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PubMed1986

Interaction of bovine complement with Fasciola hepatica.

The host-parasite relationship of Fasciola hepatica in cattle was characterized by determining the effects of the parasite on the bovine complement system. Phosphosaline extracts of F. hepatica adults inhibited both the classical and alternative pathways of complement activation in normal bovine and human sera in a protein dose-dependent manner. The in vitro reaction of viable newly excysted juveniles (NEJ) with bovine serum (NBS) and with bovine serum containing antibodies specific to F. hepatica (IBS) resulted in no detectable changes in serum hemolytic complement activity for either pathway. This lack of complement consumption occurred even though these same flukes incubated in IBS for at least 24 h developed a precipitate that adhered to the parasite tegument, a feature that may reflect antibody--antigen reactions.

Animals↗

PubMed1999

Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.

The excretory-secretory antigen of Fasciola hepatica (ESA) is involved in the suppressive phenomena of cellular immune responses in rats. The ESA can depress the proliferative response of spleen mononuclear cells and inhibit nitric oxide (NO) production by peritoneal cells. In the present study we identified ESA proteins of ca 24 kDa, which shared significant sequence homology to glutathione-S-transferase (GST) obtained from homogenates of F. hepatica adults, other helminths and different mammals. When the dimeric form of these proteins ca 48 kDa was cultured with rat spleen cells, a significant decrease of proliferative response to Con A was detected, starting from 20 micrograms/ml of ESA protein (P < 0.03). We also observed a significant inhibition of nitrite production by incubation with the dimeric form in normal peritoneal macrophages (P < 0.04). These results indicated that the GST secreted by the parasite could be involved in evasion of the parasite from the host immune response.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica.

The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

Amino Acid Sequence↗

PubMed1994

A dipeptidylpeptidase secreted by Fasciola hepatica.

A dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

Animals↗

PubMed1976

Enzyme polymorphism in Fasciola hepatica L.: esterases.

Homogenates of individual adult Fasciola hepatica, collected from bile ducts of sheep, were subjected to isoelectric focusing in polyacrylamide flat-bed gels after which zymograms were made by enzymatic and staining reactions. Seven identifiable isozymes that hydrolyzed alpha-naphthyl acetate were found. These 7 isozymes were identified by their isoelectric points as 5.10, 5.15, 5.25, 5.40, 5.55, 5.65, and 5.75. None of these isozymes was common to all F hepatica examined. Combinations of these isozymes in individual flukes revealed 7 distinct phenotypes, the frequency of which varied between local populations, i.e., populations of flukes obtained from individual sheep of different origin. In addition to these 7 isozymes, there were weak staining bands of enzyme activity, between pH 5.8 and the cathode end of the gels, which could not be accurately identified. The method of isoelectric focusing and zymogram preparation used in the present study did not reveal cholinesterase activity, even though the presence of activity in homogenates was demonstrated by polyacrylamide disc-gel electrophoresis. Strong activity of acid phosphatase between pH 5 and 6 was found, but poor resolution prevented obtaining accurate identification of isozymes. Weak activity with poor resolution of isozymes of alkaline phosphatase was observed between pH 4.4 and 5.75.

Acid Phosphatase↗

PubMed1982

The development of the host-response in juvenile Lymnaea palustris to invasion by Fasciola hepatica.

Laboratory studies indicate that miracidia of Fasciola hepatica can penetrate several species of lymnaeids in Europe, other than the natural host lymnaea truncatula. However, the production of cercariae from infections in abnormal hosts is unlimited to a small percentage of juvenile snails infected before a species-specific age. Juvenile Lymnaea palustris, of known ages, were exposed individually to F. hepatica miracidia and killed at 24 h intervals up to 14 days post exposure, and then processed for histological examination to ascertain reasons for the failure of F. hepatica infections to develop successfully in this abnormal host. The results indicate that the course of infection is rapidly halted by a cellular encapsulation response against the sporocyst. The response may be divided into two stages: first, the development of a cellular capsule and the concomitant degeneration of the sporocyst; second, the removal of the remains of the parasite and dispersal of the capsule. The efficiency of the response appears to increase with increasing age of the snail; statistical manipulation of the data obtained from histological investigations suggest that the defence mechanisms develop quickly in the first 6 days post hatching with little subsequent development during the experimental period. However, as sporocysts were encapsulated and killed regardless of age of the snail at exposure to infection, it is unlikely that in the host-parasite system used any of the infections would have survived.

Aging↗

PubMedDate not supplied

Rearing of Lymnaea columella (Say, 1817), intermediate host of Fasciola hepatica (Linnaeus, 1758).

The intermediate host of Fasciola hepatica, Lymnaea columella, collected in Belo Horizonte, Minas Gerais, Brazil, was reared in our laboratory. The aim of the current study was to standardize a rearing and maintenance technique. Two kinds of diet were tested: fresh lettuce (A) and rodent ration + 10% CaCO3 plus fresh lettuce (B). The age for the beginning of oviposition ranged from 27 to 57 days. Ten days after oviposition at 24.7 degrees C, 100% eclosion occurred. The complete life cycle varied from 37 to 67 days. The average numbers of eggs per egg mass were 26.3 and 31.1 with diets (A) and (B), respectively. The lettuce and ration fed snails presented a increased growth although the difference was not statistically significant (p > 0.05). The mortality rate varied from 40 to 64% after 90 days. The maximum longevity was 183 days, 21.5 mm length and 11 mm wide. The methodology to mass breed and maintain these snails was found to be suitable in the laboratory

Animal Feed↗

PubMed1984

Interaction of Fasciola hepatica with albendazole and its metabolites.

Adult Fasciola hepatica recovered from sheep 12 and 24 h after a single oral dose of albendazole (20 mg/kg) contained significant amounts of two oxidized metabolites of albendazole (ABZ), a sulphoxide (SX) and a sulphone (SO), but not ABZ. Flukes incubated in vitro with 10 microM SX or SO contained these metabolites at a level two to three times the level observed in flukes recovered from sheep 24 h after a curative oral dose of ABZ. The concentration of ABZ in flukes was 10-fold greater than either SX or SO after a 24 h in vitro incubation in 10 microM of the respective drug. Flukes exposed to ABZ in vitro contained two-fold higher SX levels than SX-treated flukes due to a combination of spontaneous oxidation in media and fluke-mediated oxidation of ABZ. Measurement of end-products of glucose metabolism following 24 h incubation in 10 microM of either ABZ, SX or SO did not show a significant difference between treated and untreated flukes.

Albendazole↗

PubMed2006

The presence of uninfected Omphiscola glabra in a breeding of infected Galba truncatula enhanced the characteristics of snail infections with Fasciola hepatica.

Experimental infections of Galba truncatula with Fasciola hepatica were carried out under laboratory conditions to determine if the presence of another lymnaeid, Omphiscola glabra, during snail breeding had an indirect effect on the growth of G. truncatula and to analyse consequences on cercarial production. When the two lymnaeids are raised together, the survival of G. truncatula at day 30 post-exposure (PE), the prevalence of snail infections, and the shell height of cercariae-shedding snails at day 45 PE were significantly higher. By contrast, the other parameters characterizing snail infections only showed insignificant variations. The origin of O. glabra (living in a snail community, or monospecific population) used for the breeding of G. truncatula did not have a significant effect on the values of each parameter. Even if variations in the mean numbers of metacercariae were insignificant, the greater survival of G. truncatula at day 30 PE and the higher numbers of cercariae-shedding snails in the groups living with O. glabra allowed to obtain a higher total number of larvae than in alone-raised groups of G. truncatula.

Animals↗

PubMed1981

Killing of newly excysted juveniles of Fasciola hepatica in sensitized rats.

Newly excysted juvenile Fasciola hepatica were injected intraperitoneally into previously sensitized rats. They were recovered at various intervals post-injection and examined by light and electron microscopy. The challenge flukes were rapidly coated with peritoneal cells which, in the early stages, were mainly eosinophils. The eosinophils adhered closely to the flukes and degranulated on to their surface releasing cytochemically detectable peroxidase. Vacuoles formed in the tegument of the flukes beneath the adherent eosinophils and these increased in size until they spanned the width of the tegument, thus destroying its continuity. From 4 h post-injection some of the flukes had lost their teguments and were surrounded by phagocytic cells, particularly neutrophils; at this stage they were judged to be dead. During the first five minutes post-injection degranulating mast cells were associated with the challenge flukes. Their role in eosinophil chemotaxis is discussed as are the possible mechanisms of eosinophil adherence and degranulation. Using an anti-C3 fluorescein conjugate, it was demonstrated that C3 was not bound to the surface of challenge flukes either in vivo or in vitro in immune serum.

Animals↗

PubMed2004

The use of tetraphyll as food for snails increases the intensity of cercarial shedding in Galba truncatula infected with Fasciola hepatica.

Experimental infections of Galba truncatula with Fasciola hepatica were carried out under laboratory conditions to determine why the use of Tetraphyll, when provided every 3 days as food for snails, stimulated the intensity of cercarial shedding. In snails raised on cos lettuce and Tetraphyll, the number of metacercariae was significantly higher than that recorded in controls on lettuce only. In the former group, numerous metacercariae were counted on day 1 of every 3-day period (when snails fed on Tetraphyll), while the numbers of these larvae strongly decreased on day 2 and were very low on day 3. The repetition of this experiment using microalgae or modified Boray's diet every 3 days as food for snails gave the same results. However, the number of metacercariae recorded on day 1 of every 3-day period was significantly higher in snails raised on lettuce and Tetraphyll than in those reared on lettuce and algae (or Boray's diet). According to the authors, the first contact of an infected snail with Tetraphyll induces a faster differentiation of cercariae within the rediae and the accumulation of many free cercariae in the snail's hemocoel, so that a second contact of the snail with this food 3 days after allows free cercariae to complete their glycogen and fat reserves and to rapidly exit from the snail. However, a temporary immobilization of the infected snail by Tetraphyll, or rather its degradation products, and a massed exit of cercariae cannot be excluded.

Animal Feed↗

PubMed1984

Desensitization of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica.

Incubation of the intact liver fluke Fasciola hepatica with serotonin (5-HT) resulted in a time-dependent decrease in both 5-HT-stimulated adenylated cyclase activity and specific [3H]LSD binding in the subsequently prepared cell-free fluke particles. In control fluke particles, the activation of adenylate cyclase by 5-HT was biphasic, indicating a high and low affinity form of the 5-HT receptor with half-maximal activation constants (KA) of 0.35 and 2.8 microM respectively. In contrast, 5-HT activation of desensitized particles occurred through a single set of low affinity sites having a KA value of 6.3 microM. The maximal level of 5-HT activation of adenylate cyclase was also reduced in the desensitized particles. Lysergic acid diethylamide (LSD)-stimulated adenylate cyclase activity was also less in the desensitized particles. However, unlike with 5-HT, activation by LSD occurred through a single set of sites for both control and desensitized particles. [3H]LSD binding studies showed that the affinity of LSD for the 5-HT receptors in the control and desensitized particles was similar. Thus, the decrease in [3H]LSD binding and serotonin-stimulated adenylate cyclase activity in the desensitized particles appeared to be due to a preferential loss or inactivation of the high affinity form of the 5-HT receptor. A similar time-dependent loss in 5-HT-stimulated adenylate cyclase and in [3H]LSD binding occurred in cell-free fluke particles upon the addition of 5-HT or LSD. These effects were not due to protein denaturation or metabolism of the ligand during the incubation procedure. This cell-free desensitization was reversible and temperature dependent, and was not affected by ATP or other nucleotides.

Adenosine Triphosphate↗

PubMed1981

Scintigraphic evaluation of the liver in Fasciola hepatica with radiocolloid and 67Ga-citrate.

A group of 23 patients with Fasciola hepatica have been studied in the province of Biscay. The diagnosis was reached after finding parasite eggs in the faeces or duodenal juice. The liver lesion was confirmed by means of macro and microscopic studies. Radiocolloid demonstrated the presence of cold areas in 18 patients; in 13 of them the uptake with 67Ga was positive. These isotopic findings identified Fasciola hepatica as one of the causes of 'cold areas' in traditional liver scans and positive 67Ga uptake.

Duodenum↗

PubMed1991

Production and characterization of monoclonal antibodies against excretory-secretory products of Fasciola hepatica.

Four monoclonal antibodies (mAbs) (9.49, 24.27, 46.71 and 179.57) were produced against Fasciola hepatica excretory-secretory products. Isotype analysis revealed the antibodies to be IgM, IgG3, IgG1, and IgM. In immunoblot assays, the mAbs recognized different antigenic polypeptides migrating between 29 and 180 kDa. Specificity of the mAbs was evaluated by ELISA against antigens of Fascioloides magna, Anoplocephala magna, Stichorchis subtriquetrus, Haemonchus contortus, sheep liver extract (SLE), bovine liver extract (BLE), bovine serum albumin (BSA), bovine viral diarrhea virus (BVDV), and Madin-Darby bovine kidney (MDBK) cells. Monoclonals 9.49 and 24.27 were specific, and reacted only with Fasciola hepatica antigens. However, mAb 46.71 cross-reacted with antigens of Fascioloides magna, A. magna, Stichorchis subtriquetrus, and H. contortus but not with SLE, BLE, BSA, BVDV or MDBK cells. Monoclonal antibody 179.57 cross-reacted with Fascioloides magna, A. magna, S. subtriquetrus, H. contortus, SLE, and BLE, but not with BSA, BVDV, or MDBK cells.

Animals↗

PubMed1993

Effect of early treatment with rafoxanide on antipyrine clearance in sheep infected with Fasciola hepatica.

1. The effect of the salicylanilide compound rafoxanide against immature stages of Fasciola hepatica was investigated in sheep using a series of antipyrine clearance tests and measuring glutamate dehydrogenase and gamma-glutamyl transferase activities in plasma. 2. Three animal groups were used. In two groups, sheep were infected with 200 metacercariae each. Four weeks after infection one of the two groups was treated with rafoxanide and the other was left untreated. The third group was unparasitized and also received rafoxanide. 3. Infection was confirmed by post-mortem examination of the livers of infected sheep. Infection did not alter the plasma disposition of rafoxanide. 4. The efficacy of rafoxanide, as assessed by the reduction in the number of adult flukes in treated animals compared with controls, was 85%. 5. Glutamate dehydrogenase activity fell dramatically 2 weeks after treatment but increased again at 12 weeks post-infection in the infected rafoxanide-treated group. 6. Antipyrine clearance decreased between 8 and 14 weeks post-infection in untreated sheep. In the infected rafoxanide-treated group plasma clearance of antipyrine remained unchanged until 10 weeks after rafoxanide administration when it decreased from the preinfection value of 5.09 to 3.90 ml.kg/min. Rafoxanide did not affect antipyrine disposition in uninfected sheep. 7. It was concluded that rafoxanide had an incomplete anthelmintic effect against immature stages of Fasciola hepatica, and that surviving parasites caused delayed liver damage which was reflected in an elevation in glutamate dehydrogenase activity and decreased plasma clearance of antipyrine.

Animals↗

PubMed1993

[Identification and isolation of common antigens of Fasciola hepatica].

Four antigenic polypeptides present in excretion-secretion antigens which were common to Fasciola hepatica somatic and tegumentary antigens, were identified by Western blotting and purified by affinity chromatography. Monoclonal antibody ES78 was used to that purpose. Molecular weights calculated for these polypeptides ranged from 37 to 13 kd; they proved to be highly reactive with sera from animals experimentally infected by Fasciola hepatica in periods as early as the second week of infection, with maximal values in weeks 6 and 10.

Animals↗

PubMed1997

Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components.

The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.

Amino Acid Sequence↗

PubMed1997

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay.

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of > 99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.

Animals↗