Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum . Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher Peq Pgp-11 expression was found in the gut for both genders, whereas for Peq Pgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs) causing missense mutations in the Peq Pgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM) susceptibility revealed the presence of the three SNPs and in addition statistically significant Peq Pgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P . equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance associated SNPs in populations with reduced IVM susceptibility. These SNPs are promising diagnostic candidates for detection of ML resistance with potential also for other parasitic nematode species.

P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. Expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens Pgp repertoire and provides a foundation for a better understanding of Pgp-mediated anthelmintic resistance.

Equine strongyles are a major health issue. Large strongyles can cause death of horses while cyathostomins (small strongyles) have shown increased resistance to anthelmintics worldwide. Description of strongyle communities have accumulated but little is known about the diversity of these communities and underpinning environmental factors. Strongyles were recovered after ivermectin treatment from 48 horses located in six premises in Poland. Correlation between previously published species fecundity and the observed relative abundance and prevalence were estimated. Significance of horse sex was determined at the species level (prevalence, relative abundance) and at the community level (species richness and dissimilarity between communities). Strongyle species fell into two groups, contrasted by their prevalence and relative abundance. Six to nine horses were necessary to sample at least 90% of strongyle community diversity, providing a minimal cut-off to implement sampling trial in the field. Strongyle communities entertained a network of mostly positive interactions and species co-occurrence was found more often than expected by chance. In addition, species fecundity and prevalence were negatively correlated (Pearson’s r = -0.71), suggesting functional trade-offs between species dispersal abilities and fecundity. This functional trade-off may underpin species coexistence. Horse sex was also a significant constraint shaping strongyle communities. Indeed, mares generally displayed more similar strongyle communities than stallions (P = 0.003) and Cylicostephanus calicatus was more abundant in stallions suggesting sex-specific interactions (P = 0.006). While niche partitioning is likely to explain some of the positive interactions between equine strongyle species, coexistence may also result from a functional trade-off between dispersal ability and fecundity. There is significant evidence that horse sex drives strongylid community structure, which may require differential control strategies between mares and stallions.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

The population of the European brown hare (Lepus europaeus) has been declining for the last decades in many European countries, including Poland. The goal of this study was to determine the level of endoparasite infection among hares. In 2007-2010, 83 animals were examined postmortem. The animals were weighed and analysed according to age and sex. During the dissection only the following nematodes were noticed: Trichostrongylus retortaeformis, Strongyloides papillosus, Trichuris leporis and Passalurus ambiguus in the intestinal tract and Protostrongylus pulmonalis in lungs. Body mass of hares was analysed with a general linear model (GLM) with age, sex, and presence/ absence of nematode infection as factors. The proportion of infected and uninfected hares with protozoan coccidia was compared with Fisher exact test for 2×2 contingency tables, whereas the proportion of nematode infection was compared by χ2 test. There was a significant difference in the proportion of hares infected and not infected by coccidia with the higher proportion of infected juvenile individuals (P=0.010), whereas there was no difference between males and females (P=0.41). The frequencies of hares infected vs. not infected by nematodes did not differ between sex (χ2=1.89, P=0.168) and age (χ2=0.0007, P=0.97). The mean body mass of all hares was 4.15 kg±0.40 kg. GLM model conducted for body mass of hares showed that there was a significant difference only between juvenile and adult hares (F=24.225, P=0.000005) and no significant association between the level of endoparasite infection and sex

Examination of the biodiversity of horse strongylid communities was performed on 51 domestic horses from Southern Poland and Western Ukraine by in vivo method. All horses were dewormed with macrocyclic lactone anthelmintics. Strongylids (34,715 specimens) were collected and identified by morphological criteria. In Poland, 25 strongylid species were found. Five cyathostome species dominated in the community. Large strongyles were found in 23.1 % of horses and composed 0.56 % of total strongylid number. General structure of strongylid community was multimodal with dominant, subdominant, background and rare species. In Ukraine, 19 species were found. Five cyathostome species dominated in the community. Strongylinae were found in 17.4 % of horses and composed 0.07% of community. General structures of strongylid community were multimodal or bimodal depends on type of horse keeping conditions. Regularity of anthelmintic treatment was found to be more influential on strongylid community structure than differences in horse-keeping conditions at the both countries.