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PubMed1999

Standardization of a dot immunoperoxidase assay for field diagnosis of Fasciola hepatica infected cattle.

A dot immunoperoxidase assay was developed for the detection of bovine specific antibodies to Fasciola hepatica. The standardization of the assay was done with 298 bovine sera, from seven different groups, previously studied by the reference enzyme-linked immunosorbent assay (ELISA). F. hepatica surface antigens purified by negative affinity against epitopes shared with Paramphistomum spp. and Echinococcus granulosus were used. Starting with sensitized and blocked nitrocellulose strips, the dot immunoperoxidase (DI) assay requires 2 h 15 min at room temperature. The development of a distinct brown dot indicated a positive reaction. The DI test showed a sensitivity of 82%, a specificity of 90%, with a 95% confidence level (CL), good repeatability (kappa 0.741, CL 95%), as well as a significant association with the reference ELISA (kappa 0.618, CL 95%). The DI assay is fast, simple and has a low cost - all convenient characteristics for a screening test to be used in the field.

Animals↗
PubMed1992

Eggshell precursor proteins of Fasciola hepatica, I. Structure and expression of vitelline protein B.

Antibody raised against the major eggshell protein of Fasciola hepatica (vitelline protein B, vpB) is employed to isolate cDNAs from an expression library and to localize the protein in whole worms. Two cDNAs corresponding to the protein are homologous through the N-terminal and C-terminal coding regions and widely divergent internally. No repeated regions are apparent and no significant sequence homology is seen with chorion proteins of other genera although the amino acid composition closely reflects that of other chorion proteins. Microheterogeneity observed in the vpB is due to the presence of multiple coding sequences, transcripts and a gradient of post-translational modification. Relative transcription of the vpB mRNA throughout the female reproductive tract is demonstrated.

Amino Acid Sequence↗
PubMed1984

Efficacy of clorsulon for treatment of mature naturally acquired and 8-week-old experimentally induced Fasciola hepatica infections in cattle.

In a dose-titration study against experimentally induced 8-week-old Fasciola hepatica infection (study A), 20 calves were allotted to 5 groups, each of 4 calves, and treated with different doses of an injectable formulation of clorsulon or its vehicle: group 1--controls, no drug; group 2--2 mg of clorsulon /kg; group 3--4 mg of drug/kg; group 4--8 mg/kg; and group 5--16 mg/kg. Mean numbers of flukes recovered from 4 calves in each treatment group were as follows: group 1--112.2, group 2--42, group 3--4.8, group 4--3.0, and group 5--0.2. Percentages of fluke reductions for groups 2, 3, 4, and 5 ( clorsulon -treated) calves were 62.6%, 95.7%, 97.3%, and 99.8%, respectively. Against naturally acquired mature (greater than 14-week-old) F hepatica infections (study B), a total of 161 flukes were recovered from 7 vehicle-treated control calves (group 6; mean fluke recovery = 23) and no flukes were recovered from 9 calves (group 7) given orally a formulation containing 7 mg of clorsulon /kg of body weight. Eggs were not found in the feces of clorsulon -treated calves at 20 to 21 days after treatment as compared with a mean of 7.4 eggs per gram (epg) in group 6 (control) calves. Mean bile egg recoveries were 13,532 (456 to 66,861) from group 6 calves as compared to recovery of a total of 162 (0 to 160) eggs from 3 of the 9 treated calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

An enzyme-linked immunosorbent assay for diagnosis of Fasciola hepatica infection in cattle.

An enzyme-linked immunosorbent assay (ELISA) was investigated for the diagnosis of Fasciola hepatica infection in cattle. Studies included examination of 4 antigen preparations: freshly collected fluke antigen (FFA), dead fluke antigen (DFA), lyophilized fluke antigen (LFA), and partially purified antigen (PPA) for activity and use of an ELISA with FFA and LFA for diagnosis of experimentally and naturally occurring fascioliasis in cattle. The ELISA, using FFA, detected F hepatica antibody in calves as early as 4 weeks after exposure to this parasite. The DFA exhibited slightly less activity, and LFA did not have diagnostic value until 9 to 10 weeks after exposure. The PPA produced high background readings with noninfected control sera and was not considered sufficiently specific for further use. The long incubation and short incubation ELISA procedures can be used for diagnostic work. Both were equally sensitive, with optical density readings of known positive sera routinely 2.5 times those of sera from normal controls. The repeatability of these tests was also excellent, and only slight variation in optical density was observed in ELISA was performed on representative known positive and negative sera with similar reagents in replicate tests.

Animals↗
PubMed1980

Light and electron microscopic studies on the effect of praziquantel on Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica (Trematoda) in vitro.

The fine structure of the tegument of three trematode species, Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica, was studied by means of light scanning (SEM) and transmission electron microscopy (TEM) after in vitro exposure to 0, 1, 10, and 100 micrograms/ml of the anthelmintic praziquantel for 5, 15, 30, and 60 min. In S. mansoni and D. dendriticum the resulting vacuolization of the tegument was confined to numerous small areas scattered all over the surface of the parasites and this finally led to the disruption of the apical tegumental layer. No changes were found in the tegument of F. hepatica after treatment with praziquantel.

Animals↗
PubMed1989

Immunocytochemical demonstration of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica (Trematoda, Digenea).

The localization and distribution of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica at different stages in the development of the adult fluke have been determined by an indirect immunofluorescence technique, using antisera to 19 vertebrate peptides and the invertebrate neuropeptide, FMRFamide. Positive immunoreactivity was obtained with antisera to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP) and FMRFamide. Cell bodies and nerve fibres immunoreactive to the 4 peptides are present in the anterior ganglia and the 3 pairs of longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, immunoreactivity occurs in the nerve plexuses supplying the subtegumental musculature, the oral and ventral suckers, and the muscular lining of the male and female reproductive ducts, including the ootype, uterus, cirrus pouch and gonopore. Cells displaying immunoreactivity to PYY and FMRFamide lie amongst the Mehlis' gland cells that surround the ootype. Processes from these cells extend into the wall of the ootype. One group of PP-immunoreactive cells occurs at the junction of the vitelline and ovovitelline ducts, whilst another group is situated at the entrance to the uterus from the ootype. The results are discussed in relation to the possible roles of the peptides in the neurophysiology and egg production of the fluke.

Animals↗
PubMed2001

Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda).

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined. It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S(UNC)) and tRNA(S(AGN)) lacking the DHU arm.

Animals↗
PubMedDate not supplied

The efficacy and tolerability of triclabendazole in Cuban patients with latent and chronic Fasciola hepatica infection.

Current chemotherapy for the treatment of infections caused by the liver fluke Fasciola hepatica is not satisfactory. Therefore, the efficacy and tolerability of triclabendazole (TCZ) was assessed for this indication. Eighty-two patients (51 female, 31 male, age 15-81 yr, mean 42 yr) with chronic or latent F. hepatica infection refractory to previous anti-helminthic chemotherapy were enrolled in a 60-day open, non-comparative trial. Patients received 20 mg/kg TCZ as two doses of 10 mg/kg administered after food 12 hr apart. Efficacy of treatment was assessed by stool microscopy, determination of Fasciola excretory-secretory antigen (FES) in feces, and by ultrasonography (US) which were systematically performed pre-therapy and on Days 1-7, 15, 30, and 60 post-therapy. For continuous safety assessment, patients were hospitalized during the first week after therapy and then monitored at home for the appearance of any adverse events. Clinical chemistry and hematology tests were carried out on Days 1, 3, 7, 15, and 60, and whenever an adverse effect occurred possibly related to therapy. Seventy-one (92.2%) of the 77 patients who completed the 60-day follow-up period became egg-negative. Efficacy of therapy was supported by the disappearance or decrease of FES antigen and of ultrasonography abnormalities. In the 6 remaining patients, parasitological cure was achieved by another single TCZ dose of 10 mg/kg on Day 60. A total of 74 adverse events possibly related to therapy was reported by 54 patients. The most important adverse event was colic-like abdominal pain (40 patients [49%]) consistent with the expulsion of the parasite through the bile ducts as confirmed by US on Days 2-7. Most adverse events (53) were graded as mild, 20 as moderate, and only 1 as severe (a biliary colic responding to spasmolytic therapy within two hours). Triclabendazole 20 mg/kg is an effective therapy for the treatment of F. hepatica infection in patients who have failed to respond to other antihelminthic agents. Biliary colics reflecting the expulsion of dead or damaged parasites usually occur during Day 3-7 and respond well to spasmolytic therapy.

Adolescent↗
PubMed1998

A novel ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level.

We describe an ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level. An exact number of newly excysted juveniles (NEJs) was injected into a gut segment with an intact blood supply and which was still attached to a live anaesthetized rat. NEJs that penetrated the gut wall during the following 6 h were recovered from a beaker filled with medium and were counted under a microscope. This infection model was validated and enabled us to exactly quantify the infection dose whilst at the same time exactly quantifying the number of NEJs penetrating the gut wall. The mean sum of NEJs that migrated through the gut wall into the beaker (peritoneal fraction), plus NEJs that remained in the gut wall and the gut lumen was 87% of the infective dose (+/-3.6% SEM; n=18). The function of the ex vivo segments was well-preserved, as demonstrated by only minor leakage of an inert liquid marker. The ex vivo model enabled us to measure protection against F. hepatica at the gut level. In naive rats 52% (+/-2.4% SEM; n=40) of the injected NEJs penetrated the gut wall, whereas in previously infected rats only 12% (+/-1.8% SEM; n=40) were able to do so, irrespective of the infection dose. Thus, when rats were orally primed, the migration of NEJs through the gut wall was 77% less than the migration in naive rats. We conclude that the ex vivo model should be valuable in studies of the induction and expression of protective immunity against F. hepatica in the intestine, and will aid in development and optimization of vaccines.

Animals↗
PubMed1987

Immunocytochemical demonstration of 5-hydroxytryptamine (serotonin) in the nervous system of the liver fluke, Fasciola hepatica (Trematoda, Digenea).

The localisation and distribution of 5-hydroxytryptamine (5-HT or serotonin) in the nervous system of Fasciola hepatica has been determined by an indirect immunofluorescence technique. Cell bodies and nerve fibres immunoreactive to 5-HT are present in the anterior ganglia, and the longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, similar immunoreactivity occurs in the nerve plexuses supplying the sub-tegumental muscle layers and the muscular lining of various reproductive ducts, including the ootype, uterus and cirrus pouch. The significance of these results in the light of previous studies on the role of 5-HT in F. hepatica is discussed.

Animals↗
PubMed2000

The major tegumental antigen of Fasciola hepatica contains repeated elements.

In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive lambda bacteriophage clones containing F. hepatica cDNA have been isolated. Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elementst. All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions. Antisera raised against a beta-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens. Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument. The wall of the uterus showed strong reactivity in the adult. Rats immunized with the beta-galactosidase fusion protein showed enhanced resistance to challenge infections. The role of these antigens in the host response to infection by F. hepatica is discussed.

Amino Acid Sequence↗
PubMed1981

[Immunodiffusion studies of the complete somatic antigens of Paramphistomum sp. and Fasciola hepatica].

Investigations were carried out through the agar gel immunodiffusion test of complete somatic antigens of Paramphistomum sp. and Fasciola hepatica, using the respective homologic and heterologic hyperimmune rabbit sera. Differences were established in the spectrum and number of the produced precipitation lines - 6 to Paramphistomum and 4 to Fasciola. With the identity test by the scheme in three after Zilber and Abelev (1962) no precipitation arcs were formed against the heterologic hyperimmune rabbit sera. This pointed the fact that there was no antigenic relation between the helminth species studies.

Animals↗
PubMed1982

Isolation and purification of Fasciola hepatica eggs.

The present egg isolation method is both a rapid and simple technique for recovering large numbers of Fasciola hepatica eggs (1 X 10(7) eggs/gradient) from bovine bile. Bile from infected cattle was first passed through a 45 micron screen sieve. The F. hepatica eggs were collected from the surface of the screen by backwashing with a jet of distilled water. The resultant egg suspension was layered on a 60% to 100% (v/v) linear Percoll gradient prepared in distilled water. Centrifugation at 450 g for 20 min resulted in the formation of 2 visible bands and a pellet. The top band (density of 1.075 g ml-1) contained viscous debris and crystallized bile pigments. The second visible band (density of 1.093-1.099 g ml-1) consisted of a relatively pure population of F. hepatica eggs (greater than 93%) while the pellet contained only F. hepatica egg shells.

Animals↗
PubMed1979

Effect of BCG on the resistance of rats to infection with Fasciola hepatica.

The effects of three different doses of BCG, given at various periods before infection, on the subsequent establishment of Fasciola hepatica metacercariae were studied. Although evidence was found to suggest that rats which received BCG had mounted a cell mediated immune response, there were no significant differences in worm recovery between BCG-treated rats and controls. The significance of these results in the light of current knowledge on immunity to F. hepatica in rats is discussed.

Animals↗
PubMed1987

Partial purification and characterization of cAMP-dependent protein kinase from Fasciola hepatica.

Three cyclic AMP (cAMP)-dependent protein kinases, designated A1, A2, and B, were isolated from the liver fluke Fasciola hepatica using Phenyl-Sepharose and DEAE-cellulose chromatography. These enzymes differed with respect to activation by cAMP and their molecular weights. The half-maximal activation constant for cAMP-dependent protein kinases A1 and B was 20 nM, while that of A2 was about five-fold higher (110 nM). The estimated molecular weights for cAMP-dependent protein kinases A1 and A2 (both 98,000) suggest a dimeric form for these enzymes; whereas, the higher molecular weight for cAMP-dependent protein kinase B (187,000) indicates that this enzyme is a tetramer. The physical and kinetic properties of the catalytic subunit of fluke cAMP-dependent protein kinase were similar to those reported for the mammalian enzyme. The molecular weight of the catalytic subunit was estimated to be 41,000. The pH optimum for the enzyme was 6.0, 6.5, or 7.0 when casein, histone, or protamine were used as substrates. The protein substrate specificity was in the order histone greater than arginine-rich histone greater than casein greater than protamine greater than lysine-rich histone. Free Mg2+ 'stimulated' enzyme activity at low concentrations (0.5 to 5 mM), whereas at higher concentrations (greater than 5 mM) it became inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could substitute for Mg2+. Kinetic studies indicated that the reaction mechanism of this enzyme is sequential and that MgATP and MgADP are competitive ligands. Reconstitution experiments using the subunits of fluke and bovine heart cAMP-dependent protein kinase showed that there is sufficient structural homology between these enzymes such that the catalytic subunit from one species can combine with the regulatory subunit of the other species to form inactive holoenzyme. Thus, the present results indicate that cAMP-dependent protein kinase from F. hepatica is similar but not identical to the mammalian enzyme.

Animals↗