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PubMed1994

CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.

Animals↗
PubMed1994

Appetite depression in sheep experimentally infected with Fasciola hepatica L.

The purpose of this study was to determine the effects of experimental fasciolosis at various stages of development on the daily food intake of sheep. Five male Churra sheep, 4 months of age, were infected orally with 300 Fasciola hepatica metacercariae over a 30 day period. There was a significant increase in serum glutamate dehydrogenase (GLDH) activity from 40 days post-infection and in aspartate aminotransferase (AST) activity from 60 days post-infection. Both enzyme activities reached maximum levels in the serum of infected animals at 80 days and then progressively decreased. Serum gamma-glutamyltransferase (GGT) activity was significantly increased from 80 to 120 days post-infection. Glycaemia was significantly decreased from 60 days post-infection. The average daily food intake was shown to steadily decrease until approximately 100 days. The coincidence of decreased food intake with the period of significant increase, both in AST and GLDH activities, indicated that damage caused around the time of migration of immature flukes through the liver parenchyma may be involved in appetite depression.

Animals↗
PubMed2006

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection.

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.

Animals↗
PubMed1996

Fasciola hepatica: irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke.

Irradiation has been successful in the attenuation of infective stage parasites for use as vaccines against a number of parasites including Fasciola spp. The mechanisms of action of irradiation-attenuated vaccines, however, are not clearly understood. In this study, we examined the effect of 3, 10, and 40 krad of gamma-irradiation on the expression of carbohydrates and cathepsin-B by newly excysted juvenile Fasciola hepatica (NEJ). Following irradiation of metacercariae, the expression of concanavalin A (ConA)-specific sugars was decreased on the surface of NEJ and the expression of wheat germ agglutinin (WGA)-specific sugars was increased in the gut and reduced on the surface of NEJ. Cathepsin proteases are a major component of liver fluke excretory/secretory material (ES) and can cleave host immunoglobulin (Ig). Cathepsin-B protease was localized in nonirradiated NEJ to the gut lumen and to secretory granules within the gut epithelia. Irradiation of fluke with 3, 10, and 40 krad of gamma-rays significantly reduced the tissue expression of cathepsin-B at 8 hr postirradiation in an apparently dose-dependent manner. After a further 24 hr culture tissue expression of cathepsin-B was significantly reduced in 10- and 40-krad-irradiated NEJ. Protease activity of ES samples collected over a 24-hr period from irradiated and nonirradiated NEJ cultured in vitro were tested using a rabbit Ig cleavage assay. The proteolytic activity of ES from 10- and 40-krad-irradiated NEJ was reduced during the initial 6 hr in culture and between 12 and 24 hr when compared to ES from nonirradiated controls. Biosynthetic labeling experiments using [35S]methionine and [35S]cysteine indicated that ES material was actively synthesised during 48 hr in vitro culture. Therefore, from this study, we conclude that gamma-irradiation of NEJ alters expression of cathepsin-B protease and WGA- and ConA-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host by irradiation-attenuated metacercariae of Fasciola spp.

Animals↗
PubMedDate not supplied

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein.

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44%) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Amino Acid Sequence↗
PubMed1989

Biliary tract disease due to Fasciola hepatica: report of a case.

We report a case of a 23 year old woman who presented with symptoms of biliary tract disease to our University Hospital in Bayamón, Puerto Rico. A diagnosis of chronic cholelithiasis was made and the patient was taken to surgery. An adult Fasciola hepatica was recovered from the common bile duct. We review the pathobiology of this uncommon zoonosis.

Adult↗
PubMed1987

Herd evaluation of Fasciola hepatica infection levels in Louisiana cattle by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was evaluated as a method of determining relative immunoconversion rates in calves and how immunoconversion rates and strength of optical density values correlate with prevalence of Fasciola hepatica fecal egg shedding. Ten to 55 calves and cows were examined from each of 10 separate beef cattle herds in central and southern Louisiana. Infection prevalence rates for calves averaged 8% higher when ELISA optical density values were used than those when fecal egg count data were used. Of 55 calves in 8 herds that were ELISA positive, 39 were shedding F hepatica eggs; of 53 calves that were shedding eggs, 14 were ELISA negative. Significant correlation of calf fecal and ELISA prevalence was observed for 8 herds by linear regression analysis. A chi 2 analysis showed that calf ELISA and fecal egg shedding data were not independent. Results indicate that positive ELISA reactions for as few as 10 to 15 calves from the fall calf crop of a given herd are sufficiently accurate to be used to assess F hepatica herd infection rates, the likelihood of liver condemnations at feedlot destinations, and variation between individual farms in fascioliasis infection risk. The test was less valuable as a diagnostic test when used in adult animals previously exposed to F hepatica or on an individual animal basis.

Animals↗
PubMed1994

Influence of pro-inflammatory treatments on experimental infection of rats with Fasciola hepatica: changes in serum levels of inflammatory markers during the early stages of fasciolosis.

The effect of stimulating an inflammatory reaction by injecting rats with either Freund's complete adjuvant or turpentine oil shortly before or after they were infected experimentally with Fasciola hepatica on the establishment of the parasite was studied. Freund's complete adjuvant, given either four hours before or four hours after infection with F hepatica and turpentine oil given four hours before infection resulted in a 40 to 50 per cent decrease in the rats' fluke burden. The study of markers of inflammation (acute phase proteins, leucocytes and platelets) demonstrated that the fluke had a modulating effect on the rat's inflammatory response. During the first few days after infection, F hepatica stimulated a transitory inflammatory reaction, as suggested by changes in the level of acute phase proteins (APP) in the serum. No further change in the APP titre occurred until three weeks after infection, when, together with an increase in the extent of the liver lesions, the change in the titre of APP indicated that a systemic and chronic inflammatory process had taken place.

Acute-Phase Proteins↗
PubMed2000

Fasciola hepatica: lithogenic capacity in experimentally infested rats and chemical determination of the main stone components.

A study was done of the possible association between the development of common bile-duct stones and the presence of worms in rats experimentally infected with Fasciola hepatica. A total of 157 rats were individually infected with 20 metacercariae, and another 40 animals served as controls. The rats were dissected at 100, 200, 300, and 400 days postinfection (p.i.). A significant association was observed between the observation of stones and the presence of F. hepatica adults. The global frequency of bile-duct lithiasis in the parasitized rats was 22%, with a significantly lower incidence being observed in the younger group (100 days p.i.). Different analytical techniques were used to determine the main stone components. Energy-dispersive X-ray microanalysis showed calcium to be the main component in all cases (82-94%). Scanning electron microscopy, nuclear magnetic resonance ([1H]- and [13C]-NMR), and mass spectrometry revealed the predominance of palmitic (C-16) and stearic (C-18) acid.

Animals↗
PubMed1996

Paramphistomum daubneyi and Fasciola hepatica: the redial burden and cercarial shedding in Lymnaea truncatula subjected to successive unimiracidial cross-exposures.

The development of redial burden and cercarial shedding were studied in two groups of Lymnaea truncatula subjected to successive cross-exposures to one miracidium of Paramphistomum daubneyi and one of Fasciola hepatica per snail, or vice versa. The results were compared with those obtained in controls subjected to two unimiracidial exposures to the same trematode species. The infection rate was 61% in the group cross-exposed to P. daubneyi/F. hepatica and 37% in that cross-exposed to F. hepatica/P. daubneyi; it was 37% in the control group exposed to F. hepatica and 21% in that exposed to P. doubneyi. Snails harboring larval forms of both trematodes were few in number in cross-exposed groups and the redial burden was low, with one trematode dominating over the other. Free cercariae of F. hepatica and those of P. daubneyi were significantly more numerous at day 35 in the group cross-exposed to P. daubneyi/F. hepatica than in the controls or the other cross-exposed group. Mixed cercarial sheddings were obtained from 40% of snails with emission in the group cross-exposed to P. daubneyi/F. hepatica and from 21% of those in the F. hepatica/P. daubneyi group. The numbers of P. daubneyi metacercariae were significantly greater in the group cross-exposed to P. daubneyi/F. hepatica than in the other cross-exposed group, whereas no significant difference in mean numbers was noted for the F. hepatica cysts. Repartition of metacercariae over the patent period was clearly more irregular for P. daubneyi than for the other trematode.

Animals↗
PubMed1982

Comparative efficacy of albendazole against Fasciola hepatica in sheep and calves: relationship to serum drug metabolite levels.

Calves were given albendazole (ABZ) daily in feed at levels of 0,3 or 5 mg kg-1 day-1. None of the ABZ treatment levels was significantly effective in reducing Fasciola hepatica burdens. A dose rate of 5 mg kd-1 day-1 did significantly reduce the fecal egg count. Measurements of serum drug levels from calves following a single dose of ABZ showed ABZ levels to be low, but sulfoxide and sulfone metabolites of ABZ were present in (significant) larger quantities. The total available sulfoxide present in calves, however, was much lower than in sheep receiving the same dose of ABZ. Measurements of serum metabolite levels from sheep and calves which were given daily low-level doses of ABZ also indicated that the serum sulfoxide levels of calves were much lower than those of sheep receiving the same ABZ dose. These results indicated that ABZ is not an effective prophylactic treatment for bovine fascioliasis and the differences in efficacy between sheep and cattle correlated with the differences in serum metabolite levels.

Albendazole↗
PubMed1992

Fasciola hepatica: molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein.

Immunization of mice with an antigenic polypeptide from Fasciola hepatica adult worms and having an apparent molecular mass of 12,000 Da (Fh12) has been shown to reduce the worm burden from challenge infection with Schistosoma mansoni by more than 50%. Moreover, mice infected with S. mansoni develop antibodies to Fh12 after 5-6 weeks of infection, indicating that this Fasciola-derived antigen is a cross-reactive, cross-protective protein. A lambda gt11 F. hepatica cDNA library was constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding a cross-reactive polypeptide (Fh15) was cloned by screening the F. hepatica lambda gt11 library with a monospecific, polyclonal rabbit antiserum against pure, native Fh12. The cDNA was sequenced and the predicted amino acid sequence revealed an open reading frame encoding a 132-amino-acid protein with a predicted molecular weight of 14,700 Da. This protein has significant homology to a 14-kDa S. mansoni fatty acid-binding protein. Comparison of the protective-inducing activity of recombinant Fh15 with that of purified Fh12 against schistosomes and Fasciola is warranted.

Amino Acid Sequence↗
PubMed1975

Oxidative enzymes in the development of Fasciola hepatica L. II. Dehydrogenase activity of miracidium.

Histochemical methods were used to detect succinate, isocitrate, malate, lactate, alpha-glycerol phosphate' L-glutamate, alcohol, glucose-6-phosphate, 6-phosphogluconate, and beta-hydroxybutyrate dehydrogenases in miracidium--a free living larva of Fasciola hepatica. Examinations were carried out on miracidia which had just left egg covers and on those which had survived for 18 and 24 hours, respectively. Differences were observed regarding the occurrence and intensity of the enzyme activity depending on the age of the larvae. On the basis of the localization of dehydrogenases and persistence of their activity in the miracidia in various periods of their lives, the author concludes that in young larvae there exist three parallel modes of oxidation: glycolytic, in the tricarboxylic acid cycle, and in the pentose cycle. Moreover, there occur processes of amino acid metabolism and of fatty acid oxidation. The 18-hour larvae exhibit a marked predominance of oxidation in the Krebs cycle, the remaining modes being less pronounced, and after 24 hours the only way to obtain energy in the miracidium is oxidation in the Krebs cycle. Likewise noteworthy is the enhancement of the processes of fatty acid oxidation, this being evidenced by an intensified activity of beta-hydroxybutyrate dehydrogenase. The role of each enzyme in the metabolism of the larva is discussed.

Alcohol Oxidoreductases↗
PubMed2001

Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, fasciolidae).

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. In addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic.

Animals↗