Faecal egg count reduction test in goats: Zooming in on the genus level.

The faecal egg count reduction test (FECRT) is the most widely used method to assess treatment efficacy against gastrointestinal nematodes (GIN). Information on genera composition of the GIN community is not available with this test and it is commonly obtained by identifying cultured third-stage larvae (L3) or through molecular assays in the post-treatment survey, but results provided are usually only qualitative or semi-quantitative. The updated WAAVP guidelines now recommend assessing anthelmintic efficacy for each GIN genus/species separately (genus-specific FECRT), but this approach is poorly employed in Europe and in goats especially. For this reason, four FECRT trials were conducted using oxfendazole and eprinomectin in two Italian goat farms. Samples were processed individually using the McMaster technique and then pooled to create two samples from faeces of 5 animals each. Pooled samples were analysed using the McMaster and cultured for seven days at 26°C to obtain L3s. The genus-specific FECRT was based on larval identification, integrating coproculture and FEC results. Larvae were identified as Haemonchus, Trichostrongylus, Teladorsagia, Oesophagostomum / Chabertia and Bunostomum. Molecular assays (a multiplex real-time PCR and two end-point PCRs) were also implemented on pooled samples to support the morphological identification. The Spearmann Rho test confirmed a high correlation between the two approaches (Rho = 0.941 and Rho = 0.914 respectively for Haemonchus and Trichostrongylus, the two most common genera). Both oxfendazole and eprinomectin were effective in one farm, while none in the other farm (FECR = 75.9% and 73.3% respectively). In the second farm, the genus-specific FECRT highlighted a different response to treatment among genera: oxfendazole lacked efficacy against both Haemonchus and Trichostrongylus spp., eprinomectin only against Haemonchus, while all other genera were susceptible to both drugs. This study brings new attention on the importance of adopting a genus-specific approach to identify and quantify differences in susceptibility to anthelmintics among genera in goats, providing support for FECRT interpretation, anthelmintic resistance evaluation and evidence-based GIN control.

An alien parasite in a changing world - Ashworthius sidemi has lost its traditional seasonal dynamics.

A non-native nematode Ashworthius sidemi has emerged in captive fallow deer in Central and Eastern Europe over the last decade. Although this parasite has been spreading in the wild outside it's native distributional range and colonising local European host species since the middle of the last century, limited information has been published on the seasonality of A. sidemi and its susceptibility to anthelmintics. To address this knowledge gap, we conducted a study to investigate seasonal dynamics of the non-native parasite in the current Central European climate conditions. We collected freshly voided faecal pellets at four-week intervals from February 2018 to February 2020 at a fallow deer reserve with a known history of A. sidemi presence. The faecal pellets obtained were pooled after each site visit (n = 25) and coprocultured to obtain the third stage larvae of trichostrongylid nematodes at monthly intervals. Total genomic DNA was extracted from the recovered larvae. Using real-time multiplex PCR, A. sidemi DNA was detected in 17 out of 25 larval samples (68% prevalence). During the monitoring period, the annual administration of ivermectin based premix (Cermix) took place in January 2018, 2019, and 2020, and additionally a mixture of rafoxanide and mebendazole (Rafendazol) was administered once in spring 2019. The probability of parasite presence was significantly influenced by the time since the drug administration (p = 0.048) and the mean temperature at the location (p = 0.013). Larval samples negative for A. sidemi were always identified shortly after the drug administration. However, rapid pasture contamination by the parasite eggs from two to three months after Cermix administration and within one month after Rafendazol administration suggest only a short-lived efficacy of both administered drugs. The abundance of A. sidemi DNA was positively affected by mean temperature (p = 0.044) and remained relatively stable throughout the monitoring period, with the highest peak in August 2018 and 2019. Pasture contamination with A. sidemi eggs occurred almost all year round, with the exception of the beginning of 2018, 2019, and 2020. These findings indicate adaptation of a non-native parasite to the current climatic conditions of the Czech Republic resulted in negligible seasonal patterns of parasite egg shedding.

Transcriptomic and proteomic profiling of peptidase expression in Fasciola hepatica eggs developing at host's body temperature.

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite's communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs, cultured at 37 °C to represent gall bladder retained eggs, for different time periods and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. We demonstrated that F. hepatica eggs undergo significant molecular changes when cultured at the physiological temperature of the definitive host. Egg transcriptome is subject to numerous subtle changes while their proteome is even more variable. The peptidase profile is considerably modified on both transcriptome and proteome level. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Activities of threonine peptidases were detected constantly, while the cysteine peptidases prevailing in freshly laid eggs are substituted by aspartic peptidase and metallopeptidase activities in the later stages of egg development.

The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays.

BACKGROUND: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. METHODS: Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. RESULTS: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. CONCLUSIONS: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

The use of high resolution melting analysis of ITS-1 for rapid differentiation of parasitic nematodes Haemonchus contortus and Ashworthius sidemi.

Among gastrointestinal nematodes, haematophagous strongylids Haemonchus contortus and Ashworthius sidemi belong to the most pathogenic parasites of both domestic and wild ruminants. Correct identification of parasitic taxa is of crucial importance in many areas of parasite research, including monitoring of occurrence, epidemiological studies, or testing of effectiveness of therapy. In this study, we identified H. contortus and A. sidemi in a broad range of ruminant hosts that occur in the Czech Republic using morphological/morphometric and molecular approaches. As an advanced molecular method, we employed qPCR followed by High Resolution Melting analysis, specifically targeting the internal transcribed spacer 1 (ITS-1) sequence to distinguish the two nematode species. We demonstrate that High Resolution Melting curves allow for taxonomic affiliation, making it a convenient, rapid, and reliable identification tool.

Molecular detection of Toxoplasma gondii in feathered game intended for human consumption in the Czech Republic.

Toxoplasma gondii is an important ubiquitous protozoan parasite, which can infect almost all warm-blooded vertebrates, including humans. The diagnosis of T. gondii infection is crucial for the prevention, surveillance, and control of its transmission. Here, a triplex real-time PCR assay targeting the B1 gene and 529rep element was used to determine the presence of T. gondii in feathered game (Anas platyrhynchos and Phasianus colchicus) hunted in the Czech Republic. The prevalence of T. gondii was 5.4% in wild ducks (n = 280) and 3.4% in common pheasants (n = 350). Additionally, genotyping of 28 T. gondii-positive samples revealed the presence of archetypal genotypes II and III as well as non-archetypal genotypes combining both type II and III alleles. Our results suggest that consumption of feathered game could pose a risk of T. gondii transmission to humans in the Czech Republic.

Fast and Reliable Differentiation of Eight Trichinella Species Using a High Resolution Melting Assay.

High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without sequencing. HRMA has previously been adopted to determine variability in the amplified genes of a number of organisms. However, only one work to date has focused on pathogenic parasites-nematodes from the genus Trichinella. In this study, we employed a qPCR-HRMA assay specifically targeting two sequential gene fragments-cytochrome c oxidase subunit I (COI) and expansion segment V (ESV), in order to differentiate 37 single L1 muscle larvae samples of eight Trichinella species. We show that qPCR-HRMA based on the mitochondrial COI gene allows differentiation between the sequences of PCR products of the same length. This simple, rapid and reliable method can be used to identify at the species level single larvae of eight Trichinella taxa.