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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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Roger Prichard reports financial support was provided by Zoetis. Roger Prichard reports a relationship with Zoetis that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Thavy Long reports financial support and equipment, drugs, or supplies were provided by Merck Animal Health Rahway. Dr. Roger K. Prichard reports financial support and equipment, drugs, or supplies were provided by Merck Animal Health Rahway. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

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., (Copyright © 2021. Published by Elsevier Ltd.)

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.

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., Competing Interests: I have read the journal’s policy and want to declare the following conflicts of interest. The authors DK, SH, AH were employees of Bayer Animal Health GmbH, Germany. Bayer Animal Health GmbH was a company that developed and commercialized veterinary medicines including anthelmintics. The remaining authors of this manuscript have declared no competing interests.

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.

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Vlaminck J et al.)

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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 (F ST ), 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 F ST 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.

Background: Diagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies. Molecular diagnostics are powerful tools to identify closely related species, but the requirement for costly equipment makes their implementation difficult in low-resource or field settings. Rapid, sensitive and cost-effective diagnostic tools are crucial for accurate estimation of STH infection intensity in MDA programmes in which the goal is to reduce morbidity following repeated rounds of chemotherapy., Results: In this study, colourimetric isothermal assays were developed using SmartAmp2 primer sets and reagents in loop-mediated amplification (LAMP) assays. Species-specific primer sets, designed on a specific target sequence in the β-tubulin gene, were used to identify Necator americanus, Trichuris trichiura and Ascaris lumbricoides. After initial optimization on control plasmids and genomic DNA from adult worms, assays were evaluated on field samples. Assays showed high sensitivity and demonstrated high tolerance to inhibitors in spiked faecal samples. Rapid and sensitive colourimetric assays were successfully developed to identify the STHs in field samples using hydroxy napthol blue (HNB) dye., Conclusions: Rapid and simple colourimetric diagnostic assays, using the SmartAmp2 method, were developed, with the potential to be applied in the field for detection of STH infections and the estimation of response to treatment. However, further validation on large numbers of field samples is needed.

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., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2016. Published by Elsevier Ltd.)

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., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite., (Copyright © 2016. Published by Elsevier Ltd.)

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.

Haemonchus contortus has shown a great ability to develop resistance to anthelmintic drugs. In many instances, resistance has appeared less than 10years after the introduction of a new drug class. Field populations of this species now show resistance to all major anthelmintic drug classes, including benzimidazoles (BZs), imidazothiazoles and macrocyclic lactones. In addition, resistance to the recently introduced amino-acetonitrile derivative class (monepantel) has already been reported. The existence of field populations showing resistance to all three major drug classes, and the early appearance of resistance to monepantel, threatens the sustainability of sheep and goat production systems worldwide. This chapter reviews the history of the development of resistance to the various anthelmintics in H. contortus and examines the mechanisms utilized by this species to resist the effects of these drugs. Some of these mechanisms are well understood, particularly for BZ drugs, while our knowledge and understanding of others are increasing. Finally, we summarize methods available for the diagnosis of resistance. While such diagnosis currently relies largely on the faecal egg count reduction test, which suffers from issues of expense and sensitivity, we describe past and current efforts to utilize cheaper and less laborious phenotypic assays with free-living life stages, and then describe progress on the development of molecular assays to provide sensitive resistance-detection tests., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Anthelmintic resistance in veterinary nematodes, including Haemonchus contortus, has become a limitation to maintaining high standards of animal health. Resistance in this parasite, to all drug families including the macrocyclic lactones (MLs) is a serious issue worldwide. Mechanisms of resistance to the MLs appear to be complex and to include the elimination of these compounds by ABC transporter-like proteins present in nematodes. In order to investigate the potential involvement of ABC transporters in ML resistance in H. contortus, we have characterized the functionality of the ABC transporter H. contortus P-glycoprotein-16 (Hco-PGP-16) expressed in mammalian cells. This has included a study of its interaction with different MLs, including the avermectins, abamectin (ABA) and ivermectin (IVM), and the milbemycin, moxidectin (MOX). Hco-PGP-16 transport activity was studied using the fluorophore Rhodamine 123 (Rho 123). Transfected cells expressing Hco-PGP-16 accumulated less than 50% of Rho 123 than control cells, suggesting an active transport of this tracer dye by Hco-PGP-16. The influence of the MLs on the Rho123 transport by Hco-PGP-16 was then investigated. A marked inhibition of Rho123 transport by ABA and IVM was observed. In contrast, MOX showed less effect on inhibition of Rho123 transport by Hco-PGP-16, and the inhibition was not saturable. The difference in the interaction of the avermectins and MOX with Hco-PGP-16 may help explain the slower rate of development of resistance to MOX compared with the avermectins in H. contortus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

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.

Our objective was to determine if the resistance mechanism to moxidectin (MOX) is similar of that to ivermectin (IVM) and involves P-glycoproteins (PGPs). Several Caenorhabditis elegans strains were used: an IVM and MOX sensitive strain, 13 PGP deletion strains and the IVM-R strain which shows synthetic resistance to IVM (by creation of three point mutations in genes coding for α-subunits of glutamate gated chloride channels [GluCls]) and cross-resistance to MOX. These strains were used to compare expression of PGP genes, measure motility and pharyngeal pumping phenotypes and evaluate the ability of compounds that inhibit PGP function to potentiate sensitivity or reverse resistance to MOX. The results suggest that C. elegans may use regulation of PGPs as a response mechanism to MOX. This was indicated by the over-expression of several PGPs in both drug sensitive and IVM-R strains and the significant changes in phenotype in the IVM-R strain in the presence of PGP inhibitors. However, as the inhibitors did not completely disrupt expression of the phenotypic traits in the IVM-R strain, this suggests that there likely are multiple avenues for MOX action that may include receptors other than GluCls. If MOX resistance was mediated solely by GluCls then exposure of the IVM-R strain to PGP inhibitors should not have affected sensitivity to MOX. Targeted gene deletions showed that protection of C. elegans against MOX involves complex mechanisms and depends on the PGP gene family, particularly PGP-6. While the results presented are similar to others using IVM, there were some important differences observed with respect to PGPs which may play a role in the disparities seen in the characteristics of resistance to IVM and MOX. The similarities are of concern as parasites resistant to IVM show some degree but not complete cross-resistance to MOX; this could impact nematodes that are resistant to IVM.

Background: For two decades, onchocerciasis control has been based on mass treatment with ivermectin (IVM), repeated annually or six-monthly. This drug kills Onchocerca volvulus microfilariae (mf) present in the skin and the eyes (microfilaricidal effect) and prevents for 3-4 months the release of new mf by adult female worms (embryostatic effect). In some Ghanaian communities, the long-term use of IVM was associated with a more rapid than expected skin repopulation by mf after treatment. Here, we assessed whether the embryostatic effect of IVM on O. volvulus has been altered following frequent treatment in Cameroonian patients., Methodology: Onchocercal nodules were surgically removed just before (D0) and 80 days (D80) after a standard dose of IVM in two cohorts with different treatment histories: a group who had received repeated doses of IVM over 13 years, and a control group with no history of large-scale treatments. Excised nodules were digested with collagenase to isolate adult worms. Embryograms were prepared with females for the evaluation of their reproductive capacities., Principal Findings: Oocyte production was not affected by IVM. The mean number of intermediate embryos (morulae and coiled mf) decreased similarly in the two groups between D0 and D80. In contrast, an accumulation of stretched mf, either viable or degenerating, was observed at D80. However, it was observed that the increase in number of degenerating mf between D0 and D80 was much lower in the frequently treated group than in the control one (Incidence Rate Ratio: 0.25; 95% CI: 0.10-0.63; p = 0.003), which may indicate a reduced sequestration of mf in the worms from the frequently treated group., Conclusion/significance: IVM still had an embryostatic effect on O. volvulus, but the effect was reduced in the frequently treated cohort compared with the control population.

A levamisole-sensitive acetylcholine receptor has recently been described in the parasitic nematode Haemonchus contortus. The pentameric receptor is produced from different subunit proteins, one of which is Hco-ACR-8. A truncated transcript, Hco-acr-8b, has been identified in six levamisole-resistant H. contortus isolates and was found to be absent in four levamisole-susceptible isolates, indicating Hco-acr-8b could be a potential marker for levamisole resistance. The Hco-acr-8b transcript contains exons 1 and 2 and terminates with 347bp from within the intron 2. In this work, we investigated genomic DNA sequences of the Hco-acr-8 gene, in a region including exon 2 and exon 3, from a wide range of levamisole-susceptible and resistant H. contortus isolates. Sequences potentially involved in generating the truncated splice variant within the second intron were analysed from individuals and pools of parasites. We found an insertion/deletion (indel) of 63bp located just downstream from the splice acceptor site for the alternative third exon. The sequence of the indel, when present, was similar in the 12 isolates examined. The presence or absence of this indel was statistically (Chi(2) test) correlated with levamisole resistance status. A correlation was also demonstrated between the absence of the indel and the expression of the Hco-acr-8b transcript. We believe this is the first report of a putative DNA marker for levamisole resistance detection. Using this new knowledge, we have developed a novel DNA-based assay for the detection and monitoring of levamisole resistance in parasitic nematodes of animals., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Haemonchus contortus is an important nematode of livestock that is present in most parts of the world. The life cycle comprises free living stages (egg, L1, L2 and L3 larvae), and parasitic stages (L4, adult and egg) in a ruminant. Microtubules are filamentous structures which are made from polymerization of α- and β-tubulin. In vitro polymerization of α- and β-tubulin can be achieved by increasing the temperature to 37°C under certain conditions. As part of its normal functioning, in mammals, the microtubules can be depolymerized when the temperature is reduced to 0°C. However, interestingly the microtubules of H. contortus are cold resistant i.e. they do not depolymerize at 0°C. Moreover these microtubules did not depolymerize even in the presence of 5 mM CaCl2 or 50 μM colchicine. These interesting findings may explain how larvae in the free living stages may survive cold temperatures over winter., (Copyright © 2014 Elsevier B.V. All rights reserved.)