Resistance In Nematode Parasites Research Articles

Transcriptomics of ivermectin response in Caenorhabditis elegans: Integrating abamectin quantitative trait loci and comparison to the Ivermectin-exposed DA1316 strain.

Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10-7 and 10-8 M) for 4 hours at 20°C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL.

In silico analysis of koranimine, a cyclic imine compound from Peribacillus frigoritolerans reveals potential nematicidal activity

Pine wilt disease (PWD) is a destructive vector-borne forest disease caused by the nematode Bursaphelenchus xylophilus. To date, several options are available for the management of pine wilt disease; however constant development and search for natural products with potential nematicidal activity are imperative to diversify management options and to cope with the possible future emergence of resistance in parasitic nematodes. Here, a combined metabolomics and genomics approach was employed to investigate the chemical repertoire and biosynthetic potential of the bacterial endophyte Peribacillus frigoritolerans BE93, previously characterized to exhibit nematicidal activity against B. xylophilus. Feature-based molecular networking revealed the presence of diverse secondary metabolites. A cyclic imine heptapeptide, koranimine, was found to be among the most abundant secondary metabolites produced. Genome mining displayed the presence of several putative biosynthetic gene clusters (BGCs), including a dedicated non-ribosomal peptide synthase (NRPS) BGC for koranimine. Given the non-ribosomal peptide nature of koranimine, in silico molecular docking analysis was conducted to investigate its potential nematicidal activity against the target receptor ivermectin-sensitive invertebrate α glutamate-gated chloride channel (GluCl). Results revealed the binding of koranimine at the allosteric site of the channel—the ivermectin binding site. Moreover, the ligand-receptor interactions observed were mostly shared between koranimine and ivermectin when bound to the α GluCl receptor thus, suggesting a possibly shared mechanism of potential nematicidal activity. This study highlights the efficiency of combined metabolomics and genomics approach in the identification of candidate compounds.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

SummaryLike 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.

Characterization of a P-glycoprotein drug transporter from Toxocara canis with a novel pharmacological profile

P-glycoproteins from the ATP-binding cassette transporter family are responsible for drug evasion by bacterial pathogens and neoplastic cells. More recently, these multidrug resistance transporters have been investigated for contributions to drug resistance in nematode parasites. In this study, we cloned and characterized the P-glycoprotein Tca-Pgp-11.1 from Toxocara canis, the canine intestinal ascarid. Large numbers of Tca-Pgp-11 transcripts were observed in the intestine of adult male and female worms. Heterologous expression studies confirmed sensitivity to known P-glycoprotein inhibitors. Interestingly, the competitive inhibitor verapamil had lower IC50 values than newer generation inhibitors that are designed to allosterically modulate mammalian P-glycoprotein. Consistent with other nematode P-glycoproteins, Tca-Pgp-11.1 was sensitive to ivermectin and selamectin but not moxidectin. Taken together, our data suggests that T. canis P-glycoproteins represent nematode-specific drug targets that could be exploited to enhance efficacy of existing anthelmintics.

High Throughput Screening of the NatureBank 'Marine Collection' in a Haemonchus Bioassay Identifies Anthelmintic Activity in Extracts from a Range of Sponges from Australian Waters.

Widespread resistance in parasitic nematodes to most classes of anthelmintic drugs demands the discovery and development of novel compounds with distinct mechanisms of action to complement strategic or integrated parasite control programs. Products from nature—which assume a diverse ‘chemical space’—have significant potential as a source of anthelmintic compounds. In the present study, we screened a collection of extracts (n = 7616) derived from marine invertebrates sampled from Australian waters in a high throughput bioassay for in vitro anti-parasitic activity against the barber’s pole worm (Haemonchus contortus)—an economically important parasitic nematode of livestock animals. In this high throughput screen (HTS), we identified 58 active extracts that reduced larval motility by ≥70% (at 90 h), equating to an overall ‘hit rate’ of ~0.8%. Of these 58 extracts, 16 also inhibited larval development by ≥80% (at 168 h) and/or induced ‘non-wild-type’ (abnormal) larval phenotypes with reference to ‘wild-type’ (normal) larvae not exposed to extract (negative controls). Most active extracts (54 of 58) originated from sponges, three from chordates (tunicates) and one from a coral; these extracts represented 37 distinct species/taxa of 23 families. An analysis of samples by 1H NMR fingerprinting was utilised to dereplicate hits and to prioritise a set of 29 sponge samples for future chemical investigation. Overall, these results indicate that a range of sponge species from Australian waters represents a rich source of natural compounds with nematocidal or nematostatic properties. Our plan now is to focus on in-depth chemical investigations of the sample set prioritised herein.

Three Small Molecule Entities (MPK18, MPK334 and YAK308) with Activity against Haemonchus contortus In Vitro.

Due to widespread multi-drug resistance in parasitic nematodes of livestock animals, there is an urgent need to discover new anthelmintics with distinct mechanisms of action. Extending previous work, here we screened a panel of 245 chemically-diverse small molecules for anti-parasitic activity against Haemonchus contortus—an economically important parasitic nematode of livestock. This panel was screened in vitro against exsheathed third-stage larvae (xL3) of H. contortus using an established phenotypic assay, and the potency of select compounds to inhibit larval motility and development assessed in dose-response assays. Of the 245 compounds screened, three—designated MPK18, MPK334 and YAK308—induced non-wildtype larval phenotypes and repeatedly inhibited xL3-motility, with IC50 values of 45.2 µM, 17.1 µM and 52.7 µM, respectively; two also inhibited larval development, with IC50 values of 12.3 µM (MPK334) and 6.5 µM (YAK308), and none of the three was toxic to human liver cells (HepG2). These findings suggest that these compounds deserve further evaluation as nematocidal candidates. Future work should focus on structure–activity relationship (SAR) studies of these chemical scaffolds, and assess the in vitro and in vivo efficacies and safety of optimised compounds against adults of H. contortus.

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses.

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.

Novel Equine Faecal Egg Diagnostics: Validation of the FECPAKG2.

Faecal egg counts (FECs) are the standard method of diagnosing the level of parasitic helminth egg shedding in horses and other grazing animals. Testing before treatment is an important factor in slowing the appearance of anthelmintic resistance in nematode parasites. The FECPAKG2, optimised for livestock, is reported to allow owners to perform FECs on their own animals without the need for a separate microscope or any specialist knowledge by tapping into remote expertise. However, the performance of the FECPAKG2 has yet to be assessed for equids. Therefore, a comparison of the FECPAKG2 (G2) method with an accepted equine FEC method (FECPAKG1(G1)) was performed, using faecal samples from 57 horses in Wales and 22 horses in New Zealand. There was a significant correlation between the FECs obtained by the two methods (p < 0.001) and no effect of the country of origin on the data (p = 0.157). The mean percentage accuracy compared to the control values (mean G2 count as a percentage of the mean G1 count, ±SStandard Error (SE)) was 101 ± 4%. There was no significant interaction between the method applied and the country of origin of the data (p = 0.814). The relative accuracy of the G2 method compared to the control method (FECPAKG1) was not affected by the level of infection (p = 0.124) and it was concluded that the FECPAKG2 method is a suitable method of performing FECs in horses. It is anticipated that the user-friendliness of the method will increase the uptake of FECs amongst horse owners, either by the direct use of the technology or through their veterinary practice, likely slowing the development of anthelmintic resistance.

Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the β-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.

Arylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus contortus in vitro

Due to widespread drug resistance in parasitic nematodes, there is a need to develop new anthelmintics. Given the cost and time involved in developing a new drug, the repurposing of known chemicals can be a promising, alternative approach. In this context, we tested a library (n = 600) of natural product-inspired pesticide analogues against exsheathed third stage-larvae (xL3s) of Haemonchus contortus (barber's pole worm) using a whole-organism, phenotypic screening technique that measures the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from chemical scaffolds of arylpyrrole or fipronil. The seven most promising compounds, selected based on their anthelmintic activity and/or limited cytotoxicity, are arylpyrroles that reduced the motility of fourth-stage larvae (L4s) with significant potency (IC50 values ranged from 0.04 ± 0.01 μM to 4.25 ± 0.82 μM, and selectivity indices ranged from 10.6 to 412.5). Since the parent structures of the active compounds are uncouplers of oxidative phosphorylation, we tested the effect of selected analogues on oxygen consumption in xL3s using the Seahorse XF24 flux analyser. Larvae treated with the test compounds showed a significant increase in oxygen consumption compared with the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study have identified natural product-derived molecules that are worth considering for chemical optimisation as anthelmintic drug leads.

Increased Expression of a MicroRNA Correlates with Anthelmintic Resistance in Parasitic Nematodes.

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3′ UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3′ UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species.

Targeted anthelmintic treatment of parasitic gastroenteritis in first grazing season dairy calves using daily live weight gain as an indicator

Control of parasitic gastroenteritis in cattle is typically based on group treatments with anthelmintics, complemented by grazing management, where feasible. However, the almost inevitable evolution of resistance in parasitic nematodes to anthelmintics over time necessitates a reappraisal of their use in order to reduce selection pressure. One such approach is targeted selective treatment (TST), in which only individual animals that will most benefit are treated, rather than whole groups of at-risk cattle. This study was designed to assess the feasibility of implementing TST on three commercial farms, two of which were organic. A total of 104 first-grazing season (FGS), weaned dairy calves were enrolled in the study; each was weighed at monthly intervals from the start of the grazing season using scales or weigh-bands. At the same time dung and blood samples were collected in order to measure faecal egg counts (FEC) and plasma pepsinogen, respectively. A pre-determined threshhold weight gain of 0.75kg/day was used to determine those animals that would be treated; the anthelmintic used was eprinomectin. No individual animal received more than one treatment during the grazing season and all treatments were given in July or August; five animals were not treated at all because their growth rates consistently exceeded the threshold. Mean daily live weight gain over the entire grazing season ranged between 0.69 and 0.82kg/day on the three farms. Neither FEC nor pepsinogen values were significantly associated with live weight gain. Implementation of TST at farm level requires regular (monthly) handling of the animals and the use of weigh scales or tape, but can be integrated into farm management practices. This study has shown that acceptable growth rates can be achieved in FGS cattle with modest levels of treatment and correspondingly less exposure of their nematode populations to anthelmintics, which should mitigate selection pressure for resistance by increasing the size of the refugia in both hosts and pasture.

Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mitoribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

The Effect of Macrocyclic Lactones-Ivermectin Exposure on Egg Hatching and Larval Development of Caenorhabditis elegans.

To date, the ivermectin resistance in nematode parasites has been reported and many studies are carried out to determine the causes of this problem. A free-living Caenorhabditis elegans is used as a model system for this study to investigate the response of C. elegans to ivermectin exposure by using larval development assay. Worms were exposed to ivermectin at concentration from 1 ng/mL to 10 ng/mL and dimethyl sulphoxide (DMSO) as a control. The developments of the worms were monitored for 24, 48, 72, and 96 hours until the worms become adults. Results indicated that worms' growth began to be affected by ivermectin at a concentration of 5 ng/mL, while at the concentration of 6, 7, 8, 9, and 10 ng/mL, the growth of worms were inhibited compared to control worms. Further study of the protein expression in C. elegans should be done to investigate the up-regulated and down-regulated proteins involve in ivermectin resistance.

Host ABC transporter proteins may influence the efficacy of ivermectin and possibly have broader implications for the development of resistance in parasitic nematodes

ABC transporter proteins function to extrude compounds from the cell. These proteins present an obstacle for treatment and for overcoming drug resistance as they are expressed by both host and parasite, and function similarly. The contribution of host ABC proteins to drug efficacy was examined using ivermectin and a Brugia malayi model system. Parallel in vitro and in vivo experiments were conducted using equal concentrations of ivermectin. The motilities and fecundity of B. malayi exposed to ivermectin in vitro were significantly lower than those treated in vivo. The higher motilities were correlated with low concentrations of ivermectin in worms extracted from treated hosts. The expression of ABC proteins was significantly higher in worms treated in vitro compared to those treated in vivo as well as in gerbils treated with ivermectin than in non-treated controls. The results suggest that host ABC transporters may influence the efficacy of ivermectin.

Low cost whole-organism screening of compounds for anthelmintic activity

Due to major problems with drug resistance in parasitic nematodes of animals, there is a substantial need and excellent opportunities to develop new anthelmintics via genomic-guided and/or repurposing approaches. In the present study, we established a practical and cost-effective whole-organism assay for the in vitro-screening of compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber’s pole worm). The assay is based on the use of exsheathed L3 (xL3) and L4 stages of H. contortus of small ruminants (sheep and goats). Using this assay, we screened a panel of 522 well-curated kinase inhibitors (GlaxoSmithKline, USA; code: PKIS2) for activity against H. contortus by measuring the inhibition of larval motility using an automated image analysis system. We identified two chemicals within the compound classes biphenyl amides and pyrazolo[1,5-α]pyridines, which reproducibly inhibit both xL3 and L4 motility and development, with IC50s of 14–47μM. Given that these inhibitors were designed as anti-inflammatory drugs for use in humans and fit the Lipinski rule-of-five (including bioavailability), they show promise for hit-to-lead optimisation and repurposing for use against parasitic nematodes. The screening assay established here has significant advantages over conventional methods, particularly in terms of ease of use, throughput, time and cost. Although not yet fully automated, the current assay is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation. The current assay is highly adaptable to many parasites of socioeconomic importance, including those causing neglected tropical diseases. This aspect is of major relevance, given the urgent need to deliver the goals of the London Declaration (http://unitingtocombatntds.org/resource/london-declaration) through the rapid and efficient repurposing of compounds in public–private partnerships.

Variability of the egg hatch assay to survey benzimidazole resistance in nematodes of small ruminants under field conditions

The egg hatch assay (EHA) is one of the main in vitro methods for detection of benzimidazole resistance in nematode parasites of small ruminants. However, although the EHA has been standardised at the laboratory level, the diagnostic performance of this method has not been fully characterised for field screenings. In the present work, monthly variation of benzimidazole resistance estimated by EHA was surveyed over two years in three sheep flocks and in one goat and an additional sheep flock sharing the same pastures. Resistance was measured by calculating both the effective dose of thiabendazole (TBZ) that inhibited hatching of ≥50% of parasite eggs (ED50) and the proportion (Pdd) of eggs hatching at a discriminating dose of 0.1μg/ml TBZ. Pdd exhibited higher variability than ED50, in agreement with the higher sensitivity of Pdd to changes in resistance levels. Both resistance parameters, however, were highly correlated, and their variation was similarly related to the same factors. Resistance levels differed among sheep flocks, and the resistance level of the goat flock was higher than that measured for the sheep flock sharing the same pasture. Moreover, monthly variation of resistance in goats did not mirror that recorded in sheep. Resistance levels varied seasonally, with the highest values recorded in the spring, and they were inversely related to the number of days that samples were stored under anaerobic conditions. In addition, they were directly associated with the relative abundance of Teladorsagia spp. but inversely related to the relative abundance of Haemonchus spp. After controlling for the effects of these identified factors for variation, inter-monthly sampling variation due to unknown factors was the main source of variability, accounting for more than 60–70% of variance for both resistance parameters and yielding absolute estimation errors higher than 0.06 for ED50 or 0.2 for Pdd when resistance was estimated from a single sampling. Optimum sample size, estimated from variance components, suggested that at least 4–5 samplings would be needed to halve this absolute error, whereas additional samplings would slightly increase precision but at the cost of substantially increasing sampling effort. More research is needed to identify the main factors involved in this inter-sampling variation to standardise the implementation of EHA under field conditions.

Novel assay for the detection and monitoring of levamisole resistance in Haemonchus contortus

A levamisole-sensitive acetylcholine receptor has recently been described in the parasitic nematode Haemonchus contortus. The pentameric receptor is produced from different subunit proteins, one of which is Hco-ACR-8. A truncated transcript, Hco-acr-8b, has been identified in six levamisole-resistant H. contortus isolates and was found to be absent in four levamisole-susceptible isolates, indicating Hco-acr-8b could be a potential marker for levamisole resistance. The Hco-acr-8b transcript contains exons 1 and 2 and terminates with 347bp from within the intron 2. In this work, we investigated genomic DNA sequences of the Hco-acr-8 gene, in a region including exon 2 and exon 3, from a wide range of levamisole-susceptible and resistant H. contortus isolates. Sequences potentially involved in generating the truncated splice variant within the second intron were analysed from individuals and pools of parasites. We found an insertion/deletion (indel) of 63bp located just downstream from the splice acceptor site for the alternative third exon. The sequence of the indel, when present, was similar in the 12 isolates examined. The presence or absence of this indel was statistically (Chi2 test) correlated with levamisole resistance status. A correlation was also demonstrated between the absence of the indel and the expression of the Hco-acr-8b transcript. We believe this is the first report of a putative DNA marker for levamisole resistance detection. Using this new knowledge, we have developed a novel DNA-based assay for the detection and monitoring of levamisole resistance in parasitic nematodes of animals.

Managing anthelmintic resistance – Parasite fitness, drug use strategy and the potential for reversion towards susceptibility

The rotation of different anthelmintic classes, on an approximately annual basis, has been widely promoted and adopted as a strategy to delay the development of anthelmintic resistance in nematode parasites. Part of the rationale for recommending this practice was the expectation that resistant genotype worms have a lower ecological fitness than susceptible worms, at least in the early stages of selection, and so reversion towards susceptibility could be expected in those years when an alternative class of anthelmintic was used.The routine use of combination anthelmintics might be expected to negate this opportunity for reversion because multiple classes of anthelmintic would be used simultaneously. A simulation model was used to investigate whether the optimal strategy for use of multiple drug classes (i.e. an annual rotation of two classes of anthelmintic or continuous use of two classes in combination) changed with the size of the fitness cost associated with resistance.Model simulations were run in which the fitness cost associated with each resistance gene was varied from 0% to 15% and the rate at which resistance developed was compared for each of the drug-use strategies. Other factors evaluated were the initial frequency of the resistance genes and the proportion of the population not exposed to treatment (i.e. in refugia).Increasing the proportion of the population in refugia always slowed the development of resistance, as did using combinations in preference to an annual rotation. As the fitness cost associated with resistance increased, resistance developed more slowly and this was more pronounced when a combination was used compared to a rotation. If the fitness cost was sufficiently high then resistance did not develop (i.e. the resistance gene frequency declined over time) and this occurred at lower fitness costs when a combination was used. The results, therefore, indicate that the optimal drug-use strategy to maximise the benefit of any fitness cost associated with resistance is the use of combinations of different anthelmintic classes.Manual calculations confirmed that, within the model, the only resistant genotypes capable of surviving treatment with a combination are those carrying multiple resistance genes. These individuals are less fit, resulting in the worm population surviving treatment having a lower overall ecological fitness. This is a previously unreported perspective on the use of combination anthelmintics and strengthens the argument that any new class of anthelmintic, for which resistance genes can be expected to be rare, should be brought to market in combination.