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PubMed1984

The influence of water temperature and pH on the survival of Fasciola hepatica miracidia.

Experimental studies on the survival of Fasciola hepatica miracidia show no evidence that miracidial mortality varies with the pH of the medium, at least in the range 6.0-8.0. On the other hand, miracidial mortality is shown to vary with both the temperature of the medium and the age of the larvae. The mean expected life-span of the miracidium decreases from about 35 h at 6 degrees C to about 6 h at 25 degrees C. The Gompertz survival function provides a good description of the miracidial survivorship curves over the range of temperatures used, and we describe a maximum likelihood method of estimating the mean values of the parameters of this function, together with their approximate 95% confidence limits.

Animals↗

PubMed1997

Fasciola hepatica cysteine proteinases: immunodominant antigens in human fascioliasis.

Fasciola hepatica adult worm cysteine proteinases were active-site, affinity radio-labeled with benzyloxicarbonyl-L-tyrosine-L-alanine diazomethylketone (Z-Tyr125I-Ala-CHN2). Sera from patients with fascioliasis and from rabbits experimentally infected with F. hepatica immunoprecipitated the radiolabeled parasite cysteine proteinases in immunoelectrophoresis assays. Two purified antigens were identified as part of the complex mosaic of antigens immunoprecipitated by the sera of infected patients. These antigens (Fas1 and Fas2) have been shown to be an important part of the Fharc2 precipitin band used for serologic diagnosis in humans and cattle. They showed cysteine proteinase activity with different proteolytic specificities and partial identity in double immunodiffusion assays. The results obtained in this work show that the Fas1 and Fas2 antigens are sensitive and specific antigens for diagnosis of this serious helminthic disease in humans and other susceptible hosts.

Animals↗

PubMed1993

Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica.

A cysteine proteinase released in vitro by Fasciola hepatica was purified to homogeneity by Sephacryl S-200 gel filtration chromatography followed by QAE-Sephadex chromatography. The purified enzyme resolves as a single band with an apparent molecular size of 27 kDa on reducing SDS-polyacrylamide gel electrophoresis; however, under non-reducing conditions it migrates as multiple bands, each with enzymatic activity, in the apparent molecular size range 60-90 kDa. The sequence of the first 20 N-terminal amino acids of the enzyme shows considerable homology with cathepsin L-like proteinases. Immunolocalisation studies revealed that the cathepsin L-like proteinase is concentrated within vesicles in the gut epithelial cells of liver fluke.

Amino Acid Sequence↗

PubMed2000

Partial characterization of a novel cathepsin L-like protease from Fasciola hepatica.

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CH&dbond;CH(2)-CO(2)R (where R = Me/Et/Bu(t)). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.

Amino Acid Sequence↗

PubMed1996

Immune responses of chronically infected adult cattle to Fasciola hepatica.

Eight adult cows, with an existing chronic Fasciola hepatica infection, were experimentally infected with 1300 metacercariae of F. hepatica, given as trickle infections, over two separate 10-day periods. Two fluke-naive heifers were similarly treated. Analysis of parasite-specific immunoglobulin isotypes IgM, IgG1, IgG2 and IgA showed IgG1 to be the dominant isotype in both chronically infected and previously naive animals. Lymphocyte proliferation assays demonstrated (a) an association between lymphocyte response and mature fluke burden in the chronically infected cattle and (b) no association between lymphocyte response and mature or immature fluke burden in naive heifers. There was no production of gamma-interferon (IFN gamma) by lymphocytes responding to adult fluke antigen. At post-mortem examination the burden of immature flukes in chronically infected and previously naive heifers was similar. This suggests that chronically infected animals may be tolerant to a moderate superinfection and that the prevailing immune mechanism in operation may be a non-protective response generated by the Th2 lymphocyte subset.

Animals↗

PubMed1992

Localisation of actin in the liver fluke, Fasciola hepatica.

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

Actins↗

PubMed1996

Polarization and chemokinesis of ovine and human neutrophils in response to Fasciola hepatica excretory-secretory products.

The effect of Fasciola hepatica excretory-secretory products (ES products) on the polarization and chemokinesis of sheep and human neutrophils was investigated. Flukes were cultured overnight in RPMI-1640 medium, and the resulting ES products concentrated by dialysis (cut-off 1.2 kDa) before use. At the concentrations used toxicity tests showed no effect on neutrophil viability, but ES products were capable of causing morphological changes and chemokinesis in both sheep and human neutrophils. The polarizing effect of the ES products was found to be greater in the case of sheep neutrophils with 81% of cells polarizing at 80 micrograms total ES protein/ml as opposed to 36% of human neutrophils. Chemokinesis in response to 80 micrograms total ES protein/ml was significant (P < 0.01) in both species with sheep producing the stronger response. Delipidation of the ES products with Lipidex caused no loss of polarizing activity, nor did the purified lipid fraction show any polarizing effect. ES products lost polarizing activity after heat treatment or trypsin digestion. Preliminary fractionation on an S-300 Sephacryl column suggested the presence of at least four fractions capable of causing polarization in sheep neutrophils.

Analysis of Variance↗

PubMed2002

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products.

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.

Animals↗

PubMed1981

Response of sheep to challenge infection with Fasciola hepatica.

Sheep were infected with 100 metacercariae of Fasciola hepatica and reinfected 16 weeks later with a further 100 metacercariae. Serum samples were taken weekly for 36 weeks after primary infection. Serum was assayed for the presence of the enzymes glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gamma-GT), as indicators of liver and bile duct damage respectively, and for levels of precipitating antibody. Antibody and GLDH levels rose following the primary infection but fell after patency had been reached . A peak in gamma-GT activity was associated with the onset of patency. After the challenge infection levels of both enzymes rose substantially and there were persistent fluctuations in activity. Antibody levels did not rise markedly following challenge but fluctuated at low levels until autopsy, 20 weeks after challenge. There was no resistance to challenge judged by worm recoveries at autopsy. It is suggested that the presence of adult flukes in the bile ducts suppresses the antibody response to challenge infection. Tissue damage, which is shown by fluctuations in GLDH and gamma-GT levels after adult flukes have become established in the bile ducts, is considered to be due to the feeding activity of adult flukes and the deposition of immune complexes in the liver parenchyma.

Animals↗

PubMed2006

Stage-specific differences in fecundity over the life-cycle of two characterized isolates of the liver fluke, Fasciola hepatica.

The variability inherent in different isolates of Fasciola hepatica has been evident from reports in the literature but to date there has been no systematic examination of the relationship between these differences and the fecundity of the parasite. In this study we have attempted to remedy this situation by comparing the relative efficiencies with which 2 well-characterized isolates of the liver fluke (Oberon and Fairhurst) progress through both their definitive and intermediate hosts. We did not observe a reduction in fitness in the Oberon isolate which has been reported to be triclabendazole-resistant, compared to the triclabendazole-susceptible Fairhurst isolate, but considerable inter- and intra-isolate variability at different life-cycle stages was recorded. Thus the Oberon isolate gave 4-fold the number of cercariae when 100 snails were each challenged with a single miracidium and was more successful in establishing productive infections in rats. Fairhurst metacercariae excysted at a higher rate than those from the Oberon isolate and Fairhurst flukes produced 4-fold more eggs. The extent of the intra- and inter-isolate variability revealed in this work will provide a basis for the development of models of population dynamics aimed at predicting the response of the liver fluke to changing environmental conditions such as the use of anthelmintics or climatic change.

Adaptation, Physiological↗

PubMed2004

Electrophysiological and pharmacological characterization of K+-currents in muscle fibres isolated from the ventral sucker of Fasciola hepatica.

Fibres isolated from the ventral sucker of Fasciola hepatica were identified as muscle on the basis of their contractility, and their actin and myosin staining. They were voltage-clamped at a holding potential of -40 mV and depolarization-activated outward currents were characterized both electrophysiologically and pharmacologically. Activation was well fitted by a Boltzmann equation with a half-maximal potential of + 9 mV and a slope factor of -14.3 mV, and the kinetics of activation and deactivation were voltage-sensitive. Tail current analysis showed that the reversal potential was shifted by +46+/-3 mV when E(K) was increased by 52 mV, confirming that this was a K+-current with electrophysiological characteristics similar to delayed rectifier and Ca2+-activated K+-currents in other tissues. The peak current at + 60 mV was inhibited by 76+/-6% by tetrapentylammonium chloride (1 mM) and by 84+/-7% by Ba2+ (3 mM), but was completely resistant to block by tetraethylammonium (30 mM), 3,4-diaminopyridine (100 microM) and 4-aminopyridine (10 mM). Penitrem A, a blocker of high-conductance Ca2+-activated K+-channels reduced the current at +60 mV by 23+/-5%. When the effects of Ca2+-channel blocking agents were tested, the peak outward current at + 60 mV was reduced by 71+/-7% by verapamil (30 microM) and by 59+/-4% by nimodipine (30 microM). Superfusion with BAPTA-AM (50 microM), which is hydrolysed intracellularly to release the Ca2+-buffer BAPTA, also decreased the current by 44+/-16%. We conclude that voltage-and Ca2+-sensitive K+-channels are expressed in this tissue, but that their pharmacology differs considerably from equivalent channels in other phyla.

Actins↗

PubMed1993

Pharmacological profile of the 5-hydroxytryptamine receptor of Fasciola hepatica body wall muscle.

5-HT is a candidate for the excitatory transmitter at the neuromuscular junction in trematodes including Fasciola hepatica. This study has determined the response of a muscle strip preparation from Fasciola hepatica to 5-HT and a range of agonists that distinguish between the vertebrate receptor 5-HT subtypes. 5-HT increased the resting tone and the rhythmic activity of the muscle strip. Of the 19 compounds tested, only 10 had an effect similar to 5-HT and all but 2 of these were tryptamine compounds. 5-HT was more potent than tryptamine whilst 4-OH-tryptamine had no effect, suggesting that the response is mediated by a 5-HT rather than a tryptamine receptor. 5-Fluorotryptamine and 5-carboxyamidotryptamine were the most potent agonists. 8-OH-DPAT also mimicked the effect of 5-HT, though less potently. Assuming that these agents elicit their response through a common receptor, this suggests the presence of a 5-HT receptor with similar properties in terms of agonist recognition as the vertebrate 5-HT1 class of receptor involved in controlling Fasciola muscle motility.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2001

[Extensivity of infection of Fasciola hepatica L. in cattle in Lublin's voivodship in the years 1993-2001].

Extensivity of infection of Fasciola hepatica L. among cattle in Lublin's voivodship from 1993-2001. Aim of the work was to determine extensitivity of appearance of hepatica among catlle put to slaughter in slaughtershouses in Lublin's voivodship from 1993 to 2001. Substance for the work was braught from the annual reports of official examination of slaughtered animals, reached from both main Inspectorate (department) of Veterinary in the Ministry of Agriculture and from Regional Institute of Veterinar's Hygiene in Lublin. Results of researches are as follow: extensivity of infection of Fasciola hepatica among cattle in Lublin and voivodship between 1993 - 1999 oscillates about 5%. In 2000 extensivity of invasion reached 10,9%.

Abattoirs↗

PubMed2000

Recurrent eosinophilic panniculitis associated with Fasciola hepatica infection.

Eosinophilic panniculitis is characterized by a prominent infiltration of subcutaneous fat with eosinophils. We report a case of Fasciola hepatica infection presenting with eosinophilic panniculitis successfully treated with bithionol. To our knowledge, this is the first report of recurrent eosinophilic panniculitis associated with Fasciola hepatica infection.

Animals↗

PubMed1991

Identification of phosphorylcholine containing antigens of Fasciola hepatica--successful tolerization against this epitope in experimental animals.

Phosphorylcholine containing antigens have been identified in the parasite Fasciola hepatica by immunoblotting and ELISA. Immunoblots probed with polyclonal and monoclonal antibodies indicate that the majority of antigens identified in both the immature and mature parasite contain both phosphorylcholine and non-phosphorylcholine epitopes. One antigen of 58 kDa appears to contain predominantly PC epitopes or at least this epitope is the major one responded to by host animals. Successful immunotolerization against the epitope PC was achieved by injecting the PC conjugate, ovalbumin PC, into neonatal rats. Immunotolerization against PC resulted in a 25% reduction in worm burden upon subsequent infection with Fasciola hepatica.

Animals↗

PubMed1979

The effect of Taenia hydatigena infection on existing and concurrent infections of Fasciola hepatica in sheep.

Sheep given a primary infection of Fasciola hepatica were challenged 18 weeks later with Taenia hydatigena or F hepatica, or both parasites together, or were not challenged. At the same time, control sheep were infected with T hydatigena and/or F hepatica separately or concurrently. All sheep were killed seven weeks after challenge and the number of cysts and flukes counted. Challenge infection with T hydatigena did not affect the numbers of flukes recovered from either primary or challenge F hepatica infections. On the other hand, the numbers of cysticerci were reduced in sheep previously infected with F hepatica but not in those given T hydatigena and F hepatica concurrently.

Animals↗

PubMed2006

[The occurrence of fasciola hepatica in chosen regions of Poland based on molecular and serological methods].

Fasciolosis, caused by the liver fluke (Fasciola hepatica) is an important issue for both human and animal health. The disease evokes economic losses which are a consequence of impaired animal productivity leading to higher costs of meat and milk production, as well as liver condemnation. The goals of this thesis were to: (1) elaborate a molecular method--PCR for the detection of F. hepatica DNA in intermediate and definite hosts; (2) estimate the usefulness of a recombinated cysteine proteinase produced in E. coli in the form of inclusive bodies in serological diagnosis of F. hepatica infection in definite hosts, using an enzyme-linked immunosorbent assay (ELISA); (3) conduct field research on the prevalence of infection among intermediate and definitive hosts (cattle) in chosen regions of Poland, utilizing the elaborated methods. Based on the results obtained in this study, it was established that it is possible to detect F. hepatica DNA in the feces of definite hosts with the elaborated PCR method. The amplification of a 124 base pair tandem repeat allows the detection of fluke larval stages in intermediate hosts within 12 hours of exposure and F. hepatica infection in definite hosts (by the 5th week in rats, 8th week in sheep and 10th week in cattle). Therefore, the PCR test is more sensitive than traditional microscopic methods. Furthermore, it was determined that, the recombinated cysteine proteinase in the form of inclusive bodies, after solubillization exhibits antigenic properties of the native protein and the ELISA method based on this antigen may be useful as a tool for diagnosing fasciolosis in sheep and cattle, in both serum and milk samples. The test achieves a greater sensitivity and specificity than an ELISA based on native excretory-secretory antigens. The results of field research indicate that Fasciola hepatica is a frequent parasite of cattle in central and eastern Poland. The mean prevalence was 34.86% (+/- 16.95) in all studied areas. The prevalence among intermediate hosts varied greatly (0-100%). The elaborated tests were proved to be valuable, mutually complementing diagnostic tools, applicable to different epidemiological situations.

Animals↗

PubMed2001

Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America.

Fascioliasis due to the digenean species Fasciola hepatica has recently proved to be an important public health problem, with human cases reported in countries of the five continents, including severe symptoms and pathology, with singular epidemiological characteristics, and presenting human endemic areas ranging from hypo- to hyperendemic. One of the singular epidemiological characteristics of human fascioliasis is the link of the hyperendemic areas to very high altitude regions, at least in South America. The Northern Bolivian Altiplano, located at very high altitude (3800-4100 m), presents the highest prevalences and intensities of human fascioliasis known. Sequences of the internal transcribed spacers ITS-1 and ITS-2 of the nuclear ribosomal DNA of Altiplanic Fasciola hepatica and the intermediate snail host Lymnaea truncatula suggest that both were recently introduced from Europe. Studies were undertaken to understand how the liver fluke and its lymnaeid snail host adapted to the extreme environmental conditions of the high altitude and succeeded in giving rise to high infection rates. In experimental infections of Altiplanic lymnaeids carried out with liver fluke isolates from Altiplanic sheep and cattle, the following aspects were studied: miracidium development inside the egg, infectivity of miracidia, prepatent period, shedding period, chronobiology of cercarial emergence, number of cercariae shed by individual snails, survival of molluscs at the beginning of the shedding process, survival of infected snails after the end of the shedding period and longevity of shedding and non-shedding snails. When comparing the development characteristics of European F. hepatica and L. truncatula, a longer cercarial shedding period and a higher cercarial production were observed, both aspects related to a greater survival capacity of the infected lymnaeid snails from the Altiplano. These differences would appear to favour transmission and may be interpreted as strategies associated with adaptation to high altitude conditions.

Altitude↗