Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

Genome structure and population genomics of the canine heartworm Dirofilaria immitis.

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

Functional validation of the truncated UNC-63 acetylcholine receptor subunit in levamisole resistance.

Levamisole is a broad-spectrum anthelmintic which permanently activates cholinergic receptors from nematodes, inducing a spastic paralysis of the worms. Whereas this molecule is widely used to control parasitic nematodes impacting livestock, its efficacy is compromised by the emergence of drug-resistant parasites. In that respect, there is an urgent need to identify and validate molecular markers associated with resistance. Previous transcriptomic analyses revealed truncated cholinergic receptor subunits as potential levamisole resistance markers in the trichostrongylid nematodes Haemonchus contortus, Telodorsagia circumcincta and Trichostrongylus colubriformis. In the present study we used the Xenopus oocyte, as well as the free-living model nematode Caenorhabditis elegans, as heterologous expression systems to functionally investigate truncated isoforms of the levamisole-sensitive acetylcholine receptor (L-AChR) UNC-63 subunit. In the Xenopus oocyte, we report that truncated UNC-63 from C. elegans has a strong dominant negative effect on the expression of the recombinant C. elegans L-AChRs. In addition, we show that when expressed in C. elegans muscle cells, truncated UNC-63 induces a drastic reduction in levamisole susceptibility in transgenic worms, thus providing the first known functional validation for this molecular marker in vivo.

The avermectin/milbemycin receptors of parasitic nematodes.

Glutamate-gated chloride channels are the most important target of ivermectin and related compounds in parasitic nematodes. A small family of genes encode subunits of these channels, allowing the assembly of multiple channel subtypes; the subunit composition of most of the native receptors is unknown. The members of the gene family vary between species, making extrapolation from C. elegans to parasites difficult. Expression of recombinant receptors in Xenopus oocytes can identify subunits that have the ability to co-assemble into novel channels, but localisation data, ideally at the single-cell level, is required to confirm that these subunits are expressed in the same cells and tissues. Fortunately, recent advances in this area are starting to make this information available; this information is adding to our understanding of how the drugs act and of the possible subunit combinations that create their targets in vivo.

Evaluation of the in vitro susceptibility of various filarial nematodes to emodepside.

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis.

Lack of detectable short-term effects of a single dose of ivermectin on the human immune system.

BACKGROUND: Ivermectin is widely used in human and animal medicine to treat and prevent parasite nematode infections. It has been suggested that its mode of action requires the host immune system, as it is difficult to reproduce its clinical efficacy in vitro. We therefore studied the effects of a single dose of ivermectin (Stromectol®-0.15 mg/kg) on cytokine levels and immune cell gene expression in human volunteers. This dose reduces bloodstream microfilariae rapidly and for several months when given in mass drug administration programmes. METHODS: Healthy volunteers with no travel history to endemic regions were given 3-4 tablets, depending on their weight, of either ivermectin or a placebo. Blood samples were drawn immediately prior to administration, 4 h and 24 h afterwards, and complete blood counts performed. Serum levels of 41 cytokines and chemokines were measured using Luminex® and expression levels of 770 myeloid-cell-related genes determined using the NanoString nCounter®. Cytokine levels at 4 h and 24 h post-treatment were compared to the levels pre-treatment using simple t tests to determine if any individual results required further investigation, taking p = < 0.05 as the level of significance. NanoString data were analysed on the proprietary software, nSolver™. RESULTS: No significant differences were observed in complete blood counts or cytokine levels at either time point between people given ivermectin versus placebo. Only three genes showed a significant change in expression in peripheral blood mononuclear cells 4 h after ivermectin was given; there were no significant changes 24 h after drug administration or in polymorphonuclear cells at either time point. Leukocytes isolated from those participants given ivermectin showed no difference in their ability to kill Brugia malayi microfilariae in vitro. CONCLUSIONS: Overall, our data do not support a direct effect of ivermectin, when given at the dose used in current filarial elimination programmes, on the human immune system. Trial registration ClinicalTrials.gov NCT03459794 Registered 9th March 2018, Retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03459794?term=NCT03459794&draw=2&rank=1 .

Evaluation of changes in drug susceptibility and population genetic structure in Haemonchus contortus following worm replacement as a means to reverse the impact of multiple-anthelmintic resistance on a sheep farm.

A population of Haemonchus contortus that was highly resistant to benzimidazoles and avermectin/milbemycins with a subpopulation that was resistant to levamisole, was replaced with a susceptible laboratory isolate of H. contortus in a flock of sheep. The anthelmintic susceptibility and population genetics of the newly established population were evaluated for 3.5 years using in vivo, in vitro, and molecular methods. Successful replacement of the resistant population with a susceptible population was confirmed using phenotypic and genotypic measurements; larval development assay indicated full anthelmintic susceptibility; albendazole treatment yielded 98.7% fecal egg count reduction; pyrosequence genotyping of single nucleotide polymorphisms in positions 167 and 200 of the isotype-1 beta tubulin gene were present at 0.0 and 1.7%, respectively; microsatellite genotyping indicated the background haplotype was similar to the susceptible isolate; and haplotypes of the isotype-1 beta tubulin gene were similar to the susceptible isolate. To sustain the susceptibility of the new population, targeted selective treatment was implemented using albendazole. Surprisingly, within 1.5 years post-replacement, the population reverted to a resistant phenotype. Resistance to albendazole, ivermectin, and moxidectin was confirmed via fecal egg count reduction test, larval development assay, and pyrosequencing-based genotyping. Targeted selective treatment was then carried out using levamisole. However, within one year, resistance was detected to levamisole. Population genetics demonstrated a gradual change in the genetic structure of the population until the final population was similar to the initial resistant population. Genetic analyses showed a lack of diversity in the susceptible isolate, suggesting the susceptible isolate had reduced environmental fitness compared to the resistant population, providing a possible explanation for the rapid reversion to resistance. This work demonstrates the power of combining molecular, in vitro, and in vivo assays to study phenotypic and genotypic changes in a field population of nematodes, enabling improved insights into the epidemiology of anthelmintic resistance.

Comparative sequences of the Wolbachia genomes of drug-sensitive and resistant isolates of Dirofilaria immitis.

The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40-75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected.Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.

Using population genetics to examine relationships of Dirofilaria immitis based on both macrocyclic lactone-resistance status and geography.

Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor.

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

Refugia and anthelmintic resistance: Concepts and challenges.

Anthelmintic resistance is a threat to global food security. In order to alleviate the selection pressure for resistance and maintain drug efficacy, management strategies increasingly aim to preserve a proportion of the parasite population in 'refugia', unexposed to treatment. While persuasive in its logic, and widely advocated as best practice, evidence for the ability of refugia-based approaches to slow the development of drug resistance in parasitic helminths is currently limited. Moreover, the conditions needed for refugia to work, or how transferable those are between parasite-host systems, are not known. This review, born of an international workshop, seeks to deconstruct the concept of refugia and examine its assumptions and applicability in different situations. We conclude that factors potentially important to refugia, such as the fitness cost of drug resistance, the degree of mixing between parasite sub-populations selected through treatment or not, and the impact of parasite life-history, genetics and environment on the population dynamics of resistance, vary widely between systems. The success of attempts to generate refugia to limit anthelmintic drug resistance are therefore likely to be highly dependent on the system in hand. Additional research is needed on the concept of refugia and the underlying principles for its application across systems, as well as empirical studies within systems that prove and optimise its usefulness.

Macrocyclic lactone anthelmintic-induced leukocyte binding to Dirofilaria immitis microfilariae: Influence of the drug resistance status of the parasite.

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10 nM), but much higher concentrations of ivermectin (>100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.

Effects of diethylcarbamazine and ivermectin treatment on Brugia malayi gene expression in infected gerbils (Meriones unguiculatus).

Lymphatic filariasis (LF) threatens nearly 20% of the world's population and has handicapped one-third of the 120 million people currently infected. Current control and elimination programs for LF rely on mass drug administration of albendazole plus diethylcarbamazine (DEC) or ivermectin. Only the mechanism of action of albendazole is well understood. To gain a better insight into antifilarial drug action in vivo, we treated gerbils harbouring patent Brugia malayi infections with 6 mg kg-1 DEC, 0.15 mg kg-1 ivermectin or 1 mg kg-1 albendazole. Treatments had no effect on the numbers of worms present in the peritoneal cavity of treated animals, so effects on gene expression were a direct result of the drug and not complicated by dying parasites. Adults and microfilariae were collected 1 and 7 days post-treatment and RNA isolated for transcriptomic analysis. The experiment was repeated three times. Ivermectin treatment produced the most differentially expressed genes (DEGs), 113. DEC treatment yielded 61 DEGs. Albendazole treatment resulted in little change in gene expression, with only 6 genes affected. In total, nearly 200 DEGs were identified with little overlap between treatment groups, suggesting that these drugs may interfere in different ways with processes important for parasite survival, development, and reproduction.

Efficacy and side effects of doxycycline versus minocycline in the three-dose melarsomine canine adulticidal heartworm treatment protocol.

BACKGROUND: The American Heartworm Society currently recommends the use of a monthly macrocyclic lactone, a 28-day course of 10 mg/kg doxycycline BID, and the 3-dose protocol of melarsomine dihydrochloride for the treatment of canine heartworm disease. Doxycycline is necessary for the reduction of the bacterium Wolbachia, found in all heartworm life-stages. Previous price increases and decreasing availability prompted us to evaluate alternative tetracycline antibiotics, i.e. minocycline, for the reduction of Wolbachia during canine heartworm treatment. METHODS: Thirty-two heartworm-positive dogs were randomized to receive 10 mg/kg or 5 mg/kg of either doxycycline or minocycline for 28 days BID, for a total of 8 dogs per experimental group. All dogs received 6 months of Heartgard Plus® (ivermectin/pyrantel) and the 3-dose protocol of 2.5 mg/kg melarsomine dihydrochloride. Blood samples were collected prior to the initiation of treatment, every 7 days throughout tetracycline treatment, and then monthly thereafter until the dog tested negative for the presence of heartworm antigen. DNA was isolated from circulating microfilarial samples and qPCR was performed on each sample. RESULTS: A greater number of dogs in the 10 mg/kg doxycycline and minocycline treated groups experienced gastrointestinal side effects as compared to the 5 mg/kg doxycycline and minocycline treated groups. All eight dogs in the 10 mg/kg doxycycline-treated group tested negative for the presence of Wolbachia DNA by 28 days post-tetracycline treatment. A total of two dogs in both the 5 mg/kg doxycycline- and 10 mg/kg minocycline-treated groups and three dogs in the 5 mg/kg minocycline-treated group remained positive for the presence of Wolbachia DNA by the end of tetracycline treatment. CONCLUSIONS: No lung pathology was assessed in this clinical trial, therefore the clinical effect of the remaining Wolbachia DNA in the 10 mg/kg minocycline-, 5 mg/kg doxycycline- and 5 mg/kg minocycline-treated groups cannot be determined. Owner compliance in the proper administration of these tetracyclines may be impacted by the increased severe gastrointestinal side effects reported for the 10 mg/kg doxycycline- and minocycline-treated groups. We recommend that veterinarians prescribe the recommended 10 mg/kg doxycycline for canine heartworm treatment and reduce the dosage to 5 mg/kg in cases of severe gastrointestinal side effects in order to improve owner compliance in administration of medications.

Anthelmintics - From Discovery to Resistance III (Indian Rocks Beach, FL, 2018).

The third scientific meeting in the series "Anthelmintics: From Discovery to Resistance" was held in Indian Rocks Beach, Florida, at the end of January 2018. The meeting focused on a variety of topics related to the title, including the identification of novel targets and new leads, the mechanism of action of existing drugs and the genetic basis of resistance against them. Throughout there was an emphasis on the exploitation of new technologies and methods to further these aims. The presentations, oral and poster, covered basic, veterinary and medical science with strong participation by both academic and commercial researchers. This special issue contains selected papers from the meeting.

Recognition and killing of Brugia malayi microfilariae by human immune cells is dependent on the parasite sample and is not altered by ivermectin treatment.

Mass administration of macrocyclic lactones targets the transmission of the causative agents of lymphatic filariasis to their insect vectors by rapidly clearing microfilariae (Mf) from the circulation. It has been proposed that the anti-filarial action of these drugs may be mediated through the host immune system. We recently developed an in vitro assay for monitoring the attachment to and killing of B. malayi Mf by human neutrophils (PMNs) and monocytes (PBMCs), however, the levels of both cell to worm attachment and leukocyte mediated Mf killing varied greatly between individual experiments. To determine whether differences in an individual's immune cells or the Mf themselves might account for the variability in survival, PMNs and PBMCs were isolated from 12 donors every week for 4 weeks and the cells used for survival assays with a different batch of Mf, thereby keeping donors constant but varying the Mf sample. Results from these experiments indicate that, overall, killing is Mf-rather than donor-dependent. To assess whether ivermectin (IVM) or diethylcarbamazine (DEC) increase killing, Mf were incubated either alone or with immune cells in the presence of IVM or DEC. Neither drug induced a significant difference in the survival of Mf whether cultured with or without cells, with the exception of DEC at 2 h post incubation. In addition, human PBMCs and PMNs were incubated with IVM or DEC for 1 h or 16 h prior to RNA extraction and Illumina sequencing. Although donor-to-donor variation may mask subtle differences in gene expression, principle component analysis of the RNASeq data indicates that there is no significant change in the expression of any genes from the treated cells versus controls. Together these data suggest that IVM and DEC have little direct effect on immune cells involved in the rapid clearance of Mf from the circulation.

Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

Does evaluation of in vitro microfilarial motility reflect the resistance status of Dirofilaria immitis isolates to macrocyclic lactones?

BACKGROUND: Several reports have confirmed that macrocyclic lactone-resistant isolates of Dirofilaria immitis are circulating in the United States; however, the prevalence and potential impact of drug resistance is unknown. We wished to assess computer-aided measurements of motility as a method for rapidly assessing the resistance status of parasite isolates. METHODS: Blood containing microfilariae (MF) from two clinical cases with a high suspicion of resistance was fed to mosquitoes and the resultant L3 injected into dogs that were then treated with six doses of Heartgard® Plus (ivermectin + pyrantel; Merial Limited) at 30-day intervals. In both cases patent heartworm infections resulted despite the preventive treatment. Microfilariae isolated from these dogs and other isolates of known resistance status were exposed to varying concentrations of ivermectin in vitro and their motility assessed 24 h later using computer-processed high-definition video imaging. RESULTS: We produced two isolates, Yazoo-2013 and Metairie-2014, which established patent infections despite Heartgard® Plus treatments. Measurements of the motility of MF of these and other isolates (Missouri, MP3 and JYD-27) following exposure to varying concentrations of ivermectin did not distinguish between susceptible and resistant heartworm populations. There was some evidence that the method of MF isolation had an influence on the motility and drug susceptibility of the MF. CONCLUSIONS: We confirmed that drug-resistant heartworms are circulating in the southern United States, but that motility measurements in the presence of ivermectin are not a reliable method for their detection. This implies that the drug does not kill the microfilariae via paralysis.

Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis.

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56µM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.

Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release.

BACKGROUND: Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases. PRINCIPAL FINDINGS: In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections. CONCLUSIONS/SIGNIFICANCE: Human innate immune cells are able to recognize, attach to and kill B. malayi microfilariae in an in vitro system. This suggests that, in vivo, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.