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.

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.

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.

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.

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.

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..

Neoparameoba perurans causes Amoebic Gill Disease (AGD), a major parasitic disease of marine-phase Atlantic salmon and rainbow trout worldwide. Treatment options are limited to freshwater baths, which are costly at scale and exhibit only limited long-term efficacy. N. perurans contains an obligate eukaryotic symbiont, Perkinsela-like organism (PLO). PLO belongs to the class Kinetoplastida, which includes medically and veterinary important parasites such as Trypanosoma and Leishmania. As such, we hypothesised that trypanocidal drugs developed against other kinetoplastids might also affect N. perurans, potentially through disruption of its PLO symbiont, and used this hypothesis as a rationale for prioritising a focused panel of candidate compounds for screening. A holographic motility-based cytotoxicity assay was established to identify promising candidates in vitro, followed by controlled host tolerance testing and finally a field efficacy sea trial using naturally AGD-exposed site in the west of Ireland. Several compounds showed activity in vitro, especially miltefosine (EC50 1.84 uM, amoebicidal) and isometamidum (EC50 4.63 uM, amoebostatic). In vivo (two intramuscular injections, two weeks apart), miltefosine (Odds Ratio (OR) 0.62), isometamidum (OR 0.61) and benznidazole (OR 0.64) significantly improved gill score over four weeks, with miltefosine showing the largest effect size. Gill parasitaemia, measured via qPCR, was not reduced. Instead, two compounds increased apparent amoeba loads. This work support trypanocidal as potential AGD treatments in the field, although optimisation of dosing, delivery and mode of action requires further study.

Soil-transmitted helminth (STH) infections are a major public health burden, and there are programmes of mass drug administration that attempt to ameliorate the harm that they cause. There has been increasing use of genomics to study STH infections and other parasitic nematodes, with particular interest in whole genome sequencing (WGS). For such studies, samples are commonly stored frozen, but in settings where these infections are endemic this can be difficult, and so there would be advantages to having ambient temperature storage methods. We investigated two ambient temperature storage methods - FTA cards and DESS buffer - for infective larvae of the rat parasites Nippostrongylus brasiliensis and Strongyloides ratti, prior to DNA extraction and then WGS. Our results showed that for individual larvae stored on FTA cards or in DESS buffer, this resulted in a lower proportion of sequence reads that mapped to the reference genomes, compared to the frozen control samples. Generally, for individual larvae, DESS-storage resulted in better sequencing results than FTA-storage. However, for pools of 10 or 50 larvae, then these ambient temperature storage methods generally resulted in comparable sequence read mapping to the frozen control samples.

Fasciola hepatica is a trematode parasite responsible for fasciolosis, a liver disease that affects humans and livestock worldwide. Together with other food-borne trematode infections, fasciolosis is considered a neglected tropical disease. Further, it imposes substantial agricultural losses due to infections in ruminants. No vaccine is currently available, and control heavily relies on drug treatment, especially with triclabendazole (TCBZ). However, the intensive use of TCBZ over the past four decades has led to increasing rates of treatment failures and the emergence of drug-resistant parasites. Therefore, the identification of new treatment options is an urgent priority. The currently available toolset for drug screening, however, is limited. To address this need, we established a novel, semi-automated, standardized, and objective screening assay based on motility monitoring of newly excysted juveniles using microscopic live imaging. The assay was validated by testing a panel of ten compounds with known anthelmintic properties, amongst them TCBZ (IC50: 1.5 {micro}M) and the new activator of the F. hepatica transient receptor potential melastatin (TRPM) ion channel, benzamidoquinazolinone (IC50: 1.05 {micro}M). In addition to these two compounds with known activity against F. hepatica, three compounds were identified as particularly promising with a fast onset of action and IC50 values in the nanomolar range: the salicylanilides MMV665807 (IC50: 44 nM), niclosamide (IC50: 32 nM), and its ethanolamine salt, niclosamide ethanolamine (IC50: 9 nM). Complementary live/dead staining revealed that only TCBZ displayed parasiticidal activity, while the other compounds, although leading to parasite paralysis, did not lead to parasite death within 72 hours. Scanning electron microscopy of drug treated parasites did not reveal any significant damage at concentrations corresponding to the IC50s, but strong phenotypes were visible at 20 {micro}M. The presented motility assay provides a robust method for the discovery of novel anthelmintic compounds and facilitates the ongoing effort to combat fasciolosis. Author SummaryFasciola hepatica, the common liver fluke, is a parasitic platyhelminth that infects the liver and biliary ducts of humans and livestock, causing fasciolosis, a Neglected Tropical Disease as defined by the World Health Organization. Triclabendazole is the drug of choice to treat humans and animals. However, its intensive use has led to the emergence of drug resistance resulting in treatment failures worldwide. The identification of novel drugs is therefore urgent. Here, we present a semi-automated and objective method to assess the activity of compounds on one of the key life stages of the parasite: the newly excysted juveniles (NEJ). This stage is highly motile and motility assessment can be exploited to screen for bioactive compounds. Using time-lapse imaging, we quantified NEJ movement after drug exposure. From a panel of ten tested reference anthelmintics, two known fasciolicides (triclabendazole and benzamidoquinazolinone) and three additional compounds (MMV665807, niclosamide, and niclosamide ethanolamine) displayed particularly strong activity and were selected for further investigation. This method represents a robust tool for drug screening and facilitates the discovery of new compounds against F. hepatica.

Parasitic flatworms, including cestodes and trematodes, are covered by a specialized syncytial tegument that mediates nutrient uptake and host-parasite interactions. While the tegument of trematodes has been extensively characterized, its molecular composition in cestodes remains largely unknown. In this work, we performed a comparative proteomic analysis of the tegument of three cestode species, including larval and adult stages: Hymenolepis microstoma, Mesocestoides corti (syn. M. vogae) and Echinococcus multilocularis. Using stringent enrichment criteria relative to whole-worm extracts, we identified hundreds of tegument-enriched proteins in each species. Comparative analyses revealed a conserved core of tegumental proteins shared among all three species, including members of the Tegument Allergen-Like (TAL) family, vesicular trafficking components and calcium-sensing proteins, and identified candidates for nutrient uptake activities such as glucose and nucleoside transporters. Further comparative analyses revealed a set of shared tegumental proteins with the trematode Schistosoma mansoni, including conserved proteins that are specific to parasitic flatworms, supporting the existence of a conserved ancestral tegumental proteome. Finally, we confirmed tegumental expression of several candidate genes in H. microstoma and E. multilocularis, and demonstrated regionally restricted gene expression among tegumental cytons, suggesting functional specialization within the syncytial tegument. Altogether, these results reveal an evolutionarily conserved composition of the tegument of parasitic flatworms, providing a foundation for future work targeting this critical host-parasite interface.

Meloidogyne incognita is a major plant-parasitic nematode responsible for substantial yield losses in tomato worldwide. Current control strategies rely heavily on chemical nematicides, which raise environmental concerns and face increasing regulatory restrictions, underscoring the need for sustainable alternatives. Here, we show that foliar application of an aqueous extract from cavalcade (Centrosema pascuorum) enhances tomato resistance against M. incognita. Pre-inoculation treatment with cavalcade extract prior to inoculation with root-knot nematodes (RKN) significantly reduced root gall formation, delayed nematode development, and limited second-stage juvenile penetration compared with untreated infected controls, whereas post-inoculation application conferred partial protection. Transcriptomic analyses revealed the activation of multiple defense-related pathways, including salicylic acid- and jasmonic acid-associated signaling and phenylpropanoid metabolism, supported by the upregulation of PR1 and PAL. Additional induction of lipid transfer proteins, leucine-rich repeat receptor-like kinases, resistance proteins, mitochondrial calcium uniporter, and GA2ox5 suggests coordinated activation of pathogen recognition, calcium signaling, and hormone-regulated defense networks. These findings demonstrate that cavalcade extract primes broad-spectrum defense responses in tomato and highlight its potential as an environmentally sustainable strategy for nematode management.

Plasmodium vivax (Pv) infections are developmentally asynchronous and often polyclonal, complicating interpretation of bulk parasite transcriptomes. Here, we analyzed paired in vivo and short-term ex vivo transcriptomes from Ethiopian clinical isolates using stage deconvolution and PvMSP1 haplotyping. Ex vivo maturation modestly increased inferred schizont representation while largely preserving the proportion of trophozoites and gametocytes. After adjustment for parasite stage composition, in vivo and ex vivo transcriptomes remained globally similar, with no genes significantly differentially expressed, indicating the absence of major culture-induced transcriptional response. In contrast, short-term culture reduced multiplicity of infection, contracted within-host haplotype diversity, and non-randomly depleted specific haplotypes, consistent with a clonal bottleneck. In a subset of low-complexity infections, residual expression patterns were clustered by dominant haplotype, suggesting genotype-associated transcriptional heterogeneity independent of developmental stage. Together, these findings indicate that short-term ex vivo culture enriches late asexual stages and selectively filters clones rather than inducing a common transcriptional program. These results shows that ex vivo cultures are reliable way to study gene expression, especially for late stages. However, these needs explicitly model developmental composition and infection complexity when interpreting Pv transcriptomes from natural infections Author summaryMalaria caused by Plasmodium vivax is difficult to study because this parasite cannot yet be grown continuously in the laboratory and infections in patients often contain parasites at different developmental stages and multiple parasite lineages at the same time. In this study, we wanted to understand how much of the parasite gene-expression signal reflects true biological differences, and how much is explained by parasite development or changes that occur during short-term laboratory maturation. We compared parasites collected directly from patients in Ethiopia with matched parasite matured briefly outside the body. We found that short-term culture mainly increased the proportion of later-stage parasites, but after accounting for developmental stage, the overall gene-expression patterns remained very similar. However, culture reduced the diversity of parasite lineages within infections, suggesting that some parasite lineages survive better than others under laboratory conditions. Our findings highlight that natural Pv infections are complex mixtures of parasite stages and lineages. Accounting for this complexity will improve how researchers interpret parasite gene-expression studies and design future studies of parasite invasion, transmission, and survival.

Neurocysticercosis (NCC), the infection of the central nervous system by Taenia solium larvae, is a leading cause of acquired epilepsy in endemic regions. While viable cysticerci can persist asymptomatically for extended periods, their spontaneous or drug-induced degradation triggers marked perilesional inflammation and severe neurological symptoms. Despite well-documented histopathological characterisation of these lesion states, the host transcriptional programmes associated with viable parasite persistence and early post-treatment lesion disruption remain poorly understood. To address this gap, we performed the first bulk RNA sequencing of pericystic brain tissue using a physiologically relevant porcine model of NCC. Comparing uninfected controls (n = 3), infected untreated pigs with intact viable cysts (n = 6), and antiparasitic-treated pigs with disrupted cysts (n = 3), we identified distinct transcriptional signatures associated with each disease state. Viable infection was associated with broad transcriptional changes (461 upregulated and 175 downregulated genes), characterised by local immune activation alongside suppression of blood-brain barrier (BBB) remodelling, vascular, and neuronal signalling molecular signatures. The post-treatment state with confirmed BBB disruption was associated with a smaller but directionally distinct response (160 upregulated and 57 downregulated genes), marked by inflammatory signalling and increased expression of genes associated with endothelial activation, vascular regulation, and BBB-associated remodelling. Together, these findings suggest that, while immune engagement is a feature shared across both lesion states, the BBB-associated transcriptional axis shifts substantially following treatment. These results provide an exploratory transcriptomic framework for understanding parasite persistence, treatment-induced neuroinflammation, and neurovascular remodelling in NCC, and highlight candidate pathways and genes for future mechanistic investigation.

Plant-parasitic nematodes are a diverse, polyphyletic group of plant pathogens which can infect most plant tissues and all major crops. Amongst the most damaging clades are the cyst nematodes, which can remain dormant in the soil for decades as infection-competent, developmentally arrested, second-stage juveniles in eggs. Hatching is stimulated by a variety of factors. However, the impact of hatching factor responsiveness on nematode morphology, physiology, gene expression, and infection biology has not been explored. We examined the impact of hatching time on the beet cyst nematode, Heterodera schachtii. We found that late hatchers invaded host roots and established feeding sites in greater numbers than early hatchers. We demonstrate variation in baseline parasitism gene expression and in responsiveness of genes to effectostimulins, small, plant-derived molecules which upregulate parasitism genes. Three quarters of effectostimulin-induced transcriptional changes were also modulated, either positively or negatively, by hatching time. While there were no observable morphological differences between early and late hatching nematodes on the day of their emergence from the egg, the late hatchers displayed signs of faster utilisation of internal energy reserves after 7 days at 4{degrees}C, as evidenced by less body area attributed to fat, than early hatchers. Finally, we found no evidence of substantive genetic differences between early and late hatchers, they were representative of a single population, despite the observed differences in infection, gene expression, and physiology. Taken together, non-genetic differences likely drive late hatchers to more rapidly utilise their internal energy reserves, to be more responsive to host-derived signals, and to be ultimately more infective than their early hatching counterparts.

Motility of Plasmodium sporozoites (SPZs) in the skin is a key determinant of successful host infection. Earlier studies have described rapid movement of both murine and human SPZs in skin following syringe inoculation. It is typical to classify SPZ trajectories into ``motile'' and ``immotile'' and restrict the analysis of movement patterns to motile SPZs. Because criteria to define motile SPZs are dependent on the study and are often qualitative, it remains unclear if sub-selection of motile tracks introduces biases in characterization of SPZ movement in vivo. We processed imaging data (22 movies) from a recent study of movement of P. falciparum (Pf) and P. yoelii (Py) SPZ in skin. We proposed a novel metric -- maximal spatial spread (MSS or S) --- that is the maximum Euclidean distance between any two recorded positions in a trajectory. We used MSS to classify SPZ trajectories as immotile (S<Sthreshold) or motile (S>Sthreshold) for a given threshold value Sthreshold. Larger Sthreshold values naturally resulted in a smaller fraction of tracks classified as motile, and subsequently, in an increased overall displacement, instantaneous and mean speeds, decreased mean turning angle, and higher initial slopes of the mean squared displacement (MSD) curves. We found that at intermediate values of Sthreshold Pf SPZs had a lower average speed than Py SPZs suggesting that host environment may impact SPZ movement. Both species exhibited a small but statistically significant decline in average speed with time after inoculation but this was also dependent on the Sthreshold value. Our analysis of MSD curves and turning angle distributions suggests that both Pf and Py SPZs undergo correlated random walks -- a type of Brownian walk with short-term superdiffusive displacement. By using a novel methodology of hidden Markov models (moveHMM package in R) we found that SPZ movement is best described by three movement states; however, none of these states corresponded to previously described circling gliding. Taking together, our results suggest that inference of SPZ movement patterns depends on the criteria used to define tracks as motile or immotile. Standardized preprocessing criteria are therefore important when comparing motility across Plasmodium species, experimental time points, or laboratories. Analysis of turning angle distributions and application of hidden Markov models provided additional metrics to quantify distinct modes of SPZ movement in vivo.

Ehrlichia ruminantium, the causative agent of heartwater disease, is an obligate intracellular bacterium that poses significant economic threats to livestock production in endemic regions. Current research models present substantial ethical, logistical, and economic constraints, particularly for studying host-pathogen interactions within arthropod vectors. Here we establish Galleria mellonella larvae as a tractable invertebrate infection model for Ehrlichia ruminantium, enabling experimental investigation of pathogen persistence and host-pathogen interactions in an arthropod system. Following infection, G. mellonella proved susceptible to E. ruminantium with moderate mortality and remarkable bacterial persistence. Using rhodamine-labeled bacteria and fluorescence microscopy, we tracked bacterial dissemination from injection sites to systemic distribution in characteristic segmental patterns throughout the larval body. Critically, we confirmed intracellular localization of E. ruminantium within hemocytes, the primary immune cells of G. mellonella. Quantitative PCR analysis revealed stable bacterial loads over the study period, indicating bacterial persistence within the host. These findings demonstrate that E. ruminantium can hijack the innate immune system of G. mellonella, similar to its behavior in natural hosts. The segmental bacterial distribution suggests exploitation of hemolymph circulation and sessile hemocyte populations, providing new insights into potential mechanisms of pathogen persistence. This model offers significant advantages: ethical acceptability, cost-effectiveness, experimental tractability, and compatibility with high-throughput screening approaches. The G. mellonella system represents a valuable complement to existing mammalian models and provides a unique platform for investigating arthropod-specific aspects of E. ruminantium biology, screening antimicrobial compounds, and understanding mechanisms of immune evasion that may inform strategies for heartwater disease control.

Freshwater snails act as intermediate hosts for parasites affecting both humans and livestock, including schistosomes. In Mauritania, however, the diversity, distribution, and infection status of these snails remain poorly documented. This study aimed to identify freshwater snail species collected from two schistosomiasis-endemic areas in southern Mauritania, characterize their spatial distribution, and assess their infection rates using molecular tools Malacological surveys were conducted in Kankossa and Oued Rawdha during the 2023 rainy season. A total of 806 snail specimens were collected and preserved in 70% ethanol at 4 {degrees}C prior to analysis. Five species were identified morphologically and confirmed by molecular analysis: Bulinus truncatus, B. forskalii, B. senegalensis, B. umbilicatus, and Melanoides tuberculata. MALDI-TOF MS generated high-quality spectra for 99.0% of specimens and correctly identified 99.9% of analyzable samples after molecular confirmation of discrepant cases. Preservation in ethanol at 4 {degrees}C markedly improved spectral quality compared with previously reported room-temperature storage conditions Distinct ecological distributions were observed according to water body type. B. senegalensis and B. umbilicatus were exclusively collected from temporary ponds, whereas B. truncatus, B. forskalii, and M. tuberculata were found in permanent water bodies. Real-time PCR screening detected Schistosoma haematobium complex DNA in 239/798 (29.9%) specimens, with substantially higher infestation rates in Kankossa than in Oued Rawdha. These findings demonstrate that MALDI-TOF MS is a rapid, accurate, and field-compatible tool for freshwater snail identification, including closely related species that are difficult to distinguish morphologically. This approach could facilitate large-scale epidemiological surveillance and improve monitoring of schistosomiasis transmission dynamics in endemic settings.

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is widely used for sand fly identification, but its potential to detect Leishmania infections in vectors remain underexplored. This pilot study evaluated whether MALDI-ToF MS protein profiles of lab-reared Lutzomyia longipalpis and Nyssomyia neivai can discriminate Leishmania infantum-infected from uninfected females. MethodologyColonies were experimentally infected with L. infantum using membrane feeding, and females were collected at different days post-blood meal. Thoraces and legs were processed individually for MALDI-ToF MS, and spectra were analysed using both Bruker software and custom R pipelines. Principal findingsUnsupervised approaches (MSP dendrograms, PCA) showed limited or inconsistent separation of infection status for Lu. longipalpis. In contrast, supervised machine-learning models built on peak-intensity matrices achieved excellent discrimination between infected and uninfected specimens for both species, with several algorithms reaching near-perfect performance on an external test set not used for training. Variable-importance analysis highlighted sets of m/z peaks, mainly showing decreased intensity in infected sand flies, as putative infection biomarkers. ConclusionThis proof-of-concept study highlights that L. infantum infection induces reproducible, species-specific alterations in sand-fly MALDI-TOF profiles, supporting further development of high-throughput, MS-based screening of infected vectors. Author summaryLeishmania infantum is a parasite responsible for visceral leishmaniasis, a severe neglected tropical disease. It is transmitted to humans by sandfly vectors. This study explored whether the MALDI-ToF mass spectrometry technique can detect infection by the L. infantum parasite in the two main sandfly vectors in Brazil: Lutzomyia longipalpis and Nyssomyia neivai. The method has already been tested to identify sandfly species, but its ability to detect infected insects had not been well studied. We infected laboratory-reared sandflies and analyzed their protein profiles to see whether infected and uninfected individuals could be distinguished. We found that infection changes the molecular fingerprints of both sandfly species. Machine-learning models were able to distinguish infected from uninfected specimens with very high accuracy. A small part of the most informative signal was shared between both species, while most of the peaks were species-specific, suggesting that infection affects each vector in a slightly different way. These results show that MALDI-ToF has promise as a rapid, low-cost tool for screening sandflies for Leishmania infection. With further validation, this approach could complement existing surveillance methods and help monitor disease transmission in endemic areas.

BackgroundMalaria poses a significant global health challenge, with over 200 million infections and around 500,000 deaths annually, predominantly affecting children under 5 in sub-Saharan Africa. Severe malarial anaemia (SMA), a major contributor to morbidity and mortality in this demographic, results from various factors including red blood cell destruction and immune-mediated clearance. The role of these mechanisms in SMA differs based on age and infection history. Recent studies indicate increased accumulation of P. falciparum in the bone marrow and spleen, highlighting the need for understanding localized host-parasite interactions. MethodsThis study examines the bone marrow response to malarial infection in young children with SMA or mild malarial malaria in Mozambique, contrasting it with responses observed in peripheral blood in the two cohorts. Results and conclusionsThe study demonstrated that SMA is associated with increased red blood cell production, iron metabolism and tissue injury, as well as higher total parasite biomass including peripheral and bone marrow parasitaemia. We also demonstrate that direct analysis of bone marrow aspirates provides far more resolution in stratifying host signatures and drivers of malarial anaemia across a spectrum of severity than systemic measures from blood samples.