Validation studies of the latex agglutination test for the detection of Trichinella larvae in meat products.

Human trichinellosis is a foodborne disease caused by ingestion of meat infected with Trichinella muscle larvae. To control Trichinella spp. infection in the European Union, all slaughtered pigs from holdings that are not officially recognized as applying controlled housing conditions and other animals susceptible to Trichinella infection and intended for human consumption should be examined by one of the approved digestion methods described in Regulation (EU) No. 2015/1375. In the past, Trichinella outbreaks due to the consumption of cured wild boar or pork products have been described in several European countries, making the identification of the larvae from these products relevant for Trichinella control. Therefore, this study aimed to validate the newly approved latex agglutination test (Trichin-L) for routine testing of cured meat products. The test was validated based on the OIE Guidelines using pork products spiked with Trichinella larvae. The sensitivity of the method varied greatly depending on the investigated meat product and was usually lower than for the gold standard, the magnetic stirrer method. The detection rate reached 80% for three larvae and 60% for one larva in cured pork sausages. A detection rate of 100% for three larvae and 50% for one larva was found in bacon. For frozen samples (-20°C) the Trichin-L kit is similarly sensitive as for cured samples. Further, to determine the performance of the test under field conditions, pork products from regions with known high Trichinella prevalences confiscated by customs authorities at two German international airports were analyzed. Problems associated with the Trichin-L test were incomplete digestion due to fatty ingredients, spices and very dry meat products, resulting in data which could not be evaluated. Therefore, the test is currently not suitable for the detection of Trichinella larvae in cured meat products and needs further adaptation steps to increase both usability and sensitivity.

Study on the occurrence of Trichinella spp. in raccoon dogs in Brandenburg, Germany.

In recent years the raccoon dog population in Germany has risen dramatically and a steady westward expansion can still be seen. In addition to the highest Trichinella prevalence in wild boar and the most reported Trichinella cases in domestic swine from backyard farms, the North-Eastern part of Germany also has the highest raccoon dog density in the country. Due to their distinct scavenging behavior, raccoon dogs play a significant role as Trichinella reservoir. Therefore, to increase the knowledge on Trichinella spp. in raccoon dogs, we performed a study on the occurrence of Trichinella in the North-Eastern federal state of Brandenburg. In total 1527 raccoon dogs were examined between 2000 and 2014. An average of 1.9% of the raccoon dogs were Trichinella spp. positive. 90% of the positive animals were infected with Trichinella spiralis and one animal each with Trichinella britovi and Trichinella pseudospiralis. In T. spiralis infected animals, the number of larvae found in the muscle tissue ranged between 0.5 and 235 larvae per gram (lpg), with a median of 14 larvae. A tentative temporal increase in Trichinella occurrence was seen between the time periods 2008 to 2010 and 2011 to 2014. Based on the size of the raccoon dog hunting bags of the past decade, the species spread in westerly and north-westerly direction is evident. An interesting question is how the raccoon dog will influence the Trichinella prevalence in the sylvatic cycle in these regions in the years to come.

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.

The sylvatic Trichinella cycle and its implications for Trichinella control in Germany.

Trichinellosis is a food-borne, zoonotic disease caused by a parasitic organism. Pork containing muscle larvae represents the most important source of human trichinellosis. In Germany, each slaughtered domestic swine is systematically sampled and examined for Trichinella spp. European Union legislation (EC (No.) 2075/2005) condones the approach of a risk-oriented meat inspection for Trichinella in pigs which is accompanied by monitoring programmes for pig holdings and reservoir animals. Here we discuss the current epidemiological situation of Trichinella in the sylvatic cycle in Germany and the implications for the implementation of risk-based sampling.

Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine.

Samples of intestinal content from thirty fattened pigs of six farms slaughtered at an abattoir in North-Western Germany, and faecal samples of four pigs kept as laboratory animals at the Federal Institute for Risk Assessment (BfR, Berlin, Germany) were investigated for the occurrence of microsporidia by light microscopy, PCR and sequencing. A modified Webers trichrome staining and the immunohistochemistry (the Avidin-Biotin-Peroxidase-Complex technique with a polyclonal anti-Encephalitozoon cuniculi-serum and monoclonal antibodies against Encephalitozoon intestinalis and Enterocytozoon bieneusi) was used as a screening method for the light microscopical detection of these pathogenic eukaryotes. By this light microscopically methods microsporidia suspected organisms were found in all samples (100%). By the use of PCR, microsporidia were identified in fourteen samples (41.2%). The prevalence of microsporidia infections among the farms diversifies from 0 to 80% as considered by PCR. E. bieneusi was the most prevalent species and was identified in twelve fattened pigs (40%) from five of the six tested farms (83.3%) and in two of the four laboratory animals (50%). Three of the E. bieneusi species belonged to the genotype O, one to the genotype E, and one to the genotype F. Two isolates were identified as novel genotypes and two samples showed a mixed infection of different genotypes. In three faecal samples of the pigs from two farms E. cuniculi genotype III was identified. One sample contained both microsporidia species. To our knowledge, this is the first time that the genotype III of E. cuniculi was identified in swine.