Welcome to the ICOPA 2022 Online Program

Abstract Body

Strongyloidiasis remains a major veterinary and public health challenge globally. Caused by infection with Strongyloides spp. helminths, this chronic and potentially life-long disease can have fatal outcomes in immunosuppressed dogs and people.

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 in several parts of the world during the 20th century suggested that S. stercoralis represents a species complex, with differing capacities for cross-species transmission. Despite recent advances in Strongyloides genotyping, tools of sufficient discriminatory power to prove transmission from one host to another have not yet been developed.

Confusion over the identity of the species, previously all attributed to S. stercoralis, confounds our understanding. Recent genetic studies of Strongyloides from dogs, cats, non-human primates and people indicate that S. stercoralis is a species complex containing at least three members, and likely more. One taxon has been demonstrated to infect cats, dogs and humans (designated S. stercoralis), a second appears to be specific to dogs (provisionally named “S. canis” by some researchers).

It remains unclear if dogs and cats act as a zoonotic reservoir for human infection, if humans may be infecting their companion animals, if no cross-species transmission occurs, or if this occurs in some regions of the world and not others. These questions must be answered before effective control strategies for strongyloidiasis in animals or people can be instituted. There are major gaps in our knowledge of human and veterinary Strongyloides species and their zoonotic potential.

Introduction

Strongyloidiasis is a neglected tropical disease affecting an estimated 600 million people. The infection can persist lifelong due to Strongyloides stercoralis peculiar auto-infective cycle. The cumbersome diagnosis and the limited knowledge of the mechanisms underpinning this chronic infection are key issues in disease management. Previously, we have shown that long-lasting strongyloidiasis is associated with a dampened immune response. To better explore the host-pathogen interaction, here we applied an untargeted approach to characterize infective larvae (iL3) and infected subjects’ serum proteomes.

Methods

iL3 larvae isolated from a fecal sample were analyzed by high-throughput (HT) qualitative proteomics; serum proteome of chronic strongyloidiasis patients (n=5) before and after treatment, and uninfected controls were quantitatively compared by SWATH mass spectrometry.

Results

An automatic search strategy combined with manual annotation identified more than 240 S. stercoralis proteins, which to our knowledge is the largest proteome ever published. Gene ontology analysis was used to select candidates for structural characterization and immunogenic epitope prediction, after exclusion of human homologous. Quantitative proteomics identified 208 serum proteins. Twenty four proteins were significantly modulated during the infection or following treatment, the most promising of which are undergoing validation as disease biomarker.

Conclusions

The study of both parasite- and host-derived proteins using HT proteomics is a promising strategy to improve our knowledge of the mechanisms of host-pathogen interaction in human strongyloidiasis and to highlight novel candidate antigens for the improvement of current serodiagnosis.

Introduction

Ascaris spp. infect ~700 million people worldwide and causes malnutrition and stunting. Current treatment is limited to a few drug classes; none offer long-term protection. Early in infection, Ascaris migrates through the host liver and lung. Recent studies suggest that this migration is guided by chemosensation. We propose that the chemosensory system of parasitic nematodes is an attractive but understudied therapeutic target. Here, we explore chemosensation in guiding larval Ascaris during infection.

Methods

We performed high-resolution confocal fluorescent imaging of the chemosensory neurons of A. suum larvae. We also developed a microfluidics system to understand their behaviour towards different stimulants. We further explored the transcriptomic, and proteomic responses of chemotactically responded A. suum larvae.

Results

We successfully stained A. suum larvae with anti-acetylated alpha-tubulin, beta-tubulin and confirmed their orthology with C. elegans with a detailed neuronal map for further studies. We fabricated and characterized a polydimethylsiloxane (PDMS) device to study the chemosensory behaviour of parasitic nematodes and showed that A. suum is more attracted to Pig’s liver extract than the other counterparts. Transcriptomic expression of chemoattracted A. suum reveals the chemosensory pathway responsible for the chemoattraction during the hepatopulmonary migration (HPM) in the early phase of infection.

Conclusions

Together, these findings will facilitate further understanding of the chemoreceptor biology of parasitic nematodes.

Introduction

Over 400 million people are infected by hookworms, of which ~85% are Necator americanus. In 2014, the first N. americanus genome assembly was published by Washington University (WashU) using a strain genetically adapted for hamsters. This assembly was crucial, but modern technologies now bring higher resolution and sequencing even from single worms and enable better assemblies.

Methods

We performed long-read, short-read, and chromosome-capture (Hi-C) sequencing and assembly on a laboratory strain of N. americanus not fully adapted to hamsters. We also isolated N. americanus hookworms directly from hunter-gathering humans in the Central African Republic for single-worm genome amplification, sequencing, and assembly using both Nanopore and Illumina.

Results

We assembled two N. americanus genomes (~234 Mb). Our reference has six scaffolded chromosomes, high genome contiguity (contig N50 ≥ 4 Mb, non-N ≥ 99%) and high completeness (BUSCO C score: ≥ 89% against nematoda_odb10). ITS2 phylogeny showed that the reference and African genomes are closely related; genomewide comparison showed significant differences between our reference and the WashU 2014 assembly.

Conclusions

For N. americanus, the first chromosomal assembly and the first assembly for hookworms naturally infecting humans in Africa will benefit future drug design and vaccine development experiments and will allow future population genetics of diverse N. americanus strains worldwide.

Introduction

Hookworms are parasitic nematodes that chronically infect over 400 million people, despite the fact that healthy human immune systems normally drive out most infections quickly. The mechanisms whereby hookworms successfully overcome host resistance are only partially known, but are thought to involve genes whose products interact with the host, either through intestinal digestion or through directly counteracting the host's immune system.

Methods

We collected excreted-secreted (ES) proteins from two sets of adult hookworms in vitro and used mass spectrophotometry to identify 500 Ancylostoma ceylanicum genes (out of a total of 37,016 genes) that encoded ES proteins in both collections. We also performed RNA-seq on both intestinal and non-intestinal tissues from adult hookworms whose hamster hosts either had normal immune systems or were immunosuppressed.

Results

We observed 1,098 intestinally and 124 non-intestinally expressed genes that were upregulated at least two-fold in normal hosts versus immunosuppressed hosts, with 215 intestinal and 11 non-intestinal genes instead being downregulated. No ES genes were found among the downregulated gene sets. However, 84 of the upregulated intestinal genes also encoded ES proteins, representing a 5.7-fold enrichment over chance overlap; similarly, 9 upregulated non-intestinal genes encoded ES proteins, a 5.4-fold enrichment.

Conclusions

These positively immunoregulated ES genes are key candidates for virulence factors during hookworm infection.

Introduction

More than 600 million people are infected with the nematode Strongyloides stercoralis. Studies by us and others showed: A) Dogs can carry S. stercoralis indistinguishable from the ones in humans in addition to dog specifioc ones; B) S. stercoralis show high within-species diversity in the 18S rDNA; C) Populations show differences in their preference for one of the three life cycles. Resulting questions: i) Is S. stercoralisone species or a species complex? ii) Do the various types differ in their pathology? iii) Are the different life cycle preferences genetically or environmentally induced? To address these questions, controlled experiments are desirable, which requires that the isolates of interest are in laboratory culture. I present our progress towards establishing a collection of S. stercoralisisolates from various places.

Methods

S. stercoralis are cultured in gerbils and frozen alive. For each isolate we determine a standard set of parameters (e.g. life cycle preference, sex ratio, genome sequence). The species status of the isolates is evaluated by crossing. Isolates that differ in interesting ways will be used for QTL and GWAS studies.

Results

Strain collection is ongoing. In addition to our own collection activities, we hope to encourage people encountering S. stercoralis to contribute their isolates to our collection.

Conclusions

This is an announcement of a new resource and a call for collaboration to establish a collection of S. stercoralis isolates available to the community.

Introduction

Hookworm disease is endemic throughout many parts of the Asia Pacific, despite targeted control programs of at-risk populations. The success of these programs has been hindered by the rapid re-infection rates linked to persistent reservoirs and the low sensitivity of conventional coprodiagnostic techniques employed.

Methods

We used standard faecal flotation (SFF) and a multiplex qPCR assay to calculate and compare species-specific cure and egg reduction rates of single dose albendazole (400 mg) against hookworm infections at community level. Individual patient data were used to inform a generalised linear mixed model to identify risk factors linked to hookworm infection(s) at baseline to the probability of being cured after albendazole administration.

Results

Overall, 13.5% of all 1,232 people tested by SFF were positive for hookworm infection(s). We revealed a substantial difference in cure rate of hookworm infection(s) following albendazole treatment using the SFF (81.5%) and mqPCR (46.4%) assays, and provide the first data on the efficacy of this drug against the zoonotic hookworm Ancylostoma ceylanicum. We estimated that as age increases by one year, the odds of being cured decreases by 0.4%–3.7% while the odds of being cured for people who boiled drinking water was estimated to be between 1·02 and 6·82.

Conclusions

These findings show that the adoption of refined diagnostic techniques is central to monitoring hookworm infections and the success of control strategies, which can ultimately aid in reducing associated morbidity in human populations. The approach taken is likely to be directly applicable to other parts of Southeast Asia and the Western Pacific, where specific epidemiological conditions might hamper the success of targeted treatment programs.