Next-generation sequencing technologies for helminth diagnostics and surveillance in ruminants: shifting diagnostic barriers.

Helminth infections in grazing ruminants are a major issue for livestock farming globally, but are unavoidable in outdoor grazing systems and must be effectively managed to avoid deleterious effects to animal health, and productivity. Next-generation sequencing (NGS) technologies are transforming our understanding of the genetic basis of anthelmintic resistance (AR) and epidemiological studies of ruminant gastrointestinal parasites. They also have the potential to not only help develop and validate molecular diagnostic tests but to be directly used in routine diagnostics integrating species-specific identification and AR into a single test. Here, we review how these developments have opened the pathway for the development of multi-AR and multispecies identification in a single test, with widespread implications for sustainable livestock farming for the future.

A set of nematode rRNA cistron databases and a primer assessment tool to enable more flexible and comprehensive metabarcoding.

The ITS-2-rRNA has been particularly useful for nematode metabarcoding but does not resolve all phylogenetic relationships, and reference sequences are not available for many nematode species. This is a particular issue when metabarcoding complex communities such as wildlife parasites or terrestrial and aquatic free-living nematode communities. We have used markerDB to produce four databases of distinct regions of the rRNA cistron: the 18S rRNA gene, the 28S rRNA gene, the ITS-1 intergenic spacer and the region spanning ITS-1_5.8S_ITS-2. These databases comprise 2645, 254, 13,461 and 10,107 unique full-length sequences representing 1391, 204, 1837 and 1322 nematode species, respectively. The comparative analysis illustrates the complementary value but also reveals a better representation of Clade III, IV and V than Clade I and Clade II nematodes in each case. Although the ITS-1 database includes the largest number of unique full-length sequences, the 18S rRNA database provides the widest taxonomic coverage. We also developed PrimerTC, a tool to assess primer sequence conservation across any reference sequence database, and have applied it to evaluate a large number of previously published rRNA cistron primers. We identified sets of primers that currently provide the broadest taxonomic coverage for each rRNA marker across the nematode phylum. These new resources will facilitate more comprehensive metabarcoding of nematode communities using either short-read or long-read sequencing platforms. Further, PrimerTC is available as a simple WebApp to guide or assess PCR primer design for any genetic marker and/or taxonomic group beyond the nematode phylum.

Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

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.

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.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine.

The faecal egg count reduction test (FECRT) remains the method of choice for establishing the efficacy of anthelmintic compounds in the field, including the diagnosis of anthelmintic resistance. We present a guideline for improving the standardization and performance of the FECRT that has four sections. In the first section, we address the major issues relevant to experimental design, choice of faecal egg count (FEC) method, statistical analysis, and interpretation of the FECRT results. In the second section, we make a series of general recommendations that are applicable across all animals addressed in this guideline. In the third section, we provide separate guidance details for cattle, small ruminants (sheep and goats), horses and pigs to address the issues that are specific to the different animal types. Finally, we provide overviews of the specific details required to conduct an FECRT for each of the different host species. To address the issues of statistical power vs. practicality, we also provide two separate options for each animal species; (i) a version designed to detect small changes in efficacy that is intended for use in scientific studies, and (ii) a less resource-intensive version intended for routine use by veterinarians and livestock owners to detect larger changes in efficacy. Compared to the previous FECRT recommendations, four important differences are noted. First, it is now generally recommended to perform the FECRT based on pre- and post-treatment FEC of the same animals (paired study design), rather than on post-treatment FEC of both treated and untreated (control) animals (unpaired study design). Second, instead of requiring a minimum mean FEC (expressed in eggs per gram (EPG)) of the group to be tested, the new requirement is for a minimum total number of eggs to be counted under the microscope (cumulative number of eggs counted before the application of a conversion factor). Third, we provide flexibility in the required size of the treatment group by presenting three separate options that depend on the (expected) number of eggs counted. Finally, these guidelines address all major livestock species, and the thresholds for defining reduced efficacy are adapted and aligned to host species, anthelmintic drug and parasite species. In conclusion, these new guidelines provide improved methodology and standardization of the FECRT for all major livestock species.

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.

Effects of naturally acquired gastrointestinal nematode infection on bovine viral diarrhea virus vaccine-directed antibody response in western Canadian feedlot cattle.

Objective: To determine how gastrointestinal nematode (GIN) infection, reflected by fecal egg counts and Ostertagia ostertagi serum antibody titers, is associated with the antibody response to bovine viral diarrhea virus type 1 (BVDV-1) vaccine antigen in fall-weaned feedlot cattle from western Canada. Animals: Cross-sectional study with 240 steer calves derived from an auction market. Procedure: At feedlot arrival, calves were given a commercial vaccine containing modified live BVDV-1. Serum neutralization antibody titers against BVDV-1 antigens were determined in individual blood samples collected pre-vaccination and 21 d after vaccination. A modified Wisconsin sugar floatation method was used to obtain individual calf GIN egg counts in fecal samples on arrival. Antibody titers against O. ostertagi were determined using an enzyme-linked immunosorbent assay in on-arrival blood samples. Results: Fecal egg counts and O. ostertagi titers were not associated with vaccine antibody-fold changes. Similarly, fecal egg counts and O. ostertagi titers were not associated with vaccine-induced seroconversion. Conclusions: The relatively low GIN burdens, reflected by the overall low fecal egg counts in these fall-weaned feedlot calves, did not have measurable adverse effects on the humoral immune response to BVDV-1 vaccine antigens. Clinical relevance: An adequate response to vaccination is important for cattle welfare and productivity. Conditions that negatively affect this response may vary regionally, such as GIN infection. Understanding this is essential. Although subclinical intestinal parasitism did not noticeably affect the antibody response in these steers, higher GIN burdens and actual immune protection from clinical disease remain to be investigated.

Effets d'une infection par des nématodes gastro-intestinaux d'origine naturelle sur la réponse en anticorps dirigés par le vaccin contre le virus de la diarrhée virale bovine chez les bovins des parcs d'engraissement de l'Ouest canadien. Objectif: Déterminer comment l'infection par les nématodes gastro-intestinaux (GIN), reflétée par le nombre d'oeufs fécaux et les titres d'anticorps sériques d'Ostertagia ostertagi, est associée à la réponse en anticorps à l'antigène du vaccin contre le virus de la diarrhée virale bovine de type 1 (BVDV-1) chez les bovins en parc d'engraissement sevrés à l'automne de l'Ouest canadien. Animaux: Étude transversale auprès de 240 veaux bouvillons issus d'un marché aux enchères. Procédure: À leur arrivée au parc d'engraissement, les veaux ont reçu un vaccin commercial contenant du BVDV-1 vivant modifié. Les titres d'anticorps sériques neutralisants contre les antigènes BVDV-1 ont été déterminés dans des échantillons de sang individuels prélevés avant la vaccination et 21 jours après la vaccination. Une méthode de Wisconsin modifiée de flottation au sucre a été utilisée pour obtenir le nombre d'oeufs GIN de chaque veau dans les échantillons fécaux à l'arrivée. Les titres d'anticorps dirigés contre O. ostertagi ont été déterminés à l'aide d'un dosage immuno-enzymatique dans des échantillons de sang à l'arrivée. Résultats: Le nombre d'oeufs fécaux et les titres d'O. ostertagi n'étaient pas associés aux modifications du titre d'anticorps vaccinaux. De même, le nombre d'oeufs fécaux et les titres d'O. ostertagi n'étaient pas associés à la séroconversion induite par le vaccin. Conclusion: Les charges relativement faibles de GIN, reflétées par le faible nombre global d'oeufs fécaux chez ces veaux d'engraissement sevrés à l'automne, n'ont pas eu d'effets indésirables mesurables sur la réponse immunitaire humorale aux antigènes du vaccin BVDV-1. Pertinence clinique: Une réponse adéquate à la vaccination est importante pour le bien-être et la productivité des bovins. Les conditions qui affectent négativement cette réponse peuvent varier selon les régions, telles que l'infection par les GIN. Comprendre cela est essentiel. Bien que le parasitisme intestinal subclinique n'ait pas sensiblement affecté la réponse en anticorps chez ces bouvillons, des charges de GIN plus élevées et une protection immunitaire réelle contre la maladie clinique restent à étudier.(Traduit par Dr Serge Messier).

It's a small world for parasites: evidence supporting the North American invasion of European Echinococcus multilocularis.

Echinococcus multilocularis (Em), the causative agent of human alveolar echinococcosis (AE), is present in the Holarctic region, and several genetic variants deem to have differential infectivity and pathogenicity. An unprecedented outbreak of human AE cases in Western Canada infected with a European-like strain circulating in wild hosts warranted assessment of whether this strain was derived from a recent invasion or was endemic but undetected. Using nuclear and mitochondrial markers, we investigated the genetic diversity of Em in wild coyotes and red foxes from Western Canada, compared the genetic variants identified to global isolates and assessed their spatial distribution to infer possible invasion dynamics. Genetic variants from Western Canada were closely related to the original European clade, with lesser genetic diversity than that expected for a long-established strain and spatial genetic discontinuities within the study area, supporting the hypothesis of a relatively recent invasion with various founder events.

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.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

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.

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.

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.

Interactions of Caenorhabditis elegans ß-tubulins with the microtubule inhibitor and anthelmintic drug albendazole.

Parasitic nematodes are major human and agricultural pests, and benzimidazoles are amongst the most important broad-spectrum anthelmintic drug class used for their control. Benzimidazole resistance is now widespread in many species of parasitic nematodes in livestock globally and an emerging concern for the sustainable control of human soil-transmitted helminths. ß-tubulin is the major benzimidazole target, although other genes may influence resistance. Among the 6 Caenorhabditis elegans ß-tubulin genes, loss of ben-1 causes resistance without other apparent defects. Here, we explored the genetics of C. elegans ß-tubulin genes in relation to the response to the benzimidazole derivative albendazole. The most highly expressed ß-tubulin isotypes, encoded by tbb-1 and tbb-2, were known to be redundant with each other for viability, and their products are predicted not to bind benzimidazoles. We found that tbb-2 mutants, and to a lesser extent tbb-1 mutants, were hypersensitive to albendazole. The double mutant tbb-2 ben-1 is uncoordinated and short, resembling the wild type exposed to albendazole, but the tbb-1 ben-1 double mutant did not show the same phenotypes. These results suggest that tbb-2 is a modifier of albendazole sensitivity. To better understand how BEN-1 mutates to cause benzimidazole resistance, we isolated mutants resistant to albendazole and found that 15 of 16 mutations occurred in the ben-1 coding region. Mutations ranged from likely nulls to hypomorphs, and several corresponded to residues that cause resistance in other organisms. Null alleles of ben-1 are albendazole-resistant and BEN-1 shows high sequence identity with tubulins from other organisms, suggesting that many amino acid changes could cause resistance. However, our results suggest that missense mutations conferring resistance are not evenly distributed across all possible conserved sites. Independent of their roles in benzimidazole resistance, tbb-1 and tbb-2 may have specialized functions as null mutants of tbb-1 or tbb-2 were cold or heat sensitive, respectively.

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.

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.

Metabarcoding in two isolated populations of wild roe deer (Capreolus capreolus) reveals variation in gastrointestinal nematode community composition between regions and among age classes.

BACKGROUND: Gastrointestinal nematodes are ubiquitous for both domestic and wild ungulates and have varying consequences for health and fitness. They exist as complex communities of multiple co-infecting species, and we have a limited understanding of how these communities vary in different hosts, regions and circumstances or of how this affects their impacts. METHODS: We have undertaken ITS2 rDNA nemabiome metabarcoding with next-generation sequencing on populations of nematode larvae isolated from 149 fecal samples of roe deer of different sex and age classes in the two isolated populations of Chizé and Trois Fontaines in France not co-grazing with any domestic ungulate species. RESULTS: We identified 100 amplified sequence variants (ASVs) that were assigned to 14 gastrointestinal nematode taxa overall at either genus (29%) or species (71%) level. These taxa were dominated by parasites classically found in cervids-e.g. Ostertagia leptospicularis, Spiculopteragia spp. Higher parasite species diversity was present in the Trois Fontaines population than in the Chizé population including the presence of species more typically seen in domestic livestock (Haemonchus contortus, Bunostomum sp., Cooperia punctata, Teladorsagia circumcincta). No differences in parasite species diversity or community composition were seen in the samples collected from three zones of differing habitat quality within the Chizé study area. Young roe deer hosted the highest diversity of gastrointestinal nematodes, with more pronounced effects of age apparent in Trois Fontaines. The effect of host age differed between gastrointestinal nematode species, e.g. there was little effect on O. leptospicularis but a large effect on Trichostrongylus spp. No effect of host sex was detected in either site. CONCLUSIONS: The presence of some livestock parasite species in the Trois Fontaines roe deer population was unexpected given the isolation of this population away from grazing domestic livestock since decades. Overall, our results illustrate the influence of host traits and the local environment on roe deer nemabiome and demonstrate the power of the nemabiome metabarcoding approach to elucidate the composition of gastrointestinal nematode communities in wildlife.