International Journal for Parasitology

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.

BackgroundLoiasis is a chronic filarial infection endemic to Central and West Africa. Although long considered benign, increasing evidence links loiasis to substantial morbidity and mortality. The infection is associated with immune modulation, including Th2-skewed responses and elevated regulatory cytokines. Clinically, loiasis is classified as microfilaremic (presence of circulating microfilariae) or amicrofilaremic ("occult") disease, the latter defined by a history of eyeworm migration without detectable microfilaremia. This study investigated how chronic L. loa infection influences antibody and T cell responses to SARS-CoV-2 following natural infection. MethodsBetween 2022 and 2024 this cross-sectional study was done in Lambarene and surrounding rural areas of Gabon. Study procedures included diagnostics for loiasis and immunological assays. Microfilaremia was confirmed by stained blood smear microscopy, and occult disease was identified using the Rapid Assessment Procedure for Loiasis. SARS-CoV-2-specific IgG responses to spike and nucleocapsid proteins were measured by ELISA, and IFN-{gamma} responses to spike antigen were assessed using an interferon-gamma release assay. ResultsOverall, 192 participants were categorized as microfilaremic (n=43), occult loiasis (n=59), or without evidence of active loiasis (n=90). IFN-{gamma} responses were reduced in microfilaremic individuals compared with other participants (p= 0.031), whereas IgG responses did not differ. Subsequent analysis across the three groups confirmed that IFN-{gamma} responses were lower in microfilaremic compared with occult participants (p= 0.012). ConclusionThese findings suggest that microfilaremic loiasis may impair proinflammatory T cell responses to viral antigens, highlighting the need for further research into the broader immunological effects of Loa loa infection in endemic populations. Authors summaryLoiasis is a parasitic infection caused by the worm Loa loa and is common in parts of Central and West Africa. Although long considered relatively benign, growing evidence indicates that loiasis is associated with substantial morbidity. The immunological consequences of chronic Loa loa infection remain poorly understood. A small number of studies suggest that Loa loa may influence immune regulation, but its broader impact on antiviral immunity is largely unknown. The COVID-19 pandemic provided a unique opportunity to examine immune responses to a newly emerging virus in a population where loiasis is endemic. We therefore investigated how different forms of Loa loa infection influence immune responses after natural SARS-CoV-2 infection. We compared individuals with circulating microfilariae in their blood (microfilaremic), individuals with occult loiasis (history of eye worm), and individuals without signs of active infection. We found that microfilaremic individuals had weaker virus-specific IFN-{gamma} T cell responses, while antibody levels were similar across groups. These findings suggest that active loiasis may dampen certain antiviral immune functions. Understanding the underlying mechanisms is important, as such immune modulation could affect responses to vaccines and other infectious diseases in endemic regions.

Three molecularly undescribed filarial species were co-detected, while screening animals for Brugia malayi, the agent of lymphatic filariasis. Single microfilariae (Mf) isolated from blood samples of crab-eating macaques (Macaca fascicularis) from Belitung, Indonesia, and from pet dogs and cats in Sabah, Malaysia, were analyzed. Among 163 macaques, 33 (20.2%) were positive for large Mf (mean length 498.9 {micro}m) similar to Dirofilaria ( Belitung I). One macaque was infected with small Mf (mean length 150.4 {micro}m) ( Belitung II), with a high density of 17,150 Mf/mL. In two cats co-infected with B. malayi, Mf of a Dirofilaria species ( Sabah) with an average length of 299.1 {micro}m were detected. Morphometric analysis of Mf showed distinct differences between these three species and other Mf described in the area. Whole genome amplification and genome sequencing of 24 individual Mf enabled phylogenetic analysis of mitochondrial genomes, and analysis of specific mitochondrial and nuclear barcode regions. The three Mf groups formed distinct clusters and did not match any currently available reference sequence. Cluster Belitung I from macaques formed a sister group to all other Dirofilaria. Cluster Belitung II included bird filariae and primate filariae of the genus Mansonella as close relatives. The cluster Sabah formed a monophyletic group with the zoonotic species D. asiatica and Dirofilaria sp. Thailand. DNA of Wolbachia endobacteria was detected in Mf of Belitung I and Sabah, but not in Belitung II. These findings highlight the limited understanding of filarial diversity in macaques and cats in Asia and underscore the need for a more comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites. Author summaryFilarial worms are parasitic nematodes that infect humans and animals and are often transmitted by the same vector mosquito. We identified three molecularly undescribed filarial species while investigating animals as reservoirs for the agent of lymphatic filariasis, Brugia malayi on Belitung Island, Indonesia, and in Sabah, Malaysia. Blood samples were collected from Indonesian macaques and Malaysian pet cats. Out of 163 macaques, 20.2% tested positive for exceptionally large microfilariae (Mf) of an unclassified Dirofilaria-like species (Belitung I). Another filarial species ( Belitung II) with very small Mf, but with a remarkably high density of 17,150 Mf/mL was detected in one macaque. Two cats harbored medium sized Mf of a Dirofilaria species (Sabah). Genetic analysis revealed unique phylogenetic clusters that did not match any reference sequence. Dirofilaria sp. Sabah was closely related to the zoonotic D. asiatica complex, whereas Belitung I clustered as a sister group to Dirofilaria. Belitung II Mf clustered next to but not within the Mansonella spp. cluster. DNA of Wolbachia endobacteria was only detected in Mf of Belitung I and Sabah. These findings highlight the limited understanding of filarial diversity in animals and underscore the need for a comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites.

Background: Female genital schistosomiasis (FGS) is a neglected gynaecological manifestation of Schistosoma haematobium (S. haematobium) infection, resulting from the deposition of parasite eggs in the female genital tract. Although urogenital schistosomiasis is highly prevalent in parts of coastal Kenya, including Kilifi County, the burden of FGS among women of reproductive age remains poorly characterised. Routine diagnosis of S. haematobium infection relies largely on urine microscopy, which may underestimate genital involvement. This study aimed to assess the prevalence, diagnostic concordance, and risk factors for FGS among women of reproductive age in Kilifi County, Kenya. Methodology: In this cross-sectional study, 320 randomly selected women aged 15-50 years were recruited from rural Kilifi County; 261 provided complete data for analysis. A structured questionnaire was administered to collect sociodemographic and behavioural information. Urinary schistosomiasis was assessed using triplicate urine microscopy over three consecutive days, and FGS was evaluated using real-time polymerase chain reaction (PCR) targeting the S. haematobium Dra1 gene sequence on self-collected high vaginal swabs. Results: Overall, the prevalence of PCR-confirmed FGS was 36.0% (94/261), while urinary egg excretion was detected in 13.0% (34/261) of participants. Concordance between urine microscopy and genital PCR was 70.9%. Notably, 72% of women with PCR-confirmed FGS had no detectable parasite eggs in their urine. In bivariate analyses, factors such as urinary infection severity, water contact behaviours, haematuria, dysuria, age group, place of residence, and prior history of schistosomiasis were found to be associated with female genital schistosomiasis (FGS). However, in the multivariable logistic regression, only sub-location and urinary infection severity remained independently associated with the infection. Additionally, PCR cycle threshold (Ct) values showed a non-linear relationship with mean urinary egg counts, indicating that the detection of genital parasite DNA does not directly correspond to the urinary egg burden. Conclusion: FGS prevalence among women in Kilifi County was substantially higher than indicated by urine microscopy alone. The majority of women with genital schistosomiasis did not exhibit detectable urinary egg excretion, highlighting the limitations of routine parasitological screening for identifying genital disease. These findings underscore the need to incorporate genital sampling and molecular diagnostics into schistosomiasis control strategies targeting women of reproductive age in endemic settings.

AimTo resolve the topological branching patterns, the timing of demographic events, and the effective population size changes associated with major demographic events. LocationMidwestern (eastern North Central) and Northeastern USA TaxonBlacklegged tick, Ixodes scapularis (Say, 1821) MethodsUsing three independent genomic datasets, single-nucleotide variants were analyzed for demographic inference. Maximum likelihood topologies and prior ecological knowledge were used to generate nested demographic hypotheses. The best-fit scenario and the associated demographic parameter estimates were determined using approximate Bayesian computation under a random forest statistical model. The topologies and parameters supported in the three independent datasets were compared to generate insights about the demographic history of blacklegged ticks in the region. ResultsThe emergence of extant northern populations of blacklegged ticks began between 10-15 k.y.a. (thousand years ago), with independent population splits from the common ancestor during the Early-Mid-Holocene, and never more recent than 4 k.y.a. All populations sustained moderately large population sizes without bottlenecks, with Michigan as the exception. Michigan appears to have an uncertain placement that depends on sampling, reflecting its admixed origin. Main conclusionsThere are multiple populations of northern blacklegged ticks that have persisted independently as deglaciated regions in the northern U.S. were recolonized following the Last Glacial Maximum (26.5 to 19 k.y.a.). The current ecological expansions across the northern U.S. are likely seeded by separate relictual populations with distinctive genomic ancestry rather than a range expansion from a single source, with important implications for vector-borne disease management.

Whipworms (Trichuris spp.) are intracellular intestinal parasites that develop within the host caecal epithelium, yet the host signals that regulate their growth and developmental progression remain poorly understood. Progress in studying these processes has been limited by the lack of physiologically relevant in vitro systems capable of supporting sustained whipworm development. Here, we established an in vitro infection system using caecal organoids (caecaloids) and evaluated their capacity to support sustained growth and morphological development of Trichuris muris larvae. To rigorously validate this system, we generated a comprehensive and up-to-date anatomical and biometrical reference dataset describing the whole-body growth and tissue-level morphogenesis of T. muris throughout its life cycle in vivo. Quantitative analysis across larval and adult stages confirmed that the trajectory of parasite growth is largely conserved across host mouse strains and provided a detailed contextualised description of the development of key anatomical structures of T. muris. Using this reference framework, we evaluated parasite growth and development in long-term T. muris-caecaloid co-cultures. Larvae invading the caecaloid epithelium remained intracellular within syncytial tunnels and exhibited sustained growth over extended culture periods. in vitro parasites developed increasing anatomical complexity, including formation of the bacillary band, stichosome, intestine, and rectum. Importantly, quantitative comparisons revealed that larvae developing within caecaloids follow growth trajectories and morphological developmental patterns closely resembling those observed in vivo. This study therefore presents the first detailed anatomical and morphometric framework for validating whipworm development in an organoid system and provides concrete evidence that the caecaloid epithelium is sufficient to trigger and sustain whipworm growth and morphogenesis, establishing caecaloids as a powerful experimental platform for investigating Trichuris infection and development.

BackgroundTrypanosoma rangeli is a non-virulent hemoflagellate protozoan parasite that infects mammals, including humans, in Central and South America. It is primarily transmitted through the bites of triatomine bugs and shares an overlapping geographical distribution with T. cruzi, as well as triatomine vectors and mammalian hosts, and various shared surface antigens. The life cycle of T. rangeli differs from those of other human-infecting trypanosomes, such as T. cruzi and T. brucei, and the molecular mechanisms underlying host-parasite and host-vector interactions are not well understood, demanding improved molecular and genomic resources. ResultsThe use of a hybrid approach to sequence and assemble the T. rangeli genome, complemented by transcriptomics and proteomics for functional gene annotation, led to the generation of the near-complete genome sequence of the parasite. Detailed intra- and inter-specific comparative genomics allowed analysis of polymorphisms, genome structure and improved resolution of genes coding for important surface molecules such as Mucins, TASV and GP63. ConclusionsThe improved T. rangeli genome assembly, combined with comparative genomics has yielded novel biological insights. These included the first description of a metalloprotease activity, attributed to specific GP63 genes that are absent in Leishmania species. In addition, a TASV gene family that is absent in T. cruzi was identified, which indicates a possible role in the T. rangeli infection process.

Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infections were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author SummarySoil-transmitted helminths (STH) such as the parasite Strongyloides, are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STH in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in stool samples from humans infected with STH, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read Illumina and long-read Oxford Nanopore Technologies, but short-read outperformed long-read shotgun metagenomics. Contamination of bacteria sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.

BackgroundGastrointestinal helminths of companion animals are neglected sources of zoonotic infection in low and middle-income countries. In Bangladesh, close humananimal contact and large free-roaming dog and cat populations may facilitate parasite transmission, yet region-specific data remain limited. This study assessed the prevalence, species diversity, and zoonotic potential of gastrointestinal helminths in companion animals in northeastern Bangladesh. MethodsA cross-sectional study was conducted between January and December 2025 across urban and rural areas of the Sylhet Division. Fecal samples from 900 animals (600 dogs and 300 cats; owned and stray) were examined using standard coproscopic techniques. Molecular confirmation of selected positive samples was performed using PCR targeting ITS-2, 18S rRNA, and mitochondrial cox1 genes, followed by sequencing. Risk factors associated with infection were evaluated using multivariable logistic regression. ResultsOverall, 45.9% (95% CI: 42.6-49.2) of animals were infected with at least one gastrointestinal helminth, with mixed infections detected in 18.4%. Prevalence was similar in dogs (45.7%) and cats (46.3%) but significantly higher in stray animals (65.7%) than in owned animals (36.6%). Predominant zoonotic helminths included Ancylostoma spp., Toxocara canis, Toxocara cati, Dipylidium caninum, and Taenia/Echinococcus spp. Molecular analysis confirmed 93% of morphologically identified infections and revealed high genetic similarity to zoonotic reference strains. Stray status, lack of deworming, young age, and outdoor roaming were significant risk factors for infection (p < 0.05). ConclusionsCompanion dogs and cats in northeastern Bangladesh harbor a high burden of zoonotic gastrointestinal helminths and represent important reservoirs for human exposure. Strengthening One Health-based surveillance, routine deworming, and stray animal management is essential to reduce zoonotic transmission.

Helminthiases remain a major global health burden, and limitations of current anthelmintic therapies highlight the need for new pharmacological targets. In this study, we examined the effects of ion-channel and cytoskeletal modulators on bovine lung protoscoleces (PSCs) of Echinococcus granulosus sensu lato. Compounds acting on ion channels (praziquantel, amiloride, and amlodipine) and cytoskeletal components (albendazole and cytochalasin D) were evaluated using a semi-automated motility assay, methylene blue exclusion to assess viability, and scanning electron microscopy (SEM) to characterize structural damage. All compounds produced concentration-dependent reductions in PSCs motility. Amlodipine was the most potent inhibitor of motility, whereas praziquantel and cytochalasin D produced pronounced tegumental alterations and strong correlations between motility impairment and parasite death. In contrast, amiloride markedly reduced motility with comparatively minor effects on viability, indicating a primarily paralytic effect. Cytoskeletal disruption induced severe structural damage and parallel declines in motility and viability. SEM analysis revealed extensive tegumental collapse, loss of glycocalyx, and microtrichial damage in PSCs exposed to cytoskeletal and calcium-modulating agents. These findings highlight cytoskeletal organization and calcium-dependent ion fluxes as key physiological vulnerabilities in E. granulosus. Comparative analysis of these pharmacological targets provides mechanistic insight into how disruptions in cytoskeletal dynamics and cation homeostasis compromise parasite motility and survival.

Trichomonas gallinae is a protozoan parasite of major concern in avian medicine, particularly in domestic pigeons (Columba livia). This study investigated the risk factors associated with the frequency of nitroimidazole resistance and T. gallinae prevalence in domesticated pigeons from Eastern Spain, kept for different competitions. A total of 220 pigeons from 11 lofts were sampled and examined by microscopy and culture, revealing a 63.6% infection prevalence. Genotyping identified genotype C as predominant, with occasional detection of genotype A, mixed A/C infections, and one isolate of Lineage III. In vitro susceptibility testing of 42 isolates showed a high prevalence (81%) of metronidazole resistance (MIC values [&ge;] 20 {micro}g/ml), with minimum inhibitory concentrations (MICs) ranging from 5 to >100 {micro}g/mL in 9/11 pigeon lofts examined. Resistance was significantly associated with the use of metronidazole and was more frequent in young and non-reproductive birds. Biannual treatments and the combination of ronidazole and dimetridazole at higher doses were associated with lower infection rates than monotherapies or annual treatments. No significant associations were found between resistance and environmental or loft management parameters, although poor hygiene and high bird density were common in lofts with resistant strains. These findings highlight the urgent need for regulated treatment protocols, improved biosecurity, and the development of alternative trichomonacidal agents to combat the emergence of drug-resistant T. gallinae in pigeon populations.

Haemosporida is a diverse order of vector-borne apicomplexan parasites infecting terrestrial vertebrates worldwide, including humans, but the evolutionary relationships among its genera remain unresolved. The phylogenetic placement of two bat-restricted genera, Nycteria and Polychromophilus, both of which lack erythrocytic schizogony, has varied across studies depending on taxon sampling and marker choice. To address this problem, an expanded dataset of near-complete mitochondrial (mtDNA) genomes together with nine nuclear loci were analyzed. Phylogenetic analyses of mtDNA recovered Nycteria and Polychromophilus as a strongly supported monophyletic clade. In contrast, analyses based only on the three mitochondrial coding genes (CDS) or a reduced nuclear dataset failed to recover their monophyly and showed low support and extensive topological conflict at deeper nodes. These results indicate that near-complete mitochondrial genomes recover phylogenetic signal that is not captured by reduced mitochondrial coding sequences or partial nuclear datasets. Molecular dating analyses further showed that divergence estimates for a putative Nycteria-Polychromophilus clade are compatible with the proposed times for bats diversification, and consistent with the broader haemosporidian timescale. When the Nycteria-Polychromophilus clade was incorporated as a calibration prior, divergence-time estimates became more precise without altering the overall evolutionary timeframe. Substantial mitochondrial gene-order rearrangements in a distinct Nycteria lineage were confirmed, highlighting structural divergence within this bat-associated group. In addition, heterogeneity in rates across mtDNA haemosporidian lineages was observed. Together, these findings support the existence of a distinct bat-associated clade whose deeper placement and evolutionary significance should be tested with broader phylogenomic sampling. Author SummaryMalaria parasites belong to a diverse group of organisms that infect many kinds of vertebrates, including birds, reptiles, and mammals (such as humans). Understanding how these parasites are related to each other is important for explaining how key biological traits have evolved. However, the relationships among major groups of haemosporidian parasites, including malaria parasites, remain unclear, particularly for those infecting bats. In this study, we focused on two groups of bat parasites, Nycteria and Polychromophilus, which share unusual biological features. The inferred evolutionary relationships of these two genera to other haemosporidians have been inconsistent across previous studies. By analyzing near-complete mitochondrial genomes, we found strong evidence that these two groups descended from a common evolutionary ancestor. In contrast, smaller datasets including nuclear genes failed to recover this relationship and produced conflicting results, suggesting that they lack sufficient information to resolve deep evolutionary relationships. We also found that this bat-associated lineage likely originated around the same time as early bats. In addition, we identified structural changes in the mitochondrial genome of one lineage, highlighting its evolutionary distinctiveness. Together, our results suggest that bats host a unique group of malaria parasites and demonstrate that more complete genetic data are essential for resolving their evolutionary history.

BackgroundLeishmania donovani (LD) is an obligate intracellular parasite that survives and replicates within macrophages. Leptomonas seymouri (LS), a traditionally monoxenous trypanosomatid, has been repeatedly co-isolated with LD from visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients in India, often together with Leptomonas seymouri narna-like virus 1 (Lepsey NLV1). Whether LS can survive and replicate within mammalian macrophages, and how co-infection influences parasite and viral dynamics, remains unresolved. Methods and FindingsUsing murine (RAW 264.7) and human (THP-1) macrophages, we systematically evaluated intracellular survival, replication, and revival of LS alone and during co-infection with LD. Quantitative ITS1 PCR demonstrated significant increases in intracellular parasite DNA over 48-168 h post-infection, with mean intracellular loads rising up to [~]7.6-fold, indicating active replication rather than persistence. Reduced extracellular parasite load suggested restricted cell lysis and enhanced macrophage survival in co-infection compared to mono-infections. In co-infection scenario, generally LS displayed higher persistence compared to LD. Giemsa staining confirmed intracellular localization of LS. Parasites recovered from infected macrophages remained viable and revived as motile promastigotes, whereas extracellular parasites failed to survive beyond 48 h, confirming macrophages as the exclusive niche for prolonged viability. Interestingly, co-infection dampened macrophage IL-12 production, suggesting altered host immune activation. Lepsey NLV1 RNA accumulated predominantly within macrophages and persisted up to 168 h post-infection. Virus load within LS in co-infection state was [~]2.5-4 and [~]7.5-23 fold higher (for RAW 264.7 and THP-1 respectively) compared to LS mono-infection. Purified virus alone failed to enter macrophages, indicating LS-dependent viral delivery. ConclusionsOur findings question the prevailing view of LS as a strictly non-infective parasite, demonstrating its capacity to replicate within mammalian macrophages and persist during mono-or LD co-infection. The identification of a stable LD-LS-virus interaction highlights a previously underappreciated "triple-pathogen" biology with potential implications for VL and PKDL pathogenesis.

Microsporidia sp. MB, a microsporidian symbiont found naturally in Anopheles mosquitoes, has potential as a novel malaria control tool since it can inhibit Plasmodium development and transmission. The most feasible MB-based Plasmodium control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught An. gambiae s.l from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the An. coluzzii host was examined using a novel duplex qPCR assay. We also examined the impact of MB on fecundity through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Three An. coluzzii colonies and one An. gambiae s.l hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in fecundity. Establishment of several stable MB carrying An. gambiae s.l colonies and understanding the impact of spores on fecundity are significant steps forward in developing MB as a malaria control tool.

Background Urogenital schistosomiasis (UGS), caused by Schistosoma haematobium (S. haematobium), disproportionately affects women in sub-Saharan Africa and can lead to haematuria, anaemia, and urinary tract morbidity. Data on the prevalence in women of reproductive age remains limited in contrast to infection among school-aged children in Kenya. This study assessed the prevalence of UGS and its socioeconomic determinants among women in Kilifi County, Kenya. Methods: Urine samples (20-50 mL) were collected from each participant over three consecutive days. Day-one samples were tested for haematuria, proteinuria, nitrites, leukocytes, and pregnancy using dipsticks. On the other hand, 10 mL of urine was examined for S. haematobium eggs via urine filtration on all three days. Results: A total of 599 women aged 15-50 years were enrolled, with complete data available for 336. The mean age was 33 years; 57.7% were <35 years. Most participants were from rural Magarini Sub-county (63%) and engaged in crop farming (62.5%). Primary education was the highest level attained by 59.8% of participants. Frequent contact with stagnant water was reported by 92%. The overall prevalence of S. haematobium infection was 13.7% (95% CI: 10.2-17.8), higher in Magarini (14.9%) than in Rabai (12.0%), though not statistically significant. Younger age, primary education, and frequent water contact were associated with higher infection rates; however, after adjustment for covariates, haematuria showed the strongest independent association with infection. Women with haematuria were 25.2 times more likely to be infected (OR: 25.24, 95% CI: 7.07-82.63, p < 0.001); multivariate analysis confirmed haematuria as the sole significant predictor (OR: 20.83, 95% CI: 5.45-79.57, p < 0.001). Conclusion: UGS prevalence among women in Kilifi County is substantial, with variation between sub-counties. Haematuria demonstrated the strongest independent association with infection and may serve as a simple, non-invasive diagnostic marker. These findings underscore the pressing need for the integration of UGS screening into the reproductive health services and targeted interventions. Authors Summary UGS, caused by the parasitic worm Schistosoma haematobium, is a neglected tropical disease and remains a major public health problem in sub-Saharan Africa. Although control programmes in Kenya primarily target school-aged children, women of reproductive age are frequently exposed through daily water contact and may develop chronic urinary and reproductive health complications. However, data on the infection burden among adult women are limited. In this study, we assessed the prevalence of urogenital schistosomiasis and associated risk factors among women aged 15-50 years in Kilifi County, Kenya. Urine samples were collected over three consecutive days and examined for parasite eggs and indicators of urinary tract disease. We found that urogenital schistosomiasis affected more than one in ten women in the rural sub-counties where the study was conducted. Haematuria was strongly associated with infection and remained the most reliable predictor after accounting for other social and behavioural factors. These findings demonstrate that UGS is an under-recognised health issue among women and highlight the potential value of simple urine-based screening tools. Integrating UGS screening into existing reproductive health services could improve early detection and contribute to more inclusive disease control strategies.

Root-knot nematodes (RKN; Meloidogyne spp.) are among the most damaging plant-parasitic nematodes worldwide and present a growing threat to global food production as their geographical ranges expand. Despite their importance, the geographical distributions, proliferation risks and crop-level impacts of major RKN species remain poorly understood. Here we assess global habitat suitability for six economically significant or regulated tropical RKN species: M. arenaria, M. incognita, M. javanica, M. enterolobii, M. ethiopica and M. luci. We integrated statistical species distribution models (SDMs), mechanistic thermal-time development models and experimentally derived host-suitability metrics. Ensemble SDMs, parameterised using global occurrence records and soil-derived bioclimatic variables, indicate broad climatically suitable ranges for the four well-represented species (M. arenaria, M. incognita, M. javanica and M. enterolobii) across much of the tropics, subtropics and warm temperate regions. Suitability was lower in cooler northern latitudes and, to a degree, in the humid tropics. Predictions for M. ethiopica and M. luci remain uncertain due to limited occurrence data. Thermal-time phenological models, built from published estimates of base temperatures and Growing Degree-Day (GDD) requirements, revealed a strong latitudinal gradient in potential generation number, with life-cycle completion unlikely at higher latitudes. The risk to 26 globally important crops was evaluated by combining crop-specific reproduction factor and gall index data with global crop distribution maps, indicating highest potential impacts in southern Brazil, the central United States, parts of West and East Africa, eastern India and northern China. Together, our analyses show that most global agricultural land is suitable for the focal species establishment and proliferation, with risk shaped jointly by climate, soil conditions and crop hosts. Strengthening distributional surveys and experimentally quantifying species-specific thermal and host responses will be essential to anticipate and mitigate future threats from these crop pests under climate change.

Phlebotomine sand flies are major vectors of Leishmania parasites, yet the mechanisms underlying their blood-feeding behavior remain poorly understood. In Lutzomyia longipalpis, the primary vector of Leishmania infantum in the Americas, feeding occurs via telmophagy, a pool-feeding method which is known by involving dermal laceration, salivation, and the creation of a blood pool. While the biochemical effects of sand fly saliva on host hemostasis, inflammation, and immunity are well studied, the dynamics of mouthpart movements and saliva at the feeding site remain to be systematically explored. Using intravital microscopy, fluorescent saliva labelling and image analysis, we characterized the mechanical actions of mouthparts and the spatial-temporal patterns of salivation during feeding on mammalian skin. Our recordings indicate that the labrum and hypopharynx are the most prominent mouthparts during feeding and exhibit scissor-like movements during probing. At specific moments, these structures close forcefully, generating small blood splashes in multiple directions. Feeding occurred in two distinct phases: an initial probing phase, often distinguished by ineffective blood intake, and a subsequent engorgement phase that was initiated exclusively upon the activation of small dermal "feeder vessels."Acridine Orange labelling showed abundant early salivation that penetrated progressively deeper into the dermis and remained detectable for over an hour, reflecting both the tissue damage and enzymatic effects. The analysis of images demonstrated the sequential salivation events, highlighting an initial high-frequency phase followed by a more gradual pattern during engorgement. These findings provide the first real-time, detailed view of the coordinated interactions between mouthpart mechanics, targeted salivation, and host microvascular responses in Lu. longipalpis. This study redefines sand fly telmophagy as a non-passive and coordinated process integrating mouthpart mechanics, salivation, and modulation of host vasculature. This work advances our understanding of sand fly vector-host interactions and underscores the potential of salivary molecules as targets for transmission-blocking strategies. Author SummaryPhlebotomine sand flies are the main vectors of Leishmania infantum, the parasite responsible for visceral leishmaniasis in the Americas. Although sand flies are traditionally classified as "pool feeders," meaning they lacerate the skin and feed from small pools of blood, the mechanics of how they obtain blood and deliver saliva into host skin have remained poorly understood. In this study, we used image analysis, intravital microscopy and fluorescent labeling of saliva to visualize, in real time, the feeding behavior of Lutzomyia longipalpis on mammalian skin. We show that blood feeding is not a passive process based solely on blood pooling. Instead, it involves coordinated movements of the mouthparts, modulation of host microvessels with the saliva contribution, and the recruitment of small dermal "feeder vessels" that supply blood directly to the insect. Our findings reveal that sand fly feeding is a highly orchestrated interaction between vector and host, integrating mechanical tissue disruption, salivary secretion, and vascular responses. These processes likely create a favorable microenvironment for Leishmania establishment and transmission. By providing a detailed characterization of mouthpart and salivation dynamics, this study advances our understanding of sand fly biology and highlights salivary components and feeding-site events as potential targets for transmission-blocking strategies.

Helminths are parasitic worms that secrete a variety of immune-regulating molecules to modulate the hosts inflammatory responses, enabling them to persist within the host over a long period of time, even decades. Their capacity to control host responses has prompted research into helminth-derived molecules as potential therapies for controlling excessive immune and inflammatory activity across a range of diseases. This systematic review with cross-study quantitative analysis aims to synthesize the published data on helminth-derived immunomodulatory peptides/polypeptides/proteins (HDIPs) with a focus on determining the extent of their disease-modifying and anti-inflammatory potential in in vivo animal models of inflammatory disease. In accordance with PRISMA 2020 guidelines, a predefined systematic search of the PubMed, Web of Science and Medline databases identified relevant studies published up to February 2026, and 65 articles were included after screening. We found that, although the HDIPs were assessed in multiple different disease models, most published studies assessed their potential in mouse models of colitis, asthma, arthritis and sepsis. Twenty species from which >65 isolated HDIPs were derived were tested in these models, with the trematode, Fasciola hepatica, and the nematode, Acanthocheilonema viteae, the most explored species. A common property of the HDIPs was the ability to significantly reduce disease severity across the in vivo animal models of inflammatory disease, underpinned by a decrease in pro-inflammatory cytokine levels and an increase in anti-inflammatory cytokine levels. Overall, this systematic review with cross-study quantitative analysis not only synthesizes the existing literature in this field but also highlights the disease-modifying and anti-inflammatory potential of HDIPs for a range of diseases in which immunoregulatory therapeutics may improve disease outcomes. It also encourages accelerated advancement of these helminth-derived molecules into first-in-human clinical trials.

Cochliomyia hominivorax, New world Screwworm (NWS), has become a reemerging veterinary concern in the United States due to the recent northward expansion of fly detections as far as northern Mexico. Rapid, accurate and validated detection pipelines need to be developed in the case of an incursion into the United States. Confirmatory cases are evaluated by morphological identification with no paired test to verify identifications. With the frequency of submissions of non-ideal samples, particularly from fly traps, a molecular tool would be necessary for species identification. In this manuscript, we develop and assess a pipeline including three real-time PCR assays targeting the ribosomal RNA and five sets of Sanger primers targeting the mitochondrial genome that would be used as a paired tool with morphological identification. Two of the assessed real-time PCR assays are highly specific, sensitive and repeatable requiring <1 copy per reaction for detection. Four of the five Sanger primer sets were assessed, optimized and results evaluated for potential use in preliminary geographic analysis of specimens. This workflow will expedite screening of samples, provide a method to verify results using different tools and help understand genetic variations within the mitochondria for NWS outbreaks.

Plasmodium vivax transmission from humans to mosquitoes depends on the density of gametocytes that in turn depends on asexual parasite replication and gametocyte commitment. These processes are often analyzed separately, despite being biologically linked and measured with substantial uncertainty. We used a joint Bayesian latent-variable model to simultaneously analyze parasite density, gametocyte density, and mosquito infectivity while accounting for measurement error and propagating uncertainty across linked processes. The model was applied to individual-level data from three P. vivax transmission studies conducted in Ethiopia (n = 455). A tenfold increase in gametocyte density was associated with more than a twofold increase in the odds of mosquito infection (odds ratio [OR] = 2.32, 95% credible interval [CrI]: 2.12-2.54). Asexual parasite density was also positively associated with infectivity after accounting for gametocyte density (OR = 1.74, 95% CrI: 1.60-1.90), and inclusion of parasite density improved predictive performance. Pathway decomposition within the joint model indicated that approximately 41% of the parasite-infectivity association operated through gametocyte density. Increasing age was associated with lower asexual parasite density but higher gametocyte density, resulting in minimal overall association with infectivity. Predicted infection probability increased sigmoidally with gametocyte density, remaining low at lower densities before increasing sharply and approaching a plateau at higher densities. Gametocyte density produced the largest predicted changes in the proportion of infected mosquitoes, while asexual parasite density added predictive information not fully captured by measured gametocyte density alone. This approach links molecular parasite measurements with mosquito infection risk while accounting for measurement uncertainty and provides an interpretable framework for studying the P. vivax infectious reservoir. Author SummaryMalaria transmission occurs when mosquitoes ingest sexual-stage parasites, called gametocytes, during a blood meal. In Plasmodium vivax infections, human-to-mosquito transmission depends on linked biological stages, including asexual parasite replication, gametocyte production, and mosquito infection. These processes are closely connected and often measured with uncertainty, making them difficult to study using standard approaches that analyze them separately. In this study, we applied a joint Bayesian model that analyzes parasite density, gametocyte density, and mosquito infectivity together while accounting for uncertainty in laboratory measurements. Using data from three studies in Ethiopia, we quantified how parasite density, gametocyte density, and host characteristics relate to mosquito infection. The analysis showed that measured gametocyte density alone did not fully explain variation in infectivity, and that asexual parasite density provided additional predictive information. We also found that age was associated differently with asexual parasite and gametocyte densities, resulting in little overall association with infectivity. This approach helps link molecular parasite measurements with transmission outcomes and improves understanding of the P. vivax infectious reservoir in endemic settings.