S. Reckinger

Distribution of Cryptosporidium parvum subtypes in calves in Germany.

Cryptosporidium DNA was extracted from 134 faecal specimens from pre-weaned calves from different German Federal States (age range, 3-15 days old), which tested positive for oocysts by microscopic analysis. The 18S rDNA gene and the oocyst wall protein gene (COWP) were used as targets for PCR and RFLP techniques. Cryptosporidium species were identified by using SspI, MboII and RsaI endonucleases for the digestion of 18S rDNA and COWP amplified fragments, respectively. In all samples, restriction patterns corresponding to Cryptosporidium parvum were identified, which is in agreement with abundant literature data indicating C. parvum as the most common species in pre-weaned calves. In order to estimate the genetic heterogeneity among C. parvum calf isolates, 53 samples chosen to represent different German Federal States were successfully subtyped by sequence analysis of the highly polymorphic 60-kDa glycoprotein gene. All isolates belonged to the allele IIa (with seven subtypes), with the exception of one isolate that belonged to the allele IId. Moreover, three novel subtypes of the allele family IIa have been found. This study confirms the utility of genotyping and subtyping tools in characterizing the transmission of Cryptosporidium spp. This is the first molecular epidemiological report about subtyping of Cryptosporidium bovine isolates in Germany.

Evaluation of a Western Blot and ELISA for the detection of anti-Trichinella-IgG in pig sera

Human trichinellosis is a foodborne disease caused by ingestion of infective Trichinella muscle larvae via pork or meat of other food animals which are susceptible to this zoonotic parasite. There are new approaches for a risk-oriented meat inspection for Trichinella in pigs which are accompanied by monitoring programmes on herd level to control freedom from this parasite. For this purpose, testing schemes utilizing serological tests with a high sensitivity and specificity are required. This study aimed at the evaluation of an ELISA and a Western Blot (WB) for the detection of anti-Trichinella-IgG in terms of sensitivity and specificity taking results of artificial digestion as gold standard. For this purpose, 144 field sera from pigs confirmed as Trichinella-free as well as 159 sera from pigs experimentally infected with T. spiralis (123), T. britovi (19) or T. pseudospiralis (17) were examined by ELISA (excretory-secretory antigen) and WB (crude worm extract). Sera from pigs experimentally infected with four other nematode species were included to investigate the cross-reactivity of the antigen used in the WB. For all Trichinella-positive pig sera, band pattern profiles were identified in the WB and results were analysed in relation to ELISA OD% values. Testing of pig sera revealed a sensitivity of 96.8% for the ELISA and 98.1% for the WB whereas the methods showed a specificity of 97.9 and 100%, respectively. WB analysis of Trichinella-positive pig sera revealed five specific band patterns of 43, 47, 61, 66, and 102 kDa of which the 43 kDa protein was identified as the predominant antigen. The frequency of the band pattern profile was irrespective of the dose and the period of infection as well as the Trichinella species investigated. In conclusion, monitoring in swine farms for Trichinella antibodies should be based on screening pig sera by means of ELISA followed by confirmatory testing through WB analysis.

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.

Polymerase chain reaction - Restriction fragment length polymorphism analysis for the differentiation of Trichinella nativa and Trichinella britovi

Recently, Trichinella nativa was identified in foxes in Germany and Poland, indicating that the geographical distribution of T. nativa is not restricted to areas north of the isotherm -4°C in January. In the European Union, legislation requires that a regular monitoring of the occurrence of Trichinella spp. in indicator animals such as foxes or raccoon dogs be carried out. The Trichinella isolates must also be identified on a species level. The multiplex PCR recommended by the Community Reference Laboratory for Trichinella allows species identification, yet the differentiation of T. nativa and Trichinella britovi, a widespread Trichinella species in the temperate regions of Europe, is unstable. We therefore describe an easy and reliable method for the differentiation of the two species, which can be utilised to monitor a potential spread of T. nativa in Central Europe.

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.

A study on the suitability of inactivated Trichinella spiralis larvae for proficiency samples.

The consumption of raw or undercooked Trichinella infected meat, especially pork and horse meat, can have important implications for public health. Therefore each animal carcass from a Trichinella susceptible species intended for human consumption must be examined for Trichinella. Laboratories carrying out testing of official control samples must undergo a quality assurance program and should regularly participate in proficiency testing schemes. To date, Trichinella proficiency samples are prepared with live larvae, which, as a level 2 pathogen, require specific shipping and disinfection procedures. Therefore, the suitability of using inactivated Trichinella larvae as proficiency samples was tested. We found that Trichinella larvae treated with 2% formaldehyde for 24h had lost their infectivity and showed a comparable recovery rate to naïve larvae after artificial digestion, albeit with a prolonged sedimentation time.

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.

Comparison of three artificial digestion methods for detection of non-encapsulated Trichinella pseudospiralis larvae in pork

In a ring trial involving five laboratories (A, B, C, D, and E), three different methods of artificial digestion were compared for the detection of non-encapsulated Trichinella pseudospiralis larvae in minced meat. Each sample panel consisted of ten 1g minced pork samples. All samples in each panel were derived from a bulk meat preparation with a nominal value of either 7 or 17 larvae per g (lpg). Samples were tested for the number of muscle larvae using the magnetic stirrer method (labs A, B, and E), stomacher method (lab B), and Trichomatic 35 (labs C and D). T. pseudospiralis larvae were found in all 120 samples tested. For samples with 7 lpg, larval recoveries were significantly higher using the stomacher method versus the magnetic stirrer method, but there were no significant differences for samples with 17 lpg. In comparing laboratory results irrespective of the method used, lab B detected a significantly higher number of larvae than lab E for samples with 7 lpg, and lab E detected significantly less larvae than labs A, B, and D in samples with 17 lpg. The lowest overall variation for quantitative results (i.e. larval recoveries which were outside the tolerance range) was achieved by using the magnetic stirrer method (22%), followed by the stomacher method (25%), and Trichomatic 35 (30%). Results revealed that T. pseudospiralis larvae in samples with a nominal value of 7 and 17 lpg can be detected by all three methods of artificial digestion.

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.