Lack of correlation between parasite burden and key weight metrics in poultry infected with intestinal ascarids.

Ascaridia galli and Ascaridia dissimilis are the most common and economically impactful nematode parasites of commercial poultry. These infections rarely cause clinical disease, but reduction in feed conversion efficiency is detected. To determine if feed conversion efficiency reductions correlate with any physiological measures independent of clinical disease, we determined if ascarid infections correlate with changes in the weights of the small intestine, liver, or total animal weight (quantitative measures of animal health). No correlation between parasite burden and these metrics were observed, supporting the concept that feed conversion is the only production metric impacted by ascarid infections.

Development of non-sedating benzodiazepines with in vivo antischistosomal activity.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad-spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970s. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) is not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

Development of non-sedating antischistosomal benzodiazepines.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970's. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) are not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index, and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

Wolbachia-mediated resistance to Zika virus infection in Aedes aegypti is dominated by diverse transcriptional regulation and weak evolutionary pressures.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the wMel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of wMel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia-infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia. Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia-mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.

Mapping resistance-associated anthelmintic interactions in the model nematode Caenorhabditis elegans.

Parasitic nematodes infect billions of people and are mainly controlled by anthelmintic mass drug administration (MDA). While there are growing efforts to better understand mechanisms of anthelmintic resistance in human and animal populations, it is unclear how resistance mechanisms that alter susceptibility to one drug affect the interactions and efficacy of drugs used in combination. Mutations that alter drug permeability across primary nematode barriers have been identified as potential resistance mechanisms using the model nematode Caenorhabditis elegans. We leveraged high-throughput assays in this model system to measure altered anthelmintic susceptibility in response to genetic perturbations of potential cuticular, amphidial, and alimentary routes of drug entry. Mutations in genes associated with these tissue barriers differentially altered susceptibility to the major anthelmintic classes (macrocyclic lactones, benzimidazoles, and nicotinic acetylcholine receptor agonists) as measured by animal development. We investigated two-way anthelmintic interactions across C. elegans genetic backgrounds that confer resistance or hypersensitivity to one or more drugs. We observe that genetic perturbations that alter susceptibility to a single drug can shift the drug interaction landscape and lead to the appearance of novel synergistic and antagonistic interactions. This work establishes a framework for investigating combinatorial therapies in model nematodes that can potentially be translated to amenable parasite species.

Molecular Surveillance Detects High Prevalence of the Neglected Parasite Mansonella ozzardi in the Colombian Amazon.

BACKGROUND: Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions. METHODS: We deployed molecular and classical approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. RESULTS: Loop-mediated isothermal amplification (LAMP) assays on whole-blood samples detected a much higher prevalence of Mansonella ozzardi infection (approximately 40%) compared to blood smear microscopy or LAMP performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias and occult infections. Mansonella infection rates increased with age and were higher among men. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. CONCLUSION: These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

A high-throughput sensory assay for parasitic and free-living nematodes.

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes. Insight box Nematodes are powerful model organisms for understanding the sensory biology of multicellular eukaryotes, and many parasitic species cause disease in humans. Simple sensory assays performed on agarose plates have been the bedrock for establishing the neuronal, genetic, and developmental foundations for many sensory modalities in nematodes. However, these classical assays are poorly suited for translational movement of many parasitic nematodes and the sensation of water-soluble molecules (gustation). We have designed a device for high-throughput nematode sensory assays in a gel matrix. This 'gustatory microplate' is amenable to several species and reveals novel responses by free-living and parasitic nematodes to cues and drugs.

Wolbachia -mediated resistance to Zika virus infection in Aedes aegypti is dominated by diverse transcriptional regulation and weak evolutionary pressures.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the w Mel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of w Mel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia -infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia . Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia -mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system. Author Summary: When Wolbachia bacteria infect Aedes aegypti mosquitoes, they dramatically reduce the mosquitoes' susceptibility to infection with a range of arthropod-borne viruses, including Zika virus (ZIKV). Although this pathogen-blocking effect has been widely recognized, its mechanisms remain unclear. Furthermore, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to Wolbachia -mediated blocking. Here, we use host transcriptomics and viral genome sequencing to examine the mechanisms of ZIKV pathogen blocking by Wolbachia and viral evolutionary dynamics in Ae. aegypti mosquitoes. We find complex transcriptome patterns that do not suggest a single clear mechanism for pathogen blocking. We also find no evidence that Wolbachia exerts detectable selective pressures on ZIKV in coinfected mosquitoes. Together our data suggest that it may be difficult for ZIKV to evolve Wolbachia resistance, perhaps due to the complexity of the pathogen blockade mechanism.

Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.

Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite Brugia malayi to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.

A high-throughput nematode sensory assay reveals an inhibitory effect of ivermectin on parasite gustation.

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes.

Mapping resistance-associated anthelmintic interactions in the model nematode Caenorhabditis elegans.

Parasitic nematodes infect billions of people and are mainly controlled by anthelmintic mass drug administration (MDA). While there are growing efforts to better understand mechanisms of anthelmintic resistance in human and animal populations, it is unclear how resistance mechanisms that alter susceptibility to one drug affect the interactions and efficacy of drugs used in combination. Mutations that alter drug permeability across primary nematode barriers have been identified as potential resistance mechanisms using the model nematode Caenorhabditis elegans. We leveraged high-throughput assays in this model system to measure altered anthelmintic susceptibility in response to genetic perturbations of potential cuticular, amphidial, and alimentary routes of drug entry. Mutations in genes associated with these tissue barriers differentially altered susceptibility to the major anthelmintic classes (macrocyclic lactones, benzimidazoles, and nicotinic acetylcholine receptor agonists) as measured by animal development. We investigated two-way anthelmintic interactions across C. elegans genetic backgrounds that confer resistance or hypersensitivity to one or more drugs. We observe that genetic perturbations that alter susceptibility to a single drug can shift the drug interaction landscape and lead to the appearance of novel synergistic and antagonistic interactions. This work establishes a framework for investigating combinatorial therapies in model nematodes that can potentially be translated to amenable parasite species.

Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host.

Vector-borne, filarial nematode diseases cause significant disease burdens in humans and domestic animals worldwide. Although there is strong direct evidence of parasite-driven immunomodulation of mammalian host responses, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a filarial nematode and causative agent of Lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae-derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an Ae. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs. AAEL002590, an Ae. aegypti gene encoding a serine protease, was shown to be downregulated when cells were treated with biologically relevant EV concentrations in vitro. Injection of adult female mosquitoes with biologically relevant concentrations of EVs validated these results in vivo, recapitulating the downregulation of AAEL002590 transcript. This gene was predicted to be involved in the mosquito phenoloxidase (PO) cascade leading to the canonical melanization response and correspondingly, both suppression of this gene using RNAi and parasite EV treatment reduced PO activity in vivo. Our data indicate that parasite-derived EVs interfere with critical immune responses in the vector host, including melanization.

Nemacol is a small molecule inhibitor of C. elegans vesicular acetylcholine transporter with anthelmintic potential.

Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol enhances the ability of the anthelmintic Ivermectin to paralyze C. elegans and the ruminant nematode parasite Haemonchus contortus. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through a validated anthelmintic target.

Pharmacological Profiling of a Brugia malayi Muscarinic Acetylcholine Receptor as a Putative Antiparasitic Target.

The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor of B. malayi (Bma-GAR-3) that is highly expressed across intramammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Finally, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

Multivariate chemogenomic screening prioritizes new macrofilaricidal leads.

Development of direct acting macrofilaricides for the treatment of human filariases is hampered by limitations in screening throughput imposed by the parasite life cycle. In vitro adult screens typically assess single phenotypes without prior enrichment for chemicals with antifilarial potential. We developed a multivariate screen that identified dozens of compounds with submicromolar macrofilaricidal activity, achieving a hit rate of >50% by leveraging abundantly accessible microfilariae. Adult assays were multiplexed to thoroughly characterize compound activity across relevant parasite fitness traits, including neuromuscular control, fecundity, metabolism, and viability. Seventeen compounds from a diverse chemogenomic library elicited strong effects on at least one adult trait, with differential potency against microfilariae and adults. Our screen identified five compounds with high potency against adults but low potency or slow-acting microfilaricidal effects, at least one of which acts through a novel mechanism. We show that the use of microfilariae in a primary screen outperforms model nematode developmental assays and virtual screening of protein structures inferred with deep learning. These data provide new leads for drug development, and the high-content and multiplex assays set a new foundation for antifilarial discovery.

Phenotypic Profiling of Macrocyclic Lactones on Parasitic Schistosoma Flatworms.

Macrocyclic lactones are front-line therapies for parasitic roundworm infections; however, there are no comprehensive studies on the activity of this drug class against parasitic flatworms. Ivermectin is well known to be inactive against flatworms. However, the structure-activity relationship of macrocyclic lactones may vary across phyla, and it is entirely possible other members of this drug class do in fact show antiparasitic activity on flatworms. For example, there are several reports hinting at the anti-schistosomal activity of doramectin and moxidectin. To explore this class further, we developed an automated imaging assay combined with measurement of lactate levels from worm media. This assay was applied to the screening of 21 macrocyclic lactones (avermectins, milbemycins, and others such as spinosyns) against adult schistosomes. These in vitro assays identified several macrocyclic lactones (emamectin, milbemycin oxime, and the moxidectin metabolite 23-ketonemadectin) that caused contractile paralysis and lack of lactate production. Several of these were also active against miracidia, which infect the snail intermediate host. Hits prioritized from these in vitro assays were administered to mice harboring patent schistosome infections. However, no reduction in worm burden was observed. Nevertheless, these data show the utility of a multiplexed in vitro screening platform to quantitatively assess drug action and exclude inactive compounds from a chemical series before proceeding to in vivo studies. While the prototypical macrocyclic lactone ivermectin displays minimal activity against adult Schistosoma mansoni, this family of compounds does contain schistocidal compounds which may serve as a starting point for development of new anti-flatworm chemotherapies.

wrmXpress: A modular package for high-throughput image analysis of parasitic and free-living worms.

Advances in high-throughput and high-content imaging technologies require concomitant development of analytical software capable of handling large datasets and generating relevant phenotypic measurements. Several tools have been developed to analyze drug response phenotypes in parasitic and free-living worms, but these are siloed and often limited to specific instrumentation, worm species, and single phenotypes. No unified tool exists to analyze diverse high-content phenotypic imaging data of worms and provide a platform for future extensibility. We have developed wrmXpress, a unified framework for analyzing a variety of phenotypes matched to high-content experimental assays of free-living and parasitic nematodes and flatworms. We demonstrate its utility for analyzing a suite of phenotypes, including motility, development/size, fecundity, and feeding, and establish the package as a platform upon which to build future custom phenotypic modules. We show that wrmXpress can serve as an analytical workhorse for anthelmintic screening efforts across schistosomes, filarial nematodes, and free-living model nematodes and holds promise for enabling collaboration among investigators with diverse interests.

Making sense of sensory behaviors in vector-borne helminths.

Migrations performed by helminths are impressive and diverse, and accumulating evidence shows that many are controlled by sophisticated sensory programs. The migrations of vector-borne helminths are particularly complex, requiring precise, stage-specific regulation. We review the contrasting states of knowledge on snail-borne schistosomes and mosquito-borne filarial nematodes. Rich observational data exist for the chemosensory behaviors of schistosomes, while the molecular sensory pathways in nematodes are well described. Recent investigations on the molecular mechanisms of sensation in schistosomes and filarial nematodes have revealed some features conserved within their respective phyla, but adaptations correlated with parasitism are pronounced. Technological developments are likely to extend these advances, and we forecast how these technologies may be applied.

Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi.

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

High-content approaches to anthelmintic drug screening.

Most anthelmintics were discovered through in vivo screens using animal models of infection. Developing in vitro assays for parasitic worms presents several challenges. The lack of in vitro life cycle culture protocols requires harvesting worms from vertebrate hosts or vectors, limiting assay throughput. Once worms are removed from the host environment, established anthelmintics often show no obvious phenotype - raising concerns about the predictive value of many in vitro assays. However, with recent progress in understanding how anthelmintics subvert host-parasite interactions, and breakthroughs in high-content imaging and machine learning, in vitro assays have the potential to discern subtle cryptic parasite phenotypes. These may prove better endpoints than conventional in vitro viability assays.