Haemonchus contortus Adopt Isolate-Specific Life History Strategies to Optimize Fitness and Overcome Obstacles in Their Environment: Experimental Evidence.

Gastrointestinal nematodes (GIN) use flexible life history strategies to maintain their fitness under environmental challenges. Costs incurred by a challenge to one life trait can be recouped by increasing the expression of subsequent life traits throughout their life cycle. Anticipating how parasites respond to the challenge of control interventions is critical for the long-term sustainability of the practice and to further ensure that the parasites withstand favourable adaptive responses. There is currently limited information on whether distinct populations of a GIN species respond to the same environmental challenge in a consistent manner, with similar alterations to their life history strategies or comparable fitness outcomes. This study compared the life history traits and experimental fitness of three distinct Haemonchus contortus isolates exposed to environmental challenges at both the parasitic (i.e., passage through resistant or susceptible sheep) and free-living (i.e., exposure to diverse climatic conditions) life stages. The key findings show that H. contortus maintain their fitness under challenge with isolate-specific alterations to their life history strategies. Further, partial exploration of the H. contortus isolates transcriptomes using cDNA-AFLP methods confirmed disparate expression profiles between them. These results bring fresh insights into our understanding of the non-genetic adaptive processes of GIN that may hinder the efficacy of parasite control strategies.

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

Drug-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.

Integrative biology defines novel biomarkers of resistance to strongylid infection in horses.

The widespread failure of anthelmintic drugs against nematodes of veterinary interest requires novel control strategies. Selective treatment of the most susceptible individuals could reduce drug selection pressure but requires appropriate biomarkers of the intrinsic susceptibility potential. To date, this has been missing in livestock species. Here, we selected Welsh ponies with divergent intrinsic susceptibility (measured by their egg excretion levels) to cyathostomin infection and found that their divergence was sustained across a 10-year time window. Using this unique set of individuals, we monitored variations in their blood cell populations, plasma metabolites and faecal microbiota over a grazing season to isolate core differences between their respective responses under worm-free or natural infection conditions. Our analyses identified the concomitant rise in plasma phenylalanine level and faecal Prevotella abundance and the reduction in circulating monocyte counts as biomarkers of the need for drug treatment (egg excretion above 200 eggs/g). This biological signal was replicated in other independent populations. We also unravelled an immunometabolic network encompassing plasma beta-hydroxybutyrate level, short-chain fatty acid producing bacteria and circulating neutrophils that forms the discriminant baseline between susceptible and resistant individuals. Altogether our observations open new perspectives on the susceptibility of equids to strongylid infection and leave scope for both new biomarkers of infection and nutritional intervention.

Nicotine-sensitive acetylcholine receptors are relevant pharmacological targets for the control of multidrug resistant parasitic nematodes.

The control of parasitic nematodes impacting animal health relies on the use of broad spectrum anthelmintics. However, intensive use of these drugs has led to the selection of resistant parasites in livestock industry. In that respect, there is currently an urgent need for novel compounds able to control resistant parasites. Nicotine has also historically been used as a de-wormer but was removed from the market when modern anthelmintics became available. The pharmacological target of nicotine has been identified in nematodes as acetylcholine-gated ion channels. Nicotinic-sensitive acetylcholine receptors (N-AChRs) therefore represent validated pharmacological targets that remain largely under-exploited. In the present study, using an automated larval migration assay (ALMA), we report that nicotinic derivatives efficiently paralyzed a multiple (benzimidazoles/levamisole/pyrantel/ivermectin) resistant field isolate of H. contortus. Using C. elegans as a model we confirmed that N-AChRs are preferential targets for nornicotine and anabasine. Functional expression of the homomeric N-AChR from C. elegans and the distantly related horse parasite Parascaris equorum in Xenopus oocytes highlighted some striking differences in their respective pharmacological properties towards nicotine derivative sensitivity. This work validates the exploitation of the nicotine receptors of parasitic nematodes as targets for the development of resistance-breaking compounds.

Strongyle Infection and Gut Microbiota: Profiling of Resistant and Susceptible Horses Over a Grazing Season.

Gastrointestinal strongyles are a major threat to horses' health and welfare. Given that strongyles inhabit the same niche as the gut microbiota, they may interact with each other. These beneficial or detrimental interactions are unknown in horses and could partly explain contrasted susceptibility to infection between individuals. To address these questions, an experimental pasture trial with 20 worm-free female Welsh ponies (10 susceptible (S) and 10 resistant (R) to parasite infection) was implemented for 5 months. Fecal egg counts (FEC), hematological and biochemical data, body weight and gut microbiological composition were studied in each individual after 0, 24, 43, 92 and 132 grazing days. R and S ponies displayed divergent immunological profiles and slight differences in microbiological composition under worm-free conditions. After exposure to natural infection, the predicted R ponies exhibited lower FEC after 92 and 132 grazing days, and maintained higher levels of circulating monocytes and eosinophils, while lymphocytosis persisted in S ponies. Although the overall gut microbiota diversity and structure remained similar during the parasite infection between the two groups, S ponies exhibited a reduction of bacteria such as Ruminococcus, Clostridium XIVa and members of the Lachnospiraceae family, which may have promoted a disruption of mucosal homeostasis at day 92. In line with this hypothesis, an increase in pathobionts such as Pseudomonas and Campylobacter together with changes in several predicted immunological pathways, including pathogen sensing, lipid metabolism, and activation of signal transduction that are critical for the regulation of immune system and energy homeostasis were observed in S relative to R ponies. Moreover, S ponies displayed an increase in protozoan concentrations at day 92, suggesting that strongyles and protozoa may contribute to each other's success in the equine intestines. It could also be that S individuals favor the increase of these carbohydrate-degrading microorganisms to enhance the supply of nutrients needed to fight strongyle infection. Overall, this study provides a foundation to better understand the mechanisms that underpin the relationship between equines and natural strongyle infection. The profiling of horse immune response and gut microbiota should contribute to the development of novel biomarkers for strongyle infection.

Ivermectin failure in the control of Oxyuris equi in a herd of ponies in France.

Drug resistance in equine gastro-intestinal parasitic nematodes has been reported throughout the world. While the focus is usually put on cyathostomins, observations of macrocylic lactone failure against Oxyuris equi have accumulated over the last decade. Here we report the failure of ivermectin in the control of O. equi in an experimental Welsh pony herd. In a first trial, 6 ponies previously drenched with moxidectin and showing patent O. equi infections were administered ivermectin and subsequently monitored for O. equi egg excretion over one month. This trial demonstrated a failure of ivermectin to control O. equi egg excretion as half of ponies demonstrated recurrent egg excretion in the peri-anal region during 21days after treatment. One year later, six female Welsh ponies drenched with moxidectin demonstrated signs of itching and scratching in their peri-anal region with worms being found transiently in fecal materials three weeks later. Ponies were allocated to three treatment groups, i.e. ivermectin, pyrantel embonate and fenbendazole and monitored for egg excretion over five weeks. Fenbendazole and pyrantel embonate broke ivermectin suboptimal efficacy as soon as 8 and 14days respectively after treatment, while egg excretion remained constant throughout the 41-day long trial in the ivermectin-treated ponies. This is the first report of ivermectin failure against O. equi in France. In the absence of critical efficacy test, it remains unclear whether true resistance is at stake or if these observations confound a constitutive suboptimal efficacy of ivermectin against O. equi.

Economic assessment of FEC-based targeted selective drenching in horses.

In the face of an increased prevalence of drug-resistant cyathostomin populations, a targeted selective treatment (TST) strategy based on Faecal Egg Counts (FECs) has been proposed as an alternative management strategy. However, associated costs may be a barrier to the uptake of this strategy. Our study aims to provide an economic assessment of FEC-based TST. FECs were determined in a Welsh pony herd thrice a year from 2010 to 2014. This database was used to explore the impact of FEC price, sampling strategy (individual or pooled) and labour-associated costs. Drug price was set at the cheapest level, hence providing a conservative framework to determine the maximum viable FEC price in the context of a cost-driven horse industry. The maximum viable FEC price for a cost-efficient individual based strategy was determined by an in silico bootstrap approach consisting of randomly sampling 1000 virtual pony herds of various sizes (1 to 100 ponies) from the available database and estimating the associated costs (FEC price ranging from € 1 to € 10, anthelmintic costs and labour-associated costs). The costs and benefits of the pooling strategy that consists of basing the decision to treat on group FEC values were also investigated. This is thought to reduce FEC-based costs but may result in highly infected individuals being left undrenched, i.e. in false-negatives, as a result of FEC overdispersion. For various pool-sizes (1-20 ponies) and various cut-off thresholds (50-200 eggs/g), we sampled 1000 pony herds in silico to estimate the associated costs and determine the number of positive ponies within a negative pool. Following these simulations, pool-based FECs of various sizes were performed on 40 ponies to compare predictions with real data. Within 4 years, anthelmintic costs were cut by 80%, albeit with free FECs. In silico estimations suggested that an individual FEC-based TST would not be cost-efficient in this context for an FEC price above € 5. With a pooled FEC strategy, the proportion of false-negatives never exceeded 15% of the pool size. The combination of a 14-pony pool and a cut-off value of 150 eggs/g minimized total costs while keeping the number of false negatives to a reasonable level. Real data obtained from infected ponies however, suggested that pool size should not exceed 10 individuals, since the inhomogeneous mixing of faeces of larger pools probably reduced the correlation between average pooled FECs and the mean of individual FECs. Our study provides an economic framework that could be valuable for emphasizing the use of FEC-based approaches in the field.

Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

A suite of genes expressed during transition to parasitic lifestyle in the trichostrongylid nematode Haemonchus contortus encode potentially secreted proteins conserved in Teladorsagia circumcincta.

The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, however spreading of anthelmintic resistance has reduced their efficacy. The genes involved in the transition to parasitic lifestyle could constitute targets of interest to develop alternative control strategies. In the trichostrongylid nematode Haemonchus contortus, we have used a SSH (Suppressive Subtractive Hybridization) based approach to generate two distinct subtracted cDNA libraries specifically enriched in cDNA expressed during the early parasitic fourth stage larvae L4 (5 days post-infection). A total of 200 clones were subjected to dot-blot experiments and 46 clones were selected for further characterization. The 46 corresponding expressed sequence tags (EST) were found to cluster into nine contigs. The corresponding full-length cDNA was obtained for all candidates. The genes encoding potentially secreted proteins were investigated in more detail. RT-PCR experiments confirmed their specific expression or over expression from the early L4 larvae to the adult stages and search for homologs in the trichostrongylid species T. circumcincta was performed in order to investigate whether they may be novel cross-specific targets.

Genetic diversity of levamisole receptor subunits in parasitic nematode species and abbreviated transcripts associated with resistance.

OBJECTIVES: The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely unknown in parasitic nematodes. In contrast, genetic screens for mutants that survive exposure to LEV in the free-living nematode Caenorhabditis elegans have led to the identification of five genes (unc-38, unc-63, unc-29, lev-1 and lev-8) that encode a LEV-sensitive acetylcholine receptor (L-AChR). Loss of these genes leads to LEV resistance. In this study, orthologues of these genes were identified in three species of trichostrongylid nematodes that have a major impact on small ruminants: Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Polymorphism associated with LEV resistance have been investigated by comparing transcripts of these subunits in LEV susceptible and LEV-resistant isolates of the three strongylid species. BASIC METHODS: Partial sequences were identified by PCR experiments and full-length cDNA sequences corresponding to AChR subunits in the three trichostrongylid species were obtained using 3'-rapid amplification of cDNA ends-PCR and 5' rapid amplification of cDNA ends anchored with the spliced leader sequence, SL1. Expression of L-AChR subunits was investigated in LEV-resistant and LEV-susceptible isolates of H. contortus, T. circumcincta and T. colubriformis using reverse transcription PCR. MAIN RESULTS: We have identified a total of 20 full-length cDNA sequences corresponding to L-AChR subunits in three parasitic trichostrongylid species of which 14 correspond to novel sequences. Genes orthologous to unc-29, unc-63, unc-38 and lev-1 were found in each trichostrongylid species, whereas no gene corresponding to lev-8 has yet been identified. We have found 11 distinct paralogous sequences corresponding to the C. elegans unc-29 gene clustered in four groups revealing an unexpected diversity of unc-29-like genes. Complete coding sequences of the L-AChR subunits in two LEV-resistant and three susceptible isolates of H. contortus, T. circumcincta and T. colubriformis were essentially unchanged, but abbreviated transcripts of the unc-63 subunit were specifically expressed in resistant isolates of all three species. CONCLUSION: The candidate gene strategy developed in this study revealed an unexpectedly high diversity of L-AChR subunits specific to the trichostrongylid parasites that are a principal target for the drug LEV. Abbreviated variants, predicted to produce nonfunctional unc-63, were associated with LEV resistance. This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.

cDNA-AFLP analysis in levamisole-resistant Haemonchus contortus reveals alternative splicing in a nicotinic acetylcholine receptor subunit.

The cDNA-AFLP (cDNA-amplified fragment length polymorphism) method comparing transcripts from levamisole-resistant and susceptible Haemonchus contortus isolates has led to the successful identification of a number of potentially useful levamisole-resistance markers. In the present study, we report the characterization of the transcript-derived fragment (TDF) named HAX which was confirmed to be specifically expressed in three levamisole-resistant isolates by RT-PCR experiments. Cloning and sequencing of the full-length cDNA sequence of HAX revealed high similarity to the Caenorhabditis elegans acr-8 gene and its putative H. contortus orthologue encoding a nicotinic acetylcholine receptor subunit. This Hco-acr-8b short isoform corresponded to a spliced variant of Hco-acr-8 mRNA containing the two first exons and a part of intron 2. As nicotinic acetylcholine receptors constitute the pharmacological target of levamisole, Hco-acr-8b may potentially be involved in the molecular mechanisms leading to levamisole-resistance acquisition in H. contortus.

Benzimidazole resistance in Trichostrongylus axei in sheep: long-term monitoring of affected sheep and genotypic evaluation of the parasite.

This is the first report of benzimidazole (BZ) resistance in the nematode Trichostrongylus axei in sheep. Trichostrongylus axei infects several species of herbivores including sheep, cattle and horses, and the emergence of anthelmintic resistance could lead to significant problems in its control. Benzimidazole resistance in two sheep flocks in central France was detected by post-treatment worm counts. The sequencing of a central region of the isotype 1 beta-tubulin gene from adult T. axei recovered post-mortem revealed only one, non-synonymous single nucleotide polymorphism at position 200 (Phe200Tyr), which had already been reported for other nematodes. Seven years after BZ treatment ceased, T. axei helminths present were still resistant to BZ suggesting these parasites do not revert to susceptibility to this anthelmintic, even when the selection pressure had been removed for many years. The findings also highlight major changes in the make-up of the nematode burden in sheep flocks that accompanies the emergence of BZ resistance.

Nematode parasites and scrapie: experiments in sheep and mice.

To demonstrate the possible role of nematode parasites in the modification of host susceptibility to scrapie, experiments were conducted using sheep naturally exposed to scrapie, chosen by their genotype at the PrP gene, and infected with Teladorsagia circumcincta. Two 4-year duration experiments demonstrated that the nematode infection shortened the development of scrapie with a significant regression between the level of infection and age at first scrapie symptoms (P < 0.006). Investigations by ELISA tests in different species of nematode parasites of the digestive tract collected from scrapie infected ewes did not reveal the presence of PrPSc. In scrapie-infected C57BL mice, infected or not with Heligmosoides polygyrus at various times, parasitized animals showed a slight but significantly longer survival period. Assays on transmission by the larvae hatching from eggs collected from scrapie-infected mice were unsuccessful. We concluded that nematodes modify host susceptibility to scrapie, but their role in the horizontal transmission of the disease was not demonstrated.

Regulation of Teladorsagia circumcincta and Trichostrongylus colubriformis worm populations by grazing sheep with differing resistance status.

In an experiment lasting 4 years, changes in the Teladorsagia circumcincta and Trichostrongylus colubriformis populations were compared in lambs and adult sheep with differing resistance statuses. Two flocks of 30 rams (resistant R and susceptible S) grazed separate pastures and 8 rams were slaughtered in the middle and at the end of each grazing season. Five groups of tracer lambs were added each year to estimate the pasture infectivity and were killed for worm counts. The availability of animals with differing resistance statuses (rams and tracer lambs) and differing levels of infection made it possible to investigate the number, size and fecundity of worms of these two species. The inflammatory response was measured in the rams by counting the globule leukocytes, mast cells and eosinophils in the fundic, pyloric and intestinal mucosa. In the tracer lambs, the daily egg production by the female worms of both species was negatively correlated with the worm burden. Worm length accounted for 60 and 70% of the variation in the number of eggs in utero for T. circumcincta and T. colubriformis respectively. Worm length was closely associated with the resistance status of the host; there were greater differences between lambs, and S and R rams for T. colubriformis. T. circumcincta worm lengths were not affected by the worm number. Globule leukocyte counts were related to the worm burdens, and mast cell counts to worm length in the R and S rams. The number, size and fecundity of the worms may well be regulated by similar mechanisms in both species, but T. colubriformis seemed to be more intensively regulated than T. circumcincta. This finding could be useful in devising more effective methods of parasite control.

A short-term divergent selection for resistance to Teladorsagia circumcincta in Romanov sheep using natural or artificial challenge.

This experiment was conducted to assess the efficiency of selection on the basis of response to artificial challenges in order to breed sheep resistant to natural infection. A short-term divergent selection process was designed to estimate the genetic parameters of these two traits. Two flocks, including 100 Romanov ram lambs each, were challenged in 1990 when they were 6 months old. One flock received three artificial infections with 20 000 third-stage Teladorsagia circumcincta larvae, at intervals of 7 weeks. Faecal egg counts (FEC) were performed on Days 22, 25 and 28 post infection (p.i.) and the animals were drenched on Day 28 p.i. The other flock was grazed for 5 months on a pasture contaminated with the same species. Faecal samples were taken from the lambs at similar ages. About 5 rams with the lowest FEC and 5 with the highest FEC were selected in each flock and mated with unselected ewes. Their offspring (200 animals) were challenged in 1992, half in the same way as their sires, and the other half by the other method. Because of a drought in the summer of 1990, it was necessary to repeat part of the experiment, and in 1992 the 5 and 8 rams with the lowest and highest FEC, respectively, were selected from the offspring challenged on the pasture in 1992 and were mated with unselected ewes. Their progeny (about 80 animals) were challenged in 1994, half by natural infection, half by artificial infection. The mean FEC of the flock increased from the first to the third artificial infection. The natural infection was highly variable in different years, reflecting the difficulty of assessing resistance using this mode of challenge. Genetic parameters were estimated using animal models and REML solutions. The repeatabilities of the FEC following artificial and natural infection were 0.49 and 0.70 respectively within a period of one week, and 0.22 and 0.41 respectively for periods separated by intervals of 7 weeks; the heritabilities of the single egg count were 0.22 and 0.38 respectively. The genetic correlation was 0.87: the FEC recorded under natural or artificial infection appear to depend on the same genetic potential.