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Dose-dependent pharmacokinetics and efficacy of MK-401 against old, and young-mature infections of Fasciola hepatica in the rat.

The dose-dependent pharmacokinetics and the efficacy of MK-401 (4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide) against old and young-mature infections of Fasciola hepatica were studied in experimentally infected rats. Fractionation of the host's blood after administration of 14C-MK-401 (0.77-15.8 mg/kg) showed that MK-401 was bound predominately to erythrocytes at doses below 4 mg/kg and at higher doses was distributed equally between the red cells and the plasma. Maximum amounts of MK-401 in the blood occurred 2 to 4 hr postadministration and were a hyperbolic function of dose, increasing almost linearly with dose up to 6 mg/kg and then beginning to saturate. Drug uptake by F. hepatica occurred at all doses and increased in direct proportion to the blood level. A single oral dose of MK-401 at 5 mg/kg was found to be highly effective (89%) against older infections (39-44 wk) but was virtually ineffective (1.5%) against younger flukes (9-16 wk). After administration of 14C-MK-401 at 5 mg/kg, drug concentrations in the blood and flukes of rats harboring older infections were significantly higher than those in the blood and flukes of rats with younger infections. Virtually identical differences in the blood level of MK-401 were observed in young and in old, noninfected rats after administration of 14C-MK-401 at 5 mg/kg. The increased efficacy of MK-401 against older infections of F. hepatica in the rat may be related to the age of the host rather than the parasite.

Age Factors↗

Partial characterization of the epitope on excretory-secretory products of Fasciola hepatica recognized by monoclonal antibody ES78.

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

Animals↗

A fatty acid binding protein from Fasciola hepatica induced protection in C57/BL mice from challenge infection with Schistosoma bovis.

Three strains of mice (NMRI, C57/BL, BALB/c) were each immunized with a 12 kDa purified, native Fasciola hepatica fatty acid binding protein (Fh12) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh12 had significant reductions in S. bovis worm burden recoveries (96 and 87% reductions over controls in two separate experiments). When using NMRI or BALB/c mice, Fh12 alone or in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice vaccinated against Fh 12, antibodies to the IgG2a isotype, but not to the IgG1 isotype, increased by 2 weeks after the second immunization and remained high through 8 weeks of S. bovis infection. Antibodies to S. bovis increased after 4 weeks of infection. Regarding cytokine production by spleen mononuclear cells, C57/BL mice vaccinated with Fh12 in adjuvant, and having the highest protective response against challenge infection with S. bovis, had an increase of IFN-gamma production with Concanavalin A but no increase of IL-4 in similarly stimulated cells. These results suggest that the protection obtained in this group of mice is mediated by a Th1 immune response.

Animals↗

Effect of inoculum on the size and location of Fasciola hepatica subsequently recovered from the livers of rats.

Two outbred strains of Sprague-Dawley rats were given, by intraperitoneal injection, 5, 10, 20, 30 or 50 newly excysted juvenile (NEJ) Fasciola hepatica. Ninety days post-infection all rats were killed and their livers teased apart under 10 X magnification for quantitation of the flukes present. There was no significant difference in worm numbers between the rat strains. However, several significant differences (P less than 0.05) between the various infection groups were observed. As the size of the infective dose was increased from 5 to 50 NEJ, the percentage of the infective dose recovered from the livers of the infected rats decreased from 36.7 to 12.1%. With the 5 and 10 NEJ infective doses, the worms recovered were large mature flukes (1.52 and 1.68 cm, respectively) and were found in the common bile ducts. In the rats receiving infections of 30 and 50 NEJ, the flukes were smaller (0.88 and 0.55 cm, respectively), immature, and were primarily located in the liver parenchyma. These findings are important in light of previous studies on the development of resistance in the rat to challenge infections in which immunity was based on both the size and the location of the flukes recovered. The results from our study indicate that in a primary infection of 20 or greater NEJ, many small immature flukes remain in the liver parenchyma, even after several months. When testing for resistance to F. hepatica in rats, these flukes may erroneously be thought to comprise a portion of the challenge infection.

Animals↗

Fasciola hepatica: tegumental changes induced in vitro by the deacetylated (amine) metabolite of diamphenethide.

The effect of the deacetylated (amine) metabolite of diamphenethide (10 mugm/ml) on the tegument of Fasciola hepatica over a 24 hr period in vitro has been determined by means of transmission electron microscopy. In the tegumental syncytium, there is an initial accumulation of T2 secretory bodies at the apical surface (after 6 hr), together with increased exocytosis of secretory bodies and blebbing of the surface membrane. After 9 hr, the two surfaces of the fluke show different tegumental responses to drug treatment with a marked swelling of the basal infolds in the dorsal tegument, while the ventral tegument remains normal. By 18 hr, the swelling in the dorsal tegument is very severe, the entire basal region becoming edematous. In some areas, the tegument becomes detached to expose the basal lamina. The ventral tegument retains a fairly normal morphology, although there is a slight swelling of the basal infolds. The edema spreads internally to the cell bodies, beginning after 9 hr on the dorsal side of the fluke and 18 hr on the ventral side. By 18 hr, the flooding on the dorsal side is very severe and the cells attenuated, retaining few contacts with the surrounding parenchyma. From 9 hr onwards, there are progressive changes in cell structure, including a decrease in amount of granular endoplasmic reticulum and extent of its ribosomal covering, a decrease in numbers of secretory bodies, a swelling of the trans-most Golgi cisternae and disruption of the release of secretory bodies, and a swelling and disorganization of the mitochondria. The results are discussed in relation to the postulated activity of the deacetylated (amine) metabolite of diamphenethide as a Na+ ionophore.

Acetanilides↗

Characterization and partial purification of a leucine aminopeptidase from Fasciola hepatica.

An aminopeptidase activity capable of hydrolyzing different aminomethylcoumarin amino acids, but mainly leucine-7-amino-4-methylcoumarin (Leu-NHMc), was detected in deoxycholic soluble extracts from adult Fasciola hepatica. The enzyme (EC 3.4.11.1) was partially purified by gel filtration and EAH-Sepharose affinity chromatography using bestatin as a ligand. Results obtained by gel filtration, direct fluorogenic substrate analysis in polyacrylamide gel, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that in a native form the enzyme might be aggregated as a high molecular weight complex. By affinity chromatography on concanavalin A Sepharose, the enzyme did not bond to the column showing that it lacks mannose residues. The F. hepatica aminopeptidase was characterized as a metalloproteinase based on its activation by Mn2+ and Mg2+, and its inhibition by EDTA, 1,10-phenanthroline, and bestatin. It has an optimal activity at a pH between 8.0 and 8.5. Histochemical localization revealed strong leucine naphthylamidase activity at the cells lining the gut epithelium of the parasite.

Animals↗

Fasciola hepatica: ultrastructural changes to the tegument of juvenile flukes following incubation in vitro with the deacetylated (amine) metabolite of diamphenethide.

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 microgramsml-1) were examined by transmission electron microscopy. A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds. The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal. Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased. The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down. Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

Animals↗

Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke.

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.

Amino Acid Sequence↗

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.

Animals↗

Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens.

Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.

Animals↗

Redial growth and cercarial productivity of Fasciola hepatica in three species of young lymnaeid snails.

Experimental infections of 1-mm high snails using three populations of Lymnaea (L. glabra, L. ovata and L. truncatula) and a cattle strain of Fasciola hepatica miracidia were carried out under laboratory conditions to determine if the snail species had an effect on the number of free rediae, their growth, and cercarial productivity in relation to each redial category (R1a, R1b, R2a, or R2b/R3a). The total number of rediae ranged from 6.4 to 7.5 per snail. The mean body length of rediae varied from 1-1.2 mm (R1a) to 0.3-0.4 mm (R2b/R3a). The width of the intrapharyngeal lumen also varied from 26.0-38.8 microm to 3.0-4.2 microm, respectively. The redial category had a significant effect on both measurements, whereas snail species only had a significant influence on body length. The mean number of cercariae produced by all living rediae at day 49 post-exposure ranged from 63.0 in L. glabra to 87.2 in L. truncatula. In L. ovata and L. truncatula, 55.8% and 58.6% of cercariae, respectively, were produced by R2a rediae, whereas 53.9% of cercariae in L. glabra were formed by the R1b rediae. When young snails were infected with F. hepatica, the species of snail had an effect on the number of living rediae, their length and their cercarial productivity.

Animals↗

Oxidative responses during bacterial phagocytosis of polymorphonuclear leucocytes in primarily and secondarily Fasciola hepatica infected goats.

The polymorphonuclear leukocyte (PMN) function, in terms of oxidative response during bacterial phagocytosis, was studied using a Luminol-Dependant Chemiluminiscence (LDCL) assay in primarily and secondarily Fasciola hepatica infected goats. Polymorphonuclear leukocytes of F. hepatica infected goats displayed lower LDCL responses than naive goats. The lowest responses were observed in secondarily infected animals that had higher parasitic burdens and more prominent hepatic lesions. The reduced responses were induced by a functional defect of the circulating PMN but also by a direct involvement of serum factors. Both circulating parasite products and the non protective immune response that occurred during secondary F. hepatica infection of goats could be implied in the alteration of PMN function. These findings suggest the existence of an important mechanism for impairment of the host immune system during goat fasciolosis.

Animals↗

Gastrointestinal hormones: environmental cues for Fasciola hepatica?

The effects of pharmacological concentrations of several gastrointestinal hormones on the rate of sucker activity and the frequency and the amplitude of spontaneous longitudinal muscle contractions have been examined in adult Fasciola hepatica. Caerulein and serum decrease the rate of oral sucker activity; motilin decreases and CCK-PZ increases ventral sucker activity when compared to controls. Caerulein, serum and motilin significantly inhibit the frequency of contractions while bile, caerulein and motilin decrease the amplitude of contractions. These results suggest that F. hepatica can recognize and respond to certain gastrointestinal hormones and there may be adaptive value in these behavioural responses.

Animals↗

The combined effect of isolation and Fasciola hepatica infection on the life history traits of Fossaria cubensis.

Life history traits of Fossaria cubensis were compared between isolated and paired snails after infection with three miracidia of Fasciola hepatica. Four experimental groups were tested: isolated-unexposed, paired-unexposed, isolated-infected, and paired-infected. A repeated-measures ANOVA showed statistically significant interactions among isolation, infection, and age effects for shell size, number of egg masses per snail, number of eggs per snail, and number of viable eggs per snail. Isolated-unexposed snails exhibited the higher values of these variables and those of survival and finite and intrinsic rates of natural increase. Infection stimulated shell growth during the prepatent period, but differences were present only in paired snails since isolation causes a similar effect. Reproduction, in terms of the number of egg masses per snail and the number of eggs per mass per snail, decreases in the presence of parasitic infection, whereas isolation stimulates it. These effects were observed from early stages of infection.

Animals↗

Fasciola hepatica: effects on blood constituents and liver minerals in fallow deer (Dama dama).

Haematological and serum biochemical constituents as well as the liver mineral concentrations can be affected by Fasciola hepatica. The present study compares the blood and liver constituents in 19 fallow deer with fascioliosis and in 43 without it. Blood samples, gastro-intestinal tract, lungs and liver were taken from 62 male and female fallow deer that were shot, aged 6 months and over, in enclosures, between 1999 and 2001 in Slovenia. Samples were analysed and the means of various parameters of blood and liver were determined. Significant differences in blood were found in the mean cell volume, total serum proteins, sodium, and calcium; and in liver and calcium and copper were higher in animals without fascioliosis. Mean cell haemoglobin concentration, eosinophils, basophils, lymphocytes, monocytes, alanine aminotransferase, urea, glucose and copper in blood were significantly higher in animals with fascioliosis. It has been concluded that some of the values in fallow deer like in other domestic ruminants are the sensitive indicators of liver cell damage in fascioliosis.

Animals↗

The development of Fasciola hepatica parthenitae in Lymnaea truncatula by modification of Muellerius capillaris infection.

Adult and juvenile Lymnaea truncatula (height 4 and 1 mm, respectively) were subjected to a dual infection with either Fasciola hepatica and Muellerius capillaris, or with M. capillaris and F. hepatica at 4-h, 7-, 14- and 30-day intervals between the two exposures. The results were compared with those of snails which had been infected with a single parasite species. In dually infected snails, there was a high mortality at the second exposure to both parasite species, and it was strongly increased by each reinfection. The interexposure time had a significant influence on these mortality rates. Histological examination demonstrated a decreased number of rediae (50% or greater) in dually infected snails beginning with the 4-h interval in adults and the 7-day interval in juveniles. The exposure order and the time interval between exposures had a significant influence on the number of rediae: the number of parasites per snail decreased as the time interval lengthened. Delayed maturity occurred in live free rediae in snails infected by F. hepatica and M. capillaris at the 14- and 30-day intervals. In those infected by M. capillaris and F. hepatica, time to reach maturity was not modified compared to that in snails infected with a single species. Prostronglylid larvae were clearly less numerous in dually infected adult snails than in corresponding mono-infected snails. Third-stage larvae were found in the 14- and 30-day groups, regardless of exposure order and were absent in mono-infected snails.

Animals↗

Eggshell precursor proteins of Fasciola hepatica, II. Microheterogeneity in vitelline protein B.

At least 3 structural protein precursors of the eggshell are synthesized and stockpiled in the extensive vitelline cells of the liver fluke Fasciola hepatica L. One of these, vitelline protein B, consists of a closely related family of proteins that owes its apparent electrophoretic heterogeneity to variations in the Tyr to DOPA conversion as well as to subtle variations in the primary sequence. The efficiency of the Tyr to DOPA conversion ranges from a maximum of about 90% to a minimum of 55% in the protein. Trypsin digestion in borate buffer at pH 8 was used to produce DOPA-peptides for sequencing. Notably, trypsin does not cleave Arg/Lys-DOPA sequences at borate concentrations greater than 0.15 M. Peptides with DOPA-containing sequences most frequently have flanking amino acids such as Lys, Ser, or Asp on the N-terminal side and Gly or Asp on the C-terminal side. All protein variants fall within a narrow molecular weight range (30-33 kDa), a pI range of 6.9 to 8.3, and the collective majority would appear to share a common N-terminal sequence up to residue 28. The results suggest some combination of the following: variations in post-translational hydroxylation, alternative post-transcriptional splicing and/or the existence of multiple gene copies of eggshell precursors. The latter have been shown to occur in the blood fluke Schistosoma mansoni [15].

Amino Acid Sequence↗

Dynamics of Fasciola hepatica infection in two species of snails in a rural locality of Mexico.

The aim of the present study was to identify the species that are natural intermediate hosts of Fasciola hepatica in a Research Centre in Chapa de Mota, State of Mexico, Mexico, where temperate and humid conditions prevail. Also, the magnitude and dynamics of the natural infection in four biotopes were analysed. The study was performed between October 2000 and November 2001. The molluscs were collected, identified and studied for trematode infection. Soil humidity and temperature were determined monthly. A total of 4042 snails were collected 3372 (83%) were Fossaria humilis and 670 (17%) F. bulimoides. F. hepatica was present in 2537 (75.2%) and 515 (76.9%) of the two snail species, respectively. Temperature, soil humidity and microenvironment affected the dynamics of the host population and the parasite infestation, with a significant increase from August to November 2001. The highest prevalence was observed in the warmer and more humid biotopes.

Animals↗