A Large-Scale Outbreak of Trichinellosis from Infected Wild Boar Meat in Croatia and the Role of Real-Time PCR Assays in Confirming the Source of the Disease.

BACKGROUND: Trichinellosis in Croatia posed a significant health concern during the 1990s, followed by a notable improvement in the epidemiological situation. However, in 2017, there was a resurgence, with 37 recorded cases in 3 outbreaks and 3 sporadic cases. The source of this epidemic was homemade meat products derived from wild boar meat, leading to 26 infections. METHODS: At the beginning of the outbreak and during the treatment of the patients, the medical and epidemiological records prepared throughout the investigation and over the course of patient treatment were reviewed. The recovery of the first-stage (L1) larvae from suspect meat products was achieved by artificial digestion. The molecular identification of the isolated larvae was performed by multiplex PCR. The molecular identification of the meat used to prepare the meat products was performed by real-time PCR assays. RESULTS: The epidemic started in early 2017. In total, 71 exposed persons were documented: 26 with clinical symptoms and 3 hospitalised in two cities in different counties. The L1 burden in three different meat products was from 5.25 to 7.08 larvae per gram (LPG), and T. spiralis was determined as the aetiological agent of the outbreak. The molecular and biological identification confirmed that implicated meat products were made solely from wild boar meat. CONCLUSIONS: Although trichinellosis is no longer a frequent occurrence in Croatia, several cases are still registered nearly every year. Wild boar meat poses an important risk factor for human health if compulsory testing is not conducted before consumption, especially if the meat products are consumed without proper thermal processing.

Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentication of Meat Products.

Single-nucleotide polymorphisms (SNPs) are powerful molecular markers for the identification and differentiation of closely related organisms. A variety of methods can be used to determine the allele that is present at a specific locus in the genome, including real-time PCR by using an allele-specific primer. In order to increase the selectivity for the target allele, deliberate mismatch bases at the 3' end of the allele-specific primer may be introduced. This strategy has already been used for the identification and differentiation of microorganisms and plants. We have recently developed real-time PCR assays involving mismatch primers for the identification and differentiation of closely related deer species (red deer, fallow deer, sika deer) or the discrimination of wild boar and domestic pig in game meat products. These methods are applicable to detect meat species adulteration in food products.In this chapter, we offer a protocol for the design of PCR primer/probe systems suitable for meat species authentication in food. We address the retrieval and alignment of sequences, primer design by using a commercial software and the introduction of deliberate mismatch bases. In addition, we describe how the suitability of primer/probe systems can be tested in silico and in practice. We use the design of PCR primer/probe systems for wild boar and domestic pig as example.

Applicability of a duplex and four singleplex real-time PCR assays for the qualitative and quantitative determination of wild boar and domestic pig meat in processed food products.

Appropriate analytical methods are needed for the detection of food authentication. We investigated the applicability of a duplex real-time PCR assay targeting chromosome 1 and two singleplex real-time PCR assays targeting chromosome 9, both published recently, for the qualitative and quantitative determination of wild boar and domestic pig in processed food products. In addition, two singleplex real-time PCR assays targeting chromosome 7 were tested for their suitability to differentiate the two subspecies. Even by targeting the three genome loci, the probability of misclassification was not completely eliminated. Application of the real-time PCR assays to a total of 35 commercial meat products, including 22 goulash products, revealed that domestic pig DNA was frequently present, even in 14 out of 15 products declared to consist of 100% wild boar. Quantitative results obtained with the real-time PCR assays for wild boar (p < 0.001) and those for domestic pig (p < 0.001) were significantly different. However, the results obtained with the real-time PCR assays for wild boar (r = 0.673; p < 0.001) and those for domestic pig (r = 0.505; p = 0.002) were found to be significantly correlated. If the rules given in the paper are followed, the real-time PCR assays are applicable for routine analysis.

Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR.

Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krskopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.

Tetraplex real-time PCR assay for the simultaneous identification and quantification of roe deer, red deer, fallow deer and sika deer for deer meat authentication.

Analytical methods are needed for the identification and quantification of meat species to detect food adulteration. Since game meat is more expensive than meat from domesticated animal species, it is a potential target for adulteration. We present a tetraplex real-time PCR assay that allows the simultaneous determination of the content of roe deer, red deer, fallow deer and sika deer. The tetraplex assay showed only moderate cross-reactivity with closely related species. After optimization the tetraplex assay had a limit of detection of 0.1% (w/w) and a limit of quantification of 0.5% (w/w) for each of the four deer species. The tetraplex assay was found to be robust, slight modifications of the experimental setup did not lower its performance. Recoveries obtained by analyzing DNA mixtures and DNA isolates from model game sausages were similar to those obtained with the singleplex assays.

Sika deer (Cervus nippon)-specific real-time PCR method to detect fraudulent labelling of meat and meat products.

Since game meat is more valuable and expensive than meat from domesticated animal species it is a potential target for adulteration. Analytical methods must allow the identification and quantification of meat species to be applicable for the detection of fraudulent labelling. We developed a real-time PCR assay for the authentication of sika deer (Cervus nippon) and products thereof. The primer/probe system amplifies a 71 bp fragment of the kappa-casein precursor gene. Since the target sequence contained only one sika deer-specific base, we introduced a deliberate base mismatch in the forward primer. The real-time PCR assay did not show cross-reactivity with 19 animal and 49 plant species tested. Low cross-reactivity was observed with red deer, fallow deer, reindeer and moose. However, with a ΔCt value of ≥11.79 between sika deer and the cross-reacting species, cross-reactivity will not affect the accuracy of the method. LOD and LOQ, determined by analysing serial dilutions of a DNA extract containing 1% (w/w) sika deer DNA in pig DNA, were 0.3% and 0.5%, respectively. The accuracy was evaluated by analysing DNA mixtures and DNA isolates from meat extract mixtures and meat mixtures. In general, recoveries were in the range from 70 to 130%.

Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration.

The aim of the present study was to develop a real-time PCR assay for the identification and quantification of fallow deer (Dama dama) in food to detect food adulteration. Despite high sequence homology among different deer species, a fallow deer-specific primer/probe system targeting a fragment of the nuclear MC1-R gene was designed. This primer/probe system did not amplify DNA from 19 other animals and 50 edible plant species. Moderate cross-reactivity was observed for sika deer, red deer, roe deer, reindeer and wild boar. The LOD and LOQ of the real-time PCR assay were 0.1% and 0.4%, respectively. To validate the assay, DNA mixtures, meat extract mixtures, meat mixtures and model game sausages were analyzed. Satisfactory quantitative results were obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest.

Effects of terminal dimethylation and metal coordination of proline-2-formylpyridine thiosemicarbazone hybrids on lipophilicity, antiproliferative activity, and hR2 RNR inhibition.

The nickel(II), copper(II), and zinc(II) complexes of the proline-thiosemicarbazone hybrids 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (L-Pro-FTSC or (S)-H2L(1)) and 3-methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (D-Pro-FTSC or (R)-H2L(1)), as well as 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine 4,4-dimethyl-thiosemicarbazone (dm-L-Pro-FTSC or (S)-H2L(2)), namely, [Ni(L-Pro-FTSC-2H)]2 (1), [Ni(D-Pro-FTSC-2H)]2 (2), [Ni(dm-L-Pro-FTSC-2H)]2 (3), [Cu(dm-L-Pro-FTSC-2H)] (6), [Zn(L-Pro-FTSC-2H)] (7), and [Zn(D-Pro-FTSC-2H)] (8), in addition to two previously reported, [Cu(L-Pro-FTSC-2H)] (4), [Cu(D-Pro-FTSC-2H)] (5), were synthesized and characterized by elemental analysis, one- and two-dimensional (1)H and (13)C NMR spectroscopy, circular dichroism, UV-vis, and electrospray ionization mass spectrometry. Compounds 1-3, 6, and 7 were also studied by single-crystal X-ray diffraction. Magnetic properties and solid-state high-field electron paramagnetic resonance spectra of 2 over the range of 50-420 GHz were investigated. The complex formation processes of L-Pro-FTSC with nickel(II) and zinc(II) were studied in aqueous solution due to the excellent water solubility of the complexes via pH potentiometry, UV-vis, and (1)H NMR spectroscopy. The results of the antiproliferative activity in vitro showed that dimethylation improves the cytotoxicity and hR2 RNR inhibition. Therefore, introduction of more lipophilic groups into thiosemicarbazone-proline backbone becomes an option for the synthesis of more efficient cytotoxic agents of this family of compounds.