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PubMed1983

Antibodies to Fasciola hepatica antigens during experimental infections in cattle measured by ELISA.

Chromatography of a soluble PBS extract of adult Fasciola hepatica on Sephadex G200 produced 7 fractions, F1-F7. The antigenicity of these fractions varied with F1 being the most antigenic. Antibody responses to these fractions showed different time courses. Antibodies to F1, F2 and F3 appeared early on in infection and, for the most part, persisted throughout infection, whereas antibodies to F4, and F5 increased gradually during infection. A standardised assay was developed to measure anti-F1 antibodies. The assay was reproducible and able to detect antibodies by 3 weeks post-infection in experimentally-infected calves.

Animals↗

PubMed1995

A bolus for the administration to cattle of metacercariae of the liver fluke Fasciola hepatica.

A gelatin-agar bolus, designed and developed for the administration of metacercariae of the liver fluke Fasciola hepatica, was evaluated in adult Holstein Friesian cattle. The metacercariae, enclosed within a gelatin capsule, were placed inside the bolus and delivered to each animal using an oesophageal balling gun. At slaughter, 13 weeks after challenge, an average of 25% of the challenge dose was recovered from each liver. This percentage recovery is similar to that obtained with other known methods. The new bolus, however, offers improved handling qualities. In addition, the bolus also has potential for improving a number of other techniques including those for the administration of other parasites, compounds or chemotherapeutic agents.

Agar↗

PubMed1984

Fasciola hepatica: site of resistance to reinfection in cattle.

The effective site of resistance to reinfection of cattle with Fasciola hepatica was examined by recovery of challenge flukes from either the liver or body cavity. Calves infected 18 or 26 weeks previously with F. hepatica showed levels of resistance to reinfection of 56 and 94%, respectively, as assessed by recovery of flukes from the liver 15-16 weeks after challenge. Plasma glutamate dehydrogenase (EC 1.4.1.3; GLDH) enzyme activity estimations revealed only a marginal increase in these latter resistant calves compared with previously naive controls, indicating minimal liver damage as a result of migrating flukes. By comparison, when immature challenge flukes were recovered from the body cavity 4 or 14 days after infection of corresponding previously infected or naive calves, there was no significant difference in numbers. It appears, therefore, that, in cattle, resistance mechanisms are effective against challenge flukes at or soon after penetration of the liver.

Animals↗

PubMed1993

Analysis of the results obtained using a technic of experimental primary infestation with Fasciola hepatica in the rat.

In this work, we analyzed the results obtained using a technic of infestation with Fasciola hepatica in the rat. The infestations were carried out with 30 metacercariae in a gelatin capsule. The humoral immune response, measured by ELISA, and the liver necroses, spleen size, number and length of the flukes recovered at necropsy were followed during the course of four experimental primary infestations. The results showed that the magnitude of the parasitic burden did not correlate with the humoral immune response of the rat. The study of the liver necroses, spleen size, fluke length and antibody titres showed that the use of 30 metacercariae in a capsule as a technic of infestation in the rat ensured the homogeneity of the parasite development and the humoral immune response of the host.

Animals↗

PubMed1996

Protection of cattle against Fasciola hepatica infection by vaccination with glutathione S-transferase.

Glutathione S-transferase (GST) from the liver fluke Fasciola hepatica was assessed as a vaccine immunogen in cattle in a number of immunological adjuvants. Significant reductions in fluke burdens (49-69%) were only observed in cattle vaccinated with GST in Quil Alsqualene Montanide (SM) and PLG microspheres in SM but there was no correlation between anti-GST IgG titres and protection. In separate experiments, animals vaccinated with GST in Quil AlSM were still significantly protected (48%, P < 0.05) 6 months after boosting and no significant differences in protection were seen when the metacercarial challenge was given over 1 month instead of as a single bolus. Inhibition of GST enzyme activity in vitro by cattle antisera did not correlate with reduced fluke burdens.

Adjuvants, Immunologic↗

PubMed2004

A native 13-kDa fatty acid binding protein from the liver fluke Fasciola hepatica.

A 13-kDa fatty acid binding protein (FABP) (Fh13) has been isolated from the cytosol of adult Fasciola hepatica and its physicochemical and binding characteristics determined. Fh13 appears to exist as a dimer in native solution. Binding of the fluorescent fatty acid analogue 11-((5-dimethyl aminonaphthalene-1-sulfonyl) amino) undecanoic acid (DAUDA) to Fh13 results in changes in the emission spectrum, which are reversed by oleic acid. The binding activity for DAUDA determined from titration experiments revealed a single binding site per monomeric unit with Kd of 1.5 microM. The displacement of DAUDA by competitive nonfluorescent ligands allowed Kd values for oleic (2.5 microM), retinoic (2.8 microM), palmitic (4.1 microM) and arachidonic acid (6.1 microM) to be calculated. Ten commonly used anthelmintics were evaluated for binding to Fh13, but only bithionol showed binding activity commensurate with those of the putative natural ligands (Kd 6.8 microM).

Animals↗

PubMed1975

The efficiency of clioxanide and rafoxanide against Fasciola hepatica in sheep by different routes of administration.

Groups of sheep were infected with 100 viable metacercariae of Fasciola hepatica. The efficiencies of clioxanide and rafoxanide were evaluated against infections aged 6 and 12 weeks, by using 3 dose rates of each compound by each of 3 routes of administration. Clioxanide, at 40 mg/kg, administered orally and intra-ruminally against 6-week-old liver fluke was 85 and 90% efficient respectively. At 80 mg/kg intra-abomasally its efficiency was 43%. Clioxanide at 20 mg/kg was 96% efficient against 12-week-old liver fluke when given orally or intra-ruminally and 82% efficient when given intra-abomasally. Because of its lower efficiency intra-abomasally, clioxanide may be unsatisfactory in a proportion of sheep, for use against immature liver fluke at a dose rate of 40 mg/kg. It may be expected to give a moderate to high efficiency against mature infections at 20 mg/kg.

Abomasum↗

PubMed1997

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae.

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.

Animals↗

PubMed2007

Evidence for multiple mitochondrial lineages of Fasciola hepatica (liver fluke) within infrapopulations from cattle and sheep.

The economic, veterinary, and medical impact of the parasite Fasciola hepatica, liver fluke, is difficult to alleviate due to increasing incidences of resistance to the principal anthelmintic drugs. These have occurred in widely separated regions. The rate of response to selection imposed by such drugs will be dependent on the genetic variation present in the F. hepatica gene pool, but this is at present unknown. We have assessed the genetic diversity of mitochondrial haplotypes found in the infrapopulation of flukes recovered from a calf of known provenance and from six other cattle and sheep hosts located in Ireland and four from elsewhere. Our results revealed that at least ten different mitochondrial composite PCR-restriction fragment length polymorphism haplotypes had been acquired by a single animal in 1 year, and there was comparable diversity in six other definitive hosts carrying field-acquired infections. The extent of divergence between these fluke lineages suggests that they predate the last ice age and, thus, cannot have developed in Northern Europe. A consequence of this high level of diversity is that there will be frequent selection for anthelmintic resistance and rapid responses to climatic changes.

Animals↗

PubMed1996

Antigenic components of excretory-secretory products of adult Fasciola hepatica recognized in human infections.

The antigenic components of excretory-secretory products (ESP) of adult worms of Fasciola hepatica were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using sera from 20 patients infected with F. hepatica. Sera from 184 other parasitic infections and 20 healthy volunteers were also analyzed. It was found that the ESP were composed of more than 11 polypeptides; five components detected in fascioliasis sera had molecular weights of 12.4, 16.4, 19.4, 25, and 27 kilodaltons (kD). Only the 25- and 27-kD components were recognized by all 20 fascioliasis sera. Using the ESP as antigen, it was possible to perform an enzyme-linked immunosorbent assay with a sensitivity of 95% and a specificity of 97%. Sera from other parasitic infections had antibodies to antigenic components with apparent molecular weights of 37, 38.4, 52, 63, 73, 87, 109, and 116 kD that were also found in sera from fascioliasis patients. These findings suggested that the 25- and 27-kD antigenic components may be sensitive and specific for the diagnosis of human fascioliasis.

Animals↗

PubMed1998

Fasciola hepatica development in the experimentally infected black rat Rattus rattus.

The finding of natural infection of Rattus rattus by Fasciola hepatica on Corsica has stimulated further research into the role of the black rat in the epidemiology of fascioliasis. Corsican black rats were experimentally individually infected with 20 metacercariae from cattle and murine isolates obtained from naturally infected bovines and black rats. The following results were obtained: (a) in R. rattus infected with the cattle isolate, normal adult fluke development took place and infection persisted for a long period, with emission of eggs showing embryogenic capacity; (b) the development of F. hepatica adults paralleled the ontogenetic trajectories observed in other rodent-F. hepatica models; and (c) fluke adults obtained in R. rattus infected with the murine isolate exhibited a similar pattern. These findings strongly suggest that the black rat may be one of the wild reservoirs of F. hepatica and may have contributed to the large geographical extent of the disease on Corsica.

Animals↗

PubMed1994

Effects of luxabendazole on the tegument of Fasciola hepatica.

The effects in vivo of 5, 10, and 20 mg/kg of luxabendazole (LBZ) on the tegument of Fasciola hepatica have been examined 48 h, 7 days and 14 days post-treatment of experimentally-infected rats. As early as 48 h post-treatment, the drug is shown to provoke significant damage to the tegument. The pathological phenomena characterizing LBZ damage are blebbing of the apical plasmalemma, formation of microvillus-like projections over the free surface, swelling of the basal infolds and stimulation of autophagy. The spines are often fractured; the tegument in the vicinity of spines seems more strongly altered than that in other foci. The basal layer is often changed, from increase of electron density to lack of integrity with the apical cytoplasm. The progress of the ultrastructural damage with time is not expressed. However, cytochemical data show that a longer post-treatment intervals the surface-coat structure becomes irregular and patches of ruthenium red positive material of variable thickness are focally accumulated. Only a slight dose-effect is noted 48 h after LBZ application when the alterations provoked by 5 mg/kg are less evident than those by 10 and 20 mg/kg.

Acid Phosphatase↗

PubMed1996

Fasciola hepatica: rapid switching of stage-specific antigen expression after infection.

Early developmental stages of the trematode parasite Fasciola hepatica were collected from the peritoneal cavity and liver of mice during a ten day infection period. Using one dimensional SDS-PAGE, differences in protein expression profiles were observed in stages collected on the same day post-infection in different physiological locations and also in juvenile parasites collected from the same location on different days post-infection. Four rat monoclonal antibodies were raised against the parasite using lymph nodes draining infected tissues. Three monoclonal antibodies, FY3-1, FY3-2 and FY4-7, were generated using cells from the mesenteric lymph node of recently challenged immune rats, while FY1-6 was derived from hepatic lymph node cells of a chronically infected rat. The epitope recognized by FY3-2 appeared to be carbohydrate in nature and was present on the surface of newly excysted juveniles. Immunoblots revealed that the antigens recognized by FY3-1, FY3-2 and FY4-7 were only expressed for two days after infection. In contrast, FY1-6 recognized epitopes expressed across all developmental stages screened. The rapid changes in protein and antigen expression observed during the early stages of infection may assist the parasite to evade the host immune response.

Animals↗

PubMed2004

A scanning electron microscope study on the route of entry of clorsulon into the liver fluke, Fasciola hepatica.

Three experiments were carried out in vitro to determine the roles of the tegument and gut of Fasciola hepatica in the uptake of the flukicidal drug, clorsulon. Changes to the two surfaces were assessed by scanning electron microscopy. In the first experiment, the flukes were ligatured to prevent the oral ingestion of drug and treated for 24 h in clorsulon (10 microg/ml). The gastrodermal surface remained normal and few changes to the tegumental surface were observed. In the second experiment, flukes were fed for 24 h on red blood cells isolated from rats dosed with clorsulon at 12.5 mg/kg body weight; this experiment was designed to prevent the exposure of the tegumental surface to the drug. The gastrodermal surface was severely disrupted and the gut lamellae were disorganised and necrotic. Swelling of the tegument and blebbing on the tegumental surface were evident, but the changes were not severe. More severe swelling of the tegument was observed in the third experiment, in which flukes were incubated for 24 h in clorsulon (10 microg/ml), with both absorptive surfaces being available for drug uptake. The gastrodermal surface was badly disrupted and the gut lamellae were disorganised and necrotic. Taking the results of the three experiments together, the gastrodermal surface was more affected than the tegument and the greatest disruption to the two surfaces was seen when both routes of entry were available to the fluke. The data support a previous study which indicated that entry of clorsulon into the fluke in vivo is principally by the oral ingestion of drug bound to the red blood cells.

Animals↗

PubMed1987

Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme.

Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.

Adenosine Triphosphate↗

PubMed2004

Response of two isolates of Fasciola hepatica to treatment with triclabendazole in vivo and in vitro.

In this study, the susceptibility of two isolates of Fasciola hepatica--the Fairhurst and Oberon isolates--to treatment with triclabendazole was investigated, both in vivo and in vitro. The Fairhurst isolate originated in England, but has since been maintained in Australia; the Oberon isolate originated in Australia. Triclabendazole had a very high efficacy against the Fairhurst isolate. In sheep (dose: 10 mg/kg), the efficacy ranged from 78.4% at 2 weeks post-infection to 98.5% at 6 weeks post-infection. In cattle (dose: 12 mg/kg) efficacy was 89% at 2 weeks post-infection and 100% at 12 weeks. In contrast, against the Oberon isolate, triclabendazole had 0% efficacy against 2-week-old flukes in sheep (dose: 10 mg/kg) and 5% against 4-week-old flukes. Surface changes to flukes of the two isolates were assessed by scanning electron microscopy following treatment in vitro for 24 h in triclabendazole sulphoxide (15 and 50 microg/ml). Disruption took the form of blebbing, swelling and furrowing of the tegument and was greater in the Fairhurst than the Oberon isolate. Surface changes generally were more severe in the anterior than posterior region of the fluke and the dorsal surface was also consistently more severely affected than the ventral surface. Disruption was more severe at the higher drug concentration for both isolates. The morphological data is consistent with the efficacy data, which indicates that the Fairhurst isolate of F. hepatica is susceptible to triclabendazole treatment, whilst the Oberon isolate is refractory.

Animals↗

PubMedDate not supplied

Correlation of specific antibody titre and avidity with protection in cattle immunized against Fasciola hepatica.

Cattle produce specific serum antibody mainly of the IgG1 isotype in response to infection with the liver fluke, Fasciola hepatica. In these animals a positive correlation between fluke-specific serum IgG1 levels and fluke-burden in non-immunized infected animals was observed. In contrast, immunization of cattle with a combination of the fluke-derived antigens cathepsin L2 (CL2) and fluke haemoglobin (FHb) in Freund's complete/incomplete adjuvant (FCA/FLA) induced a specific antibody response involving IgG2, as well as IgG1. These immunized animals also exhibited very high (72%) levels of protection against a subsequent challenge infection. When the vaccine was administered in FIA alone the specific antibody response, while still involving IgG1 and IgG2, was of lower magnitude (10-fold and 100-fold, respectively) and no significant reduction in fluke burden was observed following challenge. Nevertheless, in these animals, a strong IgG2 response was associated with low fluke burdens. These results provide further evidence of the non-protective nature of specific immune responses in cattle following F. hepatica infection, and demonstrate that vaccination can induce a qualitatively different, and protective, response.

Animals↗

PubMed1975

Trials with rafoxanide. 7. Efficacy against Fasciola hepatica, Haemonchus placei and Bunostomum phlebotomum in cattle.

Anthelmintic efficacy studies involving 49 cattle either naturally or artificially infested with Fasciola hepatica are described. Some of the animals were also artificially infested with Haemonchus placei and Bunostomum phlebotomum. At dosage rates of 3,75 and 5,0 mg/kg live mass rafoxanide was 64,6 and 92,6 per cent effective respectively against adult naturally acquired infestations of F. hepatica. At 7,5 and 10,0 mg/kg it was 37,9 and 55,7 per cent effective against 42 day old liver fluke. At 7,5 mg/kg it was 100, 87,4 and 94,6 per cent effective against mature F. hepatica, fourth stage H. placei and adult B. phlebotomum respectively.

Animals↗