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Background Current measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation. Methods Dirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR). Results In vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro. Conclusions The arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae. Graphical Abstract

Anthelmintic resistance is a significant threat to livestock production systems worldwide and is emerging as an important issue in companion animal parasite management. It is also an emerging concern for the control of human soil-transmitted helminths and filaria. An important aspect of managing anthelmintic resistance is the ability to utilise diagnostic tests to detect its emergence at an early stage. In host-parasite systems where resistance is already widespread, diagnostics have a potentially important role in determining those drugs that remain the most effective. The development of molecular diagnostics for anthelmintic resistance is one focus of the Consortium for Anthelmintic Resistance and Susceptibility (CARS) group. The present paper reflects discussions of this issue that occurred at the most recent meeting of the group in Wisconsin, USA, in July 2019. We compare molecular resistance diagnostics with in vivo and in vitro phenotypic methods, and highlight the advantages and disadvantages of each. We assess whether our knowledge on the identity of molecular markers for resistance towards the different drug classes is sufficient to provide some expectation that molecular tests for field use may be available in the short-to-medium term. We describe some practical aspects of such tests and how our current capabilities compare to the requirements of an ‘ideal’ test. Finally, we describe examples of drug class/parasite species interactions that provide the best opportunity for commercial use of molecular tests in the near future. We argue that while such prototype tests may not satisfy the requirements of an ‘ideal’ test, their potential to provide significant advances over currently-used phenotypic methods warrants their development as field diagnostics., Graphical abstract Image 1, Highlights • Management of anthelmintic resistance requires reliable, sensitive and cost-effective diagnostic methods. • Molecular anthelmintic resistance tests have a number of advantages over currently-used phenotypic tests. • However, there are a number of challenges impacting on the use of molecular tests in the field. • There are also clear opportunities for proof-of-concept use of molecular tests in the short term.

Background: Dirofilaria immitis is a parasitic filarial nematode. It is the causative agent of dirofilariosis, a potentially fatal pulmonary infection which primarily infects canids and felines. dirofilariosis infections are primarily controlled with a prophylactic macrocyclic lactone (ML) regimen. Recent evidence has confirmed the development of ML-resistant isolates in the US which are genetically distinct from wild-type populations. Previous research clinically validated 9 single nucleotide polymorphism (SNP) molecular markers associated with these ML resistance phenotypes isolated from the USA. Methods: In this study, three D. immitis US laboratory-maintained isolates: two susceptible isolates, Berkeley, and Georgia II, one resistant isolate, WildCat; and eleven European D. immitis clinical samples, from Italy, Spain, and Hungary were analyzed. The samples tested were fresh microfilaria (mf) in blood or adult female worms shipped in ethanol and rehydrated in phosphate buffered saline (PBS). After DNA extraction, each sample underwent MiSeq sequencing of regions encompassing the 9 SNP sites previously associated with macrocyclic lactone susceptibility and resistance. The nucleotide frequencies of the 9 SNP sites were analyzed and the pairwise fixation index (FST) of the top 2 SNP molecular markers were calculated to estimate the probability of identity with known susceptible and resistant isolates.Results: In the three laboratory-maintained US D. immitis isolates Berkeley had a 2-SNP pairwise FST of 0.00, Georgia II had a 2-SNP pairwise FST of 0.07 indicating two ML-susceptible genotypes. WildCat had a 2-SNP pairwise FST of 0.33 indicating a ML-resistant genotype. The genotype analysis of the European clinical samples showed that all eleven had 2-SNP pairwise FST of 0.00, which indicates their genotypes, at these SNP sites, are consistent with ML susceptibility. Conclusions: The results of the current study provides a genotypic analysis of European clinical samples with US SNP molecular markers associated with ML susceptibility and resistance. The eleven clinical samples tested show no genomic evidence of ML-resistance. Vigilance is needed to maintain susceptibility to heartworm preventives in regions of the world so far without resistance. The early adoption of genotyping of clinical D. immitis samples could provide an early indication of the potential development of ML-resistance and aid to distinguish clinical cases of heartworm infection due to ML resistance from those due to a lack of prevention or inadequate compliance, as has been seen in North America.

Background: Dirofilaria immitis is a parasitic filarial nematode. It is the causative agent of dirofilariosis, a potentially fatal pulmonary infection which primarily infects canids and felines. dirofilariosis infections are primarily controlled with a prophylactic macrocyclic lactone (ML) regimen. Recent evidence has confirmed the development of ML-resistant isolates in the US which are genetically distinct from wild-type populations. Previous research clinically validated 9 single nucleotide polymorphism (SNP) molecular markers associated with these ML resistance phenotypes isolated from the USA. Methods: In this study, three D. immitis US laboratory-maintained isolates: two putative susceptible isolates, Berkeley, and Georgia II, one putative resistant isolate, WildCat; and eleven European D. immitis clinical samples, from Italy, Spain, and Hungary were analyzed. The samples tested were fresh microfilaria (mf) in blood or adult female worms shipped in ethanol and rehydrated in phosphate buffered saline (PBS). After DNA extraction, each sample underwent MiSeq sequencing of regions encompassing the 9 SNP sites previously correlated with ML resistance. The nucleotide frequencies of the 9 SNP sites were analyzed and the pairwise fixation index (FST) of the top 2 SNP molecular markers were calculated in order to estimate the probability of identity with known resistant isolates.Results: In the three laboratory-maintained US D. immitis isolates Berkeley had a 2-SNP pairwise FST of 0.00, indicating a ML-susceptible genotype, WildCat had a 2-SNP pairwise FST of 0.33 indicating a ML-resistant genotype, and Georgia II had a 2-SNP pairwise FST of 0.07, which may indicate early selection for ML resistance. The genotype analysis of the European clinical samples showed that all eleven European samples had 2-SNP pairwise FST of 0.00, which indicates their genotypes are consistent with ML susceptibility. Conclusions: Prior to genotyping the European samples, it was possible that the positive heartworm infections could have arisen because of the development of ML-resistance or due to lack of owner compliance or incomplete use of heartworm preventives. Our results indicate no genomic evidence of ML-resistance and suggests that resistance has not developed, so far, in Europe, or been introduced via movement of infected dogs. However, vigilance is needed to maintain susceptibility to heartworm preventives in regions of the world so far without resistance.

Heartworm disease, caused by Dirofilaria immitis, can be lethal in dogs and cats. It is transmitted by mosquitoes, and occurs in many parts of the world. Prevention relies on macrocyclic lactones. Macrocyclic lactones used are ivermectin, selamectin, abamectin, eprinomectin, milbemycin oxime and moxidectin, administered at 30-day intervals during the transmission season. Some moxidectin formulations are long-acting injectables. In the USA, preventives are recommended throughout the year. Loss of efficacy of macrocyclic lactone preventives was reported in 2005 and proof of resistance in the USA was published a decade later. Understanding factors which promote resistance is important to maintain control. Factors important for resistance development are discussed. Better, inexpensive tests to confirm resistance are needed. Infection in animals under chemoprophylaxis per se does not imply resistance because lack of compliance in preventive use could be the reason. In vivo confirmation of resistance is expensive, slow and ethically questionable. A microfilariae suppression test can be a surrogate test, but requires a high dose of a macrocyclic lactone and repeated blood microfilaria counts 2–4 weeks later. DNA single nucleotide polymorphism markers have been successfully used. However, the specific genetic changes which cause resistance are unknown. Surveys to map and follow the extent of resistance are needed. Long acting mosquito repellants and insecticides can play a useful role. High dose rate formulations of moxidectin, coupled with mosquito biting mitigation may reduce transmission of resistant genotypes. Doxycycline, daily for 28 days, as anti-Wolbachia treatment, can reduce transmission and remove adult parasites. However, new classes of heartworm preventives are needed. While any preventive strategy must be highly effective, registration requirements for 100% efficacy may hinder development of useful new classes of preventives. Continued reliance on macrocyclic lactone preventives, when they do not work against resistant genotypes, will spread resistance, and allow for more disease.

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer–DNDi partnership is an outstanding example of “One World Health,” in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area., Author summary Onchocerca volvulus is the causative agent of human river blindness, and current elimination programs rely on the use of ivermectin to kill microfilariae. Since no adulticidal drug is available and adult worms have a life span of up to 15 years, elimination programs need to be sustained over several decades. Emodepside is an anthelmintic that is licensed as a dewormer for cats and dogs. Due to its ability to eliminate nematodes located in various extraintestinal host tissues, including migrating larvae and adult filarial worms, it is considered to be an excellent candidate for the treatment of onchocerciasis. Intense collaboration between academia and the pharmaceutical industry has led to a deep understanding of the novel mode of action of the drug and of its parasite target spectrum. Phase I clinical trials with emodepside have demonstrated its safety and adulticide activity against the closely related cattle parasite Onchocerca ochengi. Currently, Phase II clinical trials are planned to confirm that emodepside, developed initially to improve animal health, has also the potential to improve human health by tackling a very important neglected tropical disease (NTD).

BackgroundCurrent Dirofilaria immitis prevention relies on macrocyclic lactones but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, this study aimed to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development. MethodsD. immitis microfilariae extracted from dog blood were incubated in various media conditions to identify suitable conditions for in vitro culture and development, and to determine the effects of FBS, mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript level of the ecdysone signaling pathway components were measured with droplet digital PCR. ResultsIn vitro conditions that best promote early development of D. immitis microfilariae to the “late sausage stage” have been identified, although shedding of the cuticle was not observed. FBS showed inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells was favorable to microfilarial growth. Transcript level study using droplet digital PCR also showed that developing microfilariae displayed upregulation of the ecdysone signaling system components, and 20‑hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro.ConclusionsThe arthropod host environment provides cues required for the rapid development of the D. immitis microfilariae and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding the developmental process of D. immitis microfilariae.

No abstract available.

To eliminate soil-transmitted helminth (STH) infections as a public health problem, the administration of benzimidazole (BZ) drugs to children has recently intensified. But, as drug pressure increases, the development of anthelmintic drug resistance (AR) becomes a major concern. Currently, there is no global surveillance system to monitor drug efficacy and the emergence of AR. Consequently, it is unclear what the current efficacy of the used drugs is and whether AR is already present. The aim of this study is to pilot a global surveillance system to assess anthelmintic drug efficacy and the emergence of AR in STH control programs. For this, we will incorporate drug efficacy trials into national STH control programs of eight countries (Bangladesh, Cambodia, Lao PDR, Vietnam, Ghana, Rwanda, Senegal and a yet to be defined country in the Americas). In each country, one trial will be performed in one program implementation unit to assess the efficacy of BZ drugs against STHs in school-aged children by faecal egg count reduction test. Stool samples will be collected before and after treatment with BZs for Kato-Katz analysis and preserved to purify parasite DNA. The presence and frequency of known single nucleotide polymorphisms (SNPs) in the β-tubulin genes of the different STHs will subsequently be assessed. This study will provide a global pattern of drug efficacy and emergence of AR in STH control programs. The results will provide complementary insights on the validity of known SNPs in the ß-tubulin gene as a marker for AR in human STHs as well as information on the technical and financial resources required to set up a surveillance system. Finally, the collected stool samples will be an important resource to validate different molecular technologies for the detection of AR markers or to identify novel potential molecular markers associated with AR in STH.

International audience; prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections. Nuclear receptors (NRs) are transcription factors involved in key physiological processes such as growth and development, aging and reproduction, as well as metabolism and detoxification of endo-and xenobiotics 1-4. They typically bind small lipophilic molecules, resulting in their activation, which allows the receptor to recruit co-regulators of expression of specific genes 5. Because of their central role in critical metabolic processes, including development, NRs are relevant targets for drug discovery 2. It is estimated that 10-20% of the pharmaceutical market targets these receptors in humans 5. NRs are conserved among metazoans including helminths 6. While humans harbor 49 NRs in their genome, Caenorhabditis elegans has 284 NRs 7 and some are essential for development and reproduction but also survival by enabling this free-living worm to adapt to variable environmental conditions. DAF-12 was described as a NR in C. elegans, due to the discovery of its ligands, dafachronic acids (Δ4-and Δ7-DA) 8-10. In this nematode, DAF-12 acts as a molecular switch in the signaling pathway that regulates dauer formation, or alternative progression to reproductive development and the adult stage, by sensing the environmental quality and bacterial nutrient availability 9,11. When environmental conditions are favorable, and bacteria are available, C. elegans stimulates the production of DA steroid hormones, which activate DAF-12 7,10,12. CelDAF-12 then induces transcription of development genes that promote growth and reproduction 13-15. In contrast, in unfavorable conditions, DAs are not synthesized and the DAF-12 apo-receptor interacts with a co-repressor, preventing target gene expression and resulting in arrest of development at dauer diapause larvae, a non-feeding, non-reproducing, stress-resistant, open

Background Macrocyclic lactone (ML) anthelmintics are used for chemoprophylaxis for heartworm infection in dogs and cats. Cases of dogs becoming infected with heartworms, despite apparent compliance to recommended chemoprophylaxis with approved preventives, has led to such cases being considered as suspected lack of efficacy (LOE). Recently, microfilariae collected from a small number of LOE isolates were used as a source of infection of new host dogs and confirmed to have reduced susceptibility to ML in controlled efficacy studies using L3 challenge in dogs. A specific Dirofilaria immitis laboratory isolate named JYD-34 has also been confirmed to have less than 100% susceptibility to ML-based preventives. For preventive claims against heartworm disease, evidence of 100% efficacy is required by FDA-CVM. It was therefore of interest to determine whether JYD-34 has a genetic profile similar to other documented LOE and confirmed reduced susceptibility isolates or has a genetic profile similar to known ML-susceptible isolates. Methods In this study, the 90Mbp whole genome of the JYD-34 strain was sequenced. This genome was compared using bioinformatics tools to pooled whole genomes of four well-characterized susceptible D. immitis populations, one susceptible Missouri laboratory isolate, as well as the pooled whole genomes of four LOE D. immitis populations. Fixation indexes (FST), which allow the genetic structure of each population (isolate) to be compared at the level of single nucleotide polymorphisms (SNP) across the genome, have been calculated. Forty-one previously reported SNP, that appeared to differentiate between susceptible and LOE and confirmed reduced susceptibility isolates, were also investigated in the JYD-34 isolate. Results The FST analysis, and the analysis of the 41 SNP that appeared to differentiate reduced susceptibility from fully susceptible isolates, confirmed that the JYD-34 isolate has a genome similar to previously investigated LOE isolates, and isolates confirmed to have reduced susceptibility, and to be dissimilar to the susceptible isolates. Conclusions These results provide additional evidence for the link between genotype and the reduced susceptibility phenotype observed in such isolates as JYD-34. Further work on other isolates showing reduced susceptibility to ML is required to demonstrate the value of genetic analysis in predicting the response to ML chemoprophylaxis. The authors suggest that genetic analysis may be useful in helping to interpret the results of in vivo efficacy testing of ML heartworm preventives against D. immitis isolates. Electronic supplementary material The online version of this article (10.1186/s13071-017-2437-5) contains supplementary material, which is available to authorized users.

Dirofilaria immitis, a filarial nematode, causes dirofilariasis in dogs, cats and occasionally in humans. Prevention of the disease has been mainly by monthly use of the macrocyclic lactone (ML) endectocides during the mosquito transmission season. Recently, ML resistance has been confirmed in D. immitis and therefore, there is a need to find new classes of anthelmintics. One of the mechanisms associated with ML resistance in nematodes has been the possible role of ATP binding cassette (ABC) transporters in reducing drug concentrations at receptor sites. ABC transporters, mainly from sub-families B, C and G, may contribute to multidrug resistance (MDR) by active efflux of drugs out of the cell. Gene products of ABC transporters may thus serve as the targets for agents that may modulate susceptibility to drugs, by inhibiting drug transport. ABC transporters are believed to be involved in a variety of physiological functions critical to the parasite, such as sterol transport, and therefore may also serve as the target for drugs that can act as anthelmintics on their own. Knowledge of polymorphism in these ABC transporter genes in nematode parasites could provide useful information for the process of drug design. We have identified 15 ABC transporter genes from sub-families A, B, C and G, in D. immitis, by comparative genomic approaches and analyzed them for polymorphism. Whole genome sequencing data from four ML susceptible (SUS) and four loss of efficacy (LOE) pooled populations were used for single nucleotide polymorphism (SNP) genotyping. Out of 231 SNPs identified in those 15 ABC transporter genes, 89 and 75 of them were specific to the SUS or LOE populations, respectively. A few of the SNPs identified may affect gene expression, protein function, substrate specificity or resistance development and may be useful for transporter inhibitor/anthelmintic drug design, or in order to anticipate resistance development., Graphical abstract Image 1, Highlights • In the D. immitis genome, all ABC-A, -B, -C and -G transporter genes were identified. • Within 15 ABC transporter genes identified in D. immitis, 231 SNP loci were found. • Four exonic SNPs caused changes in predicted secondary structure of ABC proteins. • D. immitis populations have low genetic variability among ABC transporter genes.

Background A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. Methodology and principal findings In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. Conclusions The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs., Author summary DNA-based tools are increasingly being used for the diagnosis of intestinal worm infections in both clinical and research laboratories. However, recovering DNA from intestinal worm eggs in stool remains a challenge since this DNA is protected by a very rigid egg shell. Furthermore, stool contains inhibitors that can affect test results and these should be removed during DNA extraction. Prior to DNA extraction, samples are often preserved, but the impact of the type of preservatives and the duration of preservation remains poorly studied. In the present study, we assessed the impact of four DNA extraction and three preservation protocols on the downstream performance of a DNA-based diagnostic tool for intestinal worms. We found significant differences in DNA recovery across the DNA and preservation protocols, but DNA from worm eggs in stool proved to be stable over time in all preservatives.

Dirofilaria immitis, a filarial parasite, causes cardiopulmonary dirofilariasis in dogs, cats and wild canids. The macrocyclic lactone (ML) class of drugs has been used to prevent heartworm infection. There is confirmed ML resistance in D. immitis and thus there is an urgent need to find new anthelmintics that could prevent and/or control the disease. Targeting ion channels of D. immitis for drug design has obvious advantages. These channels, present in the nematode nervous system, control movement, feeding, mating and respond to environmental cues which are necessary for survival of the parasite. Any new drug that targets these ion channels is likely to have a motility phenotype and should act to clear the worms from the host. Many of the successful anthelmintics in the past have targeted these ion channels and receptors. Knowledge about genetic variability of the ion channel and receptor genes should be useful information for drug design as receptor polymorphism may affect responses to a drug. Such information may also be useful for anticipation of possible resistance development. A total of 224 ion channel genes/subunits have been identified in the genome of D. immitis. Whole genome sequencing data of parasites from eight different geographical locations, four from ML-susceptible populations and the other four from ML-loss of efficacy (LOE) populations, were used for polymorphism analysis. We identified 1762 single nucleotide polymorphic (SNP) sites (1508 intronic and 126 exonic) in these 224 ion channel genes/subunits with an overall polymorphic rate of 0.18%. Of the SNPs found in the exon regions, 129 of them caused a non-synonymous type of polymorphism. Fourteen of the exonic SNPs caused a change in predicted secondary structure. A few of the SNPs identified may have an effect on gene expression, function of the protein and resistance selection processes., Graphical abstract, Highlights • In the Dirofilaria immitis genome, 126 ion channel genes were identified. • Within 126 ion channel genes, 1762 polymorphic loci were identified. • Fourteen exonic SNPs caused a change in predicted secondary structure. • SNPs may effect gene expression, protein function or resistance selection. • D. immitis populations have low genetic variability among ion channel genes.

Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of macrocyclic lactones. We postulate that Dim-PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the 'R' site for Rhodamine 123 and the 'H' site for Hoechst 33342. The avermectins appear to bind the 'R' binding site unlike the milbemycins, whereas both sub-classes of macrocyclic lactones might interact with the 'H' site of D. immitis PGP-11.

Prophylaxis with macrocyclic lactone (ML) endectocides is the primary strategy for heartworm control. Recent evidence has confirmed that ML-resistant Dirofilaria immitis isolates have evolved. Comparison of genomes of ML-resistant isolates show they are genetically distinct from wild-type populations. Previously, we identified single nucleotide polymorphisms (SNPs) that are correlated with phenotypic ML resistance. Since reliable in vitro assays are not available to detect ML resistance in L3 or microfilarial stages, the failure to reduce microfilaraemia in infected dogs treated with an ML has been proposed as a surrogate clinical assay for this purpose. The goal of our study was to validate the genotype-phenotype correlation between SNPs associated with ML resistance and failure to reduce microfilaraemia following ML treatment and to identify a minimal number of SNPs that could be used to confirm ML resistance. In this study, 29 participating veterinary clinics received a total of 148 kits containing supplies for blood collection, dosing and prepaid shipping. Patients recruited after a diagnosis of heartworm infection were treated with a single standard dose of Advantage Multi® and a blood sample taken pre- and approximately 2–4 weeks post-treatment. Each sample was processed by performing a modified Knott's Test followed by isolation of microfilariae, genomic DNA extraction and MiSeq sequencing of regions encompassing 10 SNP sites highly correlated with ML resistance. We observed significant correlation of SNP loci frequencies with the ML microfilaricidal response phenotype. Although all predictive SNP combination models performed well, a 2-SNP model was superior to other models tested. The predictive ability of these markers for ML-resistant heartworms should be further evaluated in clinical and epidemiological contexts., Graphical abstract Image 1, Highlights • Recent evidence has confirmed that heartworm isolates have evolved the ability to survive ML prophylaxis. • SNPs are highly correlated with phenotypic ML resistance, but none identifies a specific “resistance” gene. • SNPs associated with ML resistance correlate with failure to reduce MF following ML treatment. • A 2-SNP model could be employed to diagnose clinical cases of ML resistance.

Haemonchus contortus is a nematode of livestock that can cause severe disease and mortality. Ivermectin, an anti-parasitic drug that targets glutamate-gated chloride channels, is widely used in humans, livestock, companion animals and agriculture. Although an association between genetic changes to β-tubulin and exposure to ivermectin has been previously reported, direct binding between ivermectin and tubulin has not been demonstrated to date. Tubulin/microtubules are key targets for many anti-mitotic drugs used in anti-parasite and cancer therapies. We now report that ivermectin exposure increased the rate and extent of polymerisation of H. contortus recombinant α- and β-tubulin, and protected the parasitic α- and β-tubulins from limited trypsin proteolysis. Direct binding between ivermectin and the tubulin monomers exhibited low micromolar affinities, as determined using surface plasmon resonance. Subsequent equilibrium dialysis indicated that ivermectin and Taxol compete for binding to tubulin, supporting our molecular modelling that predicts ivermectin interacts with the Taxol binding pocket of both parasitic and mammalian tubulins. Collectively, our data indicate that ivermectin can bind to and stabilise microtubules (i.e., alter the tubulin polymerisation equilibrium) and this can then lead to mitotic arrest. This work extends the range of known pharmacological effects of ivermectin, and reveals its potential as an anti-mitotic agent.

Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the macrocyclic lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the macrocyclic lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the macrocyclic lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm’s intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of macrocyclic lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus .

The diversity and uniqueness of nematode heterotrimeric G-protein-coupled receptors (GPCRs) provides impetus for identifying ligands that can be used as therapeutics for treating diseases caused by parasitic nematode infections. In human medicine, GPCRs have represented the largest group of 'drugable' targets exploited in the market today. In the filarial nematode Dirofilaria immitis, which causes heartworm disease, the macrocyclic lactones (ML) have been used as the sole preventatives for more than 25 years and now there is confirmed ML resistance in this parasite. A novel anthelmintic emodepside, with antifilarial activity, can act on a GPCR. In view of the ML resistance, there is an urgent need to identify new drug targets and GPCRs of D. immitis may be promising receptors. Knowledge of polymorphism within the GPCR superfamily is of interest. A total of 127 GPCR genes have been identified, so far, in the genome of D. immitis. Whole genome sequencing data from four ML susceptible and four ML loss of efficacy populations was used to identify 393 polymorphic loci in 35 D. immitis GPCR genes. Out of 57 SNPs in exonic regions, 36 of them caused a change in an amino acid, out of which 2 changed the predicted secondary structure of the protein. Knowledge about GPCR genes and their polymorphism is valuable information for drug design processes. Further studies need to be carried out to more fully understand the implications of each of the SNPs identified by this study.

Background For dogs and cats, chemoprophylaxis with macrocyclic lactone (ML) preventives for heartworm disease is widely used in the United States and other countries. Since 2005, cases of loss of efficacy (LOE) of heartworm preventives have been reported in the U.S. More recently, ML-resistant D. immitis isolates were confirmed. Previous work identified 42 genetic markers that could predict ML response in individual samples. For field surveillance, it would be more appropriate to work on microfilarial pools from individual dogs with a smaller subset of genetic markers. Methods MiSeq technology was used to identify allele frequencies with the 42 genetic markers previously reported. Microfilaria from ten well-characterized new isolates called ZoeKY, ZoeMI, ZoeGCFL, ZoeAL, ZoeMP3, ZoeMO, ZoeAMAL, ZoeLA, ZoeJYD-34, and Metairie were extracted from fresh blood from dogs. DNA were extracted and sequenced with MiSeq technology. Allele frequencies were calculated and compared with the previously reported susceptible, LOE, and resistant D. immitis populations. Results The allele frequencies identified in the current resistant and susceptible isolates were in accordance with the allele frequencies previously reported in related phenotypes. The ZoeMO population, a subset of the ZoeJYD-34 population, showed a genetic profile that was consistent with some reversion towards susceptibility compared with the parental ZoeJYD-34 population. The Random Forest algorithm was used to create a predictive model using different SNPs. The model with a combination of three SNPs (NODE_42411_RC, NODE_21554_RC, and NODE_45689) appears to be suitable for future monitoring. Conclusions MiSeq technology provided a suitable methodology to work with the microfilarial samples. The list of SNPs that showed good predictability for ML resistance was narrowed. Additional phenotypically well characterized D. immitis isolates are required to finalize the best set of SNPs to be used for large scale ML resistance screening. Electronic supplementary material The online version of this article (10.1186/s13071-017-2428-6) contains supplementary material, which is available to authorized users.

Graphical abstract, Highlights • We report on the Consortium for Anthelmintic Resistance and Susceptibility 2013 meeting. • Recent advances in the identification of markers for anthelmintic resistance are described. • The use of markers for benzimidazole resistance in field studies with veterinary and human nematodes. • The application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance., Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.

Heavy reliance on macrocyclic lactones to treat parasitic nematodes has resulted in the evolution of widespread drug resistance that threatens human and animal health. Management strategies have been proposed that would slow the rise of resistance, however testing these strategies has been hampered by the lack of identified strong-effect resistance markers in parasites. We show that the Caenorhabditis elegans gene Cel_dyf-7, necessary for amphid sensory neuron development, also confers macrocyclic lactone sensitivity. In the sheep parasite Haemonchus contortus: (i) strains selected for macrocyclic lactone resistance were enriched in a Hco_dyf-7 haplotype that was rare in the drug-naïve population, (ii) the resistant haplotype correlated with the sensory neuron defects, and (iii) the resistant haplotype was associated with decreased Hco_dyf-7 expression. Resistant field isolates of H. contortus from five continents were enriched for the resistant haplotype, demonstrating the relevance of the Hco_dyf-7 haplotype to practise and indicating that it is a locus of strong effect. Hemizygosity resulting from sex linkage of dyf-7 likely contributes to the rise of resistance in treated populations.

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.

Background Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana—exposed to more than a decade of regular ivermectin treatment—have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread. Methodology/Principal findings Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR. Conclusions/Significance This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations., Author summary Onchocerciasis is a human parasitic disease endemic across large areas of Sub-Saharan Africa, where more than 99% of the estimated 100 million people globally at-risk live. The microfilarial stage of Onchocerca volvulus causes pathologies ranging from mild itching to visual impairment and ultimately, irreversible blindness. Mass administration of ivermectin kills microfilariae and has an anti-fecundity effect on adult worms by temporarily inhibiting the development in utero and/or release into the skin of new microfilariae, thereby reducing morbidity and transmission. Phenotypic and genetic changes in some parasite populations that have undergone multiple ivermectin treatments in Cameroon and Ghana have raised concern that sub-optimal response to ivermectin's anti-fecundity effect may increase in frequency, reducing the impact of ivermectin-based control measures. We used next generation sequencing of small pools of parasites to define genome-wide genetic differences between phenotypically characterised good and sub-optimal responder parasites from Cameroon and Ghana, and identified multiple regions of the genome that differentiated the response types. These regions were largely different between parasites from these two countries but revealed common molecular pathways that might be involved in determining the extent of response to ivermectin's anti-fecundity effect. These data reveal a more complex than previously described pattern of genetic diversity among O. volvulus populations that differ in their geography and response to ivermectin treatment.

Background Soil-transmitted helminths (STHs) are the most prevalent intestinal helminths of humans, and a major cause of morbidity in tropical and subtropical countries. The benzimidazole (BZ) drugs albendazole (ABZ) and mebendazole (MBZ) are used for treatment of human STH infections and this use is increasing dramatically with massive drug donations. Frequent and prolonged use of these drugs could lead to the emergence of anthelmintic resistance as has occurred in nematodes of livestock. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing. However, these techniques are complicated and time consuming and not suitable for resource-constrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs. Methods To address this problem, single nucleotide polymorphism (SNP) detection assays were developed based on the Smart amplification method (SmartAmp2) to target codons 167, 198, and 200 in the β-tubulin isotype 1 gene for the hookworm Necator americanus. Findings Diagnostic assays were developed and applied to analyze hookworm samples by both SmartAmp2 and conventional sequencing methods and the results showed high concordance. Additionally, fecal samples spiked with N. americanus larvae were assessed and the results showed that the Aac polymerase used has high tolerance to inhibitors in fecal samples. Conclusion The N. americanus SmartAmp2 SNP detection assay is a new genotyping tool that is rapid, sensitive, highly specific and efficient with the potential to be used as a field tool for monitoring SNPs associated with BZ resistance. However, further validation on large numbers of field samples is required., Author Summary Hookworms are amongst the major STHs and the second most prevalent intestinal helminth of humans. Large-scale treatment with the benzimidazoles (BZs) albendazole or mebendazole is the major control strategy against STHs in mass drug administration (MDA) programs. Prolonged and repeated treatment with the same anthelmintics has led to the emergence of widespread BZ resistance in veterinary parasites which is caused by a single nucleotide polymorphism at codon 200, 167 or 198 in the β-tubulin gene. There is a considerable concern that prolonged use of the same anthelmintics with suboptimal efficacy against hookworms, may select for resistant parasites and favour the development of resistance. We developed a novel genotyping assay to screen for β-tubulin polymorphisms in N. americanus, using the SmartAmp2 method. SmartAmp2 is a unique genotyping technology that detects a mutation under isothermal conditions with high specificity and sensitivity. The N. americanus SNP detection assay is rapid, sensitive and highly specific and has the potential to be used in the field for the detection of SNPs associated with BZ resistance.

Background The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trichiura to BZ treatment. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established. Methods The objective of this study was to develop new genotyping assays to screen for the presence of β-tubulin SNPs in T. trichiura and Ascaris lumbricoides. Rapid, simple and accurate genotyping assays were developed based on the SmartAmp2 method. Primer sets were optimized and selected to distinguish the SNP-variant genotypes. After initial optimization on control plasmids, the feasibility of the assay was assessed in field samples from Haiti and Panama. Finally, spiked fecal samples were assessed to determine the tolerance of Aac polymerase to fecal inhibitors. Findings Rapid SNP genotyping assays were developed to target β-tubulin polymorphisms in T. trichiura and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to PCR inhibitors in fecal samples. Conclusion These assays proved to be robust and efficient with the potential to be used as field tools for monitoring SNPs that could be associated with BZ resistance. However, further work is needed to validate the assays on large numbers of field samples before and after treatment., Author Summary The soil-transmitted helminths (STHs) Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale, are the most prevalent intestinal helminths of humans, causing morbidity in developing countries. Large-scale preventive chemotherapy with albendazole or mebendazole is the major control strategy against STHs in mass drug administration (MDA) programs. In veterinary parasites, intensive reliance on the same anthelmintics has led to the emergence of benzimidazole (BZ) resistance, which is caused by a single nucleotide polymorphism at either codon 200, 167 or 198 in the β-tubulin isotype 1 gene. A great concern is that the intensive use of anthelmintics with suboptimal efficacy may lead to the development of resistance in human STHs. We developed novel genotyping assays to screen for β-tubulin polymorphisms in T. trichiura and A. lumbricoides based on the SmartAmp2 method. SmartAmp2 is a unique genotyping technology that detects a mutation under isothermal conditions with high specificity and sensitivity. The SNP detection assays were rapid, sensitive and highly specific for the detection of SNPs associated with BZ resistance with the potential to be used for monitoring in the field.

Macrocyclic lactones (ML) are important anthelmintics used in animals and humans against parasite nematodes, but their therapeutic success is compromised by the spread of ML resistance. Some ABC transporters, such as p-glycoproteins (Pgps), are selected and overexpressed in ML-resistant nematodes, supporting a role for some drug efflux proteins in ML resistance. However, the role of such proteins in ML transport remains to be clarified at the molecular level. Recently, Caenorhabditis elegans Pgp-1 (Cel-Pgp-1) has been crystallized, and its drug-modulated ATPase function characterized in vitro revealed Cel-Pgp-1 as a multidrug transporter. Using this crystal structure, we have developed an in silico drug docking model in order to study the binding of ML and other anthelmintic drugs to Cel-Pgp-1. All tested ML bound with high affinity in a unique site, within the inner chamber of the protein, supporting that ML may be transported by Cel-Pgp-1. Interestingly, interacting residues delineate a ML specific fingerprint involving H-bonds, including T1028. In particular, benzofurane and spiroketal moieties bound to specific sub-sites. When compared with the aglycone ML, such as moxidectin and ivermectin aglycone, avermectin anthelmintics have significant higher affinity for Cel-Pgp-1, likely due to the sugar substituent(s) that bind to a specific area involving H-bonds at Y771. Triclabendazole, closantel and emodepside bound with good affinities to different sub-sites in the inner chamber, partially overlapping with the ML binding site, suggesting that they could compete for Cel-Pgp-1-mediated ML transport. In conclusion, this work provides novel information on the role of nematode Pgps in transporting anthelmintics, and a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of clinically-relevant nematode Pgps, to improve anthelmintic therapeutics., Graphical abstract Ivermectin (red spheres) binding to Cel-Pgp-1 (blue ribbon: N-term, yellow ribbon: C-term).Image 1, Highlights • Cel-Pgp-1 cristal provides a valuable tool to study anthelmintic interactions. • Cel-Pgp-1 recognizes a broad panel of anthelmintics, including macrocyclic lactones. • Cel-Pgp-1 is predicted to transport MLs and contribute to anthelmintic resistance. • Sugars in avermectins increase the affinity to Cel-Pgp-1 compared with moxidectin. • These data provide a basis for modelling ABC homologs in parasitic nematodes.

Background The existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health. Macrocyclic lactone resistance in H. contortus seems to be multigenic including the active efflux of these drugs by P-glycoproteins, members of the ABC transporter family, present in this parasite. The goals of the present work were to determine the activity of H. contortus P-glycoprotein 9.1 (Hco-PGP-9.1) and its interaction with the avermectins, ivermectin, abamectin, and also the milbemycin, moxidectin. Additionally, the localisation of Hco-PGP-9.1 was sought in adult worms. Methods Hco-Pgp-9.1 was cloned and expressed in mammalian cells and its expression profile was determined at the transcriptional and protein level by qRT-PCR and Western-blot, respectively. The nematode transport activity was assessed using the tracer dye Rhodamine 123. A ligand competition assay between different macrocyclic lactones and Rhodamine 123 was used to establish whether or not there was interaction between Hco-PGP-9.1 and the avermectins (abamectin and ivermectin) or moxidectin. In addition, immunostaining was carried out to localise Hco-PGP-9.1 expression in the transgenic cells and in adult female parasites. Results Hco-PGP-9.1 was expressed in the cell membrane of the transfected host cells and was able to extrude Rhodamine 123. Ivermectin and abamectin, but not moxidectin, had a pronounced inhibitory effect on the ability of Hco-PGP-9.1 to transport Rhodamine 123. Antibodies raised against Hco-PGP-9.1 epitopes localised to the uterus of adult female H. contortus. Conclusions These results suggest a strong interaction of the avermectins with Hco-PGP-9.1. However, possibly due to its physico-chemical properties, moxidectin had markedly less effect on Hco-PGP-9.1. Because of the greater interaction of the avermectins than moxidectin with this transporter, it is more likely to contribute to avermectin resistance than to moxidectin resistance in H. contortus. Possible over expression of Hco-PGP-9.1 in the female reproductive system in resistant worms could reduce paralysis of the uterus by macrocyclic lactones, allowing continued egg release in drug challenged resistant worms. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1317-8) contains supplementary material, which is available to authorized users.

The functions of nine ATP-binding cassette (ABC) transporter genes, mrp-1 , mrp-4 , mrp-6 , pgp-2 , pgp-3 , pgp-4 , pgp-5 , haf-2 and haf-9 , in an ivermectin (IVM) resistant strain of Caenorhabditis elegans were screened by comparing transcription levels between the resistant (IVR10) and wild-type (Bristol N2) strains, and by measuring the effects of RNA interference (RNAi) on the IVM resistant strain, on motility, pharyngeal pumping, egg production and death in the presence or varying concentrations of IVM (0–20 ng/ml). mRNA levels of mrp-1 , 2 , 4 , 5 , 6 , 7 , pgp-1 , 2 , 4 , 12 , 14 , haf-1 , 2 and 3 were significantly increased in IVR10 compared with the N2 strain. At 15 or 20 ng/ml IVM, down regulation of mrp-1 , pgp-4 , haf-2 and haf-9 significantly increased the effect of IVM to reduce egg production. At low to moderate IVM concentrations, down regulation of mrp-1 and haf-2 reduced the motility of C. elegans . However, at high IVM concentrations motility was increased by down regulation of transcription of pgp-3 , pgp-4 and haf-9 . Down regulation of expression of mrp-1 , pgp-2 and pgp-5 resulted in reduced pharyngeal pumping in the presence of varying concentrations of IVM, while down regulation of mrp-6 and haf-2 increased pharyngeal pumping of the resistant strain irrespective of the IVM concentration used. Although the IVR10 strain was markedly resistant to IVM, compared with the unselected N2 strain, IVM led to the death of the C. elegans in a concentration dependent manner. However, differences in the IVM induced death rate, following RNAi, were not significantly different from the IVR10 strain without RNAi. The study shows that different ABC transporter genes may play a role in modulating the effects of IVM on pharyngeal pumping, motility and egg production, with down regulation of mrp-1 and haf-2 perhaps having the greatest effects. However, down regulation of expression of no individual ABC transporter gene profoundly affected the effect of IVM on mortality in the IVR10 strain. This suggests that some of these ABC transporter genes and their products may play a role in modulating the effects of IVM, but are not, individually, the critical gene responsible for IVM resistance. This study provides a model that may help to understand drug resistance in parasitic nematodes.