Old friends and friendly fire: Pregnancy, hookworm infection, and anemia among tropical horticulturalists.

OBJECTIVES: Despite public health concerns about hookworm infection in pregnancy, little is known about immune profiles associated with hookworm (Necator americanus and Ancylostoma duodenale) infection during pregnancy. Fetal tolerance requirements may constrain maternal immune response to hookworm, thereby increasing susceptibility to new infections or increasing hemoglobin loss. To explore this possibility, we study systemic immune response and hemoglobin levels in a natural fertility population with endemic helminthic infection. METHODS: We used Bayesian multilevel models to analyze mixed longitudinal data on hemoglobin, hookworm infection, reproductive state, eosinophils, and erythrocyte sedimentation rate (ESR) to examine the effects of pregnancy and hookworm infection on nonspecific inflammation, cellular parasite response, and hemoglobin among 612 Tsimane women aged 15-45 (1016 observations). RESULTS: Pregnancy is associated with lower eosinophil counts and lower eosinophil response to hookworm, particularly during the second and third trimesters. Both hookworm and pregnancy are associated with higher ESR, with evidence for an interaction between the two causing further increases in the first trimester. Pregnancy is moderately associated with higher odds of hookworm infection (OR: 1.23, 95% CI: 0.83 to 1.83). Pregnancy and hookworm both decrease hemoglobin and may interact to accentuate this effect in the first-trimester of pregnancy (Interaction: ß: -0.30 g/dL; CI: -0.870 to 0.24). CONCLUSIONS: Our findings are consistent with a possible trade-off between hookworm immunity and successful pregnancy, and with the suggestion that hookworm and pregnancy may have synergistic effects, particularly in the first trimester.

No impact of strongylid infections on the detection of Plasmodium spp. in faeces of western lowland gorillas and eastern chimpanzees.

BACKGROUND: Although a high genetic diversity of Plasmodium spp. circulating in great apes has been revealed recently due to non-invasive methods enabling detection in faecal samples, little is known about the actual mechanisms underlying the presence of Plasmodium DNA in faeces. Great apes are commonly infected by strongylid nematodes, including hookworms, which cause intestinal bleeding. The impact of strongylid infections on the detection of Plasmodium DNA in faeces was assessed in wild, western, lowland gorillas from Dzanga Sangha Protected Areas, Central African Republic and eastern chimpanzees from Kalinzu Forest Reserve, Uganda. METHODS: Fifty-one faecal samples from 22 habituated gorillas and 74 samples from 15 habituated chimpanzees were analysed using Cytochrome-b PCR assay and coprological methods. RESULTS: Overall, 26.4% of the analysed samples were positive for both Plasmodium spp. and strongylids. However, the results showed no significant impact of intensity of infections of strongylids on detection of Plasmodium DNA in gorilla and chimpanzee faeces. CONCLUSION: Bleeding caused by strongylid nematode Necator spp. cannot explain the presence of Plasmodium DNA in ape faeces.

The roundworm Strongyloides stercoralis in children, dogs, and soil inside and outside a segregated settlement in Eastern Slovakia: frequent but hardly detectable parasite.

A comparative study was carried out to evaluate the Strongyloides stercoralis infections in children and dogs inside and outside the segregated settlement in Medzev, Eastern Slovakia, and a survey of the soil within the settlement was included. Applying the Koga agar plate (KAP) culture method and microscopy examination of stool samples collected from 60 Roma and 21 nonRoma children, no larvae of S. stercoralis were detected but eggs of three nematodes (Ascaris lumbricoides, Trichuris trichiura, and Enterobius vermicularis) and cysts of two protozoan endoparasites (Giardia duodenalis and Cryptosporidium spp.) were often found. However, immunoenzymatic assay (ELISA) for the evidence of IgG antibodies against S. stercoralis showed 33.3% seroprevalence in Roma children and 23.8% prevalence in children from the majority population, attending the same school. Eosinophilia was regularly present in children with exclusive infection of S. stercoralis (eight cases) as well as in individuals suffering from mixed infections of S. stercoralis and some of the above listed parasites (16 cases); high eosinophil counts sometimes, but not always, occurred in parasitized children lacking S. stercoralis antibodies. A comparison of S. stercoralis in dogs from the settlement (40 dogs) and from a distant dog shelter (20 dogs) did not reveal remarkable differences: the direct microscopy of faecal samples revealed rhabditiform larvae in 13.3% of the dogs from the settlement (4/30) and in 10.0% of the dogs from the shelter (2/20). Out of blood samples collected from the second dog group, 55% of the dogs contained antibodies against S. stercoralis. In the soil collected from 14 various locations within the settlement, S. stercoralis larvae were observed in two samples (14.3%); however, 13 samples (92.9%) were positive for human or dog endoparasites of the genera Ancylostoma, Ascaris, Toxocara, Toxascaris, Trichuris, and Hymenolepis.

A novel isothermal microcalorimetry tool to assess drug effects on Ancylostoma ceylanicum and Necator americanus.

Soil-transmitted helminths, which affect the poorest communities, worldwide cause a range of symptoms and morbidity, yet few treatment options are available and drug resistance is a concern. To improve and accelerate anthelminthic drug discovery, novel drug screening tools such as isothermal microcalorimetry (IMC) have been tested with great potential. In this study, we used a novel microcalorimeter, the calScreener™, to study the viability on the hookworms Necator americanus and Ancylostoma ceylanicum as well as the whipworm Trichuris muris. Significant heat flow signals could be obtained with already one adult worm per channel for all three species. High-amplitude oscillations were observed for the hookworms; however, adult T. muris showed a twofold heat flow decrease during the first 24 h. Antinematodal effects of ivermectin and levamisole at 1, 10, and 100 µg/ml were evaluated on adult N. americanus and A. ceylanicum. Levamisole-treated hookworms showed a decline in heat flow and oscillation amplitude in a dose-response manner. Heat flow for ivermectin-treated hookworms increased proportionally with increased concentrations of ivermectin, though the wavelet analysis showed an opposite trend as observed by flatter wavelets. In conclusion, the calScreener™ is an excellent tool to study drug effects on intestinal hookworms at the adult worm stage as it offers a lower detection limit than other IMC devices and the possibility to monitor worm viability online.

Determination of Ancylostoma caninum ova viability using metabolic profiling.

Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum was used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analyses of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that metabolic profiling using GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of metabolic profiling using GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.

The crowding effect in Ancylostoma ceylanicum: density-dependent effects on an experimental model of infection.

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8% to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.

Comparison of novel and existing tools for studying drug sensitivity against the hookworm Ancylostoma ceylanicum in vitro.

The motility assay is the current gold standard for evaluating drug effects on hookworm larvae and adults, however, among other drawbacks the assay is time consuming, and prone to individual subjectivity. We evaluated six alternative in vitro assays, namely the feeding inhibition assay, the colourimetric AlamarBlue®, MTT formazan and acid phosphatase activity assays, as well as isothermal calorimetry and the xCELLigence System using Ancylostoma ceylanicum third-stage larvae, stimulated third-stage larvae and adults. The performances of the assays were compared to the motility assay using three standard drugs: albendazole, levamisole and ivermectin (100-1 µg/ml). None of the assays investigated offered an advantage over the motility assay, because they were all inapplicable to third-stage larvae, which were presumably metabolically and physically too inactive. Among all assays tested the xCELLigence System performed best on adult worms as the test was accurate, simple, required a minimal number of worms and offered the possibility for conducting a medium-throughput screening.

Larva migrans in Votuporanga, São Paulo, Brazil: Where does the danger hide?

Soil samples collected near municipal schools (public/EMEI and private/EPEI schools), clubs (CLB), public squares (PS) and residential condominiums (CND) and samples of animal faeces from the Zoonosis Control Centre (CCZ) of the municipality of Votuporanga/SP were analysed using the Baermann method for the detection of zoonotic helminth larvae. The prevalence rates of the nematode genera identified were determined, and the results were compared using Fisher's exact and chi-square frequency tests. Information about cases of larvae migrans in the population were collected from the Family Health Units and the private health plans. All sites were positive for Ancylostoma spp. and, with the exception of EPEIs and dog faeces, for Strongyloides spp. The prevalence of Ancylostoma spp. was 87.5% for CND samples, 74.29% for EMIEs, 63.64% for CLB, 61.76% for PS and 64.29% for dog's and 42.86% for cats at CCZ. The prevalence of Strongyloides spp. ranged from 14.29% (cats/CCZ) to 41.18% (PS). Cases of cutaneous larva migrans were reported during interviews. Thus, from the public health perspective, the risk of individuals that frequent recreational areas in the municipality, especially children, to be infected by helminth larvae is noteworthy, indicating the need to develop policies aimed at controlling this important zoonosis.

Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway.

The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in approximately 85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a approximately 70% reduction in feeding but had almost no effect on the level of any of the activation-associated mRNAs studied. Rapamycin was shown to have a weak effect on feeding, and several of the mRNAs studied exhibited greater than expected transcription following treatment. The complexities of activation-associated transcription could not be addressed using the current approach. A larger number of mRNAs needs to be investigated in order to predict or identify regulatory mechanisms proposed to function in the insulin-like signalling pathway in A. caninum.

The putative cyclooctadepsipeptide receptor depsiphilin of the canine hookworm Ancylostoma caninum.

The G-Protein-coupled receptor Hc110-R of Haemonchus contortus and its orthologue in Caenorhabditis elegans, the latrophilin-like protein 1 (LAT-1), were shown to play a role in the mode of action of the new anthelmintic compound emodepside. C. elegans LAT-1 knockout mutants showed a decreased paralysing effect of emodepside on the pharyngeal muscle. In the present study, the LAT-1 orthologue in the canine hookworm Ancylostoma caninum was identified and named depsiphilin. To obtain more information about the regulation of this receptor and to facilitate phylogenetic and evolutionary analyses of parasitic nematode genes, the genomic structure of A. caninum depsiphilin was investigated. High consistency regarding the position of introns in comparison to C. elegans LAT-1 was observed, providing indication of the same origin of the genes. With a view to possible differences in efficacy of emodepside on different developmental stages, we analysed the transcript level of A. caninum depsiphilin in eggs, L1, L3, male and female adult worms using quantitative real-time PCR. Depsiphilin is transcribed in all five examined stages, but we found a significantly lower transcript level in third-stage larvae. A correlation between these findings and a reduced emodepside activity remains to be investigated.

Cognitive and Microbiome Impacts of Experimental Ancylostoma ceylanicum Hookworm Infections in Hamsters.

Hookworms are one of the most prevalent and important parasites, infecting ~500 million people worldwide. Hookworm disease is among the leading causes of iron-deficiency anemia in the developing world and is associated with significant growth stunting and malnutrition. In humans, hookworms appear to impair memory and other forms of cognition, although definitive data are hard to come by. Here we study the impact of a human hookworm parasite, Ancylostoma ceylanicum, on cognition in hamsters in a controlled laboratory setting. We developed tests that measure long-term memory in hamsters. We find that hookworm-infected hamsters were fully capable of detecting a novel object. However, hookworm-infected hamsters were impaired in detecting a displaced object. Defects could be discerned at even at low levels of infection, whereas at higher levels of infection, hamsters were statistically unable to distinguish between displaced and non-displaced objects. These spatial memory deficiencies could not be attributed to defects in infected hamster mobility or to lack of interest. We also found that hookworm infection resulted in reproducible reductions in diversity and changes in specific taxanomic groups in the hamster gut microbiome. These data demonstrate that human hookworm infection in a laboratory mammal results in a specific, rapid, acute, and measurable deficit in spatial memory, and we speculate that gut alterations could play some role in these cognitive deficits. Our findings highlight the importance of hookworm elimination and suggest that finer tuned spatial memory studies be carried out in humans.

Development and optimization of a high-throughput screening method utilizing Ancylostoma ceylanicum egg hatching to identify novel anthelmintics.

Hookworms remain a major health burden in the developing world, with hundreds of millions currently afflicted by these blood-feeding parasites. There exists a vital need for the discovery of novel drugs and identification of parasite drug targets for the development of chemotherapies. New drug development requires the identification of compounds that target molecules essential to parasite survival and preclinical testing in robust, standardized animal models of human disease. This process can prove costly and time consuming using conventional, low-throughput methods. We have developed a novel high-throughput screen (HTS) to identify anthelmintics for the treatment of soil-transmitted helminths. Our high-throughput, plate reader-based assay was used to rapidly assess compound toxicity to Ancylostoma ceylanicum L1 larva. Using this method, we screened 39,568 compounds from several small molecule screening libraries at 10 µM and identified 830 bioactive compounds that inhibit egg hatching of the human hookworm A. ceylanicum by >50%. Of these, 132 compounds inhibited hookworm egg hatching by >90% compared to controls. The nematicidal activities of 268 compounds were verified by retesting in the egg hatching assay and were also tested for toxicity against the human HeLa cell line at 10 µM. Fifty-nine compounds were verified to inhibit A. ceylanicum egg hatching by >80% and were <20% toxic to HeLa cells. Half-maximal inhibitory concentration (IC50) values were determined for the 59 hit compounds and ranged from 0.05-8.94 µM. This stringent advancement of compounds was designed to 1) systematically assess the nematicidal activity of novel compounds against the egg stage of A. ceylanicum hookworms in culture and 2) define their chemotherapeutic potential in vivo by evaluating their toxicity to human cells. Information gained from these experiments may directly contribute to the development of new drugs for the treatment of human hookworm disease.

Hookworm infection aggravates metabolic disorder in obesity.

Obesity and ancylostomiasis are considered public health problems. Recent studies have shown that infection by intestinal helminths in obese individuals can ameliorate metabolic disorder and improve glucose tolerance by decreasing both insulin resistance and low-intensity inflammation. However, few helminth species have been studied in this context, and some modulation mechanisms still require deeper investigation. Therefore, the present work aimed to investigate the role of experimental infection with Ancylostoma ceylanicum in the modulation of the immune response in an obese experimental model. Four groups of hamsters were used as follows: two groups were submitted to a hyperlipidic and hypercaloric diet capable of inducing obesity, one infected and the other uninfected; and two normonourished control groups, one infected and one uninfected by A. ceylanicum. Biochemical, haematological, parasitological and immunological parameters were evaluated. The results demonstrated that A. ceylanicum infection accentuated weight loss in obese animals compared to normonourished animals. However, obesity reduced the recovery of worms and oviposition of the females, and both infected groups showed decreased levels of haemoglobin, albumin, iron and erythrocytes. Significant relations were observed for pathogenesis in the following cases: infection interfered in lipid metabolism, which increased levels of total cholesterol and triglycerides in the obese group, and caused a decrease in HDL levels in both groups. Obesity led to an increase in glucose levels, and the infection exacerbated this parameter in both the normonourished and obese groups. Inflammation was intensified in obese animals that showed elevated macrophage and neutrophil activation in adipose tissue, enlargement of the spleen and accumulation of lipids in the liver and faeces. Despite the decrease in IFN-γ levels, the infection did not potentiated the expression of the Foxp3, IL-10 and IL-2 transcription factor for any of the infected groups, markers that could positively compensate the host from the damage caused by obesity.

Hookworm: developmental biology of the infectious process.

Hookworms cause severe anemia and malnutrition in developing countries of the tropics, with an estimated one billion people infected worldwide. An in vitro system that models the early events of infection has provided new information about the linkage between the infectious process and the parasite's developmental biology. The cloning and expression of Ancylostoma secreted protein, ASP 1 - a secreted molecule associated with these developmental processes - is an example of how this system allows us to dissect the infectious process at the molecular level.

Strategies for the storage of Ancylostoma caninum third-stage larvae.

Although cryopreservation protocols for storage of hookworm larvae have been described, the circumstances under which the technique is necessary to ensure larval survival are not well defined. The motility of infective-stage larvae (as judged by observation) and their ability to migrate through canine skin in vitro were measured over a 7-mo period in worms held at room temperature and worms that had been cryopreserved at the start of the experiment. Cryopreserved worms showed motility and migration proportions of 45.6-48.0% and 26.8- 34.0%, respectively, throughout the experiment, compared with percentages of 92.7 and 84.1%, respectively, in the original fresh worms. Larvae held at room temperature showed a gradual decrease in motility and migration ability over the experimental period. Motility and migratory ability of cryopreserved larvae was only significantly higher (P < 0.01) than room temperature-stored larvae from 4 and 5 mo onward, respectively.

[Current status of human hookworm infection in Jiangxi Province in 2014].

OBJECTIVE: To understand the status of human hookworm (Ancylostoma duodenale or Necator americanus) infection in Jiangxi Province. METHODS: A stratified cluster random sampling was conducted based on the geographical location and economic condition in 2014. Kato-Katz's thick smear method was used for stool examinations, and filter paper cultivation was conducted for distinguishing the species of hookworms. RESULTS: A total of 21 615 persons were involved in the investigation, and there were 1 095 persons infected with hookworm with the prevalence of 5.07% (standardized rate of 4.05%). The infection rate of hookworm in Nanfeng County was the highest (13.96%). Most of the infected people had low worm burden (95.43%). The infection rate of hookworm of the female was higher than that of the male (χ2 = 28.03, P < 0.05). The prevalence increased with the age, while the infection rate of hookworm in people at ages of 75 years and higher was the highest (14.79%). Housewife was the highest risk occupation with the infection rate of 7.55%. The differences of infection rates among different educational level groups were significant (χ2 = 135.88, P < 0.05). The prevalence rates of hookworm in different landforms were significantly different (χ2 = 34.37, P < 0.05). The priority species was N. americanus (80.41%) in Jiangxi Province. CONCLUSIONS: The infection rate of hookworm decreases obviously in Jiangxi Province. The high risk groups of hookworm infection are the people with low educational level, female and elder age-group.

A Critical Role for Thermosensation in Host Seeking by Skin-Penetrating Nematodes.

Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are a major source of neglected tropical disease [1-6]. Their life cycle includes an infective third-larval (iL3) stage that searches for hosts to infect in a poorly understood process that involves both thermal and olfactory cues. Here, we investigate the temperature-driven behaviors of skin-penetrating iL3s, including the human-parasitic threadworm Strongyloides stercoralis and the human-parasitic hookworm Ancylostoma ceylanicum. We show that human-parasitic iL3s respond robustly to thermal gradients. Like the free-living nematode Caenorhabditis elegans, human-parasitic iL3s show both positive and negative thermotaxis, and the switch between them is regulated by recent cultivation temperature [7]. When engaging in positive thermotaxis, iL3s migrate toward temperatures approximating mammalian body temperature. Exposing iL3s to a new cultivation temperature alters the thermal switch point between positive and negative thermotaxis within hours, similar to the timescale of thermal plasticity in C. elegans [7]. Thermal plasticity in iL3s may enable them to optimize host finding on a diurnal temperature cycle. We show that temperature-driven responses can be dominant in multisensory contexts such that, when thermal drive is strong, iL3s preferentially engage in temperature-driven behaviors despite the presence of an attractive host odorant. Finally, targeted mutagenesis of the S. stercoralis tax-4 homolog abolishes heat seeking, providing the first evidence that parasitic host-seeking behaviors are generated through an adaptation of sensory cascades that drive environmental navigation in C. elegans [7-10]. Together, our results provide insight into the behavioral strategies and molecular mechanisms that allow skin-penetrating nematodes to target humans.

Contamination of selected recreational areas in Lublin Province, Eastern Poland, by eggs of Toxocara spp., Ancylostoma spp. and Trichuris spp.

INTRODUCTION: While using recreational areas, people take with them accompanying animals (dogs, cats). These animals are the main source and reservoir of dangerous zoonoses, including parasitoses caused by genera of nematodes, also called roundworms, Toxocara spp., Trichuris spp. and Ancylostoma spp. Attention should also be paid to the possibility of contamination of recreational areas with the eggs of intestinal parasites by wild animals (e.g. red fox). MATERIAL AND METHODS: Sand was collected on beaches and leisure terrains located in recreational areas in Zwierzyniec, Rudka reservoir on the river Wieprz, 'Echo' reservoir, accessible to tourists to the Roztocze National Park, and the 'Florianiecki' reservoir, chich is inaccessible to tourists due to its location in the depths of the National Park. Material for the research was also collected from the 'Zemborzycki reservoir in Lublin, A total of 300 sand samples were collected. The samples, approximately 500 g each, were collected into plastic bags from the superficial layer of sand, at a depth up to 3 cm. For egg detection, flotation with the Wasylikowa metod and the modified Quinn method were used. In the Quinn metod, the flotation solution is NaCl saturated solution, in the Wasylikowa method, 5% solution of NaOH is used. RESULTS: The most contaminated grounds were areas by the water 'Zemborzycki' reservoir in Lublin. The eggs of intestinal parasites were isolated from 13 out of 24 samples examined. In sand collected from the beach by the 'Echo', 'Florianiecki' and 'Rudka' water resevoirs in Zwierzyniec, no eggs of intestinal parasites were detected. CONCLUSIONS: Such a dissemination of results may indicate a connection between the degree of occurrence of geohelmint eggs and the intensity of human movement, together with accompanying animals, in places that can be used for recreation.

Terror in the dirt: Sensory determinants of host seeking in soil-transmitted mammalian-parasitic nematodes.

Infection with gastrointestinal parasitic nematodes is a major cause of chronic morbidity and economic burden around the world, particularly in low-resource settings. Some parasitic nematode species, including the human-parasitic threadworm Strongyloides stercoralis and human-parasitic hookworms in the genera Ancylostoma and Necator, feature a soil-dwelling infective larval stage that seeks out hosts for infection using a variety of host-emitted sensory cues. Here, we review our current understanding of the behavioral responses of soil-dwelling infective larvae to host-emitted sensory cues, and the molecular and cellular mechanisms that mediate these responses. We also discuss the development of methods for transgenesis and CRISPR/Cas9-mediated targeted mutagenesis in Strongyloides stercoralis and the closely related rat parasite Strongyloides ratti. These methods have established S. stercoralis and S. ratti as genetic model systems for gastrointestinal parasitic nematodes and are enabling more detailed investigations into the neural mechanisms that underlie the sensory-driven behaviors of this medically and economically important class of parasites.

In vivo and in vitro studies of Cry5B and nicotinic acetylcholine receptor agonist anthelmintics reveal a powerful and unique combination therapy against intestinal nematode parasites.

BACKGROUND: The soil-transmitted nematodes (STNs) or helminths (hookworms, whipworms, large roundworms) infect the intestines of ~1.5 billion of the poorest peoples and are leading causes of morbidity worldwide. Only one class of anthelmintic or anti-nematode drugs, the benzimidazoles, is currently used in mass drug administrations, which is a dangerous situation. New anti-nematode drugs are urgently needed. Bacillus thuringiensis crystal protein Cry5B is a powerful, promising new candidate. Drug combinations, when properly made, are ideal for treating infectious diseases. Although there are some clinical trials using drug combinations against STNs, little quantitative and systemic work has been performed to define the characteristics of these combinations in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Working with the hookworm Ancylostoma ceylanicum-hamster infection system, we establish a laboratory paradigm for studying anti-nematode combinations in vivo using Cry5B and the nicotinic acetylcholine receptor (nAChR) agonists tribendimidine and pyrantel pamoate. We demonstrate that Cry5B strongly synergizes in vivo with both tribendimidine and pyrantel at specific dose ratios against hookworm infections. For example, whereas 1 mg/kg Cry5B and 1 mg/kg tribendimidine individually resulted in only a 0%-6% reduction in hookworm burdens, the combination of the two resulted in a 41% reduction (P = 0.020). Furthermore, when mixed at synergistic ratios, these combinations eradicate hookworm infections at doses where the individual doses do not. Using cyathostomin nematode parasites of horses, we find based on inhibitory concentration 50% values that a strongylid parasite population doubly resistant to nAChR agonists and benzimidazoles is more susceptible or "hypersusceptible" to Cry5B than a cyathostomin population not resistant to nAChR agonists, consistent with previous Caenhorhabditis elegans results. CONCLUSIONS/SIGNIFICANCE: Our study provides a powerful means by which anthelmintic combination therapies can be examined in vivo in the laboratory. In addition, we demonstrate that Cry5B and nAChR agonists have excellent combinatorial properties-Cry5B combined with nAChR agonists gives rise to potent cures that are predicted to be recalcitrant to the development of parasite resistance. These drug combinations highlight bright spots in new anthelmintic development for human and veterinary animal intestinal nematode infections.