondii) infection in humans is largely unknown. New data on the presence of T. gondii in game hunted in the Federal State of Brandenburg, Germany, were obtained by direct and indirect detection (ELISA). DNA extracted either directly (5 g heart or foreleg muscle, DE) or after acid pepsin digestion (50 g heart, PD) or enriched by magnetic capture (50 g heart, MC) was examined by real-time PCR (qPCR). ELISA revealed seroprevalences of 20% in wild boar (Sus scrofa), 11% in roe deer (Capreolus capreolus) and 6% in red deer (Cervus elaphus). T. gondii DNA was detected by at least one direct detection method in 12% of wild boar, 6% of roe deer, 2% of fallow deer (Dama dama) and 2% of red deer. In both, positive wild boar and roe deer, T. gondii type II specific alleles were the most prevalent, as assessed by PCR-restriction fragment length polymorphism. The highest proportion of positive animals was detected by MC qPCR, followed by PD qPCR with a similar proportion of positive findings. Investigation of 50 g of heart muscle revealed a significantly higher proportion of positive qPCR results than analysis of 5 g (p = 0.048). An association between seropositivity and direct detection was evident in wild boar and roe deer (p < 0.001). Infectivity of T. gondii DNA-positive samples was confirmed by bioassay (4/4), providing evidence that game could represent a relevant source of viable T. gondii posing a risk for human infection.

Development of a Novel Method for Identification of Alaria alata Mesocercariae by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

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.

Prevalence of Alaria alata mesocercariae in wild boars from Brandenburg, Germany.

Since 2002, Alaria (A.) alata mesocercariae (AM) have been found during routine Trichinella inspection of wild boars in many European countries. To date, human infection with AM through consumption of undercooked or raw AM infested wild boar meat cannot be excluded. In Germany, data on the parasite's prevalence in wild boars are scarce. To better understand temporal and spatial fluctuations of this parasite, this study investigated the prevalence of AM in wild boars in the German federal state of Brandenburg during three hunting seasons from 2017 to 2020. In total, 28.3% (100/354, 95% CI: 23.3-33.3%) of all wild boars sampled in eight counties of Brandenburg were tested positive for AM by Alaria alata mesocercariae migration technique (AMT). AM were detected in wild boars from seven different counties. Samples from one county (Havelland) tested completely negative for AM (0/16). Prevalences of the seven AM positive counties of Brandenburg ranged from 11.5 (3/26, 95% CI: 2.5-30.1%) in Märkisch-Oderland to 64.1% (25/39, 95% CI: 47.2-78.8%) in Uckermark. An association between sex and A. alata positivity could not be determined. A statistically significant increase in frequency of older AM positive wild boars was observed (p = 0.001). For a nationwide assessment of the prevalence of A. alata in wild boars and the risk for consumers of ingesting viable AM by consumption of raw or undercooked AM infested wild boar meat, further long-term studies in different regions of Germany are needed.

Survival of Trichinella spiralis in cured meat products.

Processing of meat is one possible approach to control meat-borne parasites. Processing methods such as freezing, cooking and irradiation are recommended for the control of Trichinella in pork, horse or game meat if specific technical conditions are fulfilled. Curing is a widely used preservation process influencing product characteristics such as shelf life, food safety, and taste. As curing methods are characterized by high parameter variability and predictions about inactivation of parasitic stages in raw meat products are difficult, curing and smoking are not recommended for Trichinella control. The objective of this study was to investigate the survival of T. spiralis in cured raw sausages taking into account water activity (aw-value), pH value, temperature, and time. For this purpose, four different types of sausage (Knackwurst, vacuum packed Knackwurst, short ripened salami, long ripened salami) were produced using T. spiralis infested batter. After production, the sausages were stored at product specific conditions for up to 35 days. During storage, pH value and aw-value of the sausages were monitored over time. Further, sausages of each type were digested using the magnetic stirrer method and the viability of the isolated larvae was assessed using a previously published larval motility test as a proxy for viability and infectivity of Trichinella larvae. In this context, we also introduce a three-level rated infectivity score (RIS) with a clear categorization scheme allowing the assessment of the infectivity of larvae. Based on the RIS, larvae isolated from the salamis were regarded as potentially infective until day 2 (short ripened salami) or day 3 (long ripened salami) post ripening, whereas in Knackwurst, potentially infective larvae were still found by day 8 post ripening. In contrast potentially infective larvae were detected in vacuum-packed Knackwurst until day 24 post ripening. Finally, using the RIS approach, data from previously published studies were collected and subjected to a correlation analysis to identify matrix factors linked to short Trichinella inactivation times.

A two-step morphology-PCR strategy for the identification of nematode larvae recovered from muscles after artificial digestion at meat inspection.

To ensure that meat from livestock and game is safe for human consumption, European legislation lays down rules for mandatory testing. Helminth larvae are a category of zoonotic foodborne pathogens that can contaminate meat. Among helminths, the only zoonotic nematode regulated in Europe regarding meat inspection is Trichinella spp.. It is precisely during Trichinella testing that other potentially zoonotic larvae can be found. Due to current lack of tools, their identification is often very complicated. Nematode larvae other than Trichinella, recovered from artificial digestions of pig and wild boar muscles from France and Germany, were subjected to a newly developed two-step identification scheme, which includes both morphological examination and molecular assays. The first step is a general orientation towards a broad taxonomic group; the second step consists of targeted identification based on the results of first step. Different parasites were identified, some of which were not zoonotic such as Metastrongylus spp. and Angiostrongylus vasorum, but others are known to be zoonotic such as Toxocara cati, Ascaris suum, and Uncinaria stenocephala. The strategy is efficient for the identification of nematode larvae recovered from muscles but could also be applied for larvae from other sources.

Seroprevalence of Toxoplasma gondii in wild boar and deer in Brandenburg, Germany.

Consumption of game in Germany has increased during the past 10 years. Wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) are the most frequently hunted and consumed game animals in Germany, yet information on the occurrence of zoonotic pathogens in these animal species is scarce. To better estimate the public health risk emanating from handling and consumption of game, this study investigated seroprevalences of Toxoplasma gondii in game hunted in the German federal state of Brandenburg during two hunting seasons from 2017 to 2019. Toxoplasma gondii-specific antibodies were detected in 24.4% (44/180, 95% CI: 18.4%-31.4%) of wild boar, 12.8% (16/125, 95% CI: 7.5%-20%) of roe deer and 6.4% (3/47, 95% CI: 1.3%-17.5%) of red deer using a commercial ELISA kit. Seroprevalences were similar in the two hunting seasons. Correlation between sex and seropositivity could not be observed. A rise in seroprevalence was seen with increasing age in all studied game species. Observed seroprevalences suggest that T. gondii is endemic in the sylvatic environment in the German federal state of Brandenburg and imply that game could represent a relevant source for human T. gondii infection.

Performance of three molecular methods for detection of Toxoplasma gondii in pork.

Comparison of epidemiological data on the occurrence of Toxoplasma (T.) gondii tissue cysts in meat is hampered by the lack of standardization and a great variety of methods for molecular detection. Therefore, this study aimed to compare and validate three different polymerase chain reaction (PCR) methods for detection of T. gondii DNA in pork. Analytical performance characteristics of two real time PCRs (qPCRs; Tg-qPCR1, Tg-qPCR2) and one conventional endpoint PCR (cPCR), all targeting the 529 repeated element, were assessed using genomic DNA of three clonal T. gondii types prevailing in Europe and North America. qPCR efficiencies for all three clonal types ranged between 93.8 and 94.4% (Tg-qPCR1) and 94.3-95.6% (Tg-qPCR2). Tg-qPCR1 and Tg-qPCR2 showed an overall PCR performance score of 85% and displayed a similar 95% detection limit of 1.067 and 1.561 genome equivalents per PCR reaction (GE/PCR), respectively. However, T. gondii DNA could be detected at concentrations as low as 0.1 GE/PCR. Reliable quantification is possible over 4 log ranges from 105 to 100 GE/PCR with mean repeatability relative standard deviations of ≤11% and reproducibility relative standard deviations of ≤12.7%. Presumably, both qPCRs are similarly suitable for sensitive and specific detection of T. gondii DNA in pork. In contrast, the cPCR using primer pair TOX5/Tox-8 proved to be highly sensitive with a detection limit of 1.41 GE/PCR, but not suitable for detection of T. gondii DNA in pork as unspecific amplification of porcine DNA was observed resulting in bands with similar size to the desired T. gondii-specific PCR product.