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Background Mosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. Methods We developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi-infected Ae. aegypti. Results Approximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the proportion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12–14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae. Conclusions We provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.

Strongyloides stercoralis infection is prevalent worldwide and can cause lifelong infection in immunocompetent individuals, and potentially death in immunosuppressed patients. The diagnosis is hindered by the low sensitivity of microscopic examination, thus making serology an important complementary test to improve the detection rate. However, there were reports that some Strongyloides-infected individuals were negative with specific IgG and IgG4 assays, and other helminth infections were positive with commercial Strongyloides IgG-ELISAs. Thus, there is a need to develop better serodiagnostic methods for strongyloidiasis. We investigated the diagnostic potential of IgE-ELISAs using Strongyloides larval lysate. Sera from two groups infected with Strongyloides served as the positive reference, that is, 1) positive by commercial IgG-ELISAs and IgG4 rapid test, and stool samples positive by microscopy and/or PCR (group IA; n = 20); and 2) negative by IgG-ELISAs and IgG4 rapid test, but stool samples were PCR positive (group IB sera; n = 11). Sera from another two groups served as negative reference (controls), that is, 1) infected with other parasites (group II; n = 73) and 2) healthy donors (group III; n = 22). Results showed a 100% diagnostic sensitivity in detecting sera from groups IA and IB. The latter group of individuals probably had early infection because their IgG and IgG4 assays were negative. The optical density values of group IB sera were also significantly lower than those of group IA (P < 0.003). The IgE-ELISA was 100% specific when tested against sera from groups II and III. This study highlights the diagnostic potential of IgE-ELISA using larval lysate to detect strongyloidiasis, especially those with probable early infection.

Significance Infections with filarial nematodes, resulting from the bite of an infected mosquito, have a huge health impact worldwide. Sequencing mosquito transcripts revealed that a strain of Aedes aegypti that is refractory to infection with heartworm robustly activates immune genes, compared to a susceptible strain. These differences were detected early in infection and in the tissue important for larval development. Using a new assay, we found that bolstering mosquito immune activation through knockdown of a negative regulator resulted in a strong block in the emergence of transmission-stage filarial larvae, including those that cause human lymphatic filariasis. This work provides opportunities to block pathogen transmission by targeting filarial nematodes in the mosquito vector., Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.

Mechanisms governing morphogenesis and development of infectious third-stage larvae (L3i) of parasitic nematodes have been likened to those regulating dauer development in Caenorhabditis elegans. Dauer regulatory signal transduction comprises initial G protein-coupled receptor (GPCR) signaling in chemosensory neurons of the amphidial complex that regulates parallel insulin- and TGFβ-like signaling in the tissues. Insulin- and TGFβ-like signals converge to co-regulate steroid signaling through the nuclear receptor (NR) DAF-12. Discovery of the steroid ligands of DAF-12 opened a new avenue of small molecule physiology in C. elegans. These signaling pathways are conserved in parasitic nematodes and an increasing body of evidence supports their function in formation and developmental regulation of L3i during the infectious process in soil transmitted species. This review presents these lines of evidence for G protein-coupled receptor (GPCR), insulin- and TGFβ-like signaling in brief and focuses primarily on signaling through parasite orthologs of DAF-12. We discuss in some depth the deployment of sensitive analytical techniques to identify Δ7-dafachronic acid as the natural ligand of DAF-12 homologs in Strongyloides stercoralis and Haemonchus contortus and of targeted mutagenesis by CRISPR/Cas9 to assign dauer-like regulatory function to the NR Ss-DAF-12, its coactivator Ss-DIP-1 and the key ligand biosynthetic enzyme Ss-CYP-22a9. Finally, we present published evidence of the potential of Ss-DAF-12 signaling as a chemotherapeutic target in human strongyloidiasis.

A prevalent feature ofStrongyloides stercoralisis a life-long and potentially lethal infection that is due to the nematode parasite’s ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasite’s nuclear receptor,Ss-DAF-12, in governing infection. We identified Δ7-DA as the endogenousSs-DAF-12 ligand and elucidated the hormone’s biosynthetic pathway. Genetic loss of function of the ligand’s rate-limiting enzyme demonstrated that Δ7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for development of infectious larvae. Availability of the ligand permitsSs-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when Δ7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

Helminths are distinct from microbial pathogens in both size and complexity, and are the likely evolutionary driving force for type 2 immunity. CD4+ helper T cells can both coordinate worm clearance and prevent immunopathology, but issues of T cell antigen specificity in the context of helminth-induced Th2 and T regulatory cell (Treg) responses have not been addressed. Herein, we generated a novel transgenic line of the gastrointestinal nematode Strongyloides ratti expressing the immunodominant CD4+ T cell epitope 2W1S as a fusion protein with green fluorescent protein (GFP) and FLAG peptide in order to track and study helminth-specific CD4+ T cells. C57BL/6 mice infected with this stable transgenic line (termed Hulk) underwent a dose-dependent expansion of activated CD44hiCD11ahi 2W1S-specific CD4+ T cells, preferentially in the lung parenchyma. Transcriptional profiling of 2W1S-specific CD4+ T cells isolated from mice infected with either Hulk or the enteric bacterial pathogen Salmonella expressing 2W1S revealed that pathogen context exerted a dominant influence over CD4+ T cell phenotype. Interestingly, Hulk-elicited 2W1S-specific CD4+ T cells exhibited both Th2 and Treg phenotypes and expressed high levels of the EGFR ligand amphiregulin, which differed greatly from the phenotype of 2W1S-specific CD4+ T cells elicited by 2W1S-expressing Salmonella. While immunization with 2W1S peptide did not enhance clearance of Hulk infection, immunization did increase total amphiregulin production as well as the number of amphiregulin-expressing CD3+ cells in the lung following Hulk infection. Altogether, this new model system elucidates effector as well as immunosuppressive and wound reparative roles of helminth-specific CD4+ T cells. This report establishes a new resource for studying the nature and function of helminth-specific T cells., Author summary Intestinal parasitic helminths infect roughly one billion people worldwide, and there are currently no vaccines available for use in humans. In humans and experimental mouse infection models, CD4+ helper T cells that have differentiated into type 2 (Th2) effectors serve important roles in worm clearance and are considered essential for specific, long-lasting immunity. However, many helminth infections also drive expansion of regulatory T cells (Tregs) that can suppress inflammatory CD4+ T cell subsets. Whether Th2 and/or Treg subsets recognize helminth antigens is a question of great relevance to vaccine development, but no tools previously existed to identify and study endogenous helminth-specific CD4+ T cells. Here, we used transgenesis in the Strongyloides ratti model to engineer the first gastrointestinal (GI) nematode strain to express a tractable CD4+ T cell peptide epitope, 2W1S (Hulk). Our studies reveal that 2W1S-specific CD4+ T cells become both Th2s and Tregs in the lungs of infected mice and potentially serve protective and/or suppressive roles during Hulk infection. Development of this new model organism could be an important tool for studies designed to understand Th2 and Treg immunobiology, microenvironment-specific interactions, helminth-epitope processing/presentation, and T cell-dependent antibody responses.

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

Purpose of ReviewThis paper constitutes an update of recent studies on the general biology, molecular genetics, and cellular biology ofStrongyloidesspp. and related parasitic nematodes.Recent FindingsIncreasingly, human strongyloidiasis is considered the most neglected of neglected tropical diseases. Despite this, the last 5 years has seen remarkable advances in the molecular biology ofStrongyloidesspp. Genome sequences forS. stercoralis,S. ratti,S. venezuelensis,S. papillosus, and the related parasiteParastrongyloides trichosuriwere created, annotated, and analyzed. These genomic resources, along with a practical transgenesis platform forStrongyloidesspp., aided a major achievement, the advent of targeted mutagenesis via CRISPR/Cas9 inS. stercoralisandS. ratti. The genome sequences have also enabled significant molecular epidemiologic and phylogenetic findings on human strongyloidiasis, including the first genetic evidence of zoonotic transmission ofS. stercoralisbetween dogs and humans. Studies of molecular signaling pathways identified the nuclear receptorSs-DAF-12 as one that can be manipulated in the parasite by exogenous application of its steroid ligands. The chemotherapeutic implications of this were unscored by a study in which aSs-DAF-12 ligand suppressed autoinfection byS. stercoralisin a new murine model of human strongyloidiasis.SummarySeminal advances in genomics ofStrongyloidesspp. have transformed research into strongyloidiasis, facilitating fundamental phylogenetic and epidemiologic studies and aiding the deployment of CRISPR/Cas9 gene disruption and editing as functional genomic tools inStrongyloidesspp. Studies ofSs-DAF-12 signaling inS. stercoralisdemonstrated the potential of this pathway as a novel chemotherapeutic target in parasitic nematodes.

The human and canine parasitic nematode Strongyloides stercoralis utilizes an XX/XO sex determination system, with parasitic females reproducing by mitotic parthenogenesis and free-living males and females reproducing sexually. However, the genes controlling S. stercoralis sex determination and male development are unknown. We observed precocious development of rhabditiform males in permissive hosts treated with corticosteroids, suggesting that steroid hormones can regulate male development. To examine differences in transcript abundance between free-living adult males and other developmental stages, we utilized RNA-Seq. We found two clusters of S. stercoralis-specific genes encoding predicted transmembrane proteins that are only expressed in free-living males. We additionally identified homologs of several genes important for sex determination in Caenorhabditis species, including mab-3, tra-1, fem-2, and sex-1, which may have similar functions. However, we identified three paralogs of gld-1; Ss-qki-1 transcripts were highly abundant in adult males, while Ss-qki-2 and Ss-qki-3 transcripts were highly abundant in adult females. We also identified paralogs of pumilio domain-containing proteins with sex-specific transcripts. Intriguingly, her-1 appears to have been lost in several parasite lineages, and we were unable to identify homologs of tra-2 outside of Caenorhabditis species. Together, our data suggest that different mechanisms control male development in S. stercoralis and Caenorhabditis species.

Significance Understanding the molecular mechanisms controlling nematode parasite infection is important to developing new therapeutic strategies. The DAF-12 nuclear receptor signaling pathway in free-living nematodes regulates a process known as the dauer diapause. In nematode parasites, this signaling pathway is believed to govern the infectious stage. To investigate this hypothesis, we characterized the orthologous components of DAF-12 and a parasite-specific transcriptional coactivator in the potentially lethal parasite Strongyloides stercoralis. Using a CRISPR method developed specifically for S. stercoralis, we demonstrate that DAF-12 and its coactivator are required for both the entry into and exit from the infectious stage. These studies further highlight the potential of therapeutically targeting this pathway in S. stercoralis and other nematode parasites., DAF-12 is nematode-specific nuclear receptor that has been proposed to govern development of the infectious stage of parasitic species, including Strongyloides stercoralis. Here, we identified a parasite-specific coactivator, called DAF-12 interacting protein-1 (DIP-1), that is required for DAF-12 ligand-dependent transcriptional activity. DIP-1 is found only in Strongyloides spp. and selectively interacts with DAF-12 through an atypical receptor binding motif. Using CRISPR/Cas9-directed mutagenesis, we demonstrate that DAF-12 is required for the requisite developmental arrest and the ligand-dependent reactivation of infectious S. stercoralis infective third-stage larvae, and that these effects require the DIP-1 coactivator. These studies reveal the existence of a distinct nuclear receptor/coactivator signaling pathway that governs parasite development.

Significance Understanding the molecular mechanisms controlling nematode parasite infection is important to developing new therapeutic strategies. The DAF-12 nuclear receptor signaling pathway in free-living nematodes regulates a process known as the dauer diapause. In nematode parasites, this signaling pathway is believed to govern the infectious stage. To investigate this hypothesis, we characterized the orthologous components of DAF-12 and a parasite-specific transcriptional coactivator in the potentially lethal parasite Strongyloides stercoralis . Using a CRISPR method developed specifically for S. stercoralis , we demonstrate that DAF-12 and its coactivator are required for both the entry into and exit from the infectious stage. These studies further highlight the potential of therapeutically targeting this pathway in S. stercoralis and other nematode parasites.

Viral and parasitic coinfections are known to lead to both enhanced disease progression and altered disease states. HTLV-1 and Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. Independently, HTLV-1 induces a Th1 response and S. stercoralis infection induces a Th2 response. However, coinfection with the two pathogens has been associated with the development of S. stercoralis hyperinfection and an alteration of the Th1/Th2 balance. In this study, a model of HTLV-1 and S. stercoralis coinfection in CD34+ umbilical cord blood hematopoietic stem cell engrafted humanized mice was established. An increased level of mortality was observed in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected group. The mortality was not correlated with proviral loads or total viral RNA. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. HTLV-1 infected and coinfection groups showed a significant, yet non-clonal expansion of the CD4+CD25+ T-cell population. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group and no autoinfective larvae were found. However, infective larvae recovered from the coinfection group showed an enhancement in growth, as was seen in mice with S. stercoralis hyperinfection caused by treatment with steroids. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections in vivo in the context of human immune cells., Author summary Human infections with the virus HTLV-1 and the nematode Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. The coincident infections result in S. stercoralis hyperinfection and an alteration of the balance of the immune responses to the virus and to the worm. In this study, a novel model of HTLV-1 and S. stercoralis coinfection was established in humanized mice. Mortality was increased in the HTLV-1 and coinfected animals as compared to the S. stercoralis infected group. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group, however, infective larvae recovered from the coinfection group showed a significant enhancement in growth. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections.

Background Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used. Methods and principal findings A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing. Conclusions/Significance We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs., Author summary Tests that detect parasite DNA in human stool are increasingly being used for the diagnosis of infections with intestinal worms, including schistosomiasis. To ensure the quality in diagnostic testing of these parasitic worms, it is important that laboratories evaluate the diagnostic performance of their DNA-based tests. This can best be achieved by participating in an external quality assessment scheme (EQAS). An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. Currently, such an EQAS for parasitic worms is lacking. We therefore piloted an international EQAS for the diagnosis of parasitic worms involving 15 laboratories in Africa, Asia, Australia, Europe, and North America. Although most laboratories performed well, we could clearly identify those laboratories that may need to improve their test protocol. We found that laboratories were using many different test protocols, and further research should aim to verify whether this has an impact on the performance of the diagnostic outcomes.

BackgroundMosquitoes transmit filarial nematodes to both human and animal hosts, resulting in worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector.MethodsWe developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi infected Ae. aegypti.ResultsApproximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produce emerging third-stage larvae. Once D. immitis third-stage larvae emerge at 13 days post infection, the proportion of mosquitoes producing them, and the number produced per mosquito remain stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 days post infection. Increased uptake of D. immitis microfilariae increases the fitness cost to the mosquito but does not increase the number of emerging third-stage larvae.ConclusionsWe provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious L3 for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.Graphical Abstract

Heretofore, transgenesis in the parasitic nematode genus Strongyloides has relied on microinjecting transgene constructs into gonadal syncytia of free-living females. We now report transgenesis in Strongyloides stercoralis by microinjecting constructs into the syncytial testes of free-living males. Crosses of individual males microinjected with a construct encoding GFP with cohorts of 12 non-injected females produced a mean of 7.28 ± 2.09 transgenic progeny. Progeny of males and females microinjected with distinct reporter constructs comprised 2.6% ± 0.7% of individuals expressing both paternal and maternal transgenes. Implications of this finding for deployment of CRISPR/Cas9 mutagenesis in Strongyloides spp. are discussed.

BACKGROUND The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid ( RESULTS In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed ( CONCLUSIONS RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.

Whole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

Strongyloidiasis, caused mainly by the nematode Strongyloides stercoralis, is prevalent worldwide and potentially fatal in immunosuppressed patients. We report on a new IgE biomarker to diagnose Strongyloides infection. Sera from two groups infected with Strongyloides served as positive samples: Group 1A, in which infection was confirmed by stool-microscopy and/or stool-polymerase chain reaction (PCR) and was seropositive by an IgG-enzyme linked immunosorbent assay (ELISA) and an IgG4 rapid test, and Group 1B in which infection was confirmed by stool-PCR but was seronegative. Negative samples (controls) comprised infections with other parasites (Group II) and healthy donors (Group III). Immunoscreenings of an S. stercoralis complementary DNA (cDNA) library were performed, and the cDNA clone with the highest diagnostic potential (clone A133) was selected for recombinant protein production and then evaluated using IgE Western blot and ELISA. The Western blot showed that the recombinant protein (rA133) was 100% reactive with Group IA (n = 10) and Group IB (n = 5), and 96% non-reactive with Groups II and III (n = 25). Subsequently, the IgE-ELISA was developed and showed 100% diagnostic sensitivity in Groups IA (n = 32) and IB (n = 11), and 99.3% specificity in Groups II and III (n = 144). In conclusion, this study has identified rA133 as a novel recombinant protein with potential diagnostic value, and that the IgE-ELISA incorporating this protein may be useful for patient diagnosis and epidemiological studies.

Signaling or communication between host and parasite may occur over relatively long ranges to enable host finding and acquisition by infective parasitic nematode larvae. Innate behaviors in infective larvae transmitted from the soil that enhance the likelihood of host contact, such as negative geotaxis and hypermotility, are likely mediated by mechanoreception and neuromuscular signaling. Host cues such as vibration of the substratum, elevated temperature, exhaled CO2, and other volatile odorants are perceived by mechanosensory and chemosensory neurons of the amphidial complex. Beyond this, the molecular systems that transduce these external cues within the worm are unknown at this time. Overall, the signal transduction mechanisms that regulate switching between dauer and continuous reproductive development in Caenorhabditis elegans, and doubtless other free-living nematodes, have provided a useful framework for testing the hypotheses about how the morphogenesis and development of infective parasitic nematode larvae and the lifespan of adult parasites are regulated. In C. elegans, four major signal transduction pathways, G-protein-coupled receptor signaling, insulin/insulin-like growth factor signaling, TGFβ-like signaling, and steroid-nuclear hormone receptor signaling, govern the switch between dauer and continuous development and regulate adult lifespan. Parasitic nematodes appear to have conserved the functions of G-protein-coupled signaling, insulin-like signaling, and steroid-nuclear hormone receptor signaling to regulate larval development before and during the infective process. By contrast, TGFβ-like signaling appears to have been adapted for some other function, perhaps modulation of the host immune response. Of the three signal transduction pathways that appear to regulate development in parasitic nematodes, steroid-nuclear hormone signaling is the most straightforward to manipulate with administered small molecules and may form the basis of new chemotherapeutic strategies. Signaling between parasites and their hosts’ immune systems also occurs and serves to modulate these responses to allow chronic infection and downregulate acute inflammatory responses. Knowledge of the precise nature of this signaling may form the basis of immunological interventions to protect against parasitism or related lesions and to alleviate inflammatory diseases of various etiologies.

SUMMARYTransgenesis forStrongyloidesandParastrongyloideswas accomplished in 2006 and is based on techniques derived forCaenorhabditis elegansover two decades earlier. Adaptation of these techniques has been possible becauseStrongyloidesand related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. InStrongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes inS. stercoralisrequires their integration into the genome, and stable lines have been derived from integrants created using thepiggyBactransposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis inStrongyloidesandParastrongyloidesopens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.

Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.