Background: The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans. Results: Here we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates. Conclusions: The H. contortus genome and transcriptome provide an essential platform for postgenomic research in this and other important strongylid parasites.

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. 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. 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.

Parasitic nematodes infect hundreds of millions of people and farmed livestock. Further, plant parasitic nematodes result in major crop damage. The pipeline of therapeutic compounds is limited and parasite resistance to the existing anthelmintic compounds is a global threat. We have developed an INVertebrate Automated Phenotyping Platform (INVAPP) for high-throughput, plate-based chemical screening, and an algorithm (Paragon) which allows screening for compounds that have an effect on motility and development of parasitic worms. We have validated its utility by determining the efficacy of a panel of known anthelmintics against model and parasitic nematodes: Caenorhabditis elegans, Haemonchus contortus, Teladorsagia circumcincta, and Trichuris muris. We then applied the system to screen the Pathogen Box chemical library in a blinded fashion and identified compounds already known to have anthelmintic or anti-parasitic activity, including tolfenpyrad, auranofin, and mebendazole; and 14 compounds previously undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers an effective, high-throughput system for the discovery of novel anthelmintics.

Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. Recently, we developed an automated multiplexed-tandem (MT)-PCR platform for the diagnosis and identification patent infections with key genera/species of gastrointestinal nematodes of sheep and validated this approach in detailed experiments carried out in Australia. In the present study, we deployed this diagnostic platform in Scotland and Belgium to test samples from naturally infected sheep in these environments and to validate the MT-PCR platform relative to traditional diagnostic methods routinely used by local laboratories. MT-PCR detected all microscopy positive samples and there was a significant agreement between the results of the different test methods in terms of the species detected and their relative proportion in a test sample, however, for some samples, there were discrepancies between the results of the different test methods. Selective sequencing of purified MT-PCR products demonstrated the results to be 100% specific. The MT-PCR platform is an advanced method for the species/genus-specific diagnosis of gastrointestinal nematode infections in small ruminants and has demonstrated utility when deployed in different countries and climatic zones. The platform is user-friendly due to the largely automated procedure and has high versatility in that it can achieve a specific diagnosis from different types of sample templates, including larval culture and faecal samples. With appropriate modifications of the primers used, the MT-PCR platform also provides potential for the diagnosis of a variety of other pathogens of veterinary or medical importance.

The anthelmintic effects of extracted coriander oil and five pure essential oil constituents (geraniol, geranyl acetate, eugenol, methyl iso-eugenol, and linalool) were tested, using larval motility assay, on the third-stage larvae (L3s) of Haemonchus contortus, Trichostrongylus axei, Teladorsagia circumcincta, Trichostrongylus colubriformis, Trichostrongylus vitrinus and Cooperia oncophora. Coriander oil and linalool, a major component of tested coriander oil, showed a strong inhibitory efficacy against all species, except C. oncophora with a half maximal inhibitory concentration (IC₅₀) that ranged from 0.56 to 1.41% for the coriander oil and 0.51 to 1.76% for linalool. The coriander oil and linalool combinations conferred a synergistic anthelmintic effect (combination index [CI] <1) on larval motility comparable to positive control (20 mg/mL levamisole) within 24 h (p < 0.05), reduced IC₅₀ values to 0.11–0.49% and induced a considerable structural damage to L3s. Results of the combined treatment were validated by quantitative fluorometric microplate-based assays using Sytox green, propidium iodide and C₁₂-resazurin, which successfully discriminated live/dead larvae. Only Sytox green staining achieved IC₅₀ values comparable to that of the larval motility assay. The cytotoxicity of the combined coriander oil and linalool on Madin–Darby Canine Kidney cells was evaluated using sulforhodamine-B (SRB) assay and showed no significant cytotoxic effect at concentrations < 1%. These results indicate that testing essential oils and their main components may help to find new potential anthelmintic compounds, while at the same time reducing the reliance on synthetic anthelmintics.

Background: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. Methods: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4–80.7% before treatment, and 82.3–92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.