Dynamic stability of the cyathostomin – gut microbiota interactions in horses

International audienceThe dynamics of the helminth-microbiota assemblage in their host remain largely unexplored. Here, we used the horse – cyathostomin system to quantify the resilience of the nematode-bacteria interactions following an anthelmintic treatment. Ten infected Welsh ponies received a pyrantel treatment to eliminate adult parasites in the gut lumen. These were matched with uninfected treated individuals to isolate the treatment effect. These two groups were matched with untreated control individuals. Metabarcoding approaches were implemented to track faecal microbiota and cyathostomin community compositions over a 42-day time-course.The nemabiome approach identified 13 species overwhelmed by the abundance of Cylicocyclus nassatus that accounted for 52.6% of the overall diversity. The gut microbiota of infected horses exhibited higher Shannon entropy and bacterial species turnover, suggesting orderly rearrangements of assemblages. Enterococcus abundance could however discriminate between infected and uninfected ponies.Following pyrantel treatment, the dynamic stability of bacterial community in treated horses chiefly increased towards unstability but reached a stasis with limited variation across ponies afterwards. This would be compatible with creation of a new equilibrium between bacterial genera upon pyrantel treatment. Using a convergent cross-mapping approach, we evidenced a set of core bacterial genera, i.e. Fibrobacter, Saccharofermentans, and Aloprevotella that likely provide the stabilizing forces towards this new equilibrium. The cyathostomin community recovered quickly with a primarily unchanged structure 42 days after treatment.Our data provide the first description of the resilience of the horse gut microbiota in infected horses, indicating a heavily canalized system

Species interactions, stability, and resilience of the gut microbiota - helminth assemblage in horses

The nature and strength of interactions entertained among helminths and their host gut microbiota remain largely unexplored. Using 40 naturally infected Welsh ponies, we tracked the gut microbiota-cyathostomin temporal dynamics during parasite community removal and reassembly, and the associated host immune response. Infected ponies harboured 14 species of cyathostomins, overwhelmed by the abundance of Cylicocyclus nassatus. Parasite carriers exhibited gut environment modifications, higher Shannon entropy and orderly rearrangements of prokaryotic assemblages, with protective Clostridia species contributing to the successional nemabiome-microbiota crosstalk. Yet, the gut ecosystem was remarkably stable, and the host systemic response defined enrichment for B-cell activation and IgA production without corresponding changes in parasite burdens. Therefore, Clostridia microbial protection likely reduced fluctuating dynamics between the microbiota-parasite-host triad and favoured parasite tolerance. The system stability was disrupted by pyrantel treatment and parasite removal, with dire early consequences on the gut environment, microbiota diversity, and cytokine networks while highlighting the detrimental effect of cyathostomin burdens on Enterococcus spp. Both ecological communities were highly resilient to disturbance and recovered their pre-treatment compositions but for Cylicostephanus longibursatus in the parasite community. However, gut microbiotas failed to restore their original stability and shifted towards an interacting unstable state, with transient coexistence between Clostridia and core bacterial taxa, e.g. Fibrobacter and Prevotella, evoking their crucial role as stabilising forces for this new equilibrium. These observations highlight how anthelmintic treatment alters the gut microbiota stability and open new perspectives for adding nutritional intervention to current parasite management strategies in the field

Comparison of two molecular barcodes for the study of equine strongylid communities with amplicon sequencing

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

Evaluation of the nemabiome approach for the study of equine strongylid communities

Basic knowledge on the biology and epidemiology of equine strongylid species remains insufficient although it would contribute to the design of better parasite control strategies. Nemabiome is a convenient tool to quantify and to 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 cistron and its predictive performance and associated biases both remain unaddressed. This study aimed to bridge this knowledge gap using cyathostomin mock communities and comparing performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. The effects of bioinformatic parameters were investigated to determine the best analytical pipelines. Subsequently, barcode predictive abilities were compared across various mock community compositions. The replicability of the approach and the amplification biases of each barcode were estimated. Results were also compared between various types of biological samples, i.e. eggs, infective larvae or adults. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, a reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types, although infective larvae may remain the most tractable in the field. Additional strategies to improve the COI barcode performances are discussed. These results underscore the critical need of mock communities for metabarcoding purposes

Effect of sainfoin ( Onobrychis viciifolia ) on cyathostomin eggs excretion, larval development, larval community structure and efficacy of ivermectin treatment in horses

International audienceAlternative 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

Effect of sainfoin (Onobrychis viciifolia) on cyathostomin eggs excretion, larval development, larval community structure and efficacy of ivermectin treatment in horses

Ce poster a également été présenté sous forme de présentation Flash.National audienceAlternative 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