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Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis' gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

Background Snails of the Lymnaeidae family are the intermediate hosts of Fasciola species, the causative agents of fascioliasis. The purpose of this study was to determine the prevalence of Fasciola species in lymnaeid snails and to investigate the association of geoclimatic factors and Fasciola species distribution in northwestern provinces of Iran using geographical information system (GIS) data. Methods A total of 2000 lymnaeid snails were collected from 33 permanent and seasonal habitats in northwestern Iran during the period from June to November 2021. After identification by standard morphological keys, they were subjected to shedding and crushing methods. Different stages of Fasciola obtained from these snails were subjected to the ITS1 polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method for species identification. The associations of weather temperature, rainfall, humidity, evaporation, air pressure, wind speed, elevation, and land cover with the distribution of Fasciola species were investigated. Geographical and statistical analysis was performed using ArcMap and SPSS software, respectively, to determine factors related to Fasciola species distribution. Results Of the 2000 snails collected, 19 were infected with Fasciola hepatica (0.09%), six with F. gigantica (0.03%), and 13 with other trematodes. Among geoclimatic and environmental factors, mean humidity, maximum humidity, and wind speed were significantly higher in areas where F. hepatica was more common than F. gigantica . The altitude of F. hepatica -prevalent areas was generally lower than F. gigantica areas. No significant relationship was observed between other investigated geoclimatic factors and the distribution of infected snails. Conclusions The present study showed the relationship of humidity and wind speed with the distribution of snails infected with F. hepatica or F. gigantica in the northwestern regions of Iran. In contrast to F. gigantica , F. hepatica was more prevalent in low-altitude areas. Further research is recommended to elucidate the relationship between geoclimatic factors and the presence of intermediate hosts of the two Fasciola species. Graphical Abstract

Infection by the liver fluke, Fasciola hepatica, places a substantial burden on the global agri-food industry and poses a significant threat to human health in endemic regions. Widespread resistance to a limited arsenal of chemotherapeutics, including the frontline flukicide triclabendazole (TCBZ), renders F. hepatica control unsustainable and accentuates the need for novel therapeutic target discovery. A key facet of F. hepatica biology is a population of specialised stem cells which drive growth and development - their dysregulation is hypothesised to represent an appealing avenue for control. The exploitation of this system as a therapeutic target is impeded by a lack of understanding of the molecular mechanisms underpinning F. hepatica growth and development. Wnt signalling pathways govern a myriad of stem cell processes during embryogenesis and drive tumorigenesis in adult tissues in animals. Here, we identify five putative Wnt ligands and five Frizzled receptors in liver fluke transcriptomic datasets and find that Wnt/β-catenin signalling is most active in juveniles, the most pathogenic life stage. FISH-mediated transcript localisation revealed partitioning of the five Wnt ligands, with each displaying a distinct expression pattern, consistent with each Wnt regulating the development of different cell/tissue types. The silencing of each individual Wnt or Frizzled gene yielded significant reductions in juvenile worm growth and, in select cases, blunted the proliferation of neoblast-like cells. Notably, silencing FhCTNNB1, the key effector of the Wnt/β-catenin signal cascade led to aberrant development of the neuromuscular system which ultimately proved lethal - the first report of a lethal RNAi-induced phenotype in F. hepatica. The absence of any discernible phenotypes following the silencing of the inhibitory Wnt/β-catenin destruction complex components is consistent with low destruction complex activity in rapidly developing juvenile worms, corroborates transcriptomic expression profiles and underscores the importance of Wnt signalling as a key molecular driver of growth and development in early-stage juvenile fluke. The putative pharmacological inhibition of Wnt/β-catenin signalling using commercially available inhibitors phenocopied RNAi results and provides impetus for drug repurposing. Taken together, these data functionally and chemically validate the targeting of Wnt signalling as a novel strategy to undermine the pathogenicity of juvenile F. hepatica.

The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually. Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs. The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

Fasciolosis is a parasitic disease affecting humans and livestock, caused by digenean trematodes of the genus Fasciola , primarily F. hepatica and F. gigantica . This study investigates the coexistence of these species and their hybrids in yaks from Tibet, China. We analyzed the nuclear rDNA internal transcribed spacer (ITS) regions, including ITS1 and ITS2, through Sanger sequencing and Next-Generation Sequencing (NGS) to assess single-nucleotide polymorphisms (SNPs). Our results reveal that one specimen (NM008B) is identical to pure F. hepatica , while another (NM008A) contains genetic information from both F. hepatica and F. gigantica , indicating potential hybridization or introgression. The morphological analysis reveals that the collected adult F. hepatica specimens exhibit distinct characteristics, while the hybrid specimens display “intermediate” features of F. hepatica and F. gigantica . This study is the first to document the coexistence of F. hepatica and hybrid Fasciola forms in a single yak. The findings underscore the complexities of hybridization dynamics and the necessity for advanced molecular techniques in accurately identifying Fasciola species. Future research should focus on mitochondrial DNA and other nuclear gene analysis to further elucidate the nature of these hybrids and their ecological implications.

Triclabendazole (TCBZ) is the primary treatment for fascioliasis, a global foodborne zoonosis caused by Fasciola hepatica . Widespread resistance to TCBZ (TCBZ-R) in livestock and a rapid rise in resistant human infections are significant concerns. To understand the genetic basis of TCBZ-R, we sequenced the genomes of 99 TCBZ-sensitive (TCBZ-S) and 210 TCBZ-R adult flukes from 146 bovine livers in Cusco, Peru. We identify genomic regions of high differentiation ( F ST outliers above the 99.9th percentile) that encod genes involved in the EGFR-PI3K-mTOR-S6K pathway and microtubule function. Transcript expression differences are observed in microtubule-related genes between TCBZ-S and -R flukes, both without drug treatment and in response to treatment. Using only 30 SNPs, it is possible to differentiate between TCBZ-S and -R parasites with ≥75% accuracy. Our outlier loci are distinct from the previously reported TCBZ-R-associated QTLs in the UK, suggesting an independent evolution of resistance alleles. Effective genetics-based TCBZ-R surveillance must consider the heterogeneity of loci under selection across diverse geographical populations. The study reveals independent evolution of triclabendazole resistance in Fasciola hepatica in Peru and the UK, with distinct genetic signatures. It identifies novel resistance genes and underscores the need for geo-specific genetic surveillance.

Before the 1990s, human fascioliasis diagnosis focused on individual patients in hospitals or health centres. Case reports were mainly from developed countries and usually concerned isolated human infection in animal endemic areas. From the mid-1990s onwards, due to the progressive description of human endemic areas and human infection reports in developing countries, but also new knowledge on clinical manifestations and pathology, new situations, hitherto neglected, entered in the global scenario. Human fascioliasis has proved to be pronouncedly more heterogeneous than previously thought, including different transmission patterns and epidemiological situations. Stool and blood techniques, the main tools for diagnosis in humans, have been improved for both patient and survey diagnosis. Present availabilities for human diagnosis are reviewed focusing on advantages and weaknesses, sample management, egg differentiation, qualitative and quantitative diagnosis, antibody and antigen detection, post-treatment monitoring and post-control surveillance. Main conclusions refer to the pronounced difficulties of diagnosing fascioliasis in humans given the different infection phases and parasite migration capacities, clinical heterogeneity, immunological complexity, different epidemiological situations and transmission patterns, the lack of a diagnostic technique covering all needs and situations, and the advisability for a combined use of different techniques, at least including a stool technique and a blood technique.

Background The liver fluke Fasciola hepatica is one of the most important endoparasites in domestic ruminants worldwide and can cause considerable economic losses. This study presents the first population genetic analysis of F. hepatica in Germany and aims at providing new insights into genetic diversity and population structure. Methods A total of 774 liver flukes, collected from 60 cows of 17 herds and 13 cows of unknown herd origin, were subjected to comparative analysis of two mitochondrial genes ( cox1 and nad1 ), one nuclear region (internal transcribed spacer (ITS)-1) and eight nuclear microsatellite markers. In addition, individual fluke measurements allowed comparison of morphometric differences between genotypes. Results The nuclear ITS-1 region showed minimal variability, with 772 of 774 flukes having identical sequences, while the mitochondrial sequences revealed a high genetic diversity, with 119 distinct haplotypes, a mean haplotype diversity (Hd) of 0.81 and a mean nucleotide diversity ( π ) of 0.0041. Mitochondrial phylogenetic analysis identified two clusters with no clear association with the host or farm of origin. In the microsatellite analysis, all eight loci were highly polymorphic, with a mean allele frequency of 19.0 and a mean genotype frequency of 73.5 per locus. A total of 500 unique multilocus genotypes (MLGs) were found across all fluke samples, indicating that 68.5% of all genotypes were unique. A mean expected heterozygosity of 0.71 suggested a high potential for adaptability and the number of migrants (Nm = 3.5) indicated high gene flow between farms. Population structure analysis based on microsatellite data revealed that flukes from two farms differed genetically from the others. Linear mixed model results revealed that fluke length differed significantly between the two mitochondrial clusters, although it should be noted that fluke age could not be considered in the analyses. Conclusions Fasciola hepatica in German dairy farms showed high genetic diversity and gene flow. The differences in population structure identified by mitochondrial sequences compared with microsatellite loci highlight the benefits of analysing genetic markers of different origins. This is the first study to correlate fluke morphometry measurements with genetic markers, indicating that the identified markers can influence fluke size. Graphical Abstract

The spatial organization of gene expression dictates tissue functions in multicellular parasites. Here, we present the spatial transcriptome of a parasitic flatworm, the common liver fluke Fasciola hepatica . We identify gene expression profiles and marker genes for eight distinct tissues and validate the latter by in situ hybridization. To demonstrate the power of our spatial atlas, we focus on genes with substantial medical importance, including vaccine candidates (Ly6 proteins) and drug resistance genes (glutathione S-transferases, ABC transporters). Several of these genes exhibit unique expression patterns, indicating tissue-specific biological functions. Notably, the prioritization of tegumental protein kinases identifies a PKCβ, for which small-molecule targeting causes parasite death. Our comprehensive gene expression map provides unprecedented molecular insights into the organ systems of this complex parasitic organism, serving as a valuable tool for both basic and applied research. Fasciola hepatica is a complex multicellular pathogen and the causative agent of fascioliasis, a zoonotic disease that compromises liver function. Using spatial transcriptomics, the authors uncover the tissue-specific gene expression of F. hepatica and identify genes with expression in the surface layer for drug-target discovery.