International Journal for Parasitology

Anthelmintic drug resistance is a concern for the sustained control of gastrointestinal nematodes (GINs) in ruminant livestock globally. Evolutionary-epidemiological modelling, which considers both parasite dynamics and resistance dynamics in response to interventions, can be useful in determining which anthelmintic resistance management (ARM) strategies may be effective without compromising parasite control. We address two key questions in ARM. First, how to improve the measurement of AR in populations. Second, identifying effective ARM strategies to slow the spread of AR while maintaining effective parasite control. We developed a simulation framework which tracks the weather-dependent epidemiology of GINs and AR evolution, providing a highly flexible methodology to evaluate multiple ARM strategy options in a single modelling framework, allowing for novel insights due to direct comparisons between strategies. Simulations to refine our understanding of anthelmintic resistance management evaluated the impact of key areas of uncertainty, including transmission intensity, resistance intensity, resistance frequency, drug decay and linking faecal egg count reduction tests (FECRT) to resistance allele frequency. Large-scale simulations present a methodologically thorough evaluation of how treatment choices simultaneously impact epidemiological and evolutionary outcomes. Phenotypic classifications of resistance status using FECRT failed to capture fine scale changes in resistance allele frequency. The pharmacokinetics of drug decay strongly influenced ARM outcomes, and trade-offs between ARM and effective parasite control depends on genetic factors underpinning resistance. Combination therapies appear to be the most effective resistance management strategy evaluated. Our findings suggest practical implementations to manage anthelmintic resistance must simultaneously consider parasite transmission, pharmacology and parasite genetics to be robust and sustainable. We provide a rigorous simulation framework to enable such discussions allowing for a refinement into our understanding of parasite control in the presence of resistance evolution.

Antimicrobial Peptides (AMPs) are key constituents of the invertebrate innate immune system where they provide critical protection against microbial threat. Knowledge of AMP complements within phylum Nematoda is limited however nematodes adopt diverse life strategies and frequently reside in microbe-rich environments such that they are likely possess broad AMP profiles with bioactivity against a range of microbiota. Indeed, parasitic nematode AMPs likely have roles in defence against invading pathogens and modulation of the host microbiome. In this study the distribution and abundance of AMP-encoding genes were examined in 134 nematode genomes providing the most comprehensive profile of AMPs within phylum Nematoda. We reveal that phylum Nematoda is AMP-rich and -diverse, where 5887 genes encode AMPs. Genome and transcriptome analyses broadly reveal: (i) AMP family profiles that are influenced by nematode lifestyle where free-living nematodes appear to have an expansion of AMPs relative to parasitic species; (ii) major differences in the AMP profiles between nematode clades where Clade 9/V and 10/IV species possess expanded AMP repertoires; (iii) AMP families with highly restricted profiles (e.g. Cecropins and Diapausins) and others [e.g. Nemapores and Glycine Rich Secreted Peptides (GRSPs)] which are more widely distributed; (iv) complexity in the distribution and abundance of Defensin subfamily members; and (v) expression of AMPs in key nematode life stages. These data indicate that phylum Nematoda has a diverse array of AMPs and underscores the need to interrogate AMP function to unravel their importance to nematode biology and host-worm-microbiome interactions. Enhanced understanding of the Nematode Antimicrobial Peptidome will inform drug discovery pipelines for pathogen control.

Anthelmintic resistance in gastrointestinal nematode populations is endemic across all grazing livestock production systems, particularly in small ruminants, but also in cattle. In this study resistance in Cooperia oncophora and Ostertagia ostertagi to macrocyclic lactones and benzimidazoles (BZ) in Scotland was assessed. The faecal egg count reduction test (FECRT) remains the primary tool for assessing anthelmintic resistance in the field; however, differing methodologies and recent guideline updates complicate the interpretation of results across studies. Statistical approaches, such as Bayesian methods used by eggCounts and bayescount, produce varying confidence intervals, which further influence conclusions. The ability of the egg hatch test to detect anthelmintic resistance was evaluated under field conditions, and the EC50 values obtained enabled assessment of the BZ efficacy at both the farm and GIN species levels, without the need for treatment. Mixed amplicon sequencing was applied, and nemabiome analysis confirmed that C. oncophora and Os. ostertagi were the predominant species. Sequencing of the beta-tubulin isotype-1 gene in strongyle communities identified resistance alleles in multiple species. The high EC values and elevated frequencies of BZ-resistance alleles on some farms emphasise the importance of in vitro assays and molecular diagnostics for farm-level resistance management. The detection of resistance against both benzimidazoles and macrocyclic lactones on certain farms highlights the urgency of implementing sustainable control strategies. Although the data presented here are from Scotland, given the high rate of animal movement and similar patterns of anthelmintic usage in the UK, these findings are likely to be relevant across much of the UK. Highlights- Resistance to MLs and BZs was detected in GIN populations of cattle - Multidrug resistance found in Scottish GIN populations of cattle - Egg hatch test results suggest, and sequencing confirms high BZ-resistance allele frequencies

The liver fluke, Fasciola hepatica, is a major parasitic threat to ruminant health and productivity worldwide, with important implications for food security, animal welfare, and zoonotic risk. This study developed and validated a multiplex deep amplicon sequencing assay targeting the mitochondrial NADH dehydrogenase 1 (mt-ND1) and cytochrome c oxidase subunit 1 (mt-COX1) loci for high-throughput genotyping of F. hepatica. DNA was extracted from eggs sedimented from sheep and cattle faeces (n = 78) received from farms and from adult worm pools (n = 12) isolated at abattoirs from diverse regions across the UK. Following high-throughput sequencing, bioinformatics analysis was performed to demultiplex Illumina sequence reads and extract amplicon sequence variants (ASVs). A total of 11 ASVs were identified at each locus (mt-ND1: 264-279 bp; mt-COX1: 312-319 bp), with two or three predominant ASVs per locus, along with rare variants. Network and PCA analyses revealed two distinct clusters at the mt-ND1 locus: one primarily associated with sheep and another shared between sheep and cattle. In contrast, mt-COX1 sequence reads formed a single dominant cluster. Population analyses revealed extensive ASV sharing across regions, indicating high gene flow, likely facilitated by livestock movement and parasite adaptation.

Hybrids between Schistosoma haematobium and S. bovis are linked to both human and animal infections, highlighting the complex interspecies interactions that contribute to the spread of schistosomiasis. Additionally, S. bovis can infect multiple ruminant hosts, facilitating cross- species transmission and increasing the risk of zoonotic outbreaks. In this study, we investigated transcriptomic plasticity as a potential mechanism enabling hybrid schistosomes to adapt to alternative definitive hosts. We focused on two contexts: 1) introgressed S. haematobium x S. bovis hybrids, which demonstrated higher virulence in sheep compared to parental S. bovis, and 2) S. bovis infecting different host species. Our analysis uncovered 366 differentially expressed genes (DEGs), representing 4% of the total protein-coding genes, between introgressed hybrids and parental S. bovis in sheep. We also identified transcriptomic changes in S. bovis across different mammalian hosts (hamster and sheep), with around 30% of the total genes differentially expressed, demonstrating that S. bovis parasites display a high transcriptomic plasticity, allowing them to infect different definitive hosts. Shared enriched biological processes during introgression and host change include nuclear-transcribed mRNA catabolic processes, inner mitochondrial membrane organization, microtubule-based movement, response to endoplasmic reticulum stress, and sensory perception. These findings suggest that transcriptomic plasticity in S. bovis and hybrid worms enhance their ability to adapt and infect diverse host species, potentially increasing their zoonotic potential. This raises concerns for schistosomiasis epidemiology, as this plasticity could expand the parasites transmission capacity and complicate control efforts.

Strongyloides nematodes are parasites of livestock, and S. papillosus infects ruminant livestock that can cause disease. Recent genomic analysis of several Strongyloides species is now facilitating the population genomic analyses of natural Strongyloides infections, for example finding that S. ratti in wild UK rats exists as an assemblage of long-lived, asexual lineages. Here we have initiated an investigation into the population genomics of S. papillosus in goats in Pakistan. We find that S. papillosus is common, with a prevalence of 28%; that the population is genetically diverse and that individual goats commonly have mixed-genotype infections; and that there is evidence of only limited admixture. These results now provoke further questions about the host range of different S. papillosus genotypes that can in the future be investigated by further population genomic analyses.

Seasonality is a ubiquitous feature of wildlife disease ecology, but is determined by a complex interplay of environmental, parasitological and host factors. Gastrointestinal parasites often exhibit strong seasonal dynamics in wild vertebrate populations due to, for example, environmental influences on free-living or vectored life stages, and variation in the physiological and immune status of hosts across their annual cycle. At the same time, wild populations are typically infected with multiple parasites. The seasonal dynamics of co-infecting parasites may differ depending on age and reproductive status, and associations among parasites may be driven by short-term within-individual changes or longer-term interactions that are consistent among hosts. Here, we used faecal samples and egg counts collected repeatedly from individually marked and monitored wild Soay sheep that were part of a long-term study to investigate seasonal dynamics of six gastrointestinal parasite groups (strongyle nematodes, coccidian protozoa, Capillaria, Strongyloides, Nematodirus, and Moniezia). Prevalence and abundance generally tended to be higher spring and summer, and burdens were higher in lambs than adults. Within the highly prevalent strongyle nematode group, we found differences in seasonality of egg counts depending on adult reproductive status. Reproductive ewes had increased counts in spring around the time of birth followed by a drop in abundance in summer, while barren ewes showed little evidence of seasonality. Males showed a sustained rise in egg counts through spring and summer, and sex differences were only strongly apparent in summer. In contrast, in similarly prevalent coccidia we found a peak in faecal oocyst counts in spring but no differences in seasonality among males, barren and pregnant ewes. Using multivariate mixed-effects models, we went on to show that both strongyle and coccidia counts are moderately repeatable across seasons among individuals. We further show that apparent positive correlation between strongyle and coccidia counts was driven by short-term within-individual changes in both parasite burdens rather than long-term among-individual covariation. Overall, our results demonstrate that seasonality varies across demographic and parasite groups and highlight the value of investigating fluctuating susceptibility and exposure over time for understanding epidemiology of a population.

Over a quarter of the worlds population is at risk of infection by soil transmitted helminths (STH). Among the STHs Trichuris trichiura infects approximately 7% of people globally, causing a loss of 232,000 DALYS. The main strategy to combat T. trichiura infection focusses on mass drug administration with the benzimidazoles. Whilst albendazole and mebendazole have been effective at reducing the burden of other STHs, the cure rate for whipworm is less than 50% with resistance alleles rising. Glucose is the most studied nutrient in Trichuris spp, however we have no understanding, at the molecular level of the mechanism of uptake in Trichuris spp. We sought to identify putative glucose transporters in Trichuris and investigate how these can be inhibited with phloretin. Using the C. elegans Facilitated Glucose Transporter 1 (FGT) sequence we identified two potential homologs in T. muris (TmGLT) and T. trichiura (TtGLT). We should both proteins contained sequence similarity to FGT1 and contained multiple sequence domains associated with glucose and sugar transport. Further, using Alphafold and molecular docking we show glucose docking sites consistent with transport. To asses the ability of phloretin to inhibit glucose transport, we also performed molecular docking with phloretin, showing possible inhibition. To validate the potential inhibition in vitro we measured the 48h LC50 of phloretin which we showed to be 111 ug/ml against adult T. muris worms, around half that of mebendazole in the same conditions. In contrast phloretin exhibited no effect on worm burden or fecundity in vivo. Together these findings provide the first in silico characterisation of putative glucose transporters in Trichuris spp and have identified glucose transport inhibition as a promising avenue for anthelminthic drug discovery. Whilst further work is required to optimise in vivo efficacy, our results highlight parasite glucose acquisition pathways as potential druggable targets in whipworm.

Root-knot nematodes (RKNs) of the genus Meloidogyne are important pests in agriculture. RKNs are generalist herbivores with a wide host range including crop and wild plants. The latter are an important source of defensive metabolites that may be helpful for RKN management. Milkweeds (Asclepias spp.) produce toxic cardenolides that protect them from herbivory. However, it is unclear if cardenolides may defend milkweeds against RKNs. Here, we tested this directly through herbivory assays with the RKN M. incognita on milkweed species that produce negligible and high levels of cardenolides, A. tuberosa and A. curassavica, respectively. We found that M. incognita induces fewer galls on A. curassavica than A. tuberosa and fails to reproduce after reaching maturity on the former but not the latter species. This suggests that the predominantly polar cardenolides in A. curassavica may engender long-term reproductive toxicity. Further toxicity assays with the polar cardenolide ouabain showed that cardenolides can also have more immediate toxic effects on M. incognita at higher concentrations. Ouabain caused a coiled, paralytic phenotype in juvenile RKNs, a sign of neurotoxicity, leading to lethality in a subset of RKNs. Some nematodes recovered upon ouabain removal, confirming that neurotoxic cardenolides have a nematostatic effect that results in death when exposure persists. Taken together, our results provide further evidence that cardenolides may function as anti-herbivore defenses against RKNs. The dead end host plant A. curassaviva appears to possess useful properties that may be leveraged for control of RKNs in agriculture.

Anthelmintic resistance in cyathostomins is escalating worldwide, threatening equine health and highlighting the need for sustainable, ecology based parasite control strategies. Chicory (Cichorium intybus, Puna II) has emerged as a promising antiparasitic forage, yet its broader effects on the equine holobiont, parasites, microbiota, and host physiology remain poorly understood. We conducted a 32 day longitudinal grazing trial in young horses to assess how chicory affects parasitological outcomes, gut microbial ecology, nemabiome composition, behaviour, and host physiological and immune responses. Twenty-six naturally infected Anglo-Arabian horses were monitored weekly, with 13 grazing a chicory-based sward and 13 grazing a permanent pasture. Clinical parameters, body weight, and serum biochemistry remained stable across treatments, indicating that chicory was well tolerated. Immune profiles showed limited variation, although IL 10 increased in chicory fed horses, suggesting subtle immune modulation. Behavioural observations revealed no signs of discomfort and indicated slightly enhanced social interactions in the chicory group. Chicory grazing produced a marked reduction in cyathostomin egg excretion, accompanied by species specific shifts in nemabiome composition. Several cyathostomin taxa, including Cylicocyclus ashworthi, C. leptostomus, and C. nassatus, declined in chicory fed horses, whereas certain Cylicostephanus spp increased, indicating differential sensitivity rather than uniform suppression. Concomitantly, chicory induced profound ecological changes in the gut microbiota, including reduced alpha diversity, increased beta dispersion, and destabilised individual microbial trajectories. Several bacterial lineages, particularly Oscillospiraceae, Clostridiaceae, Lachnospiraceae, and Bacteroidales, were differentially enriched, reflecting a functional reorganisation of the intestinal ecosystem. Together, these findings demonstrate that chicory reduces parasite fitness, reshapes nemabiome composition, and alters gut microbial ecology while maintaining host physiological stability. Chicory thus emerges as a promising ecological tool for parasite control, capable of modulating the equine holobiont in ways that complement and potentially reduce reliance on conventional anthelmintic strategies. However, because its effects on gut microbial ecology remain uncertain, and may include shifts resembling dysbiosis, future studies are needed to monitor microbial dynamics more closely and clarify the long term ecological consequences of chicory grazing.

Due to their ubiquity, management of parasites is a common and important factor for profitable production of poultry. Heterakis gallinarum, the cecal nematode, is the most common nematode parasite of poultry. While typically causing no pathology on its own, H. gallinarum is the vector of Histomonas meleagridis, a protozoan parasite that causes blackhead disease. Histomonas meleagridis is highly pathogenic in turkeys, potentially causing high mortality. In contrast, disease caused by H. meleagridis is much less severe in chickens, where it primarily reduces productivity without manifestations of clinical disease. There are no approved treatments for H. meleagridis, making control reliant on control of the helminth vector through the use of fenbendazole (FBZ) the only drug labeled for treatment of H. gallinarum in the United States We were contacted by an industry veterinarian regarding health-related concerns in a broiler-breeder house due to histomoniasis, despite frequent anthelmintic treatments. Since we had recently diagnosed resistance to FBZ in Ascaridia dissimilis, a closely related nematode of turkeys, we were interested to determine if H. gallinarum had also evolved resistance to FBZ. Heterakis gallinarum eggs were isolated from litter collected from the house and used to infect 108 chickens. Treatment groups included a non-treated control, a label-dose and a 2X-label dose of FBZ, with 36 birds per group divided into two replicate pens of 18 birds each. Birds were placed at 1-day post hatch, and at 3 weeks of age were infected with 150 embryonated eggs via oral gavage. Two weeks post infection, treated birds were administered a minimum of either a label- or 2X label-dose of FBZ in water for 5 days (SafeGuard(R) Aquasol, 1mg/kg BW). To ensure that all birds consumed the full intended dose at a minimum, the dosage was calculated using 1.25 times the average body weight. One-week post treatment, birds were euthanized, ceca removed, and parasites enumerated. Efficacy was calculated by comparing the total numbers of worms recovered from each treatment group to the numbers recovered in the non-treated control group. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.81). There also was no efficacy benefit to treatment with a 2X dose; H. gallinarum worm counts were reduced by 42.7% and 41.4%, for the label and 2X dosages, respectively. These data provide strong evidence that H. gallinarum has developed resistance to FBZ. Consequently, in houses infected with FBZ-resistant H. gallinarum, H. meleagridis will be able to cycle through the birds in an unrestricted manner. Further investigation is needed to determine the prevalence of resistance in H. gallinarum on chicken farms, but it is clear this has the potential to have a large-scale economic impact on the poultry industry. These data when viewed together with our recent findings of FBZ resistance in A. dissimilis, suggest that drug resistance in ascarid nematodes may be an important emerging problem on poultry operations.

In 2012, the reduction in Onchocerca volvulus infection prevalence through long-term mass ivermectin distribution in African meso- and hyperendemic areas motivated expanding control of onchocerciasis (river blindness) as a public health problem to elimination of parasite transmission. Given the large contiguous hypo-, meso- and hyperendemic areas with an estimated population of 204 million, sustainable elimination requires an understanding of the geographic, and in turn genetic, boundaries of different parasite populations to ensure interventions are only stopped where the risk of re-introduction of the parasite through vector or human migration from areas with ongoing transmission is acceptable. These boundaries, which define the transmission zones of the parasite, may 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 Cote dIvoire. Population structure measures indicate that parasites from the Pru, Daka and Black Volta/Tombe river basins in central Ghana belong to one parasite population, showing that different river basins cannot be assumed to constitute independent transmission zones. This research forms 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 mass ivermectin administration 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 migration contributes to persisting prevalence levels during interventions.

Anthelmintic resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We described a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV) resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ) resistance SNPs within {beta}-tubulin isotype-1 and ITS-2 nemabiome. This constitutes the first multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing of LEV resistant H. contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an important anthelmintic class for small ruminants in New South Wales. The mixed amplicon metabarcoding approach described herein, paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Schistosoma mansoni is a parasitic helminth that is vectored through freshwater snails. While the anti-schistosome defense of the South American snail, Biomphalaria glabrata, is well studied, little is known about the immune response of the African snail, Biomphalaria sudanica. We measured expression of five candidate immune genes in B. sudanica 8, 24, and 72 hours post-exposure to S. mansoni using reverse transcription quantitative PCR. Expression patterns of resistant snails were compared to susceptible snails and those sham exposed. We also assessed how diet (lettuce vs. pellet) affected expression of three genes, given prior findings that pellet-fed snails were more susceptible to S. mansoni. Results indicated that resistant snails constitutively expressed higher levels of superoxide dismutase 1 (SOD1) than susceptible snails, consistent with expression patterns of resistant B. glabrata. Parasite-induced expression occurred at 8 hours in SOD1, biomphalysin, thioester protein 1 (TEP1), and granulin (GRN); however, for biomphalysin and TEP1, induced expression was only detected for susceptible snails. At 24 hours, biomphalysin expression increased in exposed resistant snails, and at 72 hours, all exposed snails decreased biomphalysin expression compared to controls. Parasite-induced expression of SOD1, biomphalysin, TEP1, and GRN supports the hypothesis that these genes play a role in B. sudanica anti-schistosome defense, however increased expression does not necessarily yield clearance of S. mansoni. SOD1 expression was higher in lettuce-fed snails at 8 and 24 hours, consistent with their greater resistance. Together, these results demonstrate the conserved and unique aspects of the B. sudanica anti-schistosome response.

The nuclear, internal transcribed spacer (ITS) and mitochondrial cox1 markers are widely used to differentiate Schistosoma haematobium from its livestock counterparts, S. bovis and S. curassoni. Schistosoma isolated from humans that have ITS and cox1 markers from livestock schistosomes are typically inferred as zoonotic infections, those with mixed species, heterozygous ITS are classified as F1s or recent hybrids, while those with discordant ITS and cox1 markers are considered to reflect older hybridization events. We evaluated the reliability of this classification scheme by genotyping ITS and cox1 from 132 parasites isolated from human urine, and from 37 adult schistosomes collected from cattle at 14 Nigerian locations. We also genome sequenced each sample to empirically determine livestock schistosome ancestry. ITS/cox1 genotyping suggested extensive recent hybridization and zoonotic infection. Among parasites from humans, 10.1% carried both S. curassoni and S. haematobium ITS, consistent with F1 or early generation hybrids, 21% had livestock schistosome markers at both cox1 and ITS suggesting zoonotic infection, while 13.7% carried S. bovis cox1 alongside mixed S. curassoni and S. haematobium ITS, suggesting more complex ancestry. Genome sequencing revealed a very different picture. All parasites from humans formed a tight cluster regardless of ITS or cox1 genotype, while all worms from cattle were well differentiated. We found no schistosomes containing 50% livestock parasite ancestry consistent with F1s. Instead, we observed regionally varying levels of S. bovis introgression, with modest levels in southern Nigeria (mean = 4.9%) and low levels in northern Nigeria (mean = 0.06%). These results demonstrate that: (i) two-locus genotyping is uninformative for detecting zoonotic infection or recent hybridization between S. haematobium and livestock schistosomes and (ii) previous data generated using this approach requires reinterpretation. These findings reveal the limitations of widely-used approaches for documenting zoonotic infection and hybridization between S. haematobium and livestock schistosome species.

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects.

Laboratory models are invaluable tools for studying parasite biology and pathogenesis, especially for helminth infections. However, the complex life cycles and frequently narrow host specificity of helminths present challenges to maintaining access to critical parasite material in a laboratory setting. This is especially true of Necator americanus, the most common species of hookworm that infects humans globally. Here we report the successful laboratory adaptation of an African strain of N. americanus, originally isolated from infected individuals in Beposo, Ghana. The Beposo strain has been successfully passaged across 9 generations in Golden Syrian hamsters maintained on oral dexamethasone. Differential susceptibility to mebendazole and albendazole was evaluated using an egg hatch assay, and DNA sequencing of the beta-tubulin isotype 1 gene did not identify known resistance-associated mutations in the endemic strain. Sequencing of the mitochondrial COX1 gene revealed that specimens of N. americanus from Ghana, along with reported sequences from Togo, are distinct from those from South America and Asia. Complementary microsatellite-based population analysis revealed substantial genetic variation in the founding parasite population. To further characterize the novel Beposo strain, a draft hybrid genome assembly was generated from genomic DNA extracted from a single adult male worm via an optimized Oxford Nanopore Technologies MinION library preparation approach tailored to low-input sample types. This high-quality assembly, including a complete mitogenome, is 202.8Mb in 950 contigs with an N50 >449 kb. It contains >95% of conserved nematode orthologs in complete single copy and is estimated by homology-based gene prediction to contain 12,804 genes. This study represents the first comprehensive characterization of a strain of N. americanus originating in Africa that has been successfully adapted to a laboratory animal model.

BackgroundCestodes have complex hermaphroditic reproductive systems that produce massive numbers of eggs. This reproductive output is made possible by the continuous production of serially repeated sets of reproductive systems (proglottids). However, their reproductive development remains poorly understood. ResultsWe characterized reproductive development in the model cestode Hymenolepis microstoma by analyzing markers of cell proliferation, meiosis, and differentiation along the series of proglottids. Reproductive development begins with the formation of a central genital primordium, from which the reproductive ducts and gonads differentiate. Development is proterandrous, and testicular development is prolonged. In contrast, female reproductive development occurs over a short interval and is characterized by the coordinated differentiation of the ovary and vitelline gland. Entry of oocytes into meiosis is almost synchronous, and paralleled by cell proliferation in the vitelline gland. Subsequent growth of arrested oocytes and differentiation of vitelline cells occur in parallel. Insemination coincides with the onset of ovarian meiosis, indicating a close temporal coordination between male and female reproductive development. Finally, we show that gametogenesis and insemination proceed in adult worms maintained in vitro. ConclusionsOur findings show the coordination of reproductive development in a self-fertile hermaphrodite, and provide an experimental system for studying reproductive development in cestodes.

Human infection with the intestinal nematode Strongyloides stercoralis is persistent unless effectively treated, and potentially fatal in immunosuppressed individuals. Epidemiological data are lacking due to inadequate diagnosis. A rapid antigen detection test is a priority for population surveillance, validating cure after treatment, and for screening prior to immunosuppression. We analysed open access omics data sets and used online predictors to identify S. stercoralis proteins that are likely to be present in infected stool, Strongyloides-specific, and antigenic. Transcriptomic data from gut and non-gut dwelling life cycle stages of S. stercoralis revealed 328 proteins that are differentially expressed. Strongyloides ratti proteomic data for excreted and secreted (E/S) proteins were matched to S. stercoralis, giving 1,057 orthologues. Five parasitism-associated protein families (SCP/TAPS, prolyl oligopeptidase, transthyretin-like, aspartic peptidase, acetylcholinesterase) were compared phylogenetically between S. stercoralis and outgroups, and proteins with least homology to the outgroups were selected. Proteins that overlapped between the transcriptomic and proteomic datasets were analysed by multiple sequence alignment, epitope prediction and 3D structure modelling to reveal S. stercoralis candidate peptide/protein coproantigens. We describe 22 candidates from seven genes, across all five protein families for further investigation as potential S. stercoralis diagnostic coproantigens, identified using open access data and freely-available protein analysis tools. This powerful approach can be applied to many parasitic infections with omic data to accelerate development of specific diagnostic assays for laboratory or point-of-care field application. Author summaryThe worm Strongyloides stercoralis causes infectious disease in people throughout tropical and sub-tropical regions, leading to an extensive reduction in quality of life and even death. Millions of people are at risk of infection with this parasite and improved diagnostic and control methods and technologies are urgently required. Currently, most diagnosis is carried out through methods involving visual inspection of patients faeces, which has a number of drawbacks, particularly its poor sensitivity. This paper presents a new method to develop improved diagnostic tests for S. stercoralis, by computational analysis of publicly available gene and protein sequences to predict proteins that may be detectable in faeces. This would enable the development of rapid diagnostic tests in the form of lateral flows or dipsticks, with better predictive ability and fewer drawbacks than current diagnostic methods. A number of potential proteins, predicted to have all the desired characteristics for use in such tests were found through the new method and have been presented in this paper. With validation, new diagnostic tests for S. stercoralis could be developed from these results and the computational approach could be used to target other parasitic diseases.

Wildlife rehabilitation plays a central role in the conservation of threatened primates, yet parasite dynamics during captivity are rarely reported, particularly in relation to release readiness. We investigated gastrointestinal helminth infection patterns in rehabilitating Javan slow lorises (Nycticebus javanicus), a Critically Endangered species heavily impacted by the illegal wildlife trade. Using repeated fecal sampling (147 samples from 19 adults) and Bayesian mixed-effects models, we examined parasite richness, Shannon diversity, infection probability, and egg-shedding intensity in relation to release readiness status, sex, housing condition, and time since anthelmintic treatment. Four nematode taxa identifiable through egg morphology were detected: Strongyloides spp., strongylids, oxyurids, and Trichuris spp.. Parasite richness and Shannon diversity showed no credible associations with release readiness or other host and management variables. In contrast, infection probability for Strongyloides spp. and strongylids increased with time since deworming, and Strongyloides egg counts exhibited a similar temporal pattern, consistent with post-treatment reinfection dynamics. Release readiness did not predict detection probability or parasite intensity for any parasite group, despite marked differences in captivity duration and health history between individuals deemed ready for release or not. These findings indicate that gastrointestinal helminth dynamics in rehabilitating slow lorises are driven primarily by treatment-related temporal processes and individual-level heterogeneity rather than coarse host classification. They also highlight the need for longitudinal parasite monitoring and for future work evaluating how infection dynamics, management interventions, and host health relate to rehabilitation and translocation outcomes..