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PubMed1993

Biogenic-amine acetylation: an additional function of the N-acetyltransferase from Fasciola hepatica.

The previously described polyamine N-acetyltransferase from Fasciola hepatica has been observed to have an additional function, the acetylation of biogenic amines. The activities for biogenic amines, diamines and polyamines were in a constant ratio throughout the purification process. Biogenic amines found to be substrates for the enzyme included tyramine, tryptamine, beta-phenylethylamine and histamine, with Km values of 0.12 mM, 0.26 mM, 0.30 mM and 0.76 mM respectively. Octopamine, 5-hydroxytryptamine and alpha-phenylethylamine were also acceptable as substrates, though to a lesser degree. The optimum pH for biogenic-amine acetylation was 7.5, and CoA was inhibitory to the process, with a Ki of 5.5 microM. N-Acetylation appears to play a major role in the amine metabolism of this trematode. We presume that acetylation represents the process by which the parasite inactivates excess amines.

Acetylation↗
PubMed1983

Composition, secretion, and fate of the glands in the miracidium and sporocyst of Fasciola hepatica L.

The anterior end of the miracidium of Fasciola hepatica contains a large flask-shaped apical gland and four unicellular lateral glands, all of which have ducts which pass to the tip of the apical papilla. These glands appear to be involved in penetration of the larva into the snail host. The apical gland secretes as the miracidium proves the epidermis of the host before attachment. It seems likely that its secretion is a chemical which lyses the epidermal cells. The lateral glands are PAS-positive and may contain a neutral mucopolysaccharide. They also secrete as the miracidium probes the snail and a layer of PAS-positive material may be seen at the leading edge of the apical papilla as the larva penetrates into the host. Both the apical gland and the lateral glands may be visible in the sporocyst for several days after penetration.

Animals↗
PubMed1981

Preparation and in vitro translation of mRNA from Fasciola hepatica.

Total RNA was extracted from mature and juvenile Fasciola hepatica by homogenizing in 5.0 M guanidine thiocyanate and centrifugation through a 5.7 M CsCl cushion. Yields of 2 mg/g and 1 mg/g wet weight starting material were obtained, respectively. Messenger RNA was separated from the bulk extracted RNA by binding to oligo(dT)-cellulose. About 25% of the extracted RNA bound in both adult and juvenile cases. This material was subsequently tested in a rabbit reticulocyte cell-free translation system. Up to a 12-fold stimulation of incorporation of [35S]methionine into trichloroacetic acid-precipitable material was observed over that where no message was added. When the in vitro translation products were analysed by autoradiography of SDS-polyacrylamide gradient gels, polypeptides ranging in apparent molecular weight from about 10,000 to 100,000 were observed. Several minor differences in the electrophoretic patterns obtained from juvenile and adult mRNA were observed.

Animals↗
PubMed1999

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.

Amino Acid Sequence↗
PubMed1977

Stereoscan studies of rediae, cercariae, cysts, excysted metacercariae and migratory stages of Fasciola hepatica.

The external surface of the redial body of Fasciola hepatica is provided with microvillus-like projections or short lamellae, and short cilium-like structures are common anteriorly. The anterior part of the cercarial body possesses a pattern of regularly arranged small depressions each containing a spine. Both long and short cilium-like structures occur anteriorly. The tail is spineless and provided with dorsolateral folds. The outer cyst wall is formed by granules secreted from the tegument all over the body apart from the ventral sucker. Most granules transform into fibrillae which form the thick outer spongy layer. The precursor of the inner cyst wall is at the beginning closely attached to the metacercarial surface, but later the membrane-like cyst wall extends, and when fully formed the metacercaria lies free in the flattened circular inner cyst. The ventral plug is formed by the ventral sucker. The tegument of newly excysted metacercariae is provided with simple pointed spines, but later during migration in the mouse the spines become flattened and multipointed. Very young migratory stages may be attached with host cells.

Animals↗
PubMed1986

The uptake and distribution of radioselenium in the larvae of Fasciola hepatica and its snail host Lymnaea columella.

Radiolabeled metacercariae of Fasciola hepatica were obtained in vivo by incubating infected Lymnaea columella snails with 20 muCi radioselenomethione (75Se-M) per snail in 5 ml of water for 5 h, or in vitro by incubating a batch of unlabeled F. hepatica metacercariae with 75Se-M for 24 h. Radioassay showed that only 5% of the 75Se-M was incorporated into maritas (juvenile flukes) from the in vivo labeled metacercariae. The inner cyst wall of in vivo labeled metacercariae contained 46% of the total activity, of which 21% was dissolved in the excysting medium. The outer, tan-colored cyst wall contained 49% of the radioactivity. Through diffusion/attachment, maritas from in vitro labeled metacercariae could occasionally be labeled with 0.4% of the total radioactivity. However, the activity was lost after inoculation into the body of mice. The outer and inner cyst walls of in vitro labeled metacercariae contained 92% and 7.6%, respectively, of the total activity. Microautoradiography demonstrated that 75Se-M was evenly distributed in the body of marita and the cyst wall of inner and outer layers from an in vivo labeled metacercaria. A 9 X 4 micron rectangularly-shaped aggregate of Ag degree grains was present on the outer periphery of the inner cyst wall. Microautoradiography of in vitro labeled metacercariae demonstrated a significant concentration of Ag degree grains on the cyst walls. The ventral plug contained fewer Ag degree grains per unit area compared to the other portion of the inner cyst wall. Uptake and distribution of 75Se-M in the snail host were also studied. It appeared that rediae and cercariae tended to concentrate the label in the foot, the mantle and the digestive gland. Little or no radioactivity was present in the areas where F. hepatica larvae were not found.

Animals↗
PubMed1984

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica.

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.

Animals↗
PubMed1982

Cellular proliferation of bile ductules and gamma-glutamyl transpeptidase in livers and sera of young cattle following a single infection with Fasciola hepatica.

During the course of experimental single infection with Fasciola hepatica in calves, the origin of the increased concentration of gamma-glutamyl transpeptidase (gamma-GT) found to be present in the serum during the biliary stage of the disease, was studied. The enzyme originates from the liver after flukes have caused intermittent and selective cholestasis in the bile-duct system. The enzyme which normally is present in the bile ducts can under these circumstances enter the surrounding interstitial tissues through broken-up junctions of the bile drainage system or be added to these from proliferating bile-ductule epithelium. The multiplication of bile-ductule cells can be looked upon as a neogenesis of cholangioles, which becomes essential when newly formed perilobular connective tissues prevent restoration of the anatomical connections of the bile capillaries with the biliary tree. The connective tissue growth is induced by bile fluid. An initially regional perilobular fibrosis is eventually distributed diffusely through the liver by migration of flukes. The continued and extending fibrosis is due to the constant recurrence of intermittent and selective cholestasis. The transient occlusions of branches of the bile ducts are very likely to be attributable to persistent cholangitis. The growth of connective tissues has a considerable effect on the gain in weight of the liver.

Animals↗
PubMed1994

Characterization of the oligomycin-sensitivity properties of the F1F0-ATPase in mitochondria from rats infected with the liver fluke Fasciola hepatica.

The F1F0-ATPase activity of liver mitochondria isolated from rats infected with Fasciola hepatica at 3 and 4 weeks post-infection showed a marked loss of sensitivity to oligomycin and to N,N'-dicyclohexylcarbodiimide. A loss of sensitivity to diethylstilbestrol was also demonstrated at 4 weeks post-infection. Recovery was apparent in most cases by 6 weeks post-infection. No significant difference in latent ATPase activity was observed between mitochondria from control and infected livers at any stage of the infection. The mitochondria from infected livers were therefore considered to have a full complement of the F1 moiety of the F1F0-ATPase complex. Purification of the mitochondrial ATPase from 4-week infected livers resulted in a very low yield of an oligomycin-insensitive complex. This was due to a failure to enrich specific activity during purification. The evidence presented indicates that infection with Fasciola hepatica gives rise to alterations in the function of the host liver mitochondrial ATPase, namely loss of inhibitor sensitivity and apparent structural alterations of the ATPase complex.

Animals↗
PubMed2000

The susceptibility of Lymnaea fuscus to experimental infection with Fasciola hepatica.

Three experiments on the infection of Lymnaea fuscus with Fasciola hepatica were carried out to determine if successful infections and maturation of the parasite were dependent on the size of snails at miracidial exposure. The first experiment was performed using 1-4-mm-high snails from 2 populations of L. fuscus and 1 population of Lymnaea palustris. In these snails each subjected to a single bimiracidial exposure, the prevalence of F. hepatica infection at day 35 postexposure ranged from 20.3% to 46.2% in snails measuring 1 mm in height at exposure; it was lower in the 2-mm snails and was 0 in higher size classes. The second experiment was performed by subjecting 1- and 4-mm L. fuscus to 1, 2, and 3 bimiracidial exposures. The prevalence of F. hepatica infection at day 35 postexposure was maximum in the 1-mm snails exposed once to miracidia and decreased with increasing number of exposures. The results were negative in 4-mm snails. Cercarial shedding of F. hepatica was studied in the third experiment using 1- and 2-mm L. fuscus each subjected to a single bimiracidial exposure. The total number of cercariae released from these snails was less than 50. From these results, it can be concluded that L. fuscus showed a partial resistance to F. hepatica infection due to snail age.

Animals↗
PubMed1984

[Four kinds of parasites in an 8-year-old boy. Therapeutic effects of praziquantel against Fasciola hepatica].

An 8-year-old Turkish boy had recurrent abdominal colics associated with marked eosinophilia. Four different kinds of parasites were isolated from stool and duodenal juice: Fasciola hepatica, Entamoeba histolytica, Ascaris lumbricoides and Lamblia intestinalis. Conventional drugs were successful in treating the latter three parasites. But because of problems connected with the treatment of F. hepatica, praziquantel was administered, 15 mg/kg five times daily for 5 days, with curative results. This is the first published successful treatment of Fasciola hepatica with this drug in Europe.

Ascariasis↗
PubMed1986

The susceptibility of the goat to Fasciola hepatica infections.

Mixed breed goats were infected with metacercariae of Fasciola hepatica and the resulting worm burdens were quantitated after primary and secondary exposure of the goats to the parasite. Mean length and width of the parasite recovered after all primary exposures were 1.91 +/- 0.2 cm and 0.91 +/- 0.2 cm, respectively. A mean of 71.8 +/- 5.9% of the flukes were recovered from all of the primary infections. In the secondary infections, the mean length and width of the flukes from the physically smaller population was 0.88 +/- 0.27 cm and 0.53 +/- 0.19 cm, respectively. A mean of 67 +/- 6.7% of the flukes were recovered from this secondary infection. It appears that the goat is susceptible to challenge infections with F. hepatica and that its response to this infection is much like that of sheep.

Animals↗
PubMed1986

31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica.

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.

Aerobiosis↗
PubMed2000

Second messengers mediating mechanical responses to the FARP GYIRFamide in the fluke Fasciola hepatica.

Spontaneous phasic contractions recorded from isolated body strips of Fasciola hepatica were increased in frequency and amplitude by GYIRFamide, an FMRFamide-related peptide (FaRP). Superfusion with guanosine 5'-O-(2-thiodiphosphate) (100 microM, n = 5) reduced the effects of GYIRFamide on both frequency (by 82%) and amplitude (by 75%). The adenylate cyclase inhibitor MDL-12330A (25 microM) increased spontaneous activity. MDL-12330A completely inhibited the frequency response to GYIRFamide and reduced the amplitude response by 66% as measured relative to this elevated basal activity (n = 6). Inhibition of phospholipase C (PLC) with neomycin sulfate (1 mM) had no direct effect on activity but reduced the frequency response to GYIRFamide by 64% and the amplitude increase by 95% (n = 9). The protein kinase C (PKC) inhibitor chelerythrine chloride (10 microM) also reduced frequency and amplitude responses by 98 and 99%, respectively, without affecting basal contractility (n = 5). Phorbol 12-myristate 13-acetate, an activator of PKC, increased contraction frequency and amplitude (n = 6). It was concluded that GYIRFamide stimulates mechanical activity in F. hepatica through a G protein, via a PLC- and PKC-dependent second messenger pathway.

Alkaloids↗
PubMed1964

Metabolism Of C(14)-Glucose By Fasciola Hepatica.

1. The glucose uptake rate by Fasciola hepatica was a mean value of 9.62 +/- 0.54 micro moles/hr/g, and total CO2 production rate by the flukes averaged 24.28 +/- 4.26 micro moles/hr/g wet wt. The relative specific activity of respiratory CO2 showed a mean value of 79.89 +/- 1.78 per cent. The rate of CO2 production derived from medium C(14)-glucose was a mean of 19.55 +/- 3.56 micro moles/hr/g of wet wt. Therefore, the average value of 32.72 +/- 4.8 per cent of glucose utilized by the flukes from the medium C(14)-glucose was oxidized to respiratory CO2. 2. The tissue concentration of glycogen in F. hepatica was a mean of 38.36 +/- 2.91 mg/g or 3.84 +/- 0.29 %/g of wet wt, and the turnover rate of glycogen pool was a mean of 1.6+/-0.22 %/hr or 0.65 +/- 0.13 mg/hr/g. The average value of 37.26 +/- 3.86 per cent of glucose utilized by the fluke from the medium C(4)-glucose was incorporated to the glycogen. 3. These data account for that approximately 70 per cent of the utilized glucose by the flukes participated in furnishing the oxidation into respiratory CO2 and the synthetic process into glycogen.

Journal Article↗
PubMed1965

[Metabolism Of C(14)-Lactate By Fasciola Hepatica And Eurytrema Pancreaticum]

The adult trematode, Fasciola hepatica and Eurytrema pancreaticum, employed in this experiment were obtained from the cattle slaughtered at the local abbatoir. The worms were selected and washed several times in normal sterilized saline solution. Each ten of intact F. hepatica and about thirty to fifty of E. pancreaticum were incubated in 50 cc volume of special incubation flasks with incubation medium consisting of 50 cc of Krebs-Ringer phosohate buffer (pH 7.4). The incubation medium was added C(14)-lactate and non-radioactive carrier Na-lactate so as to contain lactate concentration of 32 mg per cent. The worms were allowed to incubate for 3 hours in the Dubnoff metabolic shaking incubator at 38 degrees C. After incubation period, respiratory CO2 samples from central wall of incubation flask were analysed for total CO2 production rate and their specific activity of respiratory CO2. The lactate uptake rate was determined by analyzing the the difference between lactate concentration in a medium before and after the incubation period, and the pyruvate appearance rate was dertermined by analyzing the pyruvate concentration in a medium after incubation. The glycogen samples isolated from worms were analyzed for the tissue concentration and their radioactivities in order to determine the turnover rate of glycogen pool. Radioactivities of these serise of experiment were counted by an endwindow Geiger-Muller counter as an infinitely thin samples. The quantative analysis of C(14)-lactate utilized by F. hepatica and E. pancreaticum were summerized and compared as following. In F. hepatica the lactate uptake rate was a mean value of 1.04 +/- 0.15 micro M/hr/g of wet wt. and pyruvate apperance rate was a mean value of 0.132 +/- 0.005 micro M/hr/g of wet wt. The total CO2 production rate by the flukes averaged 13.82 +/- 0.75 micro M/hr/g of wet wt. The relative specific activities of respiratory CO2 R.S.A(CO2) showed a mean value of 9.93 +/- 0.62 per cent. The rate of CO2 production derived from medium C(14)-lactate was a mean of 1.38 +/- 0.13 micro M/hr/g of wet wt. Therefore the averge value of 55.27 +/- 5.78 per cent (R.L.D(CO2)) and 15.35 +/- 1.90 per cent (R.L.D(pyr)) of lactate was oxidized into respiratory CO2 and pyruvate respectively. On the other hand, in E. pancreaticum the lactate uptake rate was a mean value of 0.61 +/- 0.18 micro M/hr/g of wet wt, and pyruvate appearance rate was a mean of 0.023 +/- 0.001 micro M/hr/g of wet wt. The total CO2 production rate by the E. pancreaticum averaged 4.29 +/- 0.85 micro M/hr/g of wet wt. The relative specific activity of respiratory CO2 (R.S.A(CO2)) showed a mean value of 9.20 +/- 0.34 per cent. Thus, a mean value of 9.20 per cent of total CO2 production rates was originated from C14-lactate in a medium, therefore the rate of CO2 production derived from medium C(14)-lactate was a mean value of 0.40 +/- 0.10 micro M/hr/g of wet wt. The average value of 23.93 +/- 7.11 per cent(R.L.D(CO2)) and 3.86 +/- 0.45 per cent(R.L.D(pyr)) of lactate was oxidized into respiratory CO2 and pyruvate respectively. The tissue concentration of glycogen in F. hepatica was a mean of 2.63 per cent/g of wet wt, while in E. pancreaticum was a mean of 4.06 per cent/g of wet wt. The turnover rate of glycogen pool in F. hepatica yielded a value of 0.073 +/- 0.008 micro M/hr/g of wet wt whereas in E. pancreaticum yielded only a mean of 0.006 +/- 0.002 mg/hr/g of wet wt. Therefore, the half time (t(2/1)) of glycogen turnover, which is the time interval required to replace the half of glycogen pool with medium C(14)-lactate, gave value of a mean of 10.73+/-0.76 days in F. hepatica. However, incorporation of C(14)-lactate into glycogen was negligible in the E. pancreaticum. Theses data impressed that the carbohydrate such as lactate may play a role of major part of their oxidative metabolism in F. hepatica, whereas minor part of lactate participates in the oxidative metabolism in E. pancreaticum.

Journal Article↗
PubMed1976

Use of immunologic techniques to detect chemotherapeutic success in infections with Fasciola hepatica. I. Rabbit infections.

Rabbits infected with Fasciola hepatica develop precipitins to adult worm heomogenates, as observed by Ouchterlony double immunodiffusion (ID) and counterelectrophoresis (CEP). When they are successfully treated with a fasciolicidal drug such as rafoxanide at 5, 6, or 11 weeks of infection their precipitins drop dramatically by 2 weeks post-treatment, they are virtually negative by 4 weeks, and have no detectable precipitins by 5 or 6 weeks post-treatment. The results suggest that ID or CEP can be utilized to show chemotherapeutic success in rabbits infected with F. hepatica and warrant further studies as to their possible application in human fascioliasis.

Animals↗