Newly excysted juveniles (NEJs) of Fasciola gigantica induce mice liver fibrosis and M2 macrophage-like phenotype in vivo.

Liver flukes of animals are parasitic flatworms of major socioeconomic importance in many countries. Particularly, Fasciola gigantica is a leading cause of production losses to the livestock (mainly sheep and cattle) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Immune responses induced by helminth have been extensively studied, but there is limited information on this aspect by F. gigantica, especially on macrophages induced with this parasite. Studies have shown that host immune responses induced by parasitic infection is greatly correlated with the macrophage polarization axis. In the present study, we used the murine model of F. gigantica to explore the interaction of host and F. gigantica. We found F. gigantica NEJs promoted pathology and fibrosis of mice liver, and the enlargement of mice spleen. We also showed that macrophages were recruited to mice peritoneal cavity at 5 days post infection. By evaluating the expression of genetic markers of M2 macrophages such as Arg-1, Ym1 and RELMɑ, and genetic marker of M1 macrophages iNOS, we showed that M2 macrophages were induced by F. gigantica. M2 macrophages are central to the immune response during helminth infection, and our findings in this study provided insight into the immune interaction between F. gigantica and host.

First report of pre-Hispanic Fasciola hepatica from South America revealed by ancient DNA.

It is generally assumed that the digenean human liver fluke, Fasciola hepatica, gained entry to South America during the 15th century upon arrival of Europeans and their livestock. Nonetheless in Patagonia, Argentina, digenean eggs similar to F. hepatica have been observed in deer coprolites dating back to 2300 years B.P. The main objective of our present study was to identify and characterize these eggs using an ancient DNA (aDNA) study. Eggs were isolated and used for aDNA extraction, amplification and sequencing of partial regions from the cytochrome c oxidase subunit 1 and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 mitochondrial genes. Also, phylogenetic trees were constructed using Bayesian and maximum likelihood. Our results confirm the presence of F. hepatica in South America from at least 2300 years B.P. This is the first report and the first aDNA study of this trematode in South America prior to the arrival of the European cattle in the 15th century. The present work contributes to the study of phylogenetic and palaeobiogeographical aspects of F. hepatica and its settlement across America.

Proteomics and bioinformatics analysis of Fasciola hepatica somatic proteome in different growth phases.

Fasciola hepatica (F. hepatica) is a well-known zoonotic parasite that is crucial for economic and public health worldwide. Quantitative proteomics studies have been performed on proteins expressed by F. hepatica to investigate the differential expression of proteomes in different growth phases. And the screening of several marker proteins for use as early diagnostic antigens is essential. In this study, high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to analyze the differences in the expression of F. hepatica somatic proteins in different growth phases. Furthermore, gene ontology (GO) functional annotation, KEGG metabolic pathway, and clustering analyses were also performed. LC-MS/MS identified 629, 2286, 2254, and 2192 proteins in metacercariae, juvenile flukes 28dpi, immature flukes 59dpi, and adult phases, respectively. GO analysis revealed that differentially expressed proteins (DEPs) were mainly involved in transport, localization, metabolism, enzyme regulation, protein folding and binding, and nucleoside and nucleotide binding. The DEPs were enriched in cells, intracellular components, organelles, cytoplasm, vesicles, and membranes. KEGG pathway annotation results showed that the DEPs were involved in metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and other processes. These findings provide a theoretical basis for vaccine development and establishing early diagnostic methods in the future.

Novel triclabendazole prodrug: A highly water soluble alternative for the treatment of fasciolosis.

In this work we present the synthesis, aqueous solubility and stability, hydrolysis by alkaline phosphatase, and in vivo fasciolicidal activity in sheep of a highly water soluble phosphate salt prodrug of triclabendazole (MFR-5). The aqueous solubility of MFR-5 at pH 7 was 88,000-fold that of triclabendazole. MFR-5 showed excellent aqueous stability (>95% after 26h) at pH 7, making it ideal for developing pharmaceutical compositions in the form of solutions that can easily be hydrolyzed by the enzyme alkaline phosphatase (t=13.6s) to liberate the precursor compound. An aqueous solution of MFR-5 administered intramuscularly to sheep at concentrations of 4, 6 and 8mg/kg presented a fasciolicidal efficiency of 96.5%, 98.4% and 99.2%, respectively. In the in vivo experiments, MFR-5 reduced 100% the excretion of eggs in all of the above concentrations.

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Liver fluke infection and cholangiocarcinoma: a review.

Parasites are significant groups for carcinogenesis among which liver flukes, including Opisthorchis viverrini and Clonorchis sinensis, are typical representatives causing cholangiocarcinoma (CCA), the second most common primary hepatic malignancy with dismal prognosis. O. viverrini is prevalent in Southeast Asia, infecting 10 million people while C. sinensis has a wider distribution in East Asia and several Southeast Asian countries, affecting more than 35 million people's health. These two worms have some common characteristics and/or discrepancies in life cycle, genome, and transcriptome. As hot spots in recent years, genome and transcriptome research has extracted numerous novel fluke worm-derived proteins, which are excellent for carcinogenic exploration. However, just a handful of these studies have focused on the metabolic pathway. In this study, the main mechanisms of carcinogenesis of both worms, in terms of mechanical damage, metabolic products and immunopathology, and other possible pathways, will be discussed in detail. This review retrospectively describes the main traits of C. sinensis and O. viverrini, their molecular biology and core carcinogenic mechanisms in a contrast pattern.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

Controversial aspects of the life cycle of Fasciola hepatica.

Fasciola hepatica is a well-known helminth parasite, with significant economic and public health importance all over the world. It has been known since more than 630 years ago and a considerable research work has been carried out on the life cycle of this important parasite. In the hepatic phase of the life cycle of F. hepatica, it is assumed that the young flukes, after about 6-7 weeks of migration in the liver parenchyma, enter into the bile ducts of the definitive hosts and become sexually mature. Even though the secretion of cysteine peptidases including cathepsin L and B proteases by F. hepatica may justify this opinion, because of several scientific reasons and based on the experimental studies conducted in different animals (reviewed in this article), the entry of parasites into the bile ducts, after their migration in the liver parenchyma seems to be doubtful. However, considering all the facts relating to the hepatic and biliary phases of the life cycle of F. hepatica, two alternative ideas are suggested: 1) some of the migrating juvenile flukes may enter into the bile ducts immediately after reaching the liver parenchyma while they are still very small, or 2) when newly excysted juvenile flukes are penetrating into the intestinal wall to reach the liver through the abdominal cavity, a number of these flukes may enter into the choleduct and reach the hepatic bile ducts, where they mature. According to the previously performed natural and experimental studies in different animals and human beings, the supporting and opposing evidences for the current opinion as well as the evidences that might justify the two new ideas are reviewed and discussed briefly. In conclusion, our present knowledge about the time and quality of the entry of F. hepaticas into the bile ducts, seems to be insufficient, therefore, there are still some dark corners and unknown aspects in this field that should be clarified.

In vivo assessment of closantel ovicidal activity in Fasciola hepatica eggs.

Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.

Aptitude of Lymnaea palustris and L. stagnalis to Fasciola hepatica larval development through the infection of several successive generations of 4-mm-high snails.

Bimiracidial infections of Lymnaea palustris and Lymnaea stagnalis (shell height at exposure, 4 mm) with Fasciola hepatica were carried out during six successive snail generations to determine if prevalence and intensity of snail infection increased over time through descendants issuing from eggs laid by parents already exposed to this digenean. Controls were constituted by a French population of Galba truncatula (a single generation) infected according to the same protocol. In a first experiment performed with the F1 to F5 generations of L. palustris, the prevalence and intensity of F. hepatica infection in snails progressively increased. Immature rediae and a few cercariae-containing rediae of the digenean were observed in L. stagnalis from the F3 generation, but no free cercaria was noted in the bodies of this lymnaeid from the F4 to F6 generations. In another experiment carried out with the F6 generation of L. palustris, the prevalence of F. hepatica infection and the number of shed cercariae were significantly lower in L. palustris than in G. truncatula. This mode of snail infection suggests an explanation for cases of human fasciolosis occurring in central France after the collection of wild watercress from beds where L. palustris was the sole lymnaeid.

Fasciola hepatica: the redial generations in juvenile Pseudosuccinea columella.

Juvenile Pseudosuccinea columella, measuring 1 or 2 mm in height, were subjected to single-miracidium infections with Fasciola hepatica to determine the developmental pattern of redial generations and count free and live rediae according to their generation. Controls were constituted of juvenile Galba truncatula infected according to the same protocol. In the four groups, redial counts were performed in snails dissected every week from day 7 to day 49 post-exposure at 20 °C. Most infected snails showed a normal development of redial generations, whatever the lymnaeid species. In P. columella, the total number of live rediae on day 49 was 24.6 and 34.6 per infected snail in the 1 and 2-mm groups, respectively (instead of 11.5 and 18.8 rediae in the corresponding groups of G. truncatula). A single mother redia (R1a) producing only daughter rediae of the second generation was noted in each snail of three groups, while the 2-mm P. columella showed the presence of a single (43 snails/71) or two (28/71) R1a redia(e) within their bodies. The mean number of other mother rediae and that of daughter rediae of the second generation were low in the 1 and 2-mm groups of both lymnaeids. Besides, there was a delay in redial development. The development of two live R1a rediae in several P. columella from the 2-mm group needs to verify if this process would be specific to P. columella or would occur in other lymnaeid species known for their good susceptibility to the digenean and their larger size to allow harbouring rediae.

Differences in the compatibility of infection between the liver flukes Fascioloides magna and Fasciola hepatica in a Colombian population of the snail Galba sp.

Experimental infections of Galba sp. (origin, Colombia) with allopatric isolates of Fasciola hepatica from France or Fascioloides magna from the Czech Republic were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of either parasite. In snails exposed to F. hepatica, 7 of 400 snails harboured several rediae and only two snails contained a small number of free cercariae on day 50 post-exposure. In contrast, the intensity of F. magna infection in Galba sp. progressively increased from the F1 to F5 generations. Spontaneous cercarial shedding of F. magna occurred in 7 of 100 Galba sp. belonging to the F5 generation and the number of shed cercariae did not differ significantly from that noted in control Galba truncatula of French origin. Galba sp. from Colombia can be added to the list of potential intermediate hosts for F. magna.

Role of the lymnaeid snail Pseudosuccinea columella in the transmission of the liver fluke Fasciola hepatica in Egypt.

Experimental infections of three Egyptian Pseudosuccinea columella populations with sympatric miracidia of Fasciola sp., coming from cattle- or sheep-collected eggs, were carried out to determine the capacity of this lymnaeid to support larval development of the parasite. Using microsatellite markers, the isolates of Egyptian miracidia were identified as Fasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia of F. hepatica were significantly higher than values of 30.4 to 42.2% in snails with bi-miracidial infections. The number of metacercariae ranged from 243 to 472 per cercarial-shedding snail and was independent of snail origin, parasite origin and miracidial dose used for infection. If P. columella was subjected to two successive bi-miracidial infections with F. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported for Radix natalensis infected with the same parasite and the same protocol. Successful experimental infection of P. columella with F. hepatica suggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.

Fasciola hepatica: development of redial generations in experimental infections of Pseudosuccinea columella.

Experimental infections of Egyptian Pseudosuccinea columella with one or two miracidia of Fasciola hepatica per snail were carried out to determine the developmental pattern (normal or abnormal) of redial generations and specify the number of free rediae developing in snails according to their generation. Controls were constituted by a French population of Galba truncatula infected according to the same protocol. Most infected P. columella showed a normal development of redial generations (96.2-98.1 vs 75.5-85.7% for G. truncatula). In each redial category, free rediae were more numerous in P. columella than in G. truncatula, and their number were also greater in the two-miracidia groups than in single-miracidium infections for each lymnaeid considered separately. This increase in redial production was mainly due to the number of first mother (R1a) rediae producing daughter rediae only: 2 per P. columella (vs one redia in G. truncatula) in single-miracidium groups and 3.1 (vs 1.9) in the two-miracidia groups. In P. columella, the mean total number of free rediae developing in single-miracidium and bimiracidial infections was 77.2 and 117.6, respectively (instead of 33.5 and 52.1 rediae in G. truncatula). The number of F. hepatica rediae present in P. columella was related to the number of fully grown sporocysts and the quantity of R1a rediae which developed into the snail body.

Exploring the antigenic features of Fasciola hepatica rediae (Trematoda: Digenea) through the evaluation of different antigenic candidates for further monoclonal antibody generation.

The control of fasciolosis, as that of other vector-borne diseases, must be related to the control of the lymnaeid snails, the intermediate hosts of the parasite. Thus, an accurate epidemiological surveillance of the transmission foci where the infected mollusks occur is essential. For this purpose, immunoassays could be a useful tool. However, information regarding specific proteins of intramolluscan larvae and previous studies concerning monoclonal antibody generation against asexual stages of trematodes are scarce. Therefore, we explored the antigenic features of intramolluscan rediae of Fasciola hepatica to evaluate three antigenic preparations in order to use the most promising one for developing specific monoclonal antibodies. Mouse antiserum was generated against each antigen for assessing the polyclonal antibody response against the crude extract of rediae and the cross-reactivity against lymnaeids. The specific C-terminal of F. hepatica cytochrome c oxidase subunit I (first antigen), selected by in silico analyses, might not be the appropriate target for immunoassay detection of infected snails, due to its low representation in the total extract of rediae. The majoritarian mixture of low-molecular-weight proteins (<30 kDa) from the rediae homogenate (second antigen) revealed a significant cross-reactivity with lymnaeids. Evidence of the existence of mimetic immunogenic epitopes in this fraction of F. hepatica rediae was achieved. High immunogenicity of the crude extract of rediae (third antigen), mainly related to parasite's specific epitopes, was regarded. Therefore, the rediae homogenate is stated as the most promising antigen from those evaluated, for monoclonal antibody development with potentialities for detecting F. hepatica-infected snails.

The production of mammalian trematode infective stages by the snail Galba truncatula.

Several experiments on the breeding of trematode-infected Galba truncatula for obtaining and packaging Fasciola hepatica and Paramphistomum daubneyi metacercariae were carried out to determine the more convenient methods to use for commercial production of these infective stages. Compared to the breeding of infected snails in aquaterraria, the use of 14-cm Petri dishes allowed a greater prevalence of snail infection and a higher number of metacercariae. The production of these larvae was still 2.3-3.4 times greater if infected snails were dissected during the patent period. The aspiration of these metacercariae at the extremity of a Pasteur pipette significantly shortens the time necessary for their transfer from Petri dishes to Eppendorf tubes. Using 14-cm Petri dishes, snail dissection and metacercarial aspiration for their transfer strongly reduce the cost price for metacercarial production of the trematodes Fasciola hepatica and Paramphistomum daubneyi.

Pochonia chlamydosporia in the biological control of Fasciola hepatica in cattle in Southeastern Brazil.

Biological control with the use of nematophagous fungi has been described very successfully by many authors and presents itself as a complementary control method, acting on the free-living forms of helminths. The efficacy of a formulation containing the fungus Pochonia chlamydosporia in controlling Fasciola hepatica eggs in faeces was evaluated in an experimental field assay. Two bovine groups (six animals each) were used: A (control) and B (treated with fungus). At 30 days after deworming, the animals were separated into two similar paddocks with flooded areas and were given pellets containing 25 % mycelial mass (group B) or no fungus (group A) at a dose of 1 g/10 kg body weight, twice a week, during 18 months. Faecal samples were harvested fortnightly in the animals of groups A and B and they were submitted at examination of quantitative sedimentation. The mean count of F. hepatica eggs per grams of faeces was significantly higher in group A (1.19) compared with those from group B (0.82) (P < 0.01). After 18 months, animals from group B had gained 42.33 kg above (17.82 % more by weight) (P < 0.01), compared with the control group (A). Every month, faecal samples from paddocks A and B were collected and they were incubated. P. chlamydosporia was identified only in sample source of the paddock B. It can be concluded that the application of this fungical formulation with P. chlamydosporia 25 % mycelial mass was effective in reducing the availability of eggs in the environment and reinfections in calves in natural conditions.

The variable presentations and broadening geographic distribution of hepatic fascioliasis.

We report 2 unrelated cases of hepatic fascioliasis in travelers returning to the United States from Africa and the Middle East. The first case presented with acute infection. Prominent clinical features included abdominal pain, elevated liver transaminases, serpiginous hepatic lesions, pericapsular hematoma, and marked peripheral eosinophilia. The second case was diagnosed in the chronic stage of infection and presented with right upper quadrant abdominal pain, cystic hepatic lesions, and an adult fluke in the common bile duct. We review the life cycle of Fasciola species, the corresponding clinical features during the stages of human infection, diagnostic methods, and the evolving understanding of the epidemiology of human fascioliasis, particularly emphasizing fascioliasis in African countries.

Intermediate snail hosts of French Fasciola hepatica: Lymnaea neotropica and Lymnaea viatrix are better hosts than local Galba truncatula.

Allopatric and sympatric infections of Lymnaea neotropica and Lymnaea viatrix var. ventricosa with Argentinean and French isolates of Fasciola hepatica were carried out to determine the capacity of these snails to produce metacercariae and to verify if this capacity changed with snail generation. The same process was also made with a French population of Galba truncatula known to be highly susceptible to French isolates of the parasite. In each lymnaeid species separately considered, the survival rate at day 30 post-exposure and prevalence of F. hepatica infection in the group infected with Argentinean miracidia were significantly greater than those recorded in the corresponding French one. Compared to infected G. truncatula, both South American lymnaeids had longer patent periods and produced a higher number of metacercariae. The highest infections were noted with L. v. ventricosa. In the three snail species, metacercarial production was more important with the Argentinean isolate of miracidia than with the French one. If three successive generations of L. v. ventricosa are exposed to the same French isolate of miracidia, cercarial production significantly increased from parents to the F2 generation, while the other characteristics of infection only showed insignificant variations. L. neotropica and L. v. ventricosa are better intermediate hosts for French F. hepatica than local G. truncatula. The numerical increase of shed cercariae in the F1 and F2 generations of L. v. ventricosa demonstrates a rapid adaptation of this species to the French isolate of the parasite.