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 PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-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 PeqPgp-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 PeqPgp-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.

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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. Parasitic nematodes are important pathogens of livestock, companion animals and humans and the emergence and spread of anthelmintic resistance has compromised veterinary helminth control. In equines, Parascaris univalens poses a major threat particularly to juvenile horses 1,2. At present, chemotherapeutic metaphylaxis and therapy remain the most effective and commonly used strategies for veterinary helminth control although widespread anthelmintic resistance to one or multiple drug classes compromise their success 3-5. The frequent and unrestricted use of macrocyclic lactones (MLs) in domestic horses over decades has driven selection of resistant parasite populations 6. In Parascaris sp., ML resistance was first reported in the early 2000s 7 and since then has developed into a global challenge. Its presence has been demonstrated in several European countries 8-13 , North America 14-16 , as well as New Zealand 17 , Australia 18 and more recently Ethiopia 19 and Saudi-Arabia 20. Similarly, the development and spread of ML resistance has been reported in many other parasitic nematodes, including Teladorsagia circumcincta 21 and Haemonchus contortus in sheep 22 , Dirofilaria immitis 23 in dogs and even in human filarial nematodes such as Onchocerca volvulus 24. Prior to genome-wide approaches, P-glycoproteins (Pgp) were already presumed to be important contributors to ML resistance in a number of parasitic nematodes 25. Nonetheless, deciphering the mechanisms of ML resistance is challenging, partly due to the multi-genic nature of the resistanc...

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BackgroundEquine 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.MethodsStrongyles 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).ResultsStrongyle 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).ConclusionsWhile 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.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2858-9) contains supplementary material, which is available to authorized users.