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

Identification et séparation de 2 populations de macro-parasites par cytométrie en flux

Introduction et Objectifs :Les parasites Ascaridia galli et Heterakis gallinarum sont des parasites intestinaux appartenant à la famille desnématodes. Ils infectent les volailles mais présentent une pathogénicité différente. Les œufs de ces parasites sontretrouvés dans les fientes des animaux infestés et peuvent engendrer des co-infestions d’animaux. Ils ont desmorphologies proches avec une légère différence de taille (75/80 x 45/50 μm pour A. galli et 63/75 x 36/50 μm pourH. gallinarum). Bien que les vers vivent à des endroits différents du tractus digestif (jejunum pour A. galli / caecapour H. gallinarum), les œufs émis par les vers femelles de chaque espèce se retrouvent mélangés dans les fientes.De plus, seuls les œufs embryonnés sont infestants pour la volaille. Il est donc nécessaire de trouver un moyend’identifier et de séparer ces populations parasitaires quasi-similaires morphologiquement par cytométrie en fluxafin de pouvoir les étudier et les caractériser individuellement (infections expérimentales, biologie moléculaire…).Matériel et Méthodes :Les expériences ont été réalisées à partir d’échantillons contenant une population parasitaire pure obtenue aprèsséparation des vers adultes lors d’autopsies de poulets co-infestés, maintien des vers vivants ex vivo et récupérationdes œufs émis. Un mélange des 2 populations parasitaires : œufs d’A. galli et œufs d’H. gallinarum a ensuite étéeffectué. Dans le but d’infester expérimentalement des poulets, un tri d’œufs embryonnés d’A. galli a également étéréalisé sur une population contenant des éléments parasitaires embryonnés et non-embryonnés.La différenciation et le tri par cytométrie en flux de ces populations parasitaires ont été réalisés grâce à un trieur decellules MoFlo Astrios (BeckmanCoulter). La taille de la buse, les paramètres de pression et l’ensemble des réglagesde tri ont été adaptés pour trier ces parasites de grande taille. Il a été ainsi possible de séparer sans marqueurfluorescent les différentes populations de macroparasites et de récupérer ces oeufs de parasites en microplaques.Résultats, Discussion et Conclusion :Les œufs d’A. galli et d’H. gallinarum sont très proches morphologiquement et donc particulièrement complexes àdifférencier. Les œufs embryonnés et non-embryonnés d’A. galli sont également quasi-similaires morphologiquement.Cette étude représentait ainsi un défi technique lié à la grande taille des parasites, à l’absence de marquage spécifiqueet au manque de données pour utiliser la buse de 200 μm sur le MoFlo Astrios EQ (BeckmanCoulter). L’adaptationde l’ensemble des réglages et des paramètres de pilotage et de seuillage a été nécessaire. Pour différencier les 2populations parasitaires, l’association d’une combinaison de paramètres (de morphologie et d’autofluorescences) aété utilisée et plus particulièrement une combinaison des paramètres d’autofluorescence bleue après une excitationà l’aide du laser violet et d’autofluorescence verte après une excitation à l’aide du laser bleu. Des tris cellulaires dedifférentes quantités d’œufs de parasites par puits en microplaques de 96 et 384 ont été réalisés pour ensuite êtreinoculés à des poulets. L’étude ultérieure consistait à faire des essais sur les modalités d’infestations de pouletsen vue d’une évaluation future de l’effet d’extraits de macro-algues lors des infestations parasitaires (LabcomAlgahealth)

Ivermectin treatment in lactating mares results in suboptimal ivermectin exposure in their suckling foals

International audienceThe management of equine strongyles has become problematic over the last decade because of an increased prevalence of drug-resistant isolates worldwide. Therapeutic options are therefore limited, leaving macrocyclic lactones as the most often effective drug class. However, their lipophilic properties result in a long-lasting elimination that could favour drug resistance selection. As a result, ivermectin treatment in lactating mares could promote suboptimal exposure of their foal parasites to ivermectin, thereby selecting for more resistant worms. To test for this putative transfer, we selected two groups of six foal-mare pairs, one group of mares receiving ivermectin and the other being left untreated. We compared faecal egg count trajectories in foals from the two groups and quantified plasma ivermectin concentrations in ivermectin treated mares and their foals during seven days. Our results showed limited but sustained plasmatic exposure of foals associated with non-significant faecal egg count reduction (P = 0.69). This suggests that ivermectin treatment in lactating mares results in suboptimal exposure to the drug in their foal

Evaluation of plant commercial feed additives for equine cyathostomin control

International audienceThe increasing emergence of anthelmintic-resistant parasitic isolates prompts us to reassess the management of intestinal strongylosis in horses. Additionally, societal demand is shifting toward reducing the use of chemical treatments, aligning with environmentally-friendly practices and the exploration of natural alternatives. In this context, we provide an initial view of the antiparasitic activity and the effect on immune circulating blood cells of three commercialized plant-based feed additives in ponies. Three treatments, based either on mugwort (Artemisia vulgaris), echinacea (Echinacea purpurea) or curcumin (Curcuma longa) were administrated to 18 (six per treatment) Welsh female ponies naturally infected with cyathostomins to mimic their practical use in farming conditions. Another group of six untreated ponies was used as a control. Fecal egg count (FEC), the larval development percentage and the number of red blood cells, lymphocytes, monocytes, neutrophils, eosinophils and basophils were measured the first and the last day of each treatment, and compared with those characterizing the control group. None of the three treatments showed a significant effect on the studied parameters. Moreover, the efficacy of treatments, measured from the FEC reduction compared to the control group, was weak (≤ 38.6 %). Therefore, these results do not support the practical use of these additives in equine farming, even if the determination of Cohen's d values associated with the three treatments revealed some incidences on FEC and blood immune cell counts, as well as on larval development for mugwort

Effects of dietary yeast strains on immunoglobulin in colostrum and milk of sows

International audienceThe ban of antibiotic growth promoters in pig diet required the development of alternative strategies and reinforced the importance of maternal immunity to protect neonates from intestinal disorders. Milk from sows fed active dry yeasts during gestation and lactation exhibited higher immunoglobulin (Ig) and protein content in milk at day 21 of lactation. In this study, we investigated whether the administration of Saccharomyces cerevisiae strains of various origins (Sc01, Sc02, Sb03) to sows during late gestation and lactation could induce higher Ig content in colostrum and milk. Results show that yeast supplementation did not increase significantly sow body weight at days 112 of gestation and 18 of lactation as well as piglet body weight gain from birth to weaning. In contrast, the IgG level in colostrum was increased in comparison with the control group when sows were supplemented with Sc01 at both 0.05 and 0.5% (p<0.05) and Sb03 at 0.5% (p<0.01). During the lactation, the level of milk IgG remained significantly higher in comparison with the control group when sows were supplemented with Sc02 at 0.05% and 0.5% and with Sb03 at 0.5%. Furthermore, in comparison with the control sows, the level of milk IgA was significantly maintained in sows supplemented with the 3 yeast strains at 0.05%. The incidence of piglet diarrhoea was decreased in groups Sc01 at both 0.05% and 0.5% and Sc02 at 0.05%. Thus, these results show that the 3 yeast strains display immunostimulatory effects on maternal immunity, but only Sc01 supplementation at 0.05% allowed jointly the increase of IgG level in colostrum, the maintenance of IgA level in milk and the decrease of piglet diarrhoea incidence. This stimulation of maternal immunity could be associated with a better systemic (colostrum IgG) and local (milk IgA) protection of neonates and suggests that dietary yeasts may have stimulated the local gut immune system of sows

Evaluation of the direct and immunomodulatory activity of new plants extracts as a potential food additive

International audienceAlternative strategies to chemical anthelmintic are needed for the sustainable control of equine strongylids (cyathostomins). Some plants have anthelmintic or immune system stimulating activity which, when added to the diet as a nutraceutical or food additive, have beneficial effects on health or prevent an infection (Sandoval-Castro et al., 2012). To identify potential extract or plant with such anthelmintic or immunomodulatory effects, a literature search was realized using the United State Department of Agriculture database and published records extracted from NCBI Pubmed. The candidate list was further downsized to xx compounds or plants according to intellectual property rights and phytotherapy indication. The candidates have been subjected to in vitro assays aiming to quantify their ability to inhibit cyathostomin larval development (direct activity) and to modulate the inflammatory response of murine macrophage cell lines (RAW 264.7) and equine PBMCs. Following this screening, current evidence suggest that carvacrol and cinnamaldehyde were the most potent compounds. To further refine our understanding of their activity, a differential gene expression analysis will be performed on equine monocytes stimulated or not by parasite antigen in combination or not with these extracts. This will uncover mechanisms of the horse-parasite response while establishing the first hints on the mode of action of these two compounds

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

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

Genetic × environment variation in sheep lines bred for divergent resistance to strongyle infection

International audienceDrug-resistant parasites threaten livestock production. Breeding more resistant hosts could be a sustainable control strategy. Environmental variation linked to animal management practices or to parasite species turnover across farms may however alter the expression of genetic potential. We created sheep lines with high or low resistance to Haemonchus contortus and achieved significant divergence on both phenotypic and genetic scales. We exposed both lines to chronic stress or to the infection by another parasite Trichostrongylus colubriformis, to test for genotype-by-environment and genotype-by-parasite species interactions respectively. Between-line divergence remained significant following chronic stress exposure although between-family variation was found. Significant genotype-by-parasite interaction was found although H. contortus-resistant lambs remained more resistant against T. colubriformis. Growth curves were not altered by the selection process although resistant lambs were lighter after the second round of divergence, before any infection took place. Breeding for resistance is a sustainable strategy but allowance needs to be made for environmental perturbations and worm species

Chicory (Cichorium intybus) reduces cyathostomin egg excretion and larval development in grazing horses

Cyathostomins are the most prevalent parasitic nematodes of grazing horses. They are responsible for colic and diarrhea in their hosts. After several decades of exposure to synthetic anthelmintics, they have evolved to become resistant to most compounds. In addition, the drug-associated environmental side-effects question their use in the field. Alternative control strategies, like bioactive forages, are needed to face these challenges. Among these, chicory (Cichorium intybus, Puna II cultivar (cv.)) is known to convey anthelmintic compounds and may control cyathostomins in grazing horses. To challenge this hypothesis, we measured fecal egg counts and the rate of larval development in 20 naturally infected young saddle horses (2-year-old) grazing either (i) a pasture sown with chicory (n = 10) or (ii) a mesophile grassland (n = 10) at the same stocking rate (2.4 livestock unit (LU)/ha). The grazing period lasted 45 days to prevent horse reinfection. Horses in the chicory group mostly grazed chicory (89% of the bites), while those of the control group grazed mainly grasses (73%). Cyathostomins egg excretion decreased in both groups throughout the experiment. Accounting for this trajectory, the fecal egg count reduction (FECR) measured in individuals grazing chicory relative to control individuals increased from 72.9% at day 16 to 85.5% at the end of the study. In addition, larval development in feces from horses grazed on chicory was reduced by more than 60% from d31 compared to control individuals. Using a metabarcoding approach, we also evidenced a significant decrease in cyathostomin species abundance in horses grazing chicory. Chicory extract enriched in sesquiterpenes lactones was tested on two cyathostomins isolates. The estimated IC50 was high (1 and 3.4 mg/ml) and varied according to the pyrantel sensitivity status of the worm isolate. We conclude that the grazing of chicory (cv. Puna II) by horses is a promising strategy for reducing cyathostomin egg excretion and larval development that may contribute to lower the reliance on synthetic anthelmintics. The underpinning modes of action remain to be explored further