Assessing de novo parasite genomes assembled using only Oxford Nanopore Technologies MinION data.

In this study, we assessed the quality of de novo genome assemblies for three species of parasitic nematodes (Brugia malayi, Trichuris trichiura, and Ancylostoma caninum) generated using only Oxford Nanopore Technologies MinION data. Assemblies were compared to current reference genomes and against additional assemblies that were supplemented with short-read Illumina data through polishing or hybrid assembly approaches. For each species, assemblies generated using only MinION data had similar or superior measures of contiguity, completeness, and gene content. In terms of gene composition, depending on the species, between 88.9 and 97.6% of complete coding sequences predicted in MinION data only assemblies were identical to those predicted in assemblies polished with Illumina data. Polishing MinION data only assemblies with Illumina data therefore improved gene-level accuracy to a degree. Furthermore, modified DNA extraction and library preparation protocols produced sufficient genomic DNA from B. malayi and T. trichiura to generate de novo assemblies from individual specimens.

Altered larval activation response associated with multidrug resistance in the canine hookworm Ancylostoma caninum.

Parasitic gastrointestinal nematodes pose significant health risks to humans, livestock, and companion animals, and their control relies heavily on the use of anthelmintic drugs. Overuse of these drugs has led to the emergence of resistant nematode populations. Herein, a naturally occurring isolate (referred to as BCR) of the dog hookworm, Ancylostoma caninum, that is resistant to 3 major classes of anthelmintics is characterized. Various drug assays were used to determine the resistance of BCR to thiabendazole, ivermectin, moxidectin and pyrantel pamoate. When compared to a drug-susceptible isolate of A. caninum, BCR was shown to be significantly resistant to all 4 of the drugs tested. Multiple single nucleotide polymorphisms have been shown to impart benzimidazole resistance, including the F167Y mutation in the ß-tubulin isotype 1 gene, which was confirmed to be present in BCR through molecular analysis. The frequency of the resistant allele in BCR was 76.3% following its first passage in the lab, which represented an increase from approximately 50% in the founding hookworm population. A second, recently described mutation in codon 134 (Q134H) was also detected at lower frequency in the BCR population. Additionally, BCR exhibits an altered larval activation phenotype compared to the susceptible isolate, suggesting differences in the signalling pathways involved in the activation process which may be associated with resistance. Further characterization of this isolate will provide insights into the mechanisms of resistance to macrocyclic lactones and tetrahydropyrimidine anthelmintics.

Immunity mediates host specificity in the human hookworm Ancylostoma ceylanicum.

Hookworm infection affects millions globally, leading to chronic conditions like malnutrition and anaemia. Among the hookworm species, Ancylostoma ceylanicum stands out as a generalist, capable of infecting various hosts, including humans, cats, dogs and hamsters. Surprisingly, it cannot establish in mice, despite their close phylogenetic relationship to hamsters. The present study investigated the development of A. ceylanicum in immunodeficient NSG mice to determine the contribution of the immune system to host restriction. The infections became patent on day 19 post-infection (PI) and exhibited elevated egg production which lasted for at least 160 days PI. Infective A. ceylanicum larvae reared from eggs released by infected NSG mice were infectious to hamsters and capable of reproduction, indicating that the adults in the NSG mice were producing viable offspring. In contrast, A. ceylanicum showed limited development in outbred Swiss Webster mice. Furthermore, the closely related canine hookworm Ancylostoma caninum was unable to infect and develop in NSG mice, indicating that different mechanisms may determine host specificity even in closely related species. This is the first report of any hookworm species completing its life cycle in a mouse and implicate the immune system in determining host specificity in A. ceylanicum.

Comparative transcriptomics from intestinal cells of permissive and non-permissive hosts during Ancylostoma ceylanicum infection reveals unique signatures of protection and host specificity.

Soil-transmitted nematodes (STNs) place a tremendous burden on health and economics worldwide with an estimate of at least 1.5 billion people, or 24% of the population, being infected with at least 1 STN globally. Children and pregnant women carry the heavier pathological burden, and disease caused by the blood-feeding worm in the intestine can result in anaemia and delays in physical and intellectual development. These parasites are capable of infecting and reproducing in various host species, but what determines host specificity remains unanswered. Identifying the molecular determinants of host specificity would provide a crucial breakthrough towards understanding the biology of parasitism and could provide attractive targets for intervention. To investigate specificity mechanisms, members of the hookworm genus Ancylostoma provide a powerful system as they range from strict specialists to generalists. Using transcriptomics, differentially expressed genes (DEGs) in permissive (hamster) and non-permissive (mouse) hosts at different early time points during infection with A. ceylanicum were examined. Analysis of the data has identified unique immune responses in mice, as well as potential permissive signals in hamsters. Specifically, immune pathways associated with resistance to infection are upregulated in the non-permissive host, providing a possible protection mechanism that is absent in the permissive host. Furthermore, unique signatures of host specificity that may inform the parasite that it has invaded a permissive host were identified. These data provide novel insight into the tissue-specific gene expression differences between permissive and non-permissive hosts in response to hookworm infection.

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm's excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

MINI-BAERMANN FUNNEL, A SIMPLE DEVICE FOR CLEANING NEMATODE INFECTIVE LARVAE.

The Baermann filter method is a long-standing, simple technique for recovering nematodes from soil and charcoal coprocultures. Material containing the nematodes is placed on a mesh screen lined with several layers of tissue paper or cheesecloth, and the screen is placed in the mouth of the funnel. Rubber tubing attached to the funnel stem is clamped, and water is added to submerge the material. The filtration material allows the nematodes to swim through while holding back the substrate. Over time the nematodes settle at the clamp in the tubing. After several hours, the clamp is opened and water containing the nematodes is collected. Although recovery of the nematodes is efficient, they are often contaminated with soil or charcoal debris, requiring a secondary cleaning by sedimentation or filtration. Described here is a small, simplified version of the Baermann apparatus that can be used as a secondary cleaning device. The "mini-Baermann" is constructed from materials commonly found in the laboratory. Experiments using infective larvae of 3 nematode species demonstrated that the majority of the larvae applied to the device are collected within 2 hr, and nearly all by 4 hr. Dead larvae fail to pass through the filter and do not significantly impact the passage of living larvae. In addition to removing debris from nematode suspensions, this device can rapidly and efficiently separate living, motile larvae from dead larvae.

Culturing and Genetically Manipulating Entomopathogenic Nematodes.

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Controlled Infection of Humans with the Hookworm Parasite Necator americanus to Accelerate Vaccine Development : The Human Hookworm Vaccination/Challenge Model (HVCM).

In this chapter, we describe the scientific, technical, clinical and regulatory aspects of establishing a controlled human hookworm infection (CHHI) model in non-endemic and endemic geographical regions, to facilitate a pathway towards accelerated vaccine development. The success achieved in establishing the CHHI platform specifically allows the Human Hookworm Vaccine Initiative (HHVI) to accelerate its progress by establishing a human hookworm vaccination/challenge model (HVCM) in a hookworm endemic area of Brazil. The HVCM will permit the rapid and robust determination of clinical efficacy in adults, allowing for early selection of the most efficacious human hookworm vaccine (HHV) candidate(s) to advance into later-stage pivotal paediatric clinical trials and reduce the overall number of participants required to assess efficacy (Diemert et al. 2018).

Chemogenomic approach to identifying nematode chemoreceptor drug targets in the entomopathogenic nematode Heterorhabditis bacteriophora.

Parasitic nematodes constitute one of the major threats to human health, causing diseases of major socioeconomic importance worldwide. Recent estimates indicate that more than 1 billion people are infected with parasitic nematodes around the world. Current measures to combat parasitic nematode infections include anthelmintic drugs. However, heavy exposure to anthelmintics has selected populations of livestock parasitic nematodes that are no longer susceptible to the drugs, rendering several anthelmintics useless for parasitic nematode control in many areas of the world. The rapidity with which anthelmintic resistance developed in response to these drugs suggests that increasing the selective pressure on human parasitic nematodes will also rapidly generate resistant worm populations. Therefore, development of new anthelmintics is of major importance before resistance becomes widespread in human parasitic nematode populations. G-Protein Coupled Receptors (GPCRs) represent an important target for many pharmacological interventions due to their ubiquitous expression in various cell types. GPCRs contribute to numerous physiological processes, and their ligand binding sites located on cell surfaces make them accessible targets and attractive substrates in terms of druggability. In fact, ∼35 % of Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drugs target GPCRs and their associated proteins, with over 300 additional drugs targeting GPCRs at the clinical trial stage. Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent ideal substrates for target-based drug discovery. Here we set out to identify NemChRs that are transcriptionally active inside the host, and to use these NemChRs in a reverse pharmacological screen to impede parasitic development. Our data identified several NemChRs, and we focused on one that was expressed in neuronal cells and exhibited the highest fold change in transcription after host activation. Next, we performed homology modelling and molecular dynamics simulations of this NemChR in order to conduct a virtual screening campaign to identify candidate drug targets which were ranked and selected for experimental testing in bioassays. Taken together, our results identify and characterize a candidate NemChR drug target, and provide a chemogenomic pipeline for identifying nematicide substrates.

Secreted virulence factors from Heterorhabditis bacteriophora highlight its utility as a model parasite among Clade V nematodes.

Much of the available knowledge of entomopathogenic virulence factors has been gleaned from studies in the nematode parasite Steinernema carpocapsae, but there is good reason to complement this knowledge with similar studies in Heterorhabditis bacteriophora. Three candidate virulence factors from H. bacteriophora have recently been characterised, and each was demonstrated to contribute to infection. This information can be used not only to advance efforts in the biocontrol of insect pests, but also to make inferences about the emergence of parasitism among Clade V nematodes.

A putative lysozyme and serine carboxypeptidase from Heterorhabditis bacteriophora show differential virulence capacities in Drosophila melanogaster.

Nematode virulence factors are of interest for a variety of applications including biocontrol against insect pests and the alleviation of autoimmune diseases with nematode-derived factors. In silico "omics" techniques have generated a wealth of candidate factors that may be important in the establishment of nematode infections, although the challenge of characterizing these individual factors in vivo remains. Here we provide a fundamental characterization of a putative lysozyme and serine carboxypeptidase from the host-induced transcriptome of Heterorhabditis bacteriophora. Both factors accelerated the mortality rate following Drosophila melanogaster infections with Photorhabdus luminescens, and both factors suppressed phenoloxidase activity in D. melanogaster hemolymph. Furthermore, the serine carboxypeptidase was lethal to a subpopulation of flies and suppressed the upregulation of antimicrobial peptides as well as phagocytosis. Together, our findings suggest that this serine carboxypeptidase possess both toxic and immunomodulatory properties while the lysozyme is likely to confer immunomodulatory, but not toxic effects.

NemChR-DB: a database of parasitic nematode chemosensory G-protein coupled receptors.

Nematode Chemosensory G-Protein Coupled Receptors have expanded within nematodes, where they play important roles in foraging and host-seeking behaviour. Nematode Chemosensory G-Protein Coupled Receptors are most highly expressed during free-living stages when chemosensory signalling is required for host detection and nematode activation in various parasitic nematodes, and therefore position Nematode Chemosensory G-Protein Coupled Receptors at the transition from infective to parasitic stages, making them important regulators to study in terms of host-seeking and host specificity. To facilitate the analysis of Nematode Chemosensory G-Protein Coupled Receptors, here we describe an integrative database of nematode chemoreceptors called NemChR-DB. This database enables users to study diverse parasitic nematode chemoreceptors, functionally explore sequence entries through structural and literature-based annotations, and perform cross-species comparisons.

A putative UDP-glycosyltransferase from Heterorhabditis bacteriophora suppresses antimicrobial peptide gene expression and factors related to ecdysone signaling.

Insect pathogens have adopted an array of mechanisms to subvert the immune pathways of their respective hosts. Suppression may occur directly at the level of host-pathogen interactions, for instance phagocytic capacity or phenoloxidase activation, or at the upstream signaling pathways that regulate these immune effectors. Insect pathogens of the family Baculoviridae, for example, are known to produce a UDP-glycosyltransferase (UGT) that negatively regulates ecdysone signaling. Normally, ecdysone positively regulates both molting and antimicrobial peptide production, so the inactivation of ecdysone by glycosylation results in a failure of host larvae to molt, and probably a reduced antimicrobial response. Here, we examine a putative ecdysteroid glycosyltransferase, Hba_07292 (Hb-ugt-1), which was previously identified in the hemolymph-activated transcriptome of the entomopathogenic nematode Heterorhabditis bacteriophora. Injection of recombinant Hb-ugt-1 (rHb-ugt-1) into Drosophila melanogaster flies resulted in diminished upregulation of antimicrobial peptides associated with both the Toll and Immune deficiency pathways. Ecdysone was implicated in this suppression by a reduction in Broad Complex expression and reduced pupation rates in r Hb-ugt-1-injected larvae. In addition to the finding that H. bacteriophora excreted-secreted products contain glycosyltransferase activity, these results demonstrate that Hb-ugt-1 is an immunosuppressive factor and that its activity likely involves the inactivation of ecdysone.

Transcriptomic analysis of hookworm Ancylostoma ceylanicum life cycle stages reveals changes in G-protein coupled receptor diversity associated with the onset of parasitism.

Free-living nematodes respond to variable and unpredictable environmental stimuli whereas parasitic nematodes exist in a more stable host environment. A positive correlation between the presence of environmental stages in the nematode life cycle and an increasing number of G-protein coupled receptors (GPCRs) reflects this difference in free-living and parasitic lifestyles. As hookworm larvae move from the external environment into a host, they detect uncharacterized host components, initiating a signalling cascade that results in the resumption of development and eventual maturation. Previous studies suggest this process is mediated by GPCRs in amphidial neurons. Here we set out to uncover candidate GPCRs required by a hookworm to recognise its host. First, we identified all potential Ancylostoma ceylanicum GPCRs encoded in the genome. We then used life cycle stage-specific RNA-seq data to identify differentially expressed GPCRs between the free-living infective L3 (iL3) and subsequent parasitic stages to identify receptors involved in the transition to parasitism. We reasoned that GPCRs involved in host recognition and developmental activation would be expressed at higher levels in the environmental iL3 stage than in subsequent stages. Our results support the model that a decrease in GPCR diversity occurs as the larvae develop from the free-living iL3 stage to the parasitic L3 (pL3) in the host over 24-72 h. We find that overall GPCR expression and diversity is highest in the iL3 compared with subsequent parasitic stages. By 72 h, there was an approximately 50% decrease in GPCR richness associated with the moult from the pL3 to the L4. Taken together, our data uncover a negative correlation between GPCR diversity and parasitic development in hookworm. Finally, we demonstrate proof of principal that Caenorhabditis elegans can be used as a heterologous system to examine the expression pattern of candidate host signal chemoreceptors (CRs) from hookworm. We observe expression of candidate host signal CRs in C. elegans, demonstrating that C. elegans can be effectively used as a surrogate to identify expressed hookworm genes. We present several preliminary examples of this strategy and confirm a candidate CR as neuronally expressed.

Heterorhabditis bacteriophora Excreted-Secreted Products Enable Infection by Photorhabdus luminescens Through Suppression of the Imd Pathway.

Upon entering the hemocoel of its insect host, the entomopathogenic nematode Heterorhabditis bacteriophora releases its symbiotic bacteria Photorhabdus luminescens, which is also a strong insect pathogen. P. luminescens is known to suppress the insect immune response independently following its release, but the nematode appears to enact its own immunosuppressive mechanisms during the earliest phases of an infection. H. bacteriophora was found to produce a unique set of excreted-secreted proteins in response to host hemolymph, and while basal secretions are immunogenic with regard to Diptericin expression through the Imd pathway, host-induced secretions suppress this expression to a level below that of controls in Drosophila melanogaster. This effect is consistent in adults, larvae, and isolated larval fat bodies, and the magnitude of suppression is dose-dependent. By reducing the expression of Diptericin, an antimicrobial peptide active against Gram-negative bacteria, the activated excreted-secreted products enable a more rapid propagation of P. luminescens that corresponds to more rapid host mortality. The identification and isolation of the specific proteins responsible for this suppression represents an exciting field of study with potential for enhancing the biocontrol of insect pests and treatment of diseases associated with excessive inflammation.

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm, Ancylostoma caninum.

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the ß-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous ß-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.

Controlled Human Hookworm Infection: Accelerating Human Hookworm Vaccine Development.

BACKGROUND: Controlled human hookworm infection (CHHI) is a central component of a proposed hookworm vaccination-challenge model (HVCM) to test the efficacy of candidate vaccines. Critical to CHHI is the manufacture of Necator americanus infective larvae (NaL3) according to current Good Manufacturing Practice (cGMP) and the determination of an inoculum of NaL3 that is safe and reliably induces patent infection. METHODS: cGMP-grade NaL3 were produced for a phase 1 trial in 20 healthy, hookworm-naïve adults in the United States, who received either 25 or 50 NaL3. Participants were monitored for 12-18 weeks postinfection for safety, tolerability, and patency of N. americanus infection. RESULTS: Both NaL3 doses were well tolerated. Early manifestations of infection included pruritus, pain, and papulovesicular rash at the application site. Gastrointestinal symptoms and eosinophilia appeared after week 4 postinfection. The 50 NaL3 inoculum induced patent N. americanus infection in 90% of this dose group. CONCLUSIONS: The inoculum of 50 NaL3 was well tolerated and consistently induced patent N. americanus infection suitable for future HVCM trials. CLINICAL TRIALS REGISTRATION: NCT01940757.

Refined ab initio gene predictions of Heterorhabditis bacteriophora using RNA-seq.

Interest has recently grown in developing the entomopathogenic nematode Heterorhabditis bacteriophora as a model to genetically dissect the process of parasitic infection. Despite the availability of a full genome assembly, there is substantial variation in gene model accuracy. Here, a methodology is presented for leveraging RNA-seq evidence to generate improved annotations using ab initio gene prediction software. After alignment of reads and subsequent generation of a RNA-seq supported annotation, the new gene prediction models were verified on a selection of genes by comparison with sequenced 5' and 3' rapid amplification of cDNA ends products. By utilising a whole transcriptome for genome annotation, the current reference annotation was enriched, demonstrating the importance of coupling transcriptional data with genome assemblies.