Development of emodepside as a possible adulticidal treatment for human onchocerciasis-The fruit of a successful industrial-academic collaboration.

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.

Reaching the london declaration on neglected tropical diseases goals for onchocerciasis: an economic evaluation of increasing the frequency of ivermectin treatment in Africa.

BACKGROUND: Recently, there has been a shift in onchocerciasis control policy, changing from prevention of morbidity toward elimination of infection. Switching from annual to biannual ivermectin distribution may accelerate progress toward the elimination goals. However, the settings where this strategy would be cost effective in Africa have not been described. METHODS: An onchocerciasis transmission framework (EpiOncho) was coupled to a disease model in order to explore the impact on disability-adjusted life years averted, program cost, and program duration of biannual ivermectin treatment in different epidemiological and programmatic scenarios in African savannah. RESULTS: While biannual treatment yields only small additional health gains, its benefit is pronounced in the context of the elimination goals, shortening the time frames for and increasing the feasibility of reaching the proposed operational thresholds for stopping treatment. In settings with high precontrol endemicity (and/or poor coverage and compliance), it may not be possible to reach such thresholds even within 50 years of annual ivermectin, requiring adoption of biannual treatment. Our projections highlight the crucial role played by coverage and compliance in achieving the elimination goals. CONCLUSIONS: Biannual ivermectin treatment improves the chances of reaching the 2020/2025 elimination goals, potentially generating programmatic cost savings in settings with high precontrol endemicity. However, its benefit and cost are highly sensitive to levels of systematic noncompliance and, in many settings, it will lead to an increase in costs. Furthermore, it may not always be feasible to implement biannual treatment, particularly in hard-to-reach populations. This highlights the continued need for a macrofilaricide.

Investigating Dirofilaria immitis isolates infecting domestic canines and their susceptibility/resistance patterns to macrocyclic lactones in the northern region of the Mississippi Delta area (southeast Missouri).

Previous reports of macrocyclic lactone (ML) resistance in Dirofilaria immitis, the parasitic nematode which causes heartworm disease, have mainly been from the southern Mississippi Delta region. Southeast Missouri (SEMO), forming the northern boundary of this region, has not previously been well studied. The area is an ideal propagation region for heartworm infection and possibly for the spread of ML resistance. To assess whether D. immitis isolates infecting domestic canines in SEMO exhibit evidence of resistance to MLs, domestic canines, presented to veterinary facilities testing positive for heartworms through antigen and microfilariae (MF) examination, were utilized in the study. Using a descriptive epidemiological cross-sectional study, from March 2021 through February 2022, blood sample collection from 96 canines living in SEMO testing positive for heartworms were analyzed. MiSeq technology was utilized to sequence specific genetic markers associated with susceptibility/resistance for MLs in D. immitis isolates. Genomic data revealed most D. immitis isolates had genotypic profiles consistent with resistance to MLs. Of the 96 samples tested, 91 (94.8%) had a resistant genotype, 4 (4.2%) had a mixed genotype, and 1 sample (1%) genotyped as susceptible. While detailed and reliable medical histories were not available for most canines, detailed medical history from 2 canines indicated evidence of phenotypic resistance that was consistent with their genotypes. However, in vivo preventive tests are needed to confirm a high frequency of phenotypic ML resistance in D. immitis from this region. Increasing resistance patterns to MLs indicate the approach to heartworm prevention/treatment protocol should be reconsidered. New measures may be required to stop heartworm disease.

Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR.

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease") primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.

Droplet digital PCR as a tool to detect resistant isolates of Dirofilaria immitis.

Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance.

Identifying sub-optimal responses to ivermectin in the treatment of River Blindness.

Identification of drug resistance before it becomes a public health concern requires a clear distinction between what constitutes a normal and a suboptimal treatment response. A novel method of analyzing drug efficacy studies in human helminthiases is proposed and used to investigate recent claims of atypical responses to ivermectin in the treatment of River Blindness. The variability in the rate at which Onchocerca volvulus microfilariae repopulate host's skin following ivermectin treatment is quantified using an individual-based onchocerciasis mathematical model. The model estimates a single skin repopulation rate for every host sampled, allowing reports of suboptimal responses to be statistically compared with responses from populations with no prior exposure to ivermectin. Statistically faster rates of skin repopulation were observed in 3 Ghanaian villages (treated 12-17 times), despite the wide variability in repopulation rates observed in ivermectin-naïve populations. Another village previously thought to have high rates of skin repopulation was shown to be indistinguishable from the normal treatment response. The model is used to generate testable hypotheses to identify whether atypical rates of skin repopulation by microfilariae could result from low treatment coverage alone or provide evidence of decreased ivermectin efficacy. Further work linking phenotypic poor responses to treatment with parasite molecular genetics markers will be required to confirm drug resistance. Limitations of the skin-snipping method for estimating parasite load indicates that changes in the distribution of microfilarial repopulation rates, rather than their absolute values, maybe a more sensitive indicator of emerging ivermectin resistance.

Macrocyclic lactone resistance in Dirofilaria immitis: risks for prevention of heartworm disease.

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.

Concern for Dirofilaria immitis and Macrocyclic Lactone Loss of Efficacy: Current Situation in the USA and Europe, and Future Scenarios.

Dirofilaria immitis infection is one of the most severe parasitic diseases in dogs. Prevention is achieved by the administration of drugs containing macrocyclic lactones (MLs). These products are very safe and highly effective, targeting the third and fourth larval stages (L3, L4) of the parasite. Until 2011, claims of the ineffectiveness of MLs, reported as "loss of efficacy" (LOE), were generally attributed to owners' non-compliance, or other reasons associated with inadequate preventative coverage. There was solid argumentation that a resistance problem is not likely to occur because of (i) the great extent of refugia, (ii) the complexity of resistance development to MLs, and (iii) the possible large number of genes involved in resistance selection. Nevertheless, today, it is unequivocally proven that ML-resistant D. immitis strains exist, at least in the Lower Mississippi region, USA. Accordingly, tools have been developed to evaluate and confirm the susceptibility status of D. immitis strains. A simple, in-clinic, microfilariae suppression test, 14-28 days after ML administration, and a "decision tree" (algorithm), including compliance and preventatives' purchase history, and testing gaps, may be applied for assessing any resistant nature of the parasite. On the molecular level, specific SNPs may be used as markers of ML resistance, offering a basis for the validation of clinically suspected resistant strains. In Europe, no LOE/resistance claims have been reported so far, and the existing conditions (stray dogs, rich wildlife, majority of owned dogs not on preventive ML treatment) do not favor selection pressure on the parasites. Considering the genetic basis of resistance and the epizootiological characteristics of D. immitis, ML resistance neither establishes easily nor spreads quickly, a fact confirmed by the current known dispersion of the problem, which is limited. Nevertheless, ML resistance may propagate from an initial geographical point, via animal and vector mobility, to other regions, while it can also emerge as an independent evolutionary process in a new area. For these reasons, and considering the current chemoprophylaxis recommendations and increasing use of ML endectoparasiticides as a potential selection pressure, it is important to remain vigilant for the timely detection of any ML LOE/resistance, in all continents where D. immitis is enzootic.

Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript level using droplet digital PCR.

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.

The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12.

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.

Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance.

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.

Piloting a surveillance system to monitor the global patterns of drug efficacy and the emergence of anthelmintic resistance in soil-transmitted helminth control programs: a Starworms study protocol.

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 ß-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.

Perspectives on the utility of moxidectin for the control of parasitic nematodes in the face of developing anthelmintic resistance.

Macrocyclic lactone (ML) anthelmintics are the most important class of anthelmintics because of our high dependence on them for the control of nematode parasites and some ectoparasites in livestock, companion animals and in humans. However, resistance to MLs is of increasing concern. Resistance is commonplace throughout the world in nematode parasites of small ruminants and is of increasing concern in horses, cattle, dogs and other animals. It is suspected in Onchocerca volvulus in humans. In most animals, resistance first arose to the avermectins, such as ivermectin (IVM), and subsequently to moxidectin (MOX). Usually when parasite populations are ML-resistant, MOX is more effective than avermectins. MOX may have higher intrinsic potency against some parasites, especially filarial nematodes, than the avermectins. However, it clearly has a significantly different pharmacokinetic profile. It is highly distributed to lipid tissues, less likely to be removed by ABC efflux transporters, is poorly metabolized and has a long half-life. This results in effective concentrations persisting for longer in target hosts. It also has a high safety index. Limited data suggest that anthelmintic resistance may be overcome, at least temporarily, if a high concentration can be maintained at the site of the parasites for a prolonged period of time. Because of the properties of MOX, there are reasonable prospects that strains of parasites that are resistant to avermectins at currently recommended doses will be controlled by MOX if it can be administered at sufficiently high doses and in formulations that enhance its persistence in the host. This review examines the properties of MOX that support this contention and compares them with the properties of other MLs. The case for using MOX to better control ML-resistant parasites is summarised and some outstanding research questions are presented.

ABC transporter modulation: a strategy to enhance the activity of macrocyclic lactone anthelmintics.

The emergence of parasites resistant to anthelmintic macrocyclic lactones (MLs) threatens to severely limit current parasite control strategies. Improving the current ML-based chemotherapy to perpetuate the efficacy of this broad-spectrum class of anthelmintics would be advantageous. In recent years it has become evident that the absorption, distribution and elimination of the MLs in hosts and parasites are under the control of multidrug resistance transporters (MDRs) such as P-glycoproteins. Theoretically, the inhibition of these transporters should result in an increase of the drug concentration in the organisms and higher treatment efficiency. This opinion article will discuss the recent findings in this research field and assess the possibilities of this approach being used in the field.

Prevalence and intensity of Onchocerca volvulus infection and efficacy of ivermectin in endemic communities in Ghana: a two-phase epidemiological study.

BACKGROUND: Ivermectin has been used for onchocerciasis control since 1987. Because of the long-term use of this drug and the development of resistance in other nematodes, we have assessed Onchocerca volvulus burdens, effectiveness of ivermectin as a microfilaricide, and its effect on adult female worm reproduction. METHODS: For the first phase of the study, 2501 individuals in Ghana, from 19 endemic communities who had received six to 18 annual rounds of ivermectin and one ivermectin naive community, were assessed for microfilarial loads 7 days before the 2004 yearly ivermectin treatment, by means of skin snips, and 30 days after treatment to assess the ivermectin microfilaricidal action. For the second phase, skin snips were taken from 342 individuals from ten communities, who were microfilaria positive at pretreatment assessment, on days 90 and 180 after treatment, to identify the effects of ivermectin on female worm fertility, assessed by microfilaria repopulation. FINDINGS: 487 (19%) of the 2501 participants were microfilaria positive. The microfilaria prevalence and community microfilarial load in treated communities ranged from 2.2% to 51.8%, and 0.06 microfilariae per snip to 2.85 microfilariae per snip, respectively. Despite treatment, the prevalence rate doubled between 2000 and 2005 in two communities. Microfilaria assessment 30 days after ivermectin treatment showed 100% clearance of microfilaria in more than 99% of people. At day 90 after treatment, four of ten communities had significant microfilaria repopulation, from 7.1% to 21.1% of pretreatment counts, rising to 53.9% by day 180. INTERPRETATION: Ivermectin remains a potent microfilaricide. However, our results suggest that resistant adult parasite populations, which are not responding as expected to ivermectin, are emerging. A high rate of repopulation of skin with microfilariae will allow parasite transmission, possibly with ivermectin-resistant O volvulus, which could eventually lead to recrudescence of the disease.

P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus.

Ivermectin (IVM) is the only safe drug for mass-treatment of onchocerciasis. IVM resistance has been reported in gastrointestinal nematode parasites of animals. A reduction in response to IVM in Onchocerca volvulus could have significant consequences for the onchocerciasis control programs. We have found evidence that, in O. volvulus, repeated IVM treatment selects for specific alleles, of P-glycoprotein-like protein (PLP), a half-sized ABC transporter. In this study, O. volvulus samples were derived from a clinical trial in Cameroon, in which patients were sampled before, and following 3 years (1994-1997) of IVM treatments. There were four treatment groups: 150 microg/kg (1 x p.a. or 4 x p.a.) and 800 microg/kg (1 x p.a. or 4 x p.a.). DNA of O. volvulus macrofilariae was genotyped over a 476bp region of the PLP gene and at two control genes. Of the six polymorphic positions found in the PLP amplicon, three of them showed significant selection after 4 x p.a. treatment with IVM (total of 13 IVM treatments) in female worms, and one of the same single nucleotide polymorphisms (SNPs) showed significant selection in the male worms. One of the selected SNPs in the female worms caused an amino acid coding change in the putative protein sequence. We found a clear selection of some genotypes, a high SNPs association and a loss of polymorphism following 4 x p.a. treatment with IVM. These PLP SNPs and genotypes could be useful markers to follow selection for IVM resistance in the field.

P-glycoprotein selection in strains of Haemonchus contortus resistant to benzimidazoles.

Anthelmintic resistance in parasitic nematodes of livestock is a chronic problem in many parts of the world. Benzimidazoles are effective, broad-spectrum anthelmintics that bind to and selectively depolymerise microtubules. Resistance to the benzimidazoles, however, developed quickly and is caused by genetic changes in genes encoding beta-tubulins, subunits of microtubules. In Haemonchus contortus, resistance to avermectins has been correlated with genetic changes at a gene encoding a P-glycoprotein, a cell membrane transport protein that has a very high affinity for ivermectin. The substrate specificity of P-glycoprotein is very broad, and resistance to benzimidazoles can be modulated by lectins specific for P-glycoprotein. We investigated the possibility that genetic changes in P-glycoprotein might be correlated with benzimidazole resistance in nematodes. An analysis of restriction fragment length polymorphisms of a P-glycoprotein gene from a sensitive and a cambendazole-selected strain of H. contortus, derived from the sensitive strain, showed a significant difference in allele frequencies between strains. The frequency of one allele in particular increased substantially. The same allele was also found at a high frequency in an independently derived thiabendazole-selected field isolate. We present genetic evidence of selection at a P-glycoprotein locus during selection for benzimidzole resistance in H. contortus.

G-protein-coupled receptor genes of Dirofilaria immitis.

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.

Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13.

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.