Over the preceding decades, the widespread dependence on anthelmintic drugs for managing nematodes in grazing equids has given rise to resistance against commonly used anthelmintics in various countries. This study explores the prevalence of anthelmintic resistance across 44 horse farms in Ireland. Anthelmintic efficacy was evaluated through fecal egg count reduction (FECR) tests employing the mini-FLOTAC technique. Resistance to benzimidazoles was identified in 12 out of 14 farms (FECR range: 0.00% to 86.2%). Ivermectin resistance was observed on two farms, one with an FECR of 80.70% and another with an FECR of 96.10% (lower 95% high probability density interval (HPD) <90%, 11.70%). On the remaining six farms, the reduction with ivermectin still exceeded 95%. The reduced efficacy of moxidectin was noted on two farms (FECR = 86.90% and 93.50%) and on a third farm with an FECR of 99.50 and a lower HPD interval < 90% at 24.00%. In summary, these findings emphasize the urgent need for alternative strategies in equine strongyle control that reduce reliance on anthelmintics and prioritize effective management practices on Irish equine farms to hinder the impending development of drug-resistant parasite populations.

Changes to the faecal microbiota of horses associated with administration of anthelmintic drugs is poorly defined. This study included horses with cyathostomin infection where susceptibility and resistance to oxfendazole and abamectin was known. This study assessed the changes to the faecal microbiota associated with administration of two different anthelmintics in this population. Twenty-four adult horses were included. Faecal egg counts were performed on all horses prior to random allocation into abamectin (n=8), oxfendazole (n=8) or Control groups (n=8) and at Day 14 post treatment. Faecal samples were collected for microbiota analysis prior to anthelmintic administration and on Day 3 and Day 14. From each faecal sample, DNA was extracted prior to PCR amplification, next generation sequencing and analysis using QIIME2. Anthelmintic treatment was associated with changes in alpha diversity (p <0.05), with increased evenness and diversity at Day 14 and increased richness at Day 3 within the abamectin group. Differences in relative abundance of bacteria at the phyla, family and genus taxonomic levels occurred after treatment; indicating that the microbiota was altered with anthelmintic administration. The results support that anthelmintic administration and removal of cyathostomins from the large intestine of horses is associated with changes in the faecal microbiota. The results suggest that removal of cyathostomins is associated with greater differences in microbiota, compared to anthelmintic drug administration that is ineffective in reducing cyathostomin infection. Cyathostomin removal was supported by adequate reduction of faecal egg counts, determined by faecal egg count reduction testing.

Anthelmintic performance against equine cyathostomins can be evaluated by two different non-terminal measures; the Fecal Egg Count Reduction Test (FECRT) and the Egg Reappearance Period (ERP). Most available FECRT and ERP data have been determined in populations of young horses, and very little information is available from mature and senior horses. Furthermore, it is unknown how commonly occurring equine endocrine disorders such as Insulin dysregulation (ID) and Pituitary pars intermedia dysfunction (PPID) may interfere with these measurements, but it has been suggested that horses with these conditions could be more susceptible to parasitic infections. A research population of senior horses and horses with or without PPID, ID, or both were enrolled in this study. All strongylid egg count positive horses were included in an ivermectin (200 μg/kg) efficacy study. These were distributed among the following groups: ID: six, PPID: three, PPID and ID: seven, and healthy controls: three. Strongylid fecal egg counts were determined on the day of ivermectin administration, at two weeks post deworming, and on weekly intervals until eight weeks post treatment. Determination of FECRT and ERP were carried out following World Association for the Advancement of Veterinary Parasitology guidelines. Results revealed high ivermectin efficacy with mean egg count reduction at 99.7% or above in all groups at two weeks post treatment. Egg reappearance was documented at six and seven weeks in the ID and PPID/ID groups, respectively, whereas the PPID and healthy control groups both had ERP at 8 weeks. Statistical analysis found no significant differences in egg count levels between groups during the study. The expected ERP for ivermectin is 8-10 weeks, meaning that two of the groups displayed shortened ERPs. However, due to the small group sizes, these data should be interpreted with caution. Nonetheless, results do indicate a need for further investigation of the possible influence of endocrine disorders on anthelmintic performance in horses.

Cyathostomins are globally important equine parasites, responsible for both chronic and acute pathogenic effects. The occurrence of mixed infections with numerous cyathostomin species hinders our understanding of parasite epidemiology, host-parasite dynamics, and species pathogenicity. There have been few studies of cyathostomin species occurring in horses in Ireland, where temperate climatic conditions with year-round rainfall provide suitable conditions for infection of grazing animals with bursate nematodes. Here, we amplified and sequenced the ITS-2 region of adult worms harvested at post-mortem from eleven adult horses between August 2018 and June 2020, and recorded species prevalence and abundance of worms recovered from the caecum, right ventral colon and left dorsal colon, using both BLAST and IDTAXA for taxonomic attribution. Phylogenetic relationships and community composition were also recorded and compared with other relevant studies, including a global meta-analysis. Overall, our results agree with previous studies that there does not seem to be a major difference in cyathostomin species occurrence in equids in different geographical regions. We confirmed the results of other workers in relation to the difficulties in discriminating between Cylicostephanus calicatus and Coronocyclus coronatus on the basis of ITS-2 sequences.

BACKGROUND: There is a lack of consensus on how best to balance our need to minimise the risk of parasite-associated disease in the individual horse, with the need to limit the use of anthelmintics in the population to preserve their efficacy through delaying further development of resistance.
OBJECTIVE: To develop evidence-based guidelines utilising a modified GRADE framework.
METHODS: A panel of veterinary scientists with relevant expertise and experience was convened. Relevant research questions were identified and developed with associated search terms being defined. Evidence in the veterinary literature was evaluated using the GRADE evidence-to-decision framework. Literature searches were performed utilising CAB abstracts and PubMed. Where there was insufficient evidence to answer the research question the panel developed practical guidance based on their collective knowledge and experience.
RESULTS: Search results are presented, and recommendation or practical guidance were made in response to 37 clinically relevant questions relating to the use of anthelmintics in horses.
CONCLUSIONS: There was insufficient evidence to answer many of the questions with any degree of certainty and practical guidance frequently had to be based upon extrapolation of relevant information and the panel members\' collective experience and opinions.
CONCLUSIONS: Equine parasite control practices and current recommendations have a weak evidence base. These guidelines highlight changes in equine parasite control that should be considered to reduce the threat of parasite-associated disease and delay the development of further anthelmintic resistance.

Long-term intensive use of anthelmintics for parasite control of livestock, companion animals, and humans has resulted in widespread anthelmintic resistance, a problem of great socioeconomic significance. But anthelmintic therapy may also select for other biological traits, which could have implications for anthelmintic performance. Here, we highlight recent examples of changing parasite dynamics following anthelmintic administration, which do not fit the definition of anthelmintic resistance. We also consider other possible examples in which anthelmintic resistance has clearly established, but where coselection for other biological traits may have also occurred. We offer suggestions for collecting more information and gaining a better understanding of these phenomena. Finally, we propose research questions that require further investigation and make suggestions to help address these knowledge gaps.

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific \'serum score\' for each horse. Validation dataset results were then used to assess the optimised test\'s performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection.

Over the past three decades, equine strongylid egg reappearance periods (ERPs) have shortened substantially for macrocyclic lactone anthelmintics. The ERPs of ivermectin and moxidectin were originally reported in the 8-10 and 12-16 week ranges, respectively, but several recent studies have found them to be around 4-5 weeks for both actives. This loss of several weeks of suppressed strongylid egg output could have substantial implications for parasite control. This study made use of a computer simulation model to evaluate the impact of shortened ERPs on the anthelmintic performance of ivermectin and moxidectin against equine cyathostomins. The original ERPs were set to 7.1 and 15.4 weeks for ivermectin and moxidectin, respectively, while the reduced ERP was set to 4.6 weeks for both actives. Simulations were set to compare model outputs between original and reduced ERP scenarios and results expressed as percent increase in strongylid egg output, infective third stage larvae on herbage (L3h), and encysted early third stage larvae (EL3). For each drug, simulations were evaluated for two different treatment scenarios (2 and 4 treatments annually), two different age groups (yearlings and adults), and for four different climates (cold humid continental, temperate oceanic, humid subtropical, and hot/cold semi-arid). Across all simulations, there was a substantial increase of the three evaluated parameters. With the ivermectin simulations, all three parameters increased in the 100-300% range across climates, age groups and treatment intensities. The moxidectin simulations displayed a wider range of results with parameters increasing from a few hundred to several thousand percent. The increases were most pronounced for L3h in the two cooler climates, reaching as high as 6727%. Overall, the loss of anthelmintic performance was at a magnitude of 10 times larger for moxidectin compared to ivermectin. This performance loss was climate dependent, and was also affected by treatment intensity, but not by horse age. This is the first study to evaluate consequences of shortened ERPs in horses and demonstrated a substantial loss in anthelmintic performance resulting from this development. The results illustrate that anthelmintic efficacy is more than the percent reduction of fecal egg counts at 14 days post treatment, and that substantial anthelmintic performance can be lost despite FECRTs remaining at 100%.

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.

BACKGROUND: Equine strongyles encompass more than 64 species of nematode worms that are responsible for growth retardation and the death of animals. The factors underpinning variation in the structure of the equine strongyle community remain unknown.
METHODS: Using horse-based strongyle community data collected after horse deworming (48 horses in Poland, 197 horses in Ukraine), we regressed species richness and the Gini-Simpson index upon the horse\'s age, faecal egg count, sex and operation of origin. Using the Ukrainian observations, we applied a hierarchical diversity partitioning framework to estimate how communities were remodelled across operations, age groups and horses. Lastly, strongyle species counts collected after necropsy (46 horses in France, 150 in Australia) were considered for analysis of their co-occurrences across intestinal compartments using a joint species distribution modelling approach.
RESULTS: First, inter-operation variation accounted for > 45% of the variance in species richness or the Gini-Simpson index (which relates to species dominance in communities). Species richness decreased with horse\'s age (P = 0.01) and showed a mild increase with parasite egg excretion (P < 0.1), but the Gini-Simpson index was neither associated with parasite egg excretion (P = 0.8) nor with horse age (P = 0.37). Second, within-host diversity represented half of the overall diversity across Ukrainian operations. While this is expected to erase species diversity across communities, community dissimilarity between horse age classes was the second most important contributor to overall diversity (25.8%). Third, analysis of species abundance data quantified at necropsy defined a network of positive co-occurrences between the four most prevalent strongyle genera. This pattern was common to necropsies performed in France and Australia.
CONCLUSIONS: Taken together, these results show a pattern of β-diversity maintenance across age classes combined with positive co-occurrences that might be grounded by priority effects between the major species.