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The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica.

Karyotyping of Fasciola hepatica samples from Britain and Ireland has identified a triploid isolate which is effectively aspermic, rendering it necessarily asexually reproducing. Considering the extensive presence of asexually reproducing diploid and triploid Fasciola in Asia it is suggested that facultative gynogenesis is widespread in this parasite. This has important implications for the population genetics and evolution of Fasciola, especially in relation to the development and spread of drug resistance, and must be considered in the mathematical modelling of this process.

Animals↗

Proteomic analysis of glutathione transferases from the liver fluke parasite, Fasciola hepatica.

The parasite Fasciola hepatica causes major global disease of livestock, with increasing reports of human infection. Vaccine candidates with varying protection rates have been identified by pre-genomic approaches. As many candidates are part of protein superfamilies, sub-proteomics offers new possibilities to systematically reveal the relative importance of individual family proteins to vaccine formulations within populations. The superfamily glutathione transferase (GST) from liver fluke has phase II detoxification and housekeeping roles, and has been shown to contain protective vaccine candidates. GST were purified from cytosolic fractions of adult flukes using glutathione- and S-hexylglutathione-agarose, separated by 2-DE, and identified by MS/MS, with the support of a liver fluke EST database. All previously described F. hepatica GST isoforms were identified in 2-DE. Amongst the isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. We also describe cDNA encoding putative Omega class GST in F. hepatica.

Amino Acid Sequence↗

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.

Amino Acid Sequence↗

Isolation of Fasciola hepatica tegument antigens.

Fasciola hepatica tegument antigens were isolated from intact worms in the cold by using Nonidet P-40. Proof of the tegumental nature of the antigens was shown by the peroxidase-antiperoxidase immunocytochemical technique at the light microscope level. The potential of F. hepatica tegument antigens for the immunodiagnosis of rabbit and human fascioliasis was shown by Ouchterlony immunodiffusion, although cross-reactivity was evident in one of six serum samples from patients infected with Schistosoma mansoni. A genus-specific Fasciola antigen was found in F. hepatica tegument. Finally, F. hepatica tegument contained antigens which protected mice from challenge infection with S. mansoni.

Animals↗

Insights into the relationships of Palearctic and Nearctic lymnaeids (Mollusca: Gastropoda) by rDNA ITS-2 sequencing and phylogeny of stagnicoline intermediate host species of Fasciola hepatica.

Fascioliasis by Fasciola hepatica is the vector-borne disease presenting the widest latitudinal, longitudinal and altitudinal distribution known. F. hepatica shows a great adaptation power to new environmental conditions which is the consequence of its own capacities together with the adaptation and colonization abilities of its specific vector hosts, freshwater snails of the family Lymnaeidae. Several lymnaeid species only considered as secondary contributors to the liver fluke transmission have, however, played a very important role in the geographic expansion of this disease. Many of them belong to the so-called "stagnicoline" type group. Stagnicolines have, therefore, a very important applied interest in the Holarctic region, to which they are geographically restricted. The present knowledge on the genetics of stagnicolines and on their parasite-host interrelationships is, however, far from being sufficient. The present paper analyses the relationships between Palaearctic and Nearctic stagnicoline species on the base of the new light furnished by the results obtained in nuclear rDNA ITS-2 sequencing and corresponding phylogenetic studies of the lymnaeid taxa Lymnaea (Stagnicola) occulta, L. (S.) palustris palustris (topotype specimens) and L. (S.) p. turricula from Europe. Natural infections with F. hepatica have been reported in all of them. Surprisingly, ITS-2 length and GC content of L. occulta were similar and perfectly fitted within the respective ranges known in North American stagnicolines. Nucleotide differences and genetic distances were higher between L. occulta and the other European stagnicolines than between L. occulta and the North American ones. The ITS-2 sequence of L. p. turricula from Poland differed from the other genotypes known from turricula in Europe. The phylogenetic trees using the maximum-parsimony, distance and maximum-likelihood methods confirmed (i) the inclusion of L. occulta in the branch of North American stagnicolines, (ii) the link between the North American stagnicolines-L. occulta group with Galba truncatula, and (iii) the location of the L. p. turricula genotype from Poland closer to L. p. palustris than to other European L. p. turricula genotypes. The Palaearctic species occulta is included in the genus Catascopia, together with the Nearctic species catascopium, emarginata and elodes. The results suggest a potential of transmission capacity for C. occulta higher than that of other European stagnicolines or Omphiscola glabra. The relatively low genetic distances between C. occulta and G. truncatula and the clustering of both species in the same clade suggest that C. occulta may be potentially considered as the second lymnaeid intermediate host species of F. hepatica in importance in eastern and northern Europe, and probably also western and central Asia, after G. truncatula. L. p. turricula may be considered as a potential secondary vector of F. hepatica, at a level similar to that of L. p. palustris.

Animals↗

The shedding of the outer glycocalyx of juvenile Fasciola hepatica.

Freshly excysted Fasciola hepatica possess an outer glycocalyx which on incubation at 37 degrees C is rapidly shed. Using an ELISA technique the release of this parasite antigen was shown to be temperature-dependent and to occur in both normal bovine serum as well as in serum free conditions. The ELISA failed to detect the antibody--antigen complexes that occurred when flukes were incubated in immune serum. Release of specific parasite antigen fell slowly with in vitro cultured flukes, but increased with in vivo cultured flukes. Using a fluorescence inhibition assay, antigens with high ELISA titres inhibited surface fluorescence of the parasite suggesting that the ELISA was detecting surface antigens as well as other parasite metabolic products. Several metabolic blocking agents and commercial antihelminthics were titrated against juvenile F. hepatica to screen for inhibition of the surface--tegument shedding: there was little selective inhibition of surface shedding without a significant loss of motility.

Animals↗

Cloning and sequence analysis of genes encoding Fasciola hepatica immunodominant antigens.

Fasciola hepatica lambdagt11 cDNA expression library was immuno-screened with IPAb, two clones were isolated and identified as Fhlambda400 Fhlambda800. Both clones were sequenced, FhA400 contained 305 translated bases encoding 11.509 kDa and designated as SFh12, while Fhlambda800 contained 311 translated bases encoding protein of 11.058 kDa designated as SFh11. The DNA sequence homology search of Fhlambda400 revealed a relatively high degree of identity with F. hepatica amoebapore-like protein mRNA (GenBank accession No. AF286903). However, Fhlambda800 revealed the highest similarity with F. hepatica tegumental antigen (T1) mRNA (GenBank accession No. AF153056). The protein homology search of SFh12 gave 100% identity with amoebapore-like protein (APLP), while SFh-\11 showed 75% identity with F. hepatica tegumenttal antigen (TA). The biochemical analysis of the deduced proteins was identified; in addition the predicted T- & B-cell epitopes have been also evaluated. However, histological localization of identified antigens was achieved using the IPAb in an indirect immunoflorescent antibody assay (IFA). Results revealed that the IPAb labeled the outer glycocalyx in a characteristic, pattern, which proved that the identified antigens were tegumental in origin and that infected Fasciola subjects induced antibodies directed mainly against tegumental components.

Amino Acid Sequence↗

The interaction in vitro between bovine immunoglobulin and juvenile Fasciola hepatica.

Freshly excysted Fasciola hepatica possess an outer glycocalyx which stimulates an antibody response in cattle infected with F. hepatica metacercariae. Sera from animals receiving either a single or a double infection were examined for levels of IgM, IgG1 and IgG2 specific for the outer glycocalyx of intact live juvenile F. hepatica. High levels of specific IgG1 were found to predominate. Using positive sera in an indirect fluorescent antibody assay, the bovine immunoglobulins labelled the outer glycocalyx in a characteristic reticulated pattern. On incubation at 37 degrees C this pattern rapidly broke up into discrete clumps and progressed until the entire antibody-outer glycocalyx complex was shed. The shedding phenomenon was shown to be continuous and, by using preparations of Fab fragments, was not due to the bivalent binding of the sensitizing antibody.

Animals↗

DNA probes for the detection of Fasciola hepatica in snails.

Fasciola hepatica, also called the large liver fluke, is a trematode which can infect most mammals. Monitoring the infection rate of snails, which function as intermediate hosts and harbour larval stages of F. hepatica, is an important component of epidemiological studies on fascioliasis. For this purpose, DNA probes were generated which can be used for the detection of F. hepatica larvae in snails. Four highly repetitive DNA fragments were cloned in a plasmid vector and tested by Southern blot hybridization to the DNA of various trematodes for specificity and sensitivity. The probes Fhr-I, Fhr-II and Fhr-III hybridized only to F. hepatica DNA. Fhr-IV contained ribosomal RNA gene sequences and cross-hybridize with the DNA from various other trematode species. Squash blot analysis showed that the different probes were able to detect the parasite larvae in trematode-infected snails even as isolated single larvae. No signals were obtained in squash blots of uninfected snails. Probes Fhr-I, Fhr-II and Fhr-III are thus useful specific tools for studying the epidemiology of fascioliasis. The probe Fhr-IV, because of its broader spectrum, can be used to detect the larvae of a wide range of trematode species of waterbirds, which are the causative agents of swimmer's itch.

Animals↗

[Purification And Properties Of Branched Chain Amino Acid Aminotransferase From Fasciola Hepatica]

The distribution and properties of branched chain amino acid aminotransferase(EC 2.6.1.42) was investigated in adult Fasciola hepatica. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of branched chain amino acid aminotransferase was measured by the method of Ichihara and Koyama (1966). Isozyme patterns of this enzyme was also examined by DEAE-cellulose column chromatography. The results obtained were as follows: 1. The activity in homogenate was found to be 12.69 units/g wet tissue. The activity of this enzyme was relatively high compared with those in rat tissues. 2. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8 % of the activity was in cytosolic, 10.9 % in mitochondrial and 1.3 % was in nuclear fraction. 3. Cytosolic fraction of Fasciola hepatica contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase. Enzyme I was eluted by 50 mM phosphate buffer from DEAE-cellulose column and catalyzed the transamination of all three branched chain amino acids. 4. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. 5. The best substrate among three amino acids (leucine, isoleucine and valine) was L-isoleucine. 6. The optimal temperature of Enzyme I was 45 degrees C and the optimal pH was 8.2. 7. The Km value for leucine of Enzyme I was 4.17 mM. 8. The Km values for alpha-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41 mM and 4.76 x 10(-3) mM, respectively.

Journal Article↗

A fructose bisphosphate activated lactate dehydrogenase in the liver fluke Fasciola hepatica.

Lactate dehydrogenase of Fasciola hepatica showed typical Michaelis-Menten kinetics at pH 7.2, with respect to pyruvate. Addition of physiological levels of fructose bisphosphate activated the enzyme at all substrate concentrations tested; the response to this effector being hyperbolic in nature. As well as depending upon the fructose bisphosphate concentration, the Vmax and Km are modified by different buffers. The degree of activation is much greater using Tris-HCl than phosphate buffer. The pH optimum occurs at pH 6.5 whether using physiological levels of substrate in the presence or absence of fructose bisphosphate, or high levels of substrate. Of the potential effectors tested, significant inhibition was shown by the nucleoside triphosphates, especially ATP. The importance of this inhibition, coupled with the activation by fructose bisphosphate is discussed. Fasciola hepatica lactate dehydrogenase is unusual in that it does not catalyse the reverse reaction to any measurable extent. That is, lactate oxidation is negligible unless the effector fructose bisphosphate is present. Use was made of this fact to visualise the isoenzymes of lactate dehydrogenase separated by polyacrylamide disc gel electrophoresis. Five isoenzyme bands became apparent when stained in this manner.

Animals↗

[Cholestasis caused by Fasciola hepatica].

Human infestation with Fasciola hepatica is observed occasionally. Cholestasis due to parasitic obstruction of the common duct is an infrequent complication. A case of fascioliasis is described in a 56 year-old male with symptoms of biliary colic and biochemical cholestasis. The diagnosis was based on the indirect hemagglutination test and the evidence of Fasciola hepatica in the extrahepatic biliary tract shown by ultrasound and ERCP. The patient presented an acute pancreatic reaction during an attempt at therapeutic endoscopic sphincterotomy. Medical treatment was initiated then with Bithionol. Cholestasis as well as the parasitosis were eliminated. Recovery was demonstrated by the normality of the titres of hemagglutination and of the biliary tract by ERCP.

Cholestasis↗

[Pathological changes of liver in infection of Fasciola hepatica].

Pathological changes of liver in infection of Fasciola hepatica. Fasciolosis caused by Fasciola hepatica is a hepatic parasitic infection that affects numerous mammal species, mainly ruminants, in several countries of Europe, Asia and America. The economic significance of fasciolosis is mainly due to direct losses caused by a decrease of weight rate, milking capacity and the confiscation of altered livers in slaughterhouse. In this review we present mechanisms of damaging of host liver during invasion of Fasciola hepatica. Parasites digest hepatic tissue and cause extensive parenchymal destruction with intensive haemorrhagic lesions and immunological reactions. Mechanical liver damage is due to migrating juvenile fluke. Macerated hepatic cells have been observed inside the oral sucker and pharynx. Moreover, the process of ulceration of liver tissue was observed in regions adjacent to the spiny body of fluke tegument. On the other hand, the injury of the liver can be induced chemically by factors produced or induced by the fluke. Probably, fluke proteases and other tissue-degrading enzymes can be responsible for negative effect on liver parenchyma. There is no evidence to link fluke-enzymes with tissue penetration. The consequences of liver damage resulting from the migrating flukes compromises liver function which is reflected in changes of plasma protein concentration (albumin, globulin). Additianally, changes of levels of hepatic enzymes released into the blood as a result of damage of liver tissue are used to monitor the progress of the infection in a variety of Fasciola hepatica hosts and as a sensitive diagnostic aid in field infection.

Americas↗

[Determination of cholinesterase activity in Fasciola hepatica homogenates].

The cholinesterase activity in Fasciola hepatica homogenates was studied through biological techniques. Results depict that the contractile stimulating action of a constant acetylcholine dose on the isolated rat duodenum is withdrawn when the agent is previously incubated at 37 degrees C during 30 minutes with different dilutions (20%, 40% and 80%) of Fasciola hepatica homogenates. The action is recovered when an anticholinesterase, as neostigmine is previously added to the homogenate. Since these effects are similar to those obtained when different dilutions of human blood serum with a high content of cholinesterase are led to act upon the acetylcholine dose, it is concluded that the acetylcholine inactivation induced by Fasciola hepatica homogenates results from the existence of such enzyme within this parasite.

Animals↗

The dual effects of aluminum as activator and inhibitor of adenylate cyclase in the liver fluke Fasciola hepatica.

The liver fluke, Fasciola hepatica, has a very active adenylate cyclase which can be stimulated by NaF or by serotonin and guanine nucleotides. Micromolar amounts of AlCl3 augment the activation by F-. In contrast, when the enzyme is activated with serotonin and guanine nucleotides, AlCl3 inhibits the activation. Aluminum also inhibits the activation by forskolin. Gallium mimics the effects of aluminum.

Adenylyl Cyclase Inhibitors↗

Transport of serum IgA into bile of sheep infected with Fasciola hepatica.

Sheep infected with Fasciola hepatica were studied for their ability to transport intravenously injected radiolabelled IgA from serum to bile. The results show that there was an apparent reduction in the selectivity of transport of IgA into bile in infected animals compared with uninfected controls. However, in infected animals the biliary flow rate was approximately twice that of uninfected animals, and the total amount of radiolabelled IgA transported was similar irrespective of infection status. The possible relevance of the elevated biliary flow rate is discussed with respect to the pharmacokinetics of drugs used for the chemotherapy of helminth infections in sheep.

Animals↗

Glomerulonephritis in water buffaloes (Bubalus bubalis) naturally infected by Fasciola hepatica.

Glomerulonephritis caused by Fasciola hepatica was observed in buffaloes. Renal biopsies of 20 buffaloes, 11 with F. hepatica and 9 uninfected buffaloes (controls), were examined by light microscopy, direct and indirect immunofluorescence, and immunohistochemical analysis. The biopsies of seven (63.6%) infected buffaloes revealed membranoproliferative glomerulonephritis, three biopsies (27.3%) showed mesangioproliferative glomerulonephritis, and one kidney presented normal biopsy specimens. In the control group, seven buffaloes (77.8%) presented normal biopsy specimens, while two (22.2%) revealed glomerulonephritis-one with a membranoproliferative pattern, and the other with a mesangioproliferative pattern-with extensive inflammatory cell infiltrate. Our conclusion is that glomerulopathy is associated with fascioliasis and that buffaloes are suitable as a naturally existing experimental model of renal injury by circulating immune complexes.

Animals↗

Inhibition of spleen cell proliferative response to mitogens by excretory-secretory antigens of Fasciola hepatica.

The effect of Fasciola hepatica excretory-secretory antigen (ESA) on the proliferative response of spleen mononuclear (SpM) cells of normal rats to stimulation with mitogens has been examined. When ESA was added to normal SpM cells, there was a decrease in the proliferative response to concanavalin A (Con A) or lipo-polysaccharide (LPS) in a dose-dependent manner. The addition of indomethacin, which blocks prostaglandin synthesis, or N omega-nitro-L-arginine methyl ester (L-NAME) a specific inhibitor of nitric oxide (NO) synthase, had no effect on the ability of ESA to suppress the proliferative response to Con A. However, supplementation of the culture media with catalase, which degrades hydrogen peroxide (H2O2) or superoxide dismutase (SOD) to remove superoxide anion (O2), resulted in a restoration of proliferation to Con A. When LPS was used as mitogenic stimulus no inhibitor added to the culture restored the proliferation. These results suggest that H2O2 and O2- are involved in the suppressor phenomenon induced by ESA in the T-cell proliferative events.

Animals↗