Equids are infected by a diversity of gastrointestinal nematode parasites, including 64 species of equine strongyle nematodes from19 genera. Despite numerous surveys of horse strongyles worldwide, certain geographic regions and rare species remain understudied. In 1964, a new species of equine strongyle, Cylicocyclus pekingensis, was described from a donkey in China. Subsequently, this species was recorded in horses from Kazakhstan and reclassified as Hsiungia pekingensis (K’ung and Yang, 1964), the only species in this genus. Since then, H. pekingensis has not been reported elsewhere, with limited knowledge on its distribution and phylogeny.

This study documents the first record of H. pekingensis in North America. Adult specimens were recovered from fecal samples of a domestic horse in Alberta, Canada, following treatment with ivermectin. Species identification involved detailed morphological examination, complemented with sequencing of the internal transcribed spacer 1 (ITS1), 5.8S rRNA gene, and the internal transcribed spacer 2 (ITS2) regions of the nuclear genome. Phylogenetic analysis indicated a close evolutionary relationship with species from Poteriostomum and Parapoteriostomum genera. Nemabiome ITS2 sequencing of a paired pre-treatment sample also detected the presence of H. pekingensis in the studied horse. Re-analysis of public equine nemabiome datasets further detected H. pekingensis in feral horses in Alberta, but not in other regions considered. This study expands the known distribution of this rare species and enhances our knowledge of its placement in the phylogeny of equine strongyles. Furthermore, our re-analysis of public nemabiome datasets highlights the value of this approach for studying the global distribution of parasite species.

Gastrointestinal tract (GIT) nematode infections have a significant negative impact on the well-being and productivity of animals. While it is common for a host to be co-infected with multiple species of nematode parasites simultaneously, there is a lack of effective tools to study the composition of these complex parasite communities. We describe the application of the “nemabiome” amplicon sequencing to study parasitic GIT nematode communities in captive wildlife at the National Zoological Garden, South African National Biodiversity Institute. A total of 13 samples from Roan antelope (n=1), Eland (n=1), Sable antelope (n=9), Arabian oryx (n=1), and Blue duiker (n=1) were analysed for the presence of GIT nematode infections. A total of 256 operational taxonomical units (OTUs) were generated and compared to a curated database with ≥99% identity to sequences in the NCBI database. The OTUs found represented nematode species which are commonly associated with wild ruminants. These belonged to members of the genera Haemonchus, Trichostrongylus, and Cooperia, with a few OTUs classified as unidentified genera detected. These were further classified to species level, with Haemonchus contortus and Trichostrongylus colubriformis having high relative abundance across all captive ungulate species. Utilizing amplicon sequencing to examine the nemabiome holds great potential for enhancing our basic knowledge of the biology of gastrointestinal nematodes and monitoring infections. This improved understanding could guide the development of more efficacious preventive and control measures against these significant parasites, which negatively impact animal health and productivity on a global scale.

Identifying factors that drive among-individual variation in mixed parasitic infections is fundamental to understanding the ecology and evolution of host–parasite interactions. However, a lack of non-invasive diagnostic tools to quantify mixed infections has restricted their investigation for host populations in the wild. This study applied DNA metabarcoding on parasite larvae cultured from faecal samples to characterize mixed strongyle infections of 320 feral horses on Sable Island, Nova Scotia, Canada, in 2014 to test for the influence of host (age, sex and reproductive/social status) and environmental (location, local density and social group membership) factors on variation. Twenty-five strongyle species were identified, with individual infections ranging from 3 to 18 species with a mean richness (±1 s.d.) of 10.8 ± 3.1. Strongyle eggs shed in faeces were dominated by small strongyle (cyathostomins) species in young individuals, transitioning to large strongyles (Strongylus spp.) in adults. Egg counts were highest in young individuals and in the west or centre of the island for most species. Individuals in the same social group had similar parasite communities, supporting the hypothesis that shared environment may drive parasite assemblages. Other factors such as local horse density, sex, date and reproductive/social status had minimal impacts on infection patterns. This study demonstrates that mixed infections can be dynamic across host ontogeny and space and emphasizes the need to consider species-specific infection patterns when investigating mixed infections.

Alternative strategies to chemical anthelmintics are needed for the sustainable control of equine strongylids. Bioactive forages like sainfoin (Onobrychis viciifolia) could contribute to reducing drug use, with the first hints of in vitro activity against cyathostomin free-living stages observed in the past. We analysed the effect of a sainfoin-rich diet on cyathostomin population and the efficacy of oral ivermectin treatment. Two groups of 10 naturally infected horses were enrolled in a 78-day experimental trial. Following a 1-week adaptation period, they were either fed with dehydrated sainfoin pellets (70% of their diet dry matter) or with alfalfa pellets (control group) for 21-days. No difference was found between the average fecal egg counts (FECs) of the two groups, but a significantly lower increase in larval development rate was observed for the sainfoin group, at the end of the trial. Quantification of cyathostomin species abundances with an ITS-2-based metabarcoding approach revealed that the sainfoin diet did not affect the nemabiome structure compared to the control diet. Following oral ivermectin treatment of all horses on day 21, the drug concentration was lower in horses fed with sainfoin, and cyathostomin eggs reappeared earlier in that group. Our results demonstrated that short-term consumption of a sainfoin-rich diet does not decrease cyathostomin FEC but seems to slightly reduce larval development. Consumption of dehydrated sainfoin pellets also negatively affected ivermectin pharmacokinetics, underscoring the need to monitor horse feeding regimes when assessing ivermectin efficacy in the field.

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 β-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.