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Large areas of the province of Punjab, Pakistan are endemic for fascioliasis, resulting in high economic losses due to livestock infection but also affecting humans directly. The prevalence in livestock varies pronouncedly in space and time (1-70%). Climatic factors influencing fascioliasis presence and potential spread were analysed based on data from five meteorological stations during 1990-2010. Variables such as wet days (Mt), water-budget-based system (Wb-bs) indices and the normalized difference vegetation index (NDVI), were obtained and correlated with geographical distribution, seasonality patterns and the two-decade evolution of fascioliasis in livestock throughout the province. The combined approach by these three indices proved to furnish a useful tool to analyse the complex epidemiology that includes (i) sheep-goats and cattlebuffaloes presenting different immunological responses to fasciolids; (ii) overlap of Fasciola hepatica and F. gigantica; (iii) co-existence of highlands and lowlands in the area studied; and (iv) disease transmission following bi-seasonality with one peak related to natural rainfall and another peak related to man-made irrigation. Results suggest a human infection situation of concern and illustrate how climate and anthropogenic environment modifications influence both geographical distribution and seasonality of fascioliasis risks. Increased fascioliasis risk throughout the Punjab plain and its decrease in the northern highlands of the province became evident during the study period. The high risk in the lowlands is worrying given that Punjab province largely consists of low-altitude, highly irrigated plains. The importance of livestock in this province makes it essential to prioritise adequate control measures. An annual treatment scheme to control the disease is recommended to be applied throughout the whole province.

The diagnosis of a 22 year-old male patient from Kerabari, Morang District, Nepal led to the review of human fascioliasis cases and analysis of the epidemiological situation in that country not included in the WHO fascioliasis map. Symptom onset one month before egg detection and normal levels of ALT and AST did not agree with the 3-4-month migratory period of fascioliasis. A shorter acute phase may happen when the main biliary duct is reached by the migratory juveniles directly from the intestinal lumen. The causal agent was ascribed to [F]. [gigantica]-like worms after considering adult fluke morphology, altitude of the patient’s infection area, fasciolid characteristics in the neighbouring Bangladesh, and lymnaeid snail vector species known in Nepal and in the patient’s infection area. Previous reports of human infection by [Fasciola] in Nepal are reviewed. The patient in question proved to be the twelfth case and the first in whom a [F]. [gigantica]-like infection is reported. In Nepal, the wide geographical distribution of livestock fascioliasis, with high prevalences in buffaloes, cattle and goats, and the reports of [Fasciola]-infected schoolchildren close to the capital Kathmandu, give rise to concern on the situation in remote rural areas in a country where most of the population lives in rural areas. Moreover, the climate change impact in Nepal remembers Pakistan, where human fascioliasis emergence has been related to climate change and man-made irrigation. All in all, the present analysis suggests that human infection by [Fasciola] may be underestimated in Nepal.

We evaluated the genetic diversity of samples identified morphologically as spp. from sheep, cattle and goat from Kermanshah Province, western Iran using PCR-RFLP method. We used PCR-RFLP analysis of ribosomal ITS1 fragment using RsaI restriction enzyme to investigate the genetic characteristics of species obtained from different hosts (16 sheep, 28 cattle, 4 goats). The species of were confirmed by sequencing the 700 bp region of ribosomal ITS1 gene. In Kermanshah, was present in 96% of the samples, was found only in two cattle sample. No hybrid forms were detected in the present study. Our results contribute to clarify the dark spots of genotyping in different parts of Iran.

A total of 12103 snails examined in Central Kashmir for determining the population dynamics of cercariae revealed overall prevalence to be 4.03%. Gymnocephalus (0.13%), furcocercous (0.28%), echinostome (0.34%) and xiphido-cercaria (3.26%) were recorded. The prevalence of cercaria was recorded highest in summer (4.28%) followed by spring (4.05%) and autumn (3.32%). None of the cercaria was recorded during winter season. Morphologically identified cercariae of Veterinary importance were subjected to molecular analysis using genus (28S rDNA and ITS-2) specific primers. The isolates of gymnocephalus cercaria (FA28, FC28, FZ28, FC2) were identified as cercarial stages of Fasciola spp. The phylogenetic trees revealed that the isolates FA28 and FC28 belonged to Fasciola gigantica and FZ28 and FC2 isolate to F. hepatica . The isolate of Fasciola gigantica (FA28, FC28, FZ28) showed 2, 4 and 13 nucleotide polymorphisms. There was addition and deletion of 1 and 8 nucleotides at various positions in case of Fasciola isolates respectively. Besides this, there were nucleotide substitutions at 4 positions along with presence of nucleotide T at 475 position which confirmed it be Fasciola hepatica . The isolates of echinostome cercaria (B1, BD13 and GY) were identified as cercarial stages of Moliniella anceps , Echinoparyphium recurvatum and family Echinostomatidae respectively. The Moliniella anceps isolate showed prominent differences at 8 positions with respect to other Echinostomatidae spp. The insertion of C at position 612 confirmed it to be Moliniella anceps , while as other two isolates showed 2 nucleotide polymorphisms each after 28S gene amplification. On ITS-2 rDNA analysis, the isolate B1 showed 7 nucleotide polymorphisms and phylogenetic tree revealed that the isolate B1, also belonged to Echinoparyphium recurvatum . The study made it very clear that molecular characterization employing internal transcribed spacer (ITS-2) and 28S ribosomal DNA sequences are reliable approach for genetic differentiation of cercarial stages of trematodes. The phylogenetic taxonomy of echinostomes is still unclear and molecular diversity found in this study is perhaps the first study from India as well as in Indian subcontinent. So, focus should be made more on echinostomes for understanding their morphological, biological and molecular diversity for clarifying their taxonomic position.

Liver and intestinal flukes of the family Fasciolidae cause zoonotic food–borne infections that impact both agriculture and human health throughout the world. Their evolutionary history and the genetic basis underlying their phenotypic and ecological diversity are not well understood. To close that knowledge gap, we compared the whole genomes of Fasciola hepatica, Fasciola gigantica, and Fasciolopsis buski and determined that the split between Fasciolopsis and Fasciola took place ∼90 Ma in the late Cretaceous period, and that between 65 and 50 Ma an intermediate host switch and a shift from intestinal to hepatic habitats occurred in the Fasciola lineage. The rapid climatic and ecological changes occurring during this period may have contributed to the adaptive radiation of these flukes. Expansion of cathepsins, fatty-acid-binding proteins, protein disulfide-isomerases, and molecular chaperones in the genus Fasciola highlights the significance of excretory–secretory proteins in these liver-dwelling flukes. Fasciola hepatica and Fasciola gigantica diverged ∼5 Ma near the Miocene–Pliocene boundary that coincides with reduced faunal exchange between Africa and Eurasia. Severe decrease in the effective population size ∼10 ka in Fasciola is consistent with a founder effect associated with its recent global spread through ruminant domestication. G-protein-coupled receptors may have key roles in adaptation of physiology and behavior to new ecological niches. This study has provided novel insights about the genome evolution of these important pathogens, has generated genomic resources to enable development of improved interventions and diagnosis, and has laid a solid foundation for genomic epidemiology to trace drug resistance and to aid surveillance.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica , is a globally significant parasitic disease affecting livestock and humans. Both species exist in South Africa, however, information on their geographical distribution and genetic diversity is limited. This study investigated the genetic diversity and phylogenetic relationships of Fasciola species in South Africa using Pepck, COI and ITS markers. Two hundred and seven Fasciola spp. specimens analysed in this study showed that 74.4% of isolates identified as F. hepatica and 25.6% as F. gigantica based on the Pepck gene. The geographical distribution revealed that F. hepatica occurred in all surveyed provinces but was dominant in the temperate zones of South Africa. Fasciola gigantica was confined to subtropical zones, with co-occurrence observed in Limpopo, Mpumalanga, and KwaZulu-Natal provinces. The COI and ITS genes in both Fasciola species generated fourteen and eight haplotypes respectively, with low to moderate haplotype diversity (Hd = 0.287 – 0.509 for COI and 0.056 – 0.239 for ITS) and low nucleotide diversity (Pi = 0.00085 – 0.00284 for COI and 0.00024 – 0.00034 for ITS), suggesting recent population diversification between the two species. The study samples yielded six haplotypes based on COI, with three haplotypes comprising most samples i.e. Hap-1 ( F. gigantica) and Hap-5 and Hap-6 ( F. hepatica) . With ITS, two haplotypes represented most samples, i.e. Hap-1 ( F. gigantica ) and Hap-3 ( F. hepatica) . There was no observed unique distribution of haplotypes based on provinces or agro-ecological climatic zones for both markers. Phylogenetic analysis of both COI and ITS genes confirmed the grouping of the haplotypes and reiterated their relatedness. However, one isolate identified as F. gigantica in COI and with F. hepatica in ITS based on BLAST and further formed haplotypes with F. gigantica and F. hepatica haplogroups for COI and ITS, respectively. This suspected hybrid Fasciola form was from an animal with a mixed infection. These findings highlight the complex genetic structure of Fasciola spp. populations in South Africa and provide new insights into their epidemiology, with implications for targeted control strategies and further investigation of cryptic/hybrid lineages.

Background The major pathogenesis associated with Fasciola hepatica infection results from the extensive tissue damage caused by the tunnelling and feeding activity of immature flukes during their migration, growth and development in the liver. This is compounded by the pathology caused by host innate and adaptive immune responses that struggle to simultaneously counter infection and repair tissue damage. Results Complementary transcriptomic and proteomic approaches defined the F. hepatica factors associated with their migration in the liver, and the resulting immune-pathogenesis. Immature liver-stage flukes express ~ 8000 transcripts that are enriched for transcription and translation processes reflective of intensive protein production and signal transduction pathways. Key pathways that regulate neoblast/pluripotent cells, including the PI3K-Akt signalling pathway, are particularly dominant and emphasise the importance of neoblast-like cells for the parasite’s rapid development. The liver-stage parasites display different secretome profiles, reflecting their distinct niche within the host, and supports the view that cathepsin peptidases, cathepsin peptidase inhibitors, saposins and leucine aminopeptidases play a central role in the parasite’s destructive migration, and digestion of host tissue and blood. Immature flukes are also primed for countering immune attack by secreting immunomodulating fatty acid binding proteins (FABP) and helminth defence molecules (FhHDM). Combined with published host microarray data, our results suggest that considerable immune cell infiltration and subsequent fibrosis of the liver tissue exacerbates oxidative stress within parenchyma that compels the expression of a range of antioxidant molecules within both host and parasite. Conclusions The migration of immature F. hepatica parasites within the liver is associated with an increase in protein production, expression of signalling pathways and neoblast proliferation that drive their rapid growth and development. The secretion of a defined set of molecules, particularly cathepsin L peptidases, peptidase-inhibitors, saponins, immune-regulators and antioxidants allow the parasite to negotiate the liver micro-environment, immune attack and increasing levels of oxidative stress. This data contributes to the growing F. hepatica -omics information that can be exploited to understand parasite development more fully and for the design of novel control strategies to prevent host liver tissue destruction and pathology.

Fascioliasis is a common parasitic disease in livestock in China. However, human fascioliasis is rarely reported in the country. Here we describe an outbreak of human fascioliasis in Yunnan province. We reviewed the complete clinical records of 29 patients and performed an epidemiological investigation on the general human population and animals in the outbreak locality. Our findings support an outbreak due to Fasciola gigantica with a peak in late November, 2011. The most common symptoms were remittent fever, epigastric tenderness, and hepatalgia. Eosinophilia and tunnel-like lesions in ultrasound imaging in the liver were also commonly seen. Significant improvement of patients' condition was achieved by administration of triclabendazole®. Fasciola spp. were discovered in local cattle (28.6%) and goats (26.0%). Molecular evidence showed a coexistence of F. gigantica and F. hepatica. However, all eggs seen in humans were confirmed to be F. gigantica. Herb (Houttuynia cordata) was most likely the source of infections. Our findings indicate that human fascioliasis is a neglected disease in China. The distribution of triclabendazole®, the only efficacious drug against human fascioliasis, should be promoted.

Fasciola spp. infection is a significant zoonotic disease. Fasciola gigantica cathepsin L1H (FgCathL1H) is expressed across the life stages of Fasciola gigantica : newly excysted juvenile (NEJ), juvenile, and adult. An emerging tool for diagnosing fasciolosis in humans and cattle involves single-chain variable fragments (scFv) antibodies. These antibodies, consisting of linked variable regions of heavy chains (VHs) and light chains (VLs), retain binding specificity and affinity. This study aims to construct, express, and characterize an scFv antibody for use in a diagnostic kit for fasciolosis. The study successfully constructed and expressed recombinant scFv antibody genes derived from mouse spleen cells in Escherichia coli HB2151. Specific VH and VL fragments targeting recombinant FgCathL1H were amplified, inserted into a phagemid vector (pCANTAB5E), and transformed into E. coli TG1. Infection with the M13KO7 helper phage produced recombinant phages, and scFv clones with a high binding capacity were selected through three rounds of bio-panning. The expression of scFv proteins was induced with 1 mM IPTG, yielding antibodies detectable in the culture supernatant and periplasmic space. The indirect ELISA revealed strong binding in 10 scFv phage clones, which were sequenced and analyzed via computer-guided homology modeling and showed a similar classification to CDR1–3, consisting of VHs and VLs. The scFv DNA construct was approximately 747 bp in length. The SDS-PAGE, ELISA, and western blot confirmed the specificity of the scFv clone 1B, particularly at ~ 29 kDa. Docking studies showed epitopes on the scFv interacting with FgCathL1H. This scFv reacted specifically with F. gigantica antigens at 36 kDa (whole body (WB) of metacercaria and NEJ) and ~ 28 kDa (WB of 4-week-old juveniles and adults, and adult excretory–secretory protein (ES)). Immunolocalization showed positive staining in the cecal epithelium. Thus, scFv anti-rFgCathL1H shows promise for diagnosing fasciolosis.

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis , with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.