Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel ß-tubulin benzimidazole resistance mutation.

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 ß-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 ß-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans ß-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.

A database for ITS2 sequences from nematodes.

BACKGROUND: Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology. As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data. RESULTS: Here we present an ITS2 rDNA database for marker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is currently the most complete and up-to-date ITS2 database for nematodes and is able to reproduce previous analysis that used a smaller database. CONCLUSIONS: This database is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy.

Assessing anthelmintic resistance risk in the post-genomic era: a proof-of-concept study assessing the potential for widespread benzimidazole-resistant gastrointestinal nematodes in North American cattle and bison.

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 ß-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.

Population genomic and evolutionary modelling analyses reveal a single major QTL for ivermectin drug resistance in the pathogenic nematode, Haemonchus contortus.

BACKGROUND: Infections with helminths cause an enormous disease burden in billions of animals and plants worldwide. Large scale use of anthelmintics has driven the evolution of resistance in a number of species that infect livestock and companion animals, and there are growing concerns regarding the reduced efficacy in some human-infective helminths. Understanding the mechanisms by which resistance evolves is the focus of increasing interest; robust genetic analysis of helminths is challenging, and although many candidate genes have been proposed, the genetic basis of resistance remains poorly resolved. RESULTS: Here, we present a genome-wide analysis of two genetic crosses between ivermectin resistant and sensitive isolates of the parasitic nematode Haemonchus contortus, an economically important gastrointestinal parasite of small ruminants and a model for anthelmintic research. Whole genome sequencing of parental populations, and key stages throughout the crosses, identified extensive genomic diversity that differentiates populations, but after backcrossing and selection, a single genomic quantitative trait locus (QTL) localised on chromosome V was revealed to be associated with ivermectin resistance. This QTL was common between the two geographically and genetically divergent resistant populations and did not include any leading candidate genes, suggestive of a previously uncharacterised mechanism and/or driver of resistance. Despite limited resolution due to low recombination in this region, population genetic analyses and novel evolutionary models supported strong selection at this QTL, driven by at least partial dominance of the resistant allele, and that large resistance-associated haplotype blocks were enriched in response to selection. CONCLUSIONS: We have described the genetic architecture and mode of ivermectin selection, revealing a major genomic locus associated with ivermectin resistance, the most conclusive evidence to date in any parasitic nematode. This study highlights a novel genome-wide approach to the analysis of a genetic cross in non-model organisms with extreme genetic diversity, and the importance of a high-quality reference genome in interpreting the signals of selection so identified.

Survey of gastrointestinal nematodes in breeding-age heifers on 6 Saskatchewan dairy farms.

To determine the prevalence, mean fecal egg count intensities, and predominant gastrointestinal nematode species, fresh environmental fecal samples were collected from 30 grazing heifers at monthly intervals (June, July, and August) on 6 Saskatchewan dairy farms in 2016. The population average strongylid prevalence ranged from 67.2% [95% confidence interval (CI): 49.5% to 84.9%] in June to 88.3% (95% CI: 76.7% to 100.0%) in August (P = 0.05) while the mean fecal egg count ranged from 1.7 in June to 9.3 eggs/g of feces in August (P < 0.003). The predominant nematode species were identified using a deep-sequencing nemabiome assay targeting the internal transcribed spacer rDNA genomic locus of both L1 and L3-stage larvae. Cooperia oncophora and Ostertagia ostertagi were predominant on all farms, accounting for > 85% of the species. The results provide important previously unavailable data on the epidemiology of gastrointestinal nematodes (GIN) in fecal samples from these grazing heifers in Saskatchewan.

Examen des nématodes gastro-intestinaux chez les génisses en âge de se reproduire sur six fermes laitières de la Saskatchewan. Afin de déterminer la prévalence, l'intensité du dénombrement moyen d'oeufs dans les fèces et les espèces de nématodes gastro-intestinaux prédominants, des échantillons environnementaux de fèces fraîches furent prélevés de 30 génisses au pâturage à un intervalle mensuel (juin, juillet, et août) sur six fermes laitières de la Saskatchewan en 2016. La prévalence de la population moyenne de strongyles variait de 67,2 % [intervalle de confiance de 95 % (CI) : 49,5 % à 84,9 %] en juin à 88,3 % (CI 95 % : 76,7 % à 100,0 %) en août (P = 0,05) alors que le dénombrement moyen des oeufs dans les fèces variait de 1,7 en juin à 9,3 oeufs/g de fèces en août (P < 0,003). Les espèces de nématodes prédominantes furent identifiées à l'aide d'un test de séquençage profond du némabiome visant l'ADNr du locus génomique de l'espaceur interne transcrit des stades larvaires L1 et L3. Cooperia oncophora et Ostertagia ostertagi étaient prédominants sur toutes les fermes, représentant > 85 % des espèces. Ces résultats fournissent des données importantes non-disponibles auparavant sur l'épidémiologie de GIN dans des échantillons de fèces provenant de ces génisses au pâturage en Saskatchewan.(Traduit par Dr Serge Messier).

Trade-offs and mixed infections in an obligate-killing insect pathogen.

Natural populations of pathogens are frequently composed of numerous interacting strains. Understanding what maintains this diversity remains a key focus of research in disease ecology. In addition, within-host pathogen dynamics can have a strong impact on both infection outcome and the evolution of pathogen virulence, and thus, understanding the impact of pathogen diversity is important for disease management. We compared eight genetically distinguishable variants from Spodoptera exempta nucleopolyhedrovirus (SpexNPV) isolated from the African armyworm, Spodoptera exempta. NPVs are obligate killers, and the vast majority of transmission stages are not released until after the host has died. The NPV variants differed significantly in their virulence and could be clustered into two groups based on their dose-response curves. They also differed in their speed of kill and productivity (transmission potential) for S. exempta. The mixed-genotype wild-type (WT) SpexNPV, from which each variant was isolated, was significantly more virulent than any individual variant and its mean mortality rate was within the fastest group of individual variants. However, the WT virus produced fewer new infectious stages than any single variant, which might reflect competition among the variants. A survival analysis, combining the mortality and speed of kill data, confirmed the superiority of the genetically mixed WT virus over any single variant. Spodoptera exempta larvae infected with WT SpexNPV were predicted to die 2·7 and 1·9 times faster than insects infected with isolates from either of the two clusters of genotypes. Theory suggests that there are likely to be trade-offs between pathogen fitness traits. Across all larvae, there was a negative linear relationship between virus yield and speed of kill, such that more rapid host death carried the cost of producing fewer transmission stages. We also found a near-significant relationship for the same trend at the intervariant level. However, there was no evidence for a significant relationship between the induced level of mortality and transmission potential (virus yield) or speed of kill.

Introgression of ivermectin resistance genes into a susceptible Haemonchus contortus strain by multiple backcrossing.

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a "proof-of-concept" of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.

Integration of ITS-2 rDNA nemabiome metabarcoding with Fecal Egg Count Reduction Testing (FECRT) reveals ivermectin resistance in multiple gastrointestinal nematode species, including hypobiotic Ostertagia ostertagi, in western Canadian beef cattle.

A large-scale Fecal Egg Count Reduction Test (FECRT) was integrated with ITS-2 rDNA nemabiome metabarcoding to investigate anthelmintic resistance in gastrointestinal nematode (GIN) parasites in western Canadian beef cattle. The study was designed to detect anthelmintic resistance with the low fecal egg counts that typically occur in cattle in northern temperate regions. Two hundred and thirty-four auction market-derived, fall-weaned steer calves coming off pasture were randomized into three groups in feedlot pens: an untreated control group, an injectable ivermectin treatment group, and an injectable ivermectin/oral fenbendazole combination treatment group. Each group was divided into six replicate pens with 13 calves per pen. Individual fecal samples were taken pre-treatment, day 14 post-treatment, and at monthly intervals for six months for strongyle egg counting and metabarcoding. Ivermectin treatment resulted in an 82.4% mean strongyle-type fecal egg count reduction (95% CI 67.8-90.4) at 14 days post-treatment, while the combination treatment was 100% effective, confirming the existence of ivermectin-resistant GIN. Nemabiome metabarcoding of third-stage larvae from coprocultures revealed an increase in the relative abundance of Cooperia oncophora, Cooperia punctata, and Haemonchus placei at 14 days post-ivermectin treatment indicating ivermectin resistance in adult worms. In contrast, Ostertagia ostertagi third-stage larvae were almost completely absent from day 14 coprocultures, indicating that adult worms of this species were not ivermectin resistant. However, there was a recrudescence of O. ostertagi third stage larvae in coprocultures at three to six months post-ivermectin treatment, which indicated ivermectin resistance in hypobiotic larvae. The calves were recruited from the auction market and, therefore, derived from multiple sources in western Canada, suggesting that ivermectin-resistant parasites, including hypobiotic O. ostertagi larvae, are likely widespread in western Canadian beef herds. This work demonstrates the value of integrating ITS-2 rDNA metabarcoding with the FECRT to enhance anthelmintic resistance detection and provide GIN species- and stage-specific information.

Molecular characterization of Sarcocystis spp. as a cause of protozoal encephalitis in a free-ranging black bear.

A free-ranging juvenile male black bear (Ursus americanus), found dead in Alberta, Canada, had severe nonsuppurative encephalitis. Lesions in the brain were most severe in the gray matter of the cerebral cortex, and included perivascular cuffs of lymphocytes and plasma cells, areas of gliosis that disrupted the neuropil, and intralesional protozoan schizonts. The left hindlimb had suppurative myositis associated with Streptococcus halichoeri. Immunohistochemistry and molecular analyses (PCR and sequencing of 4 discriminatory loci: 18S rDNA, ITS-1 rDNA, cox1, rpoB) identified Sarcocystis canis or a very closely related Sarcocystis sp. in the affected muscle and brain tissues. The main lesion described in previously reported cases of fatal sarcocystosis in bears was necrotizing hepatitis. Fatal encephalitis associated with this parasite represents a novel presentation of sarcocystosis in bears. Sarcocystosis should be considered a differential diagnosis for nonsuppurative encephalitis in bears.

A multidisciplinary perioperative medicine clinic to improve high-risk patient outcomes: A service evaluation audit.

Various perioperative interventions have been demonstrated to improve outcomes for high-risk patients undergoing surgery. This audit assessed the impact of introducing a multidisciplinary perioperative medicine clinic on postoperative outcomes and resource usage amongst high-risk patients.Between January 2019 and March 2020, our institution piloted a Comprehensive High-Risk Surgical Patient Clinic. Surgical patients were eligible for referral when exhibiting criteria known to increase perioperative risk. The patient's decision whether to proceed with surgery was recorded; for those proceeding with surgery, perioperative outcomes and bed occupancy were recorded and compared against a similar surgical population identified as high-risk at our institution in 2017.Of 23 Comprehensive High-Risk Surgical Patient Clinic referrals, 11 did not proceed with the original planned surgery. Comprehensive High-Risk Surgical patients undergoing original planned surgery, as compared to high-risk patients from 2017, experienced reduced unplanned intensive care unit admission (8% versus 19%, respectively), 30-day mortality (0% versus 13%) and 30-day re-admission to hospital (0% versus 20%); had shorter postoperative lengths of stay (median (range) 8 (7-14) days versus 10.5 (5-28)) and spent more days alive outside of hospital at 30 days (median (range) 18 (0-25) versus 21 (16-23)). Cumulatively, the Comprehensive High-Risk Surgical patient cohort compared to the 2017 cohort (both n=23) occupied fewer postoperative intensive care (total 13 versus 24) and hospital bed-days (total 106 versus 212).The results of our Comprehensive High-Risk Surgical Patient pilot project audit suggest improved individual outcomes for high-risk patients proceeding with surgery. In addition, the results support potential resource savings through more appropriate patient selection.

An improved model for the population dynamics of cattle gastrointestinal nematodes on pasture: parameterisation and field validation for Ostertagia ostertagi and Cooperia oncophora in northern temperate zones.

Gastrointestinal nematodes (GIN) are amongst the most important pathogens of grazing ruminants worldwide, resulting in negative impacts on cattle health and production. The dynamics of infection are driven in large part by the influence of climate and weather on free-living stages on pasture, and computer models have been developed to predict infective larval abundance and guide management strategies. Significant uncertainties around key model parameters limits effective application of these models to GIN in cattle, however, and these parameters are difficult to estimate in natural populations of mixed GIN species. In this paper, recent advances in molecular biology, specifically ITS-2 rDNA 'nemabiome' metabarcoding, are synthesised with a modern population dynamic model, GLOWORM-FL, to overcome this limitation. Experiments under controlled conditions were used to estimate rainfall constraints on migration of infective L3 larvae out of faeces, and their survival in faeces and soil across a temperature gradient, with nemabiome metabarcoding data permitting species-specific estimates for Ostertagia ostertagi and Cooperia oncophora in mixed natural populations. Results showed that L3 of both species survived well in faeces and soil between 0 and 30 °C, and required at least 5 mm of rainfall daily to migrate out of faeces, with the proportion migrating increasing with the amount of rainfall. These estimates were applied within the model using weather and grazing data and use to predict patterns of larval availability on pasture on three commercial beef farms in western Canada. The model performed well overall in predicting the observed seasonal patterns but some discrepancies were evident which should guide further iterative improvements in model development and field methods. The model was also applied to illustrate its use in exploring differences in predicted seasonal transmission patterns in different regions. Such predictive modelling can help inform evidence-based parasite control strategies which are increasingly needed due climate change and drug resistance. The work presented here also illustrates the added value of combining molecular biology and population dynamics to advance predictive understanding of parasite infections.

Regional heterogeneity and unexpectedly high abundance of Cooperia punctata in beef cattle at a northern latitude revealed by ITS-2 rDNA nemabiome metabarcoding.

BACKGROUND: The species composition of cattle gastrointestinal nematode (GIN) communities can vary greatly between regions. Despite this, there is remarkably little large-scale surveillance data for cattle GIN species which is due, at least in part, to a lack of scalable diagnostic tools. This lack of regional GIN species-level data represents a major knowledge gap for evidence-based parasite management and assessing the status and impact of factors such as climate change and anthelmintic drug resistance. METHODS: This paper presents a large-scale survey of GIN in beef herds across western Canada using ITS-2 rDNA nemabiome metabarcoding. Individual fecal samples were collected from 6 to 20 randomly selected heifers (n = 1665) from each of 85 herds between September 2016 and February 2017 and 10-25 first season calves (n = 824) from each of 42 herds between November 2016 and February 2017. RESULTS: Gastrointestinal nematode communities in heifers and calves were similar in Alberta and Saskatchewan, with Ostertagia ostertagi and Cooperia oncophora being the predominant GIN species in all herds consistent with previous studies. However, in Manitoba, Cooperia punctata was the predominant species overall and the most abundant GIN species in calves from 4/8 beef herds. CONCLUSIONS: This study revealed a marked regional heterogeneity of GIN species in grazing beef herds in western Canada. The predominance of C. punctata in Manitoba is unexpected, as although this parasite is often the predominant cattle GIN species in more southerly latitudes, it is generally only a minor component of cattle GIN communities in northern temperate regions. We hypothesize that the unexpected predominance of C. punctata at such a northerly latitude represents a range expansion, likely associated with changes in climate, anthelmintic use, management, and/or animal movement. Whatever the cause, these results are of practical concern since C. punctata is more pathogenic than C. oncophora, the Cooperia species that typically predominates in cooler temperate regions. Finally, this study illustrates the value of ITS-2 rDNA nemabiome metabarcoding as a surveillance tool for ruminant GIN parasites.

Shortened egg reappearance periods of equine cyathostomins following ivermectin or moxidectin treatment: morphological and molecular investigation of efficacy and species composition.

Macrocyclic lactones have been the most widely used drugs for equine parasite control during the past four decades. Unlike ivermectin, moxidectin exhibits efficacy against encysted cyathostomin larvae, and is reported to have persistent efficacy with substantially longer egg reappearance periods. However, shortened egg reappearance periods have been reported recently for both macrocyclic lactones, and these findings have raised several questions: (i) are egg reappearance period patterns different after ivermectin or moxidectin treatment? (ii) Are shortened egg reappearance periods associated with certain cyathostomin species or stages? (iii) How does moxidectin's larvicidal efficacy affect egg reappearance period? To address these questions, 36 horses at pasture, aged 2-5 years old, were randomly allocated to three treatment groups: 1, moxidectin; 2, ivermectin; and 3, untreated control. Strongylid fecal egg counts were measured on a weekly basis, and the egg reappearance period was 5 weeks for both compounds. Strongylid worm counts were determined for all horses: 18 were necropsied at 2 weeks post-treatment (PT), and the remaining 18 at 5 weeks PT. Worms were identified to species morphologically and by internal transcribed spacer-2 (ITS-2) rDNA metabarcoding. Moxidectin and ivermectin were 99.9% and 99.7% efficacious against adults at 2 weeks post treatment, whereas the respective efficacies against luminal L4s were 84.3% and 69.7%. At 5 weeks PT, adulticidal efficacy was 88.3% and 57.6% for moxidectin and ivermectin, respectively, while the efficacy against luminal L4s was 0% for both drugs. Moxidectin reduced early L3 counts by 18.1% and 8.0% at 2 or 5 weeks, while the efficacies against late L3s and mucosal L4s were 60.4% and 21.2% at the same intervals, respectively. The luminal L4s surviving ivermectin treatment were predominantly Cylicocyclus (Cyc.) insigne. The ITS-2 rDNA metabarcoding was in good agreement with morphologic species estimates but suggested differential activity between moxidectin and ivermectin for several species, most notably Cyc. insigne and Cylicocyclus nassatus. This study was a comprehensive investigation of current macrocyclic lactone efficacy patterns and provided important insight into potential mechanisms behind shortened egg reappearance periods.

Generalist nematodes dominate the nemabiome of roe deer in sympatry with sheep at a regional level.

The growth of livestock farming and the recent expansion of wild ungulate populations in Europe favor opportunities for direct and/or indirect cross-transmission of pathogens. Comparatively few studies have investigated the epidemiology of gastro-intestinal nematode parasites, an ubiquitous and important community of parasites of ungulates, at the wildlife/livestock interface. In this study, we aimed to assess the influence of livestock proximity on the gastrointestinal nematode community of roe deer in a rural landscape located in southern France. Using ITS-2 rDNA nemabiome metabarcoding on fecal larvae, we analysed the gastrointestinal nematode communities of roe deer and sheep. In addition, we investigated Haemonchus contortus nad4 mtDNA diversity to specifically test parasite circulation among domestic and wild host populations. The dominant gastrointestinal nematode species found in both the roe deer and sheep were generalist species commonly found in small ruminant livestock (e.g. H. contortus), whereas the more specialised wild cervid nematode species (e.g. Ostertagia leptospicularis) were only present at low frequencies. This is in marked contrast with previous studies that found the nemabiomes of wild cervid populations to be dominated by cervid specialist nematode species. In addition, the lack of genetic structure of the nad4 mtDNA of H. contortus populations between host species suggests circulation of gastrointestinal nematodes between roe deer and sheep. The risk of contact with livestock only has a small influence on the nemabiome of roe deer, suggesting the parasite population of roe deer has been displaced by generalist livestock parasites due to many decades of sheep farming, not only for deer grazing close to pastures, but also at a larger regional scale. We also observed some seasonal variation in the nemabiome composition of roe deer. Overall, our results demonstrate significant exchange of gastrointestinal nematodes between domestic and wild ungulates, with generalist species spilling over from domestic ungulates dominating wild cervid parasite communities.

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations.

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus populations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signature of selection was detected on chromosome I, centred on the isotype-1 ß-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 annotated genes, including the isotype-2 ß-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 ß-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.

Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release.

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations.

A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses.

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

Seasonal epidemiology of gastrointestinal nematodes of cattle in the northern continental climate zone of western Canada as revealed by internal transcribed spacer-2 ribosomal DNA nemabiome barcoding.

BACKGROUND: Gastrointestinal nematode (GIN) epidemiology is changing in many regions of the world due to factors such as global warming and emerging anthelmintic resistance. However, the dynamics of these changes in northern continental climate zones are poorly understood due to a lack of empirical data. METHODS: We studied the accumulation on pasture of free-living infective third-stage larvae (L3) of different GIN species from fecal pats deposited by naturally infected grazing cattle. The field study was conducted on three organic farms in Alberta, western Canada. Grass samples adjacent to 24 fecal pats were collected from each of three different pastures on each farm. Internal transcribed spacer-2 nemabiome metabarcoding was used to determine the GIN species composition of the harvested larvae. The rotational grazing patterns of the cattle ensured that each pasture was contaminated only once by fecal pat deposition. This design allowed us to monitor the accumulation of L3 of specific GIN species on pastures under natural climatic conditions without the confounding effects of pasture recontamination or anthelmintic treatments. RESULTS: In seven out of the nine pastures, grass L3 counts peaked approximately 9 weeks after fecal deposition and then gradually declined. However, a relatively large number of L3 remained in the fecal pats at the end of the grazing season. Nemabiome metabarcoding revealed that Cooperia oncophora and Ostertagia ostertagi were the two most abundant species on all of the pastures and that the dynamics of larval accumulation on grass were similar for both species. Daily precipitation and temperature across the whole sampling period were similar for most of the pastures, and multiple linear regression showed that accumulated rainfall 1 week prior to sample collection had a significant impact on the pasture L3 population, but accumulated rainfall 3 weeks prior to sample collection did not. CONCLUSIONS: The results suggest that the pasture L3 population was altered by short-term microclimatic conditions conducive for horizontal migration onto grass. Overall, the results show the importance of the fecal pat as a refuge and reservoir for L3 of cattle GIN on western Canadian pastures, and provide an evidence base for the risk assessment of rotational grazing management in the region.