Understanding genetic diversity of the liver fluke Fasciola hepatica

Economical breeding is important to obtain maximum gain from the breeding in the animal sector. The economic loss has to be eliminated or should be minimized. The liver fluke, Fasciola hepatica, present mostly in sheep and dairy cattle affect the yield of animals and even cause their death. To eliminate or minimize the impact of these parasites on the animals, it is important to understand the genetic diversity of the liver fluke populations and the relationship between parasite and host at regional bases. This research was carried out to determine diversity by sequence analysis of the mitochondrial ND1 gene and ribosomal ITS1 region.     

Isolation and characterization of microsatellite markers in the liver fluke (Fasciola hepatica)

Six microsatellite markers were isolated from Fasciola hepatica, a re‐emerging parasite that causes important veterinary and public health problems. In a sample of 52 liver flukes from a region of hyperendemicity (Bolivian Altiplano), five microsatellite were polymorphic. Our results showed that liver flukes present important genetic variability, suggesting a preferential outcrossing reproduction mode for this hermaphroditic parasite.     

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.     

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica

This study represents the first beta-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one beta-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. beta-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke beta-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.     

A plasma membrane Ca(2+)-ATPase (PMCA) from the liver fluke, Fasciola hepatica

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an α-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.     

Quantification and partial characterisation of regulatory peptides in the liver fluke, Fasciola hepatica, from different mammalian hosts.

1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP-immunoreactivity was detected by RIA of HPLC fractions.     

Proteomic analysis of glutathione transferases from the liver fluke parasite, Fasciola hepatica

The parasite Fasciola hepatica causes major global disease of livestock, with increasing reports of human infection. Vaccine candidates with varying protection rates have been identified by pre-genomic approaches. As many candidates are part of protein superfamilies, sub-proteomics offers new possibilities to systematically reveal the relative importance of individual family proteins to vaccine formulations within populations. The superfamily glutathione transferase (GST) from liver fluke has phase II detoxification and housekeeping roles, and has been shown to contain protective vaccine candidates. GST were purified from cytosolic fractions of adult flukes using glutathione- and S-hexylglutathione-agarose, separated by 2-DE, and identified by MS/MS, with the support of a liver fluke EST database. All previously described F. hepatica GST isoforms were identified in 2-DE. Amongst the isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. We also describe cDNA encoding putative Omega class GST in F. hepatica.     

Physiological and morphological effects of genistein against the liver fluke, Fasciola hepatica

A study has been carried out to determine the activity of genistein against adult liver fluke, Fasciola hepatica. Flukes were incubated in vitro in genistein at a concentration of 0.27 mg/ml (=1 mM). They ceased to move after 3 h, at which point the experiment was terminated and the specimens prepared for examination by scanning and transmission electron microscopy. Surface changes to the flukes comprised swelling and blebbing, especially in the posterior region of the flukes, and there was particular disruption to the spines, accompanied by some spine loss. Fine structural changes to the tegumental syncytium indicated an accelerated release of secretory bodies at the surface, but a reduction in their production within the cell bodies. Autophagic activity was evident in the tegumental cells, a phenomenon that was also observed in the gastrodermal cells. Disruption to the testis and vitelline follicles was severe, with an apparent block in the normal developmental sequence of the spermatogenic and vitelline cells, respectively. Shell protein production by the vitelline cells was also disrupted. In separate experiments, somatic muscle strips were exposed to concentrations of genistein ranging from 1 microm to 1 mm. There were statistically significant increases in the frequency and/or amplitude of muscle contractions at concentrations of 10 microm, 100 microm and 1 mm. The results suggest that genistein is capable of causing severe morphological and neuromuscular disruption to adult flukes in vitro over a short time-span.     

A histidine-rich protein from the vitellaria of the liver fluke Fasciola hepatica

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hysteresis in thioredoxin-glutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica

Thioredoxin-glutathione reductase (TGR) was purified from the adult stage of the liver fluke Fasciola hepatica. At 38° C and pH 7.8, specific activity values were 10.2U mg(-1) and 64.5U mg(-1), with DTNB or GSSG as substrates, respectively. Under the same conditions, apparent Km values were 46±8 μM (DTNB) and 30 ± 5 μM (GSSG). The enzyme was also able to catalyze thiol/disulfide exchange reactions. A subunit Mr of 61,000 was obtained. Like the homologous enzyme from the tapeworms, a lag time was observed in the enzyme assays at moderate or high concentrations of the substrate GSSG. The hysteretic behavior was reverted in the presence of GSH and was notably dependent on pH, such that the magnitude of the lag time increased with the acidity of the medium. These results strongly suggest that a hysteretic kinetic is a common feature of TGR from any parasitic flatworm. A sequence comparison revealed the structural cysteine residues proposed to be in the origin of the peculiar kinetic behavior of TGR are absent from the F. hepatica enzyme. Based on these observations, the model proposed recently to explain the GSSG-dependent hysteretic kinetic of TGR, which assumes the covalent modification of specific cysteine residues through glutathionylation [Bonilla M. et al. (2008) J Biol Chem 283: 17898] needs to be reevaluated.     

On the ultrastructure of the ovary of the liver fluke (Fasciola hepatica L.)

Summary The ovary of the liver fluke has been studied with light and electron microscopy. The organ consisted of germ cells and a layer of peripheral cells suggested to be nurse cells, and was surrounded by a capsule containing muscular tissue. The peripheral cells rested on a thick basement membrane and were irregular in outline. Their nuclei were of irregular shape, the mitochondria were dark with few cristae and the endoplasmic reticulum was tubular or vesicular and partly studded with ribosomes. The germ cells were rounded or polyhedral except in the outer part of the ovary where some of them showed irregular processes. The germ cells of the outer region (oogonia) were relatively small and in close contact with the cells suggested to be nurse cells. The inner germ cells (oocytes) were large and loosely packed. Their nuclei were irregular and contained round distinct nucleoli. The nuclear envelopes showed numerous pores. The endoplasmic reticulum was very sparse, but free ribosomes were abundant in the cytoplasm. This corresponded with a strong basophilia removable with RNase. In addition round basophilic bodies formed by densely packed ribosomes and membraneous material occurred in close spatial relation to mitochondria. The latter contained dense granules and few cristae. Groups of vesicles and membraneous lamellae were found in the cytoplasm, but they were considerably smaller than vertebrate Golgi complexes. Numerous dense spherical granules were found mainly in the periphery of the large germ cells. The granules were strongly osmiophilic except in the terminal part of the ovary. They were PAS-positive, but negative to Sudan dyes.Supported by a grant from Jordbrukets Forskningsråd, Stockholm.     

Dynamics of host-parasite interactions: the example of population biology of the liver fluke (Fasciola hepatica)

Knowledge of the population dynamics of parasites and their hosts is essential to build veterinary and health programs. The example chosen is that of Fasciola hepatica, a food-borne trematode responsible for severe human and animal infections on the five continents. In this paper, we review the relationships between the liver fluke and its intermediate (mollusc) and definitive (vertebrate) hosts.