Synthesizing environmental, epidemiological and vector and parasite genetic data to assist decision making for disease elimination.

We present a framework for identifying when conditions are favourable for transmission of vector-borne diseases between communities by incorporating predicted disease prevalence mapping with landscape analysis of sociological, environmental and host/parasite genetic data. We explored the relationship between environmental features and gene flow of a filarial parasite of humans, Onchocerca volvulus, and its vector, blackflies in the genus Simulium. We generated a baseline microfilarial prevalence map from point estimates from 47 locations in the ecological transition separating the savannah and forest in Ghana, where transmission of O. volvulus persists despite onchocerciasis control efforts. We generated movement suitability maps based on environmental correlates with mitochondrial population structure of 164 parasites from 15 communities and 93 vectors from only four sampling sites, and compared these to the baseline prevalence map. Parasite genetic distance between sampling locations was significantly associated with elevation (r = .793, p = .005) and soil moisture (r = .507, p = .002), while vector genetic distance was associated with soil moisture (r = .788, p = .0417) and precipitation (r = .835, p = .0417). The correlation between baseline prevalence and parasite resistance surface maps was stronger than that between prevalence and vector resistance surface maps. The centre of the study area had high prevalence and suitability for parasite and vector gene flow, potentially contributing to persistent transmission and suggesting the importance of re-evaluating transmission zone boundaries. With suitably dense sampling, this framework can help delineate transmission zones for onchocerciasis and would be translatable to other vector-borne diseases.

Cytotaxonomic characterization and estimation of migration patterns of onchocerciasis vectors (Simulium damnosum sensu lato) in northwestern Ethiopia based on RADSeq data.

BACKGROUND: While much progress has been made in the control and elimination of onchocerciasis across Africa, the extent to which vector migration might confound progress towards elimination or result in re-establishment of endemism in areas where transmission has been eliminated remains unclear. In Northern Ethiopia, Metema and Metekel-two foci located near the Sudan border-exhibit continuing transmission. While progress towards elimination has been faster in Metema, there remains a problematic hotspot of transmission. Whether migration from Metekel contributes to this is currently unknown. METHODOLOGY/PRINCIPLE FINDINGS: To assess the role of vector migration from Metekel into Metema, we present a population genomics study of 151 adult female vectors using 47,638 RADseq markers and mtDNA CoI sequencing. From additional cytotaxonomy data we identified a new cytoform in Metema, closely related to S. damnosum s.str, here called the Gondar form. RADseq data strongly indicate the existence of two distinctly differentiated clusters within S. damnosum s.l.: one genotypic cluster found only in Metema, and the second found predominantly in Metekel. Because blackflies from both clusters were found in sympatry (in all four collection sites in Metema), but hybrid genotypes were not detected, there may be reproductive barriers preventing interbreeding. The dominant genotype in Metema was not found in Metekel while the dominant genotype in Metekel was found in Metema, indicating that (at the time of sampling) migration is primarily unidirectional, with flies moving from Metekel to Metema. There was strong differentiation between clusters but little genetic differentiation within clusters, suggesting migration and gene flow of flies within the same genetic cluster are sufficient to prevent genetic divergence between sites. CONCLUSIONS/SIGNIFICANCE: Our results confirm that Metekel and Metema represent different transmission foci, but also indicate a northward movement of vectors between foci that may have epidemiological importance, although its significance requires further study.

Genome-based tools for onchocerciasis elimination: utility of the mitochondrial genome for delineating Onchocerca volvulus transmission zones.

National programs in Africa have expanded their objectives from control of onchocerciasis (river blindness) as a public health problem to elimination of parasite transmission, motivated by the reduction of Onchocerca volvulus infection prevalence in many African meso- and hyperendemic areas due to mass drug administration of ivermectin (MDAi). Given the large, contiguous hypo-, meso-, and hyperendemic areas, sustainable elimination of onchocerciasis in sub-Saharan Africa requires delineation of geographic boundaries for parasite transmission zones, so that programs can consider the risk of parasite re-introduction through vector or human migration from areas with ongoing transmission when making decisions to stop MDAi. We propose that transmission zone boundaries can be delineated by characterising the parasite genetic population structure within and between potential zones. We analysed whole mitochondrial genome sequences of 189 O. volvulus adults to determine the pattern of genetic similarity across three West African countries: Ghana, Mali, and Côte d'Ivoire. Population genetic structure indicates that parasites from villages near the Pru, Daka, and Black Volta rivers in central Ghana belong to one parasite population, indicating that the assumption that river basins constitute individual transmission zones is not supported by the data. Parasites from Mali and Côte d'Ivoire are genetically distinct from those from Ghana. This research provides the basis for developing tools for elimination programs to delineate transmission zones, to estimate the risk of parasite re-introduction via vector or human movement when intervention is stopped in one area while transmission is ongoing in others, to identify the origin of infections detected post-treatment cessation, and to investigate whether persisting prevalence despite ongoing interventions in one area is due to parasites imported from others.

Procedure for Handling and Storage of Onchocerca volvulus Microfilariae Obtained from Skin Snips for Downstream Genetic Work.

WHO and endemic countries target elimination of transmission of Onchocerca volvulus, the parasite causing onchocerciasis. Population genetic analysis of O. volvulus may provide data to improve the evidence base for decisions on when, where, and for how long to deploy which interventions and post-intervention surveillance to achieve elimination. Development of necessary methods and tools requires parasites suitable for genetic analysis. Based on our experience with microfilariae obtained from different collaborators, we developed a microfilariae transfer procedure for large-scale studies in the Democratic Republic of Congo (DRC) comparing safety and efficacy of ivermectin, the mainstay of current onchocerciasis elimination strategies, and moxidectin, a new drug. This procedure is designed to increase the percentage of microfilariae in skin snips suitable for genetic analysis, improve assignment to metadata, and minimize time and materials needed by the researchers collecting the microfilariae. Among 664 microfilariae from South Sudan, 35.7% and 39.5% failed the mitochondrial and nuclear qPCR assay. Among the 576 microfilariae from DRC, 16.0% and 16.7% failed these assays, respectively. This difference may not only be related to the microfilariae transfer procedure but also to other factors, notably the ethanol concentration in the tubes in which microfilariae were stored (64% vs. ≥75%).

Assessing Onchocerca volvulus Intensity of Infection and Genetic Diversity Using Mitochondrial Genome Sequencing of Single Microfilariae Obtained before and after Ivermectin Treatment.

Onchocerciasis is a neglected tropical disease targeted for elimination using ivermectin mass administration. Ivermectin kills the microfilariae and temporarily arrests microfilariae production by the macrofilariae. We genotyped 436 microfilariae from 10 people each in Ituri, Democratic Republic of the Congo (DRC), and Maridi County, South Sudan, collected before and 4-5 months after ivermectin treatment. Population genetic analyses identified 52 and 103 mitochondrial DNA haplotypes among the microfilariae from DRC and South Sudan, respectively, with few haplotypes shared between people. The percentage of genotype-based correct assignment to person within DRC was ~88% and within South Sudan ~64%. Rarefaction and extrapolation analysis showed that the genetic diversity in DRC, and even more so in South Sudan, was captured incompletely. The results indicate that the per-person adult worm burden is likely higher in South Sudan than DRC. Analyses of haplotype data from a subsample (n = 4) did not discriminate genetically between pre- and post-treatment microfilariae, confirming that post-treatment microfilariae are not the result of new infections. With appropriate sampling, mitochondrial haplotype analysis could help monitor changes in the number of macrofilariae in a population as a result of treatment, identify cases of potential treatment failure, and detect new infections as an indicator of continuing transmission.

Geospatial modeling of pre-intervention nodule prevalence of Onchocerca volvulus in Ethiopia as an aid to onchocerciasis elimination.

BACKGROUND: Onchocerciasis is a neglected tropical filarial disease transmitted by the bites of blackflies, causing blindness and severe skin lesions. The change in focus for onchocerciasis management from control to elimination requires thorough mapping of pre-control endemicity to identify areas requiring interventions and to monitor progress. Onchocerca volvulus nodule prevalence in sub-Saharan Africa is spatially continuous and heterogeneous, and highly endemic areas may contribute to transmission in areas of low endemicity or vice-versa. Ethiopia is one such onchocerciasis-endemic country with heterogeneous O. volvulus nodule prevalence, and many districts are still unmapped despite their potential for onchocerciasis transmission. METHODOLOGY/PRINCIPLE FINDINGS: A Bayesian geostatistical model was fitted for retrospective pre-intervention nodule prevalence data collected from 916 unique sites and 35,077 people across Ethiopia. We used multiple environmental, socio-demographic, and climate variables to estimate the pre-intervention prevalence of O. volvulus nodules across Ethiopia and to explore their relationship with prevalence. Prevalence was high in southern and northwestern Ethiopia and low in Ethiopia's central and eastern parts. Distance to the nearest river (RR: 0.9850, 95% BCI: 0.9751-0.995), precipitation seasonality (RR: 0.9837, 95% BCI: 0.9681-0.9995), and flow accumulation (RR: 0.9586, 95% BCI: 0.9321-0.9816) were negatively associated with O. volvulus nodule prevalence, while soil moisture (RR: 1.0218, 95% BCI: 1.0135-1.0302) was positively associated. The model estimated the number of pre-intervention cases of O. volvulus nodules in Ethiopia to be around 6.48 million (95% BCI: 3.53-13.04 million). CONCLUSIONS/SIGNIFICANCE: Nodule prevalence distribution was correlated with habitat suitability for vector breeding and associated biting behavior. The modeled pre-intervention prevalence can be used as a guide for determining priorities for elimination mapping in regions of Ethiopia that are currently unmapped, most of which have comparatively low infection prevalence.

Genetic epidemiology of lymphatic filariasis in American Samoa after mass drug administration.

Over 892 million people in 48 countries are at risk of infection by nematodes that cause lymphatic filariasis. As part of the Global Programme to Eliminate Lymphatic Filariasis, mass drug administration is distributed to communities until surveillance indicates infection rates are below target prevalence thresholds. In some countries, including American Samoa, lymphatic filariasis transmission persists despite years of mass drug administration and/or has resurged after cessation. Nothing is known about the population genetics of Wuchereria bancrofti worms in Polynesia, or whether local transmission is persisting and/or increasing due to inadequate mass drug administration coverage, expansion from residual hotspots, reintroduction from elsewhere, or a combination. We extracted DNA from microfilariae on blood slides collected during prevalence surveys in 2014 and 2016, comprising 31 pools of five microfilariae from 22 persons living in eight villages. We sequenced 1104 bp across three mitochondrial markers (ND4, COI, CYTB). We quantified parasite genetic differentiation using variant calls and estimated haplotypes using principal components analysis, F-statistics, and haplotype networks. Of the variants called, all but eight were shared across the main island of Tutuila, and three of those were from a previously described hotspot village, Fagali'i. Genotypic data did not support population genetic structure among regions or villages in 2016, although differences were observed between worms collected in Fagali'i in 2014 and those from 2016. Because estimated haplotype frequency varied between villages, these statistics suggested genetic differentiation, but were not consistent among villages. Finally, haplotype networks demonstrated American Samoan sequence clusters were related to previously published sequences from Papua New Guinea. These are, to our knowledge, the first reports of W. bancrofti genetic variation in Polynesia. The resurgent parasites circulating on the main island of American Samoa represent a single population. This study is the first step towards investigating how parasite population structure might inform strategies to manage resurgence and elimination of lymphatic filariasis.

Chemosensory mechanisms of host seeking and infectivity in skin-penetrating nematodes.

Approximately 800 million people worldwide are infected with one or more species of skin-penetrating nematodes. These parasites persist in the environment as developmentally arrested third-stage infective larvae (iL3s) that navigate toward host-emitted cues, contact host skin, and penetrate the skin. iL3s then reinitiate development inside the host in response to sensory cues, a process called activation. Here, we investigate how chemosensation drives host seeking and activation in skin-penetrating nematodes. We show that the olfactory preferences of iL3s are categorically different from those of free-living adults, which may restrict host seeking to iL3s. The human-parasitic threadworm Strongyloides stercoralis and hookworm Ancylostoma ceylanicum have highly dissimilar olfactory preferences, suggesting that these two species may use distinct strategies to target humans. CRISPR/Cas9-mediated mutagenesis of the S. stercoralis tax-4 gene abolishes iL3 attraction to a host-emitted odorant and prevents activation. Our results suggest an important role for chemosensation in iL3 host seeking and infectivity and provide insight into the molecular mechanisms that underlie these processes.

Cryptic species diversity in ticks that transmit disease in Australia.

Ticks are important vectors of a broad range of pathogens in Australia. Many tick species are morphologically similar and are therefore difficult to identify using morphology alone, particularly when collected in the larval and nymphal life stages. We report here the application of molecular methods to examine the species diversity of ixodid ticks at two sites in southern New South Wales, Australia. Our taxon sampling included six morphologically characterised adult stage voucher specimens of Ixodes trichosuri, Ixodes tasmani, Ixodes fecialis and Ixodes holocyclus (the paralysis tick) and ~250 field collected specimens that were in the larva or nymph stage and thus not morphologically identifiable. One nuclear and two mitochondrial amplicons were sequenced using a combination of Sanger and Illumina MiSeq sequencing. Phylogenetic relationships were estimated using both maximum likelihood and Bayesian methods. Two clades with strong bootstrap and Bayesian support were observed across trees estimated from each of three markers and from an analysis of the concatenated sequences. One voucher specimen of I. trichosuri was located in one of these clades, while the other I. trichosuri voucher specimen was in a second clade with the remaining three identified species, suggesting these morphologically similar ticks may represent different cryptic species. Unidentified specimens were found across both clades, and molecular divergence of many of these is equal to or greater than that observed between identified species, suggesting additional unidentified species may exist. Further studies are required to understand the taxonomic status of ticks in Australia, and how this species diversity impacts disease risk for livestock, domestic animals, wildlife and humans.

Genomic Epidemiology in Filarial Nematodes: Transforming the Basis for Elimination Program Decisions.

Onchocerciasis and lymphatic filariasis are targeted for elimination, primarily using mass drug administration at the country and community levels. Elimination of transmission is the onchocerciasis target and global elimination as a public health problem is the end point for lymphatic filariasis. Where program duration, treatment coverage, and compliance are sufficiently high, elimination is achievable for both parasites within defined geographic areas. However, transmission has re-emerged after apparent elimination in some areas, and in others has continued despite years of mass drug treatment. A critical question is whether this re-emergence and/or persistence of transmission is due to persistence of local parasites-i.e., the result of insufficient duration or drug coverage, poor parasite response to the drugs, or inadequate methods of assessment and/or criteria for determining when to stop treatment-or due to re-introduction of parasites via human or vector movement from another endemic area. We review recent genetics-based research exploring these questions in Onchocerca volvulus, the filarial nematode that causes onchocerciasis, and Wuchereria bancrofti, the major pathogen for lymphatic filariasis. We focus in particular on the combination of genomic epidemiology and genome-wide associations to delineate transmission zones and distinguish between local and introduced parasites as the source of resurgence or continuing transmission, and to identify genetic markers associated with parasite response to chemotherapy. Our ultimate goal is to assist elimination efforts by developing easy-to-use tools that incorporate genetic information about transmission and drug response for more effective mass drug distribution, surveillance strategies, and decisions on when to stop interventions to improve sustainability of elimination.

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity.

BACKGROUND: Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread. METHODOLOGY/PRINCIPAL FINDINGS: Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR. CONCLUSIONS/SIGNIFICANCE: This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations.

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Significant heterogeneity in Wolbachia copy number within and between populations of Onchocerca volvulus.

BACKGROUND: Wolbachia are intracellular bacteria found in arthropods and several filarial nematode species. The filarial Wolbachia have been proposed to be involved in the immunopathology associated with onchocerciasis. Higher Wolbachia-to-nematode ratios have been reported in the savannah-ecotype compared to the forest-ecotype, and have been interpreted as consistent with a correlation between Wolbachia density and disease severity. However, factors such as geographic stratification and ivermectin drug exposure can lead to significant genetic heterogeneity in the nematode host populations, so we investigated whether Wolbachia copy number variation is also associated with these underlying factors. METHODS: Genomic DNA was prepared from single adult nematodes representing forest and savannah ecotypes sampled from Togo, Ghana, Côte d'Ivoire and Mali. A qPCR assay was developed to measure the number of Wolbachia genome(s) per nematode genome. Next-generation sequencing (NGS) was also used to measure relative Wolbachia copy number, and independently verify the qPCR assay. RESULTS: Significant variation was observed within the forest (range: 0.02 to 452.99; median: 10.58) and savannah (range: 0.01 to 1106.25; median: 9.10) ecotypes, however, no significant difference between ecotypes (P = 0.645) was observed; rather, strongly significant Wolbachia variation was observed within and between the nine study communities analysed (P = 0.021), independent of ecotype. Analysis of ivermectin-treated and untreated nematodes by qPCR showed no correlation (P = 0.869); however, an additional analysis of a subset of the nematodes by qPCR and NGS revealed a correlation between response to ivermectin treatment and Wolbachia copy number (P = 0.020). CONCLUSIONS: This study demonstrates that extensive within and between population variation exists in the Wolbachia content of individual adult O. volvulus. The origin and functional significance of such variation (up to ~ 100,000-fold between worms; ~10 to 100-fold between communities) in the context of the proposed mutualistic relationship between the worms and the bacteria, and between the presence of Wolbachia and clinical outcome of infection, remains unclear. These data do not support a correlation between Wolbachia copy number and forest or savannah ecotype, and may have implications for the development of anti-Wolbachia drugs as a macrofilaricidal treatment of onchocerciasis. The biological significance of a correlation between variation in Wolbachia copy number and ivermectin response remains unexplained.

Discrimination between Onchocerca volvulus and O. ochengi filarial larvae in Simulium damnosum (s.l.) and their distribution throughout central Ghana using a versatile high-resolution speciation assay.

BACKGROUND: Genetic surveillance of the human filarial parasite, Onchocerca volvulus, from onchocerciasis endemic regions will ideally focus on genotyping individual infective larval stages collected from their intermediate host, Simuliid blackflies. However, blackflies also transmit other Onchocerca species, including the cattle parasite O. ochengi, which are difficult to distinguish from the human parasite based on morphological characteristics alone. This study describes a versatile approach to discriminate between O. volvulus and O. ochengi that is demonstrated using parasite infective larvae dissected from blackflies. RESULTS: A speciation assay was designed based on genetic differentiation between O. volvulus and O. ochengi mitochondrial genome sequences that can be performed in high-throughput high-resolution melt (HRM)- or lower throughput conventional restriction fragment length polymorphism (RFLP) analyses. This assay was validated on 185 Onchocerca larvae dissected from blackflies captured from 14 communities in Ghana throughout 2011-2013. The frequency of O. ochengi was approximately 67 % of all larvae analysed, which is significantly higher than previously reported in this region. Furthermore, the species distribution was not uniform throughout the study region, with 25 %, 47 % and 93 % of O. volvulus being found in the western-most (Black Volta, Tain and Tombe), the central (Pru) and eastern-most (Daka) river basins, respectively. CONCLUSIONS: This tool provides a simple and cost-effective approach to determine the identity and distribution of two Onchocerca species, and will be valuable for future genetic studies that focus on parasites collected from blackflies. The results presented highlight the need to discriminate Onchocerca species in transmission studies, as the frequency of each species varied significantly between the communities studied.

Parastrongyloides trichosuri suggests that XX/XO sex determination is ancestral in Strongyloididae (Nematoda).

The parasitic roundworms Strongyloides stercoralis (in man) and Strongyloides ratti (in rats) employ environmentally controlled XX/XO sex determination with a pair of X chromosomes and two pairs of autosomes. Strongyloides papillosus (in sheep) has only two pairs of chromosomes, one of which combines the genetic material homologous to the S. ratti chromosomes X and I. This species creates males through the elimination of one copy of the portion related to the X chromosome (chromatin diminution). It is not clear which one of these two sex-determining mechanisms is ancestral. We demonstrate that Strongyloides vituli (in cattle) has two pairs of chromosomes like its very close relative S. papillosus whereas Parastrongyloides trichosuri, a closely related out-group to Strongyloides spp. in Australian brushtail possums, has three chromosome pairs and employs XX/XO sex determination. The X chromosome of P. trichosuri is homologous to the X chromosome of S. ratti. Our data strongly suggest that the last common ancestor of Strongyloides spp. and Parastrongyloides spp. had two pairs of autosomes along with two or one X chromosome in females and males, respectively. The situation with two pairs of chromosomes is likely derived and occurred through the fusion of the X chromosome with an autosome.

Evidence of evolutionary constraints that influences the sequence composition and diversity of mitochondrial matrix targeting signals.

Mitochondrial targeting signals (MTSs) are responsible for trafficking nuclear encoded proteins to their final destination within mitochondria. These sequences are diverse, sharing little amino acid homology and vary significantly in length, and although the formation of a positively-charged amphiphilic alpha helix within the MTS is considered to be necessary and sufficient to mediate import, such a feature does not explain their diversity, nor how such diversity influences target sequence function, nor how such dissimilar signals interact with a single, evolutionarily conserved import mechanism. An in silico analysis of 296 N-terminal, matrix destined MTSs from Homo sapiens, Mus musculus, Saccharomyces cerevisiae, Arabidopsis thaliana, and Oryza sativa was undertaken to investigate relationships between MTSs, and/or, relationships between an individual targeting signal sequence and the protein that it imports. We present evidence that suggests MTS diversity is influenced in part by physiochemical and N-terminal characteristics of their mature sequences, and that some of these correlated characteristics are evolutionarily maintained across a number of taxa. Importantly, some of these associations begin to explain the variation in MTS length and composition.

Dominant negative mutations of Caenorhabditis elegans daf-7 confer a novel developmental phenotype.

BACKGROUND: TGF-ß signaling pathways are involved in the control of development in every member of the animal kingdom. As such, TGF-ß ligands are widely divergent yet retain a set of core conserved features, specifically, a pre-protein cleavage site and several conserved ligand domain residues, the disruption of which produces a dominant negative phenotype. RESULTS: We have extended these observations into an invertebrate system by creating a series of loss-of-function Caenorhabditis elegans daf-7 transgenes. When we tested these mutant transgenes in a daf-7/+ background, we saw a molting and excretory canal phenotype. Members of both pathways downstream of daf-4 were required for this phenotype. CONCLUSIONS: Our results show that the basic mechanisms of TGF-ß function are conserved across the animal kingdom. A subset of our daf-7 mutations also produced an unexpected and novel phenotype. Epistasis experiments demonstrated that both daf-3/-5 and sma-4/-9 were downstream of our mutant daf-7 transgenes, which suggests not only a role for DAF-7 in the control of molting and the development of the excretory system but also that daf-7 and dbl-1 signaling may converge downstream of their shared Type II receptor, daf-4. Our approach may unveil new roles in development for other invertebrate TGF-ß ligands.

A research agenda for helminth diseases of humans: basic research and enabling technologies to support control and elimination of helminthiases.

Successful and sustainable intervention against human helminthiases depends on optimal utilisation of available control measures and development of new tools and strategies, as well as an understanding of the evolutionary implications of prolonged intervention on parasite populations and those of their hosts and vectors. This will depend largely on updated knowledge of relevant and fundamental parasite biology. There is a need, therefore, to exploit and apply new knowledge and techniques in order to make significant and novel gains in combating helminthiases and supporting the sustainability of current and successful mass drug administration (MDA) programmes. Among the fields of basic research that are likely to yield improved control tools, the Disease Reference Group on Helminth Infections (DRG4) has identified four broad areas that stand out as central to the development of the next generation of helminth control measures: 1) parasite genetics, genomics, and functional genomics; 2) parasite immunology; 3) (vertebrate) host-parasite interactions and immunopathology; and 4) (invertebrate) host-parasite interactions and transmission biology. The DRG4 was established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR). The Group was given the mandate to undertake a comprehensive review of recent advances in helminthiases research in order to identify notable gaps and highlight priority areas. This paper summarises recent advances and discusses challenges in the investigation of the fundamental biology of those helminth parasites under the DRG4 Group's remit according to the identified priorities, and presents a research and development agenda for basic parasite research and enabling technologies that will help support control and elimination efforts against human helminthiases.

A research agenda for helminth diseases of humans: towards control and elimination.

Human helminthiases are of considerable public health importance in sub-Saharan Africa, Asia, and Latin America. The acknowledgement of the disease burden due to helminth infections, the availability of donated or affordable drugs that are mostly safe and moderately efficacious, and the implementation of viable mass drug administration (MDA) interventions have prompted the establishment of various large-scale control and elimination programmes. These programmes have benefited from improved epidemiological mapping of the infections, better understanding of the scope and limitations of currently available diagnostics and of the relationship between infection and morbidity, feasibility of community-directed or school-based interventions, and advances in the design of monitoring and evaluation (M&E) protocols. Considerable success has been achieved in reducing morbidity or suppressing transmission in a number of settings, whilst challenges remain in many others. Some of the obstacles include the lack of diagnostic tools appropriate to the changing requirements of ongoing interventions and elimination settings; the reliance on a handful of drugs about which not enough is known regarding modes of action, modes of resistance, and optimal dosage singly or in combination; the difficulties in sustaining adequate coverage and compliance in prolonged and/or integrated programmes; an incomplete understanding of the social, behavioural, and environmental determinants of infection; and last, but not least, very little investment in research and development (R&D). The Disease Reference Group on Helminth Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to undertake a comprehensive review of recent advances in helminthiases research, identify research gaps, and rank priorities for an R&D agenda for the control and elimination of these infections. This review presents the processes undertaken to identify and rank ten top research priorities; discusses the implications of realising these priorities in terms of their potential for improving global health and achieving the Millennium Development Goals (MDGs); outlines salient research funding needs; and introduces the series of reviews that follow in this PLoS Neglected Tropical Diseases collection, "A Research Agenda for Helminth Diseases of Humans."

A research agenda for helminth diseases of humans: the problem of helminthiases.

A disproportionate burden of helminthiases in human populations occurs in marginalised, low-income, and resource-constrained regions of the world, with over 1 billion people in developing areas of sub-Saharan Africa, Asia, and the Americas infected with one or more helminth species. The morbidity caused by such infections imposes a substantial burden of disease, contributing to a vicious circle of infection, poverty, decreased productivity, and inadequate socioeconomic development. Furthermore, helminth infection accentuates the morbidity of malaria and HIV/AIDS, and impairs vaccine efficacy. Polyparasitism is the norm in these populations, and infections tend to be persistent. Hence, there is a great need to reduce morbidity caused by helminth infections. However, major deficiencies exist in diagnostics and interventions, including vector control, drugs, and vaccines. Overcoming these deficiencies is hampered by major gaps in knowledge of helminth biology and transmission dynamics, platforms from which to help develop such tools. The Disease Reference Group on Helminths Infections (DRG4), established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR), was given the mandate to review helminthiases research and identify research priorities and gaps. In this review, we provide an overview of the forces driving the persistence of helminthiases as a public health problem despite the many control initiatives that have been put in place; identify the main obstacles that impede progress towards their control and elimination; and discuss recent advances, opportunities, and challenges for the understanding of the biology, epidemiology, and control of these infections. The helminth infections that will be discussed include: onchocerciasis, lymphatic filariasis, soil-transmitted helminthiases, schistosomiasis, food-borne trematodiases, and taeniasis/cysticercosis.