Strongyloides: omics to worm-free populations

The paper will discuss our current understanding of the epidemiology and control of the soil transmitted helminths (STH) with special reference to Stronglyloides stercoralis. Progress in reducing the prevalence’s of STH globally over the past two decades will be highlighted. This has been achieved by employing the two key control methods which are repeated rounds of mass drug administration, largely targeted to school age children, and clean water provision, plus improvements in hygiene and sanitation (WaSH). The focus will be on the transmission dynamics of the parasite under the impact of repeated rounds of mass drug administration and what levels of coverage and individual compliance to treatment would be required to eliminate transmission in defined transmission settings (low, medium and high values of the basic reproductive number R0), employing individual based stochastic models of the known epidemiology of S stercoralis. Emphasis will be placed on what groups to treat, and how often to treat, to reduce prevalence to very low levels. Gaps in our knowledge of key biological processes and important epidemiological parameters will be highlighted, as will the challenges faced in both elimination S stercoralis as a public health problem, and eventually stopping transmission in areas of endemic infection.

S stercoralis shows considerable within-species DNA sequence diversity and various populations show differences in their life cycle preferences. Dogs can carry S stercoralis indistinguishable from the ones in humans, in addition to apparently dog-specific types. These findings lead to the questions: is S stercoralis one species or rather a species complex and if the various types differ in their pathogenicity and if the different life cycle preferences are genetically or environmentally specified? To address these questions, in addition to directly analysing worms collected from the field, controlled laboratory experiments are desirable. The latter requires that the isolates of interest are maintained in laboratory culture. The group aims at establishing a collection of S stercoralis isolates by culturing them in gerbils and cryo-preserving them alive. For each isolate, a set of parameters (eg life cycle preference, sex ratio, genome sequence) will be determined and the species status of the isolates will be evaluated by interbreeding experiments. Isolates that differ in interesting ways will be used for QTL and GWAS studies. The group hopes to encourage people encountering S stercoralis to contribute their isolates to our collection. All strains and information will be made available to the community.

In Australia, strongyloidiasis primarily affects returned travellers, Vietnam veterans and refugees or asylum seekers. Non-overseas acquired cases are seen almost exclusively in Australian Indigenous remote communities. We know that person to person transmission is low or non-existent, based on a study of wives of Vietnam veterans in Australia who did not contract the disease. The environmental reservoirs of Strongyloides stercoralis must play an important role in locally acquired strongyloidiasis. Therefore, in order to address transmission, we need to better understand the environmental components of the S. stercoralis lifecycle. This is also important as some Strongyloides species have demonstrated resistance to ivermectin, meaning that our best clinical intervention might be at risk. This talk will summarise the group’s work reviewing the global research on the environmental sources of Strongyloides. sp. and discuss their current approaches to determining the environmental reservoirs of S. stercoralis, looking at dogs, flies and wastewater. Additionally, their work has shown that strongyloidiasis is a disease of poverty. Acknowledging this is important, we need to shift the lens to socioeconomic factors, particularly environmental health hardware such as working showers and laundries, and effective wastewater and rubbish removal.

Strongyloidiasis remains a major veterinary and public health challenge globally. This chronic and potentially life-long disease has fatal outcomes in immunosuppressed people and dogs. Currently, the role of companion animals in the transmission cycle of human strongyloidiasis remains enigmatic. While zoonotic transmission to humans from companion animals has been proposed, this has not been confirmed. Cross-infection experiments between dogs and people during the 20th century suggested that S. stercoralis represents a species complex, with differing capacities for cross-species transmission. Recent genotyping studies of Strongyloides from dogs, cats, non-human primates and people indicate that S. stercoralis is indeed a species complex, containing at least three taxa, and likely more. One taxon has been demonstrated to infect cats, dogs and humans (designated S. stercoralis), a second appears specific to dogs (provisionally named “S. canis”). Despite recent advances in Strongyloides genotyping, sufficiently discriminatory tools to prove transmission from one host to another have not yet been developed. It remains unclear if dogs act as a zoonotic reservoir for human infection, or vice versa, or if this occurs only in some regions of the world and not in others. These questions must be answered before effective control strategies for strongyloidiasis can be instituted.

Understanding the epidemiology of multiple-host pathogens is considered one of the greatest challenges in infectious disease research. The close phylogenetic relationship between humans and nonhuman primates (NHPs) results in a partial overlap of their pathogens. The possibility of disease transmission between these two groups has increased in recent years due to closer and more frequent contact through habitat expansion and activities such as habituation for ecotourism. Several studies have attempted to uncover the transmission patterns of Strongyloides infections between humans and NHPs in both Africa and Asia. While S. f. fuelleborni has been described in wild NHPs and humans sharing the same environment, S. stercoralis appears to be restricted to NHPs living primarily in captivity, suggesting transmission by other animals or humans. The Dzanga-Sangha Protected Areas in the Central African Republic are known for habituated western lowland gorillas and BaAka people, traditional hunter-gatherers, while mountain gorillas in Virunga have been well known since Dian Fossey. Although humans and wild NHPs are in daily contact in DSPA, S. stercoralis has only been detected in humans, and S. f. fuelleborni found in NHPs differed from those found in humans, whereas S. stercoralis has been detected in wild mountain gorillas.

A step-change in the management of parasitic nematodes is urgently required to address global parasite disease burdens and the growing productivity demands of a rapidly expanding population. The global parasite problem is exacerbated by overreliance on a limited range of anthelmintics and a lack of sensitive, and quantitative, point-of-care diagnostics. The discovery and validation of improved diagnostic tools and novel anthelmintic control targets relies on the exploitation of platforms that harness relevant and tractable nematode parasites for research. Strongyloides parasites possess a uniquely tractable lifecycle, high-quality ‘omics’ resources, and advanced functional genomics tools, that are ripe for exploitation in fundamental helminth research and the discovery of novel parasite diagnostic and control approaches. Strongyloides related research at Queen’s University Belfast is centred on harnessing these experimental attributes to: (i) investigate uncharacterised facets of nematode neurobiology, including the role and importance of endocannabinoid signalling; (ii) better understand the role of antimicrobial peptides in parasite nematode biology and their ability to influence host microbiomes and (iii) identify putative novel parasite and/or host-derived diagnostic biomarkers associated with parasite infection. This presentation will provide an overview of how the Strongyloides platform supports these research themes and drives the improved diagnosis and management of parasitic nematode pathogens.

As well as an important cause of disease in humans and livestock, Strongyloides spp. are well suited as a laboratory model for parasitism. The genome assemblies of four Strongyloides species were published in 2016 and the S. ratti genome is one of the most contiguously assembled genomes of any nematode. There is also a growing database of transcriptome, proteome and small RNA data (published and unpublished) for Strongyloides spp. However, there is still a lot we don’t know about Strongyloides genomics and genetics. Using C. elegans as a benchmark, we can start to identify where the gaps in our knowledge are. For example, most of the available transcriptome data for Strongyloides is based on single time points and is lacking information about UTRs (important for understanding gene regulation). Here, Dr Hunt will present a summary of what we know about Strongyloides genomic and genetics including some of her group’s ongoing projects investigating gene organisation and expression of protein-coding genes and small RNAs. She will also discuss areas where data is lacking, for example, there is an underrepresentation of data from natural populations. Further improving data availability and better understanding the genomic and genetic basis of parasitism will elevate Strongyloides as a model organism for studying parasitism and could also be informative for improving strategies for treating and controlling helminth infections.

The use of proteomics to study human strongyloidiasis and its aetiological agent Strongyloides stercoralis has been so far limited, even though the well-known potential of these approaches for elucidating the molecular mechanisms associated with parasitism and to highlight novel immunological and diagnostic markers. Here the group employed targeted and untargeted proteomics approaches to investigate human strongyloidiasis at different levels. Particularly, by targeting specific host-derived circulating immune factors they have shown that long-lasting strongyloidiasis is not associated with the classical shift towards a type 2 immune response, but rather with a decrease in circulating chemokines, suggesting that immune cell recruitment to the infection site might be dampened in these patients. The untargeted proteomics investigation of the same patients revealed further systemic proteins significantly modulated during the infection or following treatment. When applied to the characterisation of infective larvae (iL3), this latter approach provided the largest iL3 proteome and experimental evidence about the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Additionally, a semi-automated proteome annotation highlighted 9 proteins with potential immunogenic properties. In conclusion, the investigation of parasite- and host-derived proteins can significantly expand our understanding of host-pathogen interaction and contribute to improve current serodiagnosis.

Small RNAs (sRNA) are short non-coding RNAs important for the regulation of gene expression via post-transcriptional gene silencing. The majority of sRNA research within nematodes has been carried out in the Clade V free-living model organism Caenorhabditis elegans which possess all three classes of sRNAs. Recent studies have shown that sRNA pathways are highly diverged in nematodes and C. elegans does not closely represent the sRNAs used by more distantly related nematodes, including parasitic species. For example, the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the C. elegans group (Clade V). Little is therefore known about how nematodes outside of this clade regulate TEs in the absence of the PIWI pathway. Here, the group investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female). They identified putative small-interfering RNAs that are differentially expressed between the two adult stages. A parasite-associated class of 21-22 nucleotide long sRNAs with a uridine 5'-monophosphate (21-22Us) were predicted to regulate and target TE activity. The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C. elegans, including an AT rich upstream sequence, overlapping loci and physical clustering in the genome. This is the first report of a piRNA-like sRNA class of 21-22Us that have been identified specifically in the PF of Strongyloides and in nematodes outside of Clade V.

As part of their drive through safari experience, Knowsley Safari (KS) offers its visitors a close-up encounter with their colony of olive baboons (Papio anubis) from the safety of their vehicles. Exiting vehicles, however, are sometimes contaminated with baboon faeces, posing a small health hazard. Coinciding with an animal welfare check, a coprological survey of baboon stool, both obtained from sleeping areas and cars, was conducted. Faecal material was examined by standard parasitological methods inclusive of: QUIK-CHEK RDT (Giardia), Kato-Katz coproscopy (Trichuris) and charcoal culture (Strongyloides). Across a four-day period, a total of 2,662 vehicles were examined with just under 700 stools obtained. Some 11.4% of vehicles were contaminated with faecal material. Overall prevalence of giardiasis was 37.4%, trichuriasis was 48.0% and strongyloidiasis was 13.7%. Since no faecal cysts of Giardia could be seen by microscopy, alongside very low levels of DNA detected by faecal PCR, these RDTs results were judged misleading. Further DNA characterisation confirmed the presence of Trichuris trichiura and Strongyloides fuelleborni. The latter observation represents this species’ most northern report of natural transmission. To minimise any public health risk, a future blanket administration of anthelminthic(s) is recommended, with later coprological inspection(s) to ascertain reinfection levels.

Skin-penetrating infective larvae actively search for hosts to infect and then invade hosts by skin penetration. We are interested in understanding host-seeking and host-invasion behaviors. We are using Strongyloides stercoralis and Strongyloides ratti for these studies because of their unique amenability to molecular genetic manipulation. We previously showed that Strongyloides infective larvae are attracted to host-emitted odorants and host body temperature. In this talk, I will focus on our recent investigations into skin-penetration behavior. We found that infective larvae penetrate skin by pushing their head into the skin while waving their mid-body and tail in the air. Infective larvae penetrate host skin more rapidly than non-host skin. When on non-host skin, they engage in repeated cycles of puncturing the skin and extracting themselves from the skin. We are now investigating the mechanosensory mechanisms that drive skin penetration. We have identified putative mechanoreceptors that are expressed in neurons resembling the touch receptor neurons of C. elegans, and we are now investigating the requirement for these proteins using CRISPR. We have also identified a role for dopamine signaling in driving skin penetration. Our work will provide insight into how skin-penetrating worms invade hosts and may enable the development of topical anthelmintics.

Human-parasitic nematodes are exposed to thermal environments that vary dramatically across their life cycle. Our work seeks to understand the role of thermosensation in enabling parasitic behaviors. Recent work indicates that thermosensation plays an essential role in host seeking by infective larvae, and that the thermosensory systems of Strongyloides species are highly specialized to enable robust host targeting. The contribution of thermosensation to other aspects of Strongyloides biology, including these species’ ability to survive environmental and intra-host thermal environments, is unknown.

To better understand the thermal physiology of Strongyloides species, we are now investigating the thermosensory responses of Strongyloides free-living adults, which do not host seek and must survive in the external environment. Preliminary experiments indicate that the thermotaxis behaviors of S. stercoralis free-living females are distinct from those of iL3s, as well as free-living, non-parasitic Caenorhabditis elegans adults. Furthermore, warm temperatures shorten the lifespan of Strongyloides free-living females, contrasting with the ability of parasitic females to survive prolonged exposure to host body heat. Together, these results suggest that 1) changing thermal preferences contribute to life-stage specific behaviors of Strongyloides species, and 2) the thermal preferences of free-living Strongyloides life stages are not equivalent to those of exclusively non-parasitic species.