Fasciola hepatica: the effect of the sodium inophore monensin on the adult tegument

The effect on the tegument of adultFasciola hepatica of incubation in the sodium ionophore monensin, the Na⁺/K⁺-ATPase inhibitor ouabain and ouabain pretreatment followed by monensin has been determined in vitro by scanning and transmission electron microscopy (SEM, TEM). With monensin incubation alone (1×10^–6 M), a flattening of the tegument with some loss of spines on the ventral surface is evident from 0.5 h onwards. Internally, the subtegumental musculature becomes grossly swollen, although there is no swelling of the infoldings of the basal plasma membrane of the tegument, even after 24 h incubation. Ouabain incubation (1×10^–3 M) induces folding of the apical surface of the tegument from 0.5 h onwards, and this is accompanied by the formation of bleds and microvilli. Brief (0.5 h) exposure to ouabain (1×10^–3 M) followed by monensin treatment (1×10^–4 M, 3 h) leads to gross vacuolation of the tegument, but this is not due to swelling of the basal infoldings. The other main feature of ouabain-pretreated flukes is the projection of basal lamina-like material into the tegumental syncytium. Monensin treatment alone (1×10^–6 M) results in the Golgi complexes of the tegumental cells becoming very diffuse from 1.5 h onwards, and relatively few secretory bodies are present in the cytoplasm. After 0.5 h incubation in ouabain (1×10^–3 M), the Golgi complexes of the tegumental cells are indistinct, although numerous secretory bodies are still present. The classical monensin-induced swelling of the Golgi cisternae is observed in the tegumental cells only when monensin treatment (1×10^–4 M, 3 h) was preceded by brief (0.5 h) exposure to ouabain (1×10^–3 M). The results are discussed in relation to the postulated osmoregulatory role of the tegument and the role of sodium pumps in membrane function in the fluke.     

The biological lavelling and 75Se of protein antigens of Fasciola hepatica

Adult liver flukes were incubated for several hours at 37° in a culture medium containing ⁷⁵Se-L-selenomethionine. Analysis of homogenates of the parasite showed that significant amounts of isotope had become incorporated into parasite proteins. Separation of the fluke proteins on Sephadex G-100 demonstrated the highest specific activity in the most serologically active protein fractions.     

Fasciola hepatica human infection

Summary Sixteen human cases of Fasciola hepatica infection are described. The liver was involved in 13 cases, the gall bladder in 9 cases and the stomach in 2 cases. Lesions containing parasitic remnants or fluke eggs were rarely seen. Surface scarring of the liver, scar tracks and granulomas within organs were the most characteristic changes seen and were the most useful for the histopathological diagnosis of the disease. The associated liver, bile and gastric lesions are briefly discussed.     

Infection with Fasciola hepatica

Fascioliasis, caused by the liver fluke Fasciola hepatica, is an infection that occurs worldwide, although humans are accidental hosts. F. hepatica infection comprises two stages, hepatic and biliary, with different signs and symptoms. Stool examination and ELISA can be used for the initial diagnosis. Radiographic techniques, such as computerised tomography and ultrasonography, as well as magnetic resonance imaging, are used widely for confirmation and follow-up of the disease. Invasive techniques, such as percutaneous cholangiography, endoscopic retrograde cholangiography and liver biopsy, may aid in the diagnosis but are not essential. Triclabendazole is recommended as the first-line agent for the treatment of F. hepatica infection, with bithionol as an alternative.     

Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms

Adult Fasciola hepatica worms contain multiple proteinases capable of degrading hemoglobin, immunoglobulins and collagen. Here we report the isolation and biochemical characterization of a cysteine proteinase from acidic extracts of these worms. The enzyme was purified to homogeneity by cation exchange and molecular sieve high-performance liquid chromatography. It eluted at a native molecular weight of approximately 14,500 and migrated as a single band at approximately 14,500 Da upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was assessed by employing synthetic peptide substrates, such as carbobenzoxy-phenylalanyl-arginyl-7-amino-4-trifluoro-methylcoumarin, commonly used to assay other cysteine proteinases. The proteinase was maximally active at pH 6.0, with 50% or more of the activity detected between pH 4.5 and 7.5. Inhibition of activity at pH 5.5 was seen only with compounds known to inhibit cysteine proteinases. No effect was seen with inhibitors of aspartic, serine, or metalloproteinases. The purified enzyme was stable at acidic pH at 4 degrees C, 25 degrees C, -20 degrees C, and in 1 M urea.     

Fasciola hepatica case with hemobilia

Fasciola hepatica (FH) can lead to important hepatobiliary diseases. Here we present a case of hemobilia associated with biliary FH, which is quite a rare case. The 41-year-old patient, who underwent common bile duct exploration due to hemobilia, was found to have arterial bleeding associated with ulcer caused by a dead parasite in the common bile duct. Hemobilia is a very rare complication associated with FH. When searching for the cause of hemobilia, FH should be considered.     

Electron microscope studies of Fasciola hepatica

Summary Mehlis' gland of F. hepatica is composed of two secretory cell types supported by parenchymal cells. The products of the secretory cells are carried by means of cytoplasmic extensions into the ootype. The most prevalent type of secretory cell is termed S ₁ type. It possesses many narrow granular endoplasmic cisternae and numerous Golgi complexes. The secretory bodies of this cell resemble slightly curved sausages and have a filamentous content radiating from a central core. The other secretory cell type is termed S₂ type. It is characterized by distended granular endoplasmic cisternae and numerous Golgi complexes which give rise to round dense secretory bodies with a crystalline or packed fibrous appearance.Both types of secretory cells are penetrated deeply by invaginations of interstitial material. The cytoplasmic extensions from the cells are lined with microtubules and anchored to the epithelial cells lining the ootype by septate desmosomes. The secretory bodies of both cell types are released into the lumen of the ootype, where they disintegrate.     

Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 μM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 μg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ–resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.     

Polyamine N-acetyltransferase from Fasciola hepatica.

A cytosolic polyamine N-acetyltransferase which catalyses polyamine and diamine acetylation has been partially purified from the liver fluke Fasciola hepatica. The enzyme has an apparent Mr of 50,000 and unlike the corresponding mammalian liver counterpart is capable of putrescine acetylation. Among the substrates tested, spermidine had the highest reaction rate but putrescine had a lower Km value. The Km values for spermidine, spermine, norspermidine, putrescine, cadaverine and 1,3-diaminopropane were 20 microM, 1.30 mM, 20 microM, 7 microM, 10 microM and 50 microM, respectively. Acetylated polyamines were also substrates for the trematode acetylase, but histones were inactive. The partially purified enzyme had no deacetylase activity. The Km for acetyl-CoA was 4.4 microM. Coenzyme A was strongly inhibitory with a Ki value of 5.3 microM. Bis(benzyl)polyamine analogue MDL 27695 was a potent competitive inhibitor of the enzyme with a Ki of 22 microM. Inhibition by 1,4-dimethyl-putrescine was non-competitive and had a Ki value of 15 microM. The trematode acetylase is highly dependent on sulfhydryl groups for its activity. As had been reported in nematodes, polyamine acetylation could represent a process by which trematodes convert excess polyamines to forms suitable for transport and excretion. On the other hand, this could be the regulatory step of a functional interconversion pathway in these parasites.     

Immunity in Schistosoma mansoni using antigens of Fasciola hepatica isolated by concanavalin A affinity chromatography.

Antigens of Fasciola hepatica adult worms were chromatographed using concanavalin A-Sepharose 4B. Two unbound peaks appeared in the inclusion volume (DT-1 and DT-2), and one peak was eluted with alpha-methylglucoside (E1-1). At least seven peaks were obtained by isoelectric focusing of E1-1. The largest of these peaks, with an average pI of 4.0, contained the antigens reactive with antibodies to Schistosoma mansoni. Mice immunized with DT-2 or E1-1 and challenged with S. mansoni cercariae developed 39 to 82% fewer worms than controls. DT-1 had no protective effect. Combining DT-1 and DT-2 abolished this protection. These experiments demonstrate that F. hepatica glycoprotein antigens induce in mice significant protection to infection with S. mansoni and offer an interesting approach to the study of vaccines in experimental schistosomiasis.     

IgE antibodies in human Fasciola hepatica distomiasis

In patients infected by Fasciola hepatica, total IgE and specific IgE antibodies have been determined by radioimmunoassays, and IgG, IgA, IgM levels by radial immunodiffusion test (Mancini, 1965). Moreover, total and specific IgE levels have been related to parasite egg burden, age, clinical features and eosinophilia. Elevated total IgE and specific IgE antibodies levels have been found respectively in 76% and 48% of the patients whereas there was no significant variations in other immunoglobulins levels. However, though the amount of total and specific IgE was lower than in other helminthic diseases, it appears to be a significant data of the immune response to parasites as it has been reported and discussed previously. It has been shown a significant relationship between total and specific IgE levels, the number of lines by immunoelectrophoresis, and the results of the indirect haemagglutination and indirect fluorescent antibody techniques; each method appeared to be in equal value to perform the early diagnosis of human Fasciola hepatica. In addition, specific IgE antibodies levels were correlated with eosinophilia specially when it exceeds 15%. This results demonstrate the availability of their measurement in the diagnosis of fascioliasis versus other diseases with marked eosinophilia.     

Detoxification reactions of Fasciola hepatica cytosolic glutathione transferases

Acidic/neutral glutathione (GSH) transferase forms have been isolated from Fasciola hepatica by a combination of GSH-affinity chromatography and chromatofocusing. Approximately 10-25% of the activity failed to interact with the GSH-affinity matrix when applied from crude cytosolic preparations. Following partial purification by chromatofocusing this GSH transferase activity did subsequently bind to the affinity matrix. The F. hepatica GSH transferases had catalytic activity with secondary lipid peroxidation products, the latter being possible natural substrates. The enzymes also interacted with a number of hydrophobic ligands including haematin and substituted phenol-based anthelmintics.     

Staining of Fasciola hepatica by natural herbal dyes

The plant kingdom is a valuable source of new medicinal agents. Traditional synthetic dyes used for staining worms may have some carcinogenic effects; therefore, the use of herbal dyes as an effective alternative may be interesting. The objective of the current study was to evaluate the staining effect of herbal dyes on Fasciola hepatica. Powdered alizarin, henna and curcuma in concentrations of 5, 10 and 20 g/100 mL of distilled water were used to stain a F. hepatica worm according to the carmine staining method. Our criteria for evaluation were the extent of staining on different parts of the worm such as suckers, intestine, testes, spines, ootype, vitelline duct, vitellaria, ovary, uterus and eggs. The results showed that alizarin (10 g/100 mL; 10%, w/v) was a better dye than henna (20 g/100 mL; 20%, w/v) and curcuma (20 g/100 mL; 20%, w/v) for the staining of suckers, branched intestine, vitelline duct and ootype. Although henna stained the whole body of the worm better than curcuma, curcuma had a better staining effect on surface spines, eggs and especially branched testes. In comparison with carmine, the present herbal dyes (alizarin and henna) are cheap and safe and could be potential alternatives for staining F. hepatica and other trematodes.     

Erratum to: inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 μM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 μg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ–resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.     

Citrate synthase from the liver fluke Fasciola hepatica

Citrate synthase catalyses the first step of the Krebs’ tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K _m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.     

Thiol proteases released in vitro by Fasciola hepatica

Immature Fasciola hepatica release 11 distinct proteases when cultured in vitro for 16 h as revealed by gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six of these proteases are active in the pH range 4.5 to 8.0. Five are acid proteases, being most active in the pH range 3.0 to 4.5. The majority of proteases released in vitro by immature flukes are also released by mature flukes; however, a 40-kDa protease released by immature flukes is a very minor protease released by mature flukes. The activity of all proteases is inhibited by leupeptin, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, phenylmethylsulfonyl fluoride and iodoacetamide and enhanced or stabilized by the reducing agents cysteine and dithiothreitol. Therefore, all F. hepatica in vitro-released proteases identified by gelatin-substrate SDS-PAGE are thiol proteases.     

Fasciola hepatica expresses multiple alpha- and beta-tubulin isotypes

We have identified five alpha-tubulin and six beta-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke alpha-tubulin and between 65 and 97% for beta-tubulin isotypes. Nucleotide sequence identity ranged between 68-77% and 62-80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the alpha-tubulins and two of the beta-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on beta-tubulin, three of the fluke isotypes had tyrosine at position 200 of beta-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.