Filarial DAF-12 sense the host serum to resume iL3 development during infection.

Nematode parasites enter their definitive host at the developmentally arrested infectious larval stage (iL3), and the ligand-dependent nuclear receptor DAF-12 contributes to trigger their development to adulthood. Here, we characterized DAF-12 from the filarial nematodes Brugia malayi and Dirofilaria immitis and compared them with DAF-12 from the non-filarial nematodes Haemonchus contortus and Caenorhabditis elegans. Interestingly, Dim and BmaDAF-12 exhibit high sequence identity and share a striking higher sensitivity than Hco and CelDAF-12 to the natural ligands Δ4- and Δ7-dafachronic acids (DA). Moreover, sera from different mammalian species activated specifically Dim and BmaDAF-12 while the hormone-depleted sera failed to activate the filarial DAF-12. Accordingly, hormone-depleted serum delayed the commencement of development of D. immitis iL3 in vitro. Consistent with these observations, we show that spiking mouse charcoal stripped-serum with Δ4-DA at the concentration measured in normal mouse serum restores its capacity to activate DimDAF-12. This indicates that DA present in mammalian serum participate in filarial DAF-12 activation. Finally, analysis of publicly available RNA sequencing data from B. malayi showed that, at the time of infection, putative gene homologs of the DA synthesis pathways are coincidently downregulated. Altogether, our data suggest that filarial DAF-12 have evolved to specifically sense and survive in a host environment, which provides favorable conditions to quickly resume larval development. This work sheds new light on the regulation of filarial nematodes development while entering their definitive mammalian host and may open the route to novel therapies to treat filarial infections.

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

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer-DNDi partnership is an outstanding example of "One World Health," in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area.

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

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

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

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

P-glycoproteins in anthelmintic safety, efficacy, and resistance.

P-glycoprotein (PGP) is a pivotal transmembrane transporter governing the cellular flux of diverse substances shielding mammals from toxics. It can thwart the effectiveness of medicines such as ivermectin (IVM) and other macrocyclic lactone (ML) anthelmintics, undermining therapeutic efforts. We analyze the role of PGPs in limiting the toxicity of these drugs in hosts, and their potential contribution to anthelmintic resistance in nematodes. Targeting nematode PGPs to increase drug sensitivity to MLs seems interesting, but is hampered by the lack of selective inhibitors. The nuclear hormone receptor (NHR)-8 should be seriously considered as a target because it upregulates multiple PGPs involved in anthelmintic resistance and it is specific to nematodes. This would advance our understanding of host-pathogen dynamics and foster innovative therapeutic strategies.

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.

First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern.

Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

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

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

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

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

Human soil-transmitted helminths: implications of mass drug administration.

PURPOSE OF REVIEW: With the London Declaration on neglected tropical disease (NTD), we are entering a new era of combating NTDs. However, the worldwide prospects of increased mass drug administration (MDA) treatments warrant caution on the development of anthelmintic resistance. In this review, we discuss the practical implications of MDA programs on the development of anthelmintic resistance in human soil-transmitted helminths (STH). RECENT FINDINGS: There is poor evidence of anthelmintic resistance in human STH. Moreover, there is presumptive evidence that the refugia in MDA programs to control human STH is currently large, suggesting that the development of anthelmintic resistance in STH will be slow or may not occur. It remains unclear whether the current MDA strategy to control STH will sufficiently delay or prevent the development of anthelmintic resistance. First, differences in efficacy across and within STH species, and seasonal transmission of STH have not yet been considered. Second, any surveillance system to monitor drug efficacy is lacking. Finally, there is still no agreed strategy on how to deal with anthelmintic resistance once it emerges. SUMMARY: Although anthelmintic resistance in human STH is currently of limited concern, various actions should be put in place for its delay and monitoring, and strategies should be developed in case anthelmintic resistance occurs.

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

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

Development of rapid in vitro colorimetric enzymatic activity assay to differentiate macrocyclic lactone susceptible and resistant Dirofilaria immitis isolates.

The filarial parasite Dirofilaria immitis causes dirofilariosis, a potentially fatal pulmonary condition in canids and felines. Dirofilariosis can be prevented by treatment with a prophylactic macrocyclic lactone (ML) regimen. Unfortunately, ML-resistant D. immitis isolates, genetically distinct from the wildtype population, have been confirmed via molecular markers. DNA-based tests for ML-resistance are costly and time-consuming. There lacks a simple and reliable in vitro biological test to differentiate D. immitis infections resulting from inadequate adherence to recommended prophylaxis regimens from those caused by truly resistant D. immitis isolates. The goal of the current study was to develop a minimally invasive rapid diagnostic in vitro biological assay to differentiate ML-susceptible from ML-resistant D. immitis isolates. The in vitro assay was developed based on the concept that MLs act on the microfilariae (mf) by paralyzing the excretory pore muscle, inhibiting the release of molecules, including enzymes, that regulate host immunity. The basis of the in vitro diagnostic assay is to assess the effects of ivermectin (IVM) exposure on the secretion of enzymes by the D. immitis mf at a concentration that distinguishes the ML-susceptible from ML-resistant isolates. The metabolic enzyme, triosephosphate isomerase (TPI), was chosen due to high abundance in the D. immitis secretome. In this study, the in vitro TPI enzymatic assay was optimized and tested in eight laboratory-maintained isolates. The ML-susceptible Missouri, Berkeley, and Georgia II isolate; the putative ML-susceptible Georgia III, and Big Head; and the ML-resistant JYD-34, Metairie, and WildCat. We observed mixed results, Missouri and Berkeley had statistically significant decreases in TPI activity following 24-hour in vitro IVM exposure. The three resistant isolates, JYD-34, Metairie, and WildCat showed no change in TPI activity following IVM exposure. The susceptible, or putative susceptible Georgia II, Georgia III, and Big Head isolates had a phenotypic response consistent with ML-resistance based on the in vitro assay. However, increasing genotypic evidence has presented a mixed genotype for the three isolates, indicating they may be partially selected for ML-resistance. The measurement of changes in enzymatic activity and the in vitro TPI enzymatic activity assay consequently does not appear to be a reliable detection method for ML-resistance but may be useful for identifying fully susceptible isolates.

Genetic polymorphism, constitutive expression and tissue localization of Dirofilaria immitis P-glycoprotein 11: a putative marker of macrocyclic lactone resistance.

BACKGROUND: Dirofilaria immitis causes dirofilariosis, a potentially fatal condition in canids. Dirofilaria infections can be prevented with a macrocyclic lactone (ML) prophylactic regimen. However, some D. immitis isolates have become resistant to MLs. Genetic changes on the P-glycoprotein 11 gene, encoding an ABCB transporter, have been linked to the ML-resistant phenotypes and have been proposed as markers of drug resistance. However, nothing is known about the expression and the localization of this transporter in D. immitis, despite its strong link to ML-resistant phenotypes. METHODS: We examined the clinically validated D. immitis P-glycoprotein 11 (DimPgp-11) single nucleotide polymorphism (SNP) via MiSeq analysis in three ML-susceptible isolates (Missouri, MP3 and Yazoo) and two ML-resistant isolates (JYD-34 and Metairie), and correlated the data with previously published MiSeq results of USA laboratory-maintained D. immitis isolates. The level of the expression of the DimPgp-11 messenger RNA transcript was analyzed by droplet digital PCR (ddPCR) and compared in the USA laboratory-maintained isolates, namely the ML-susceptible Missouri and Berkeley isolates, the putative ML-susceptible Georgia III and Big Head isolates and the ML-resistant isolate JYD-34. The immunolocalization of DimPgp-11 was visualized in the microfilaria (mf) life stage of the Missouri isolate using confocal microscopy. RESULTS: The results confirmed that the SNP found on DimPgp-11 is differentially expressed in the USA laboratory-maintained isolates. The ML-susceptible isolates had an alternate allele frequency of between 0% and 15%, while it ranged between 17% and 56% in the ML-resistant isolates. The constitutive expression of DimPgp-11 was similar in the Berkeley, Georgia III and Big Head isolates, while it was significantly decreased in the ML-resistant JYD-34 isolate (P < 0.05), when compared to the ML-susceptible Missouri isolate. The DimPgp-11 protein was distinctly localized within the excretory-secretory (ES) duct, pore cells and the excretory cell and, more faintly, along the mf body wall. CONCLUSION: Our data confirm that genetic polymorphism of DimPgp-11 is associated with ML resistance in USA laboratory-maintained D. imminits isolates. A link between DimPgp-11 and ML resistance in D. immitis is further supported by the lower protein expression in the ML-resistant JYD-34 isolate when compared with the ML-susceptible Missouri isolate. Interestingly, DimPgp-11 is strategically located surrounding the ES pore where it could play an active role in ML efflux.

Dirofilaria immitis: Genotyping Randomly Selected European Clinical Samples and USA Laboratory Isolates with Molecular Markers Associated with Macrocyclic Lactone Susceptibility and Resistance.

Dirofilaria immitis is a parasitic nematode and causes dirofilariosis, a potentially fatal pulmonary infection which primarily infects canids. Dirofilariosis infections are controlled via prophylactic macrocyclic lactone (ML) regimens. Recent evidence has confirmed the development of ML-resistant isolates in the USA, which are genetically distinct from wildtype populations. Single nucleotide polymorphisms (SNP) associated with ML-resistant phenotypes were clinically validated in USA populations. In this study, 3 USA laboratory-maintained isolates (Berkeley, Georgia II, and WildCat) and 11 randomly selected European clinical samples from 7 hosts were analyzed. The samples tested were fresh microfilaria (mf) in blood or adult worms preserved in ethanol. The samples underwent MiSeq sequencing of the top 9 SNP associated with ML resistance. The results provide the first genotypic analysis of the three USA laboratory-maintained isolates and any European samples. The European clinical samples show no genomic evidence of ML resistance based on the 9 SNP. The early adoption of genotyping of clinical D. immitis samples could provide an early indication of the potential development of ML resistance and aid to distinguish clinical cases of heartworm infection due to ML resistance from those due to a lack compliance with the recommended treatments, as has been seen in North America.

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

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

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

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

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

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

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

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

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

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

Model of Success: World Association for the Advancement of Veterinary Parasitology African Foundation (1997-2019).

No abstract available.