Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

piRNA-like small RNAs target transposable elements in a Clade IV parasitic nematode.

The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes. 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), and little is known about how nematodes regulate TEs in the absence of the PIWI pathway. Here, we 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). We identified putative small-interfering RNAs, microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages. Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21-22 nt long sRNAs with a 5' uridine (21-22Us) and a 5' monophosphate, and (ii) 27 nt long sRNAs with a 5' guanine/adenine (27GAs) and a 5' modification. 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. Overall, we have shown that an alternative class of sRNAs compensate for the loss of piRNAs and regulate TE activity in nematodes outside of Clade V.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum's phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non-life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type.

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.

Improved 18S and 28S rDNA primer sets for NGS-based parasite detection.

The development and application of next-generation sequencing (NGS) have enabled comprehensive analyses of the microbial community through extensive parallel sequencing. Current analyses of the eukaryotic microbial community are primarily based on polymerase chain reaction amplification of 18S rRNA gene (rDNA) fragments. We found that widely-used 18S rDNA primers can amplify numerous stretches of the bacterial 16S rRNA gene, preventing the high-throughput detection of rare eukaryotic species, particularly in bacteria-rich samples such as faecal material. In this study, we employed in silico and NGS-based analyses of faecal samples to evaluated the existing primers targeting eukaryotic 18S and 28S rDNA in terms of avoiding bacterial read contamination and improving taxonomic coverage for eukaryotes, with a particular emphasis on parasite taxa. Our findings revealed that newly selected primer sets could achieve these objectives, representing an alternative strategy for NGS.

Sequential Changes in the Host Gut Microbiota During Infection With the Intestinal Parasitic Nematode Strongyloides venezuelensis.

Soil-transmitted helminths (STHs) are medically important parasites that infect 1. 5 billion humans globally, causing a substantial disease burden. These parasites infect the gastrointestinal tract (GIT) of their host where they co-exist and interact with the host gut bacterial flora, leading to the coevolution of the parasites, microbiota, and host organisms. However, little is known about how these interactions change through time with the progression of infection. Strongyloidiasis is a human parasitic disease caused by the nematode Strongyloides stercoralis infecting 30-100 million people. In this study, we used a closely related rodent parasite Strongyloides venezuelensis and mice as a model of gastrointestinal parasite infection. We conducted a time-course experiment to examine changes in the fecal microbiota from the start of infection to parasite clearance. We found that bacterial taxa in the host intestinal microbiota changed significantly as the infection progressed, with an increase in the genera Bacteroides and Candidatus Arthromitus, and a decrease in Prevotella and Rikenellaceae. However, the microbiota recovered to the pre-infective state after parasite clearance from the host, suggesting that these perturbations are reversible. Microarray analysis revealed that this microbiota transition is likely to correspond with the host immune response. These findings give us an insight into the dynamics of parasite-microbiota interactions in the host gut during parasite infection.