Alan Trudgett

Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica

A cysteine proteinase released in vitro by Fasciola hepatica was purified to homogeneity by Sephacryl S-200 gel filtration chromatography followed by QAE-Sephadex chromatography. The purified enzyme resolves as a single band with an apparent molecular size of 27 kDa on reducing SDS-polyacrylamide gel electrophoresis; however, under non-reducing conditions it migrates as multiple bands, each with enzymatic activity, in the apparent molecular size range 60-90 kDa. The sequence of the first 20 N-terminal amino acids of the enzyme shows considerable homology with cathepsin L-like proteinases. Immunolocalisation studies revealed that the cathepsin L-like proteinase is concentrated within vesicles in the gut epithelial cells of liver fluke.

The comparative metabolism of triclabendazole sulphoxide by triclabendazole-susceptible and triclabendazole-resistant Fasciola hepatica

Benzimidazole anthelmintics are widely used against nematode, cestode and trematode parasites. The drugs undergo several enzyme-mediated reactions within the host animal that produce a number of metabolites. Although it has been shown that certain helminths, including Fasciola hepatica, can metabolise albendazole, nothing is known regarding the ability of the liver fluke to metabolise triclabendazole, which is the major flukicidal compound currently on the market. In the current study, adult triclabendazole-susceptible flukes were treated with triclabendazole sulphoxide in vitro, and the metabolism of the drug was monitored by high performance liquid chromatography. The data show that F. hepatica can metabolise triclabendazole sulphoxide into its relatively inert sulphone metabolite. Parallel experiments using triclabendazole-resistant flukes showed that the conversion of triclabendazole sulphoxide to triclabendazole sulphone was on average 20.29% greater in the resistant flukes compared with the susceptible flukes. The results are discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

Fasciola hepatica: surface and internal tegumental changes induced by treatment in vitro with the sulphoxide metabolite of albendazole ('Valbazen').

A morphological study has been carried out to determine the effect of the active sulphoxide metabolite of the benzimidazole anthelmintic, albendazole (ABZ-SO) on the adult liver fluke, Fasciola hepatica. Whole flukes were treated with ABZ-SO for 12 and 24 h at a concentration of 10 microg/ml. The changes in response to drug treatment were examined by scanning electron microscopy (SENI), transmission electron microscopy (TEM) and tubulin immunocytochemistry (ICC). No surface changes were apparent following 12 h ABZ-SO treatment, but localized blebbing was observed after 24 h, which became more extensive towards the posterior region of both surfaces. TEM of sections from the posterior midbody region revealed that ABZ-SO caused the accumulation of secretory bodies in the tegumental cells and in their cytoplasmic connections and, after 24 h, just above the basal plasma membrane. Localized blebbing of the apical membrane also occurred. The morphology of the Golgi complexes within the tegumental cells began to change after 12 h treatment with ABZ-SO and, by 24 h, few complexes were observed. A distinct increase in tubulin immunoreactivity occurred after 12 h treatment, but this decreased after 24 h. The results obtained are consistent with those expected for microtubule inhibition. They are discussed in relation to the action of established microtubule inhibitors, as well as the benzimidazole derivative, triclabendazole.

Relative activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica

A study has been carried out to determine the relative activity of triclabendazole (TCBZ) and its sulphoxide (TCBZSO) and sulphone (TCBZSO(2)) metabolites against the adult stage of the liver fluke, Fasciola hepatica. Flukes were incubated for 24h in vitro in 15mug/ml of each of the compounds and prepared for scanning and transmission electron microscopy. All three compounds induced changes to the surface morphology of the fluke, the changes comprising swelling and blebbing to a greater or lesser extent in different regions of the fluke. TCBZSO(2) was more disruptive anteriorly and TCBZSO posteriorly. Internal ultrastructural changes were evident following incubation with each of the compounds, with an order of severity TCBZSO(2)>TCBZSO>TCBZ. Swelling of the basal infolds and mitochondria were observed in the tegumental syncytium. In the tegumental cell bodies, there was a reduction in the number of secretory bodies, disruption of the Golgi complexes and swelling of the mitochondria. Severe flooding of the internal tissues was observed with TCBZSO(2) and, to a lesser extent, with TCBZSO and TCBZ. The results demonstrate that both TCBZ and TCBZSO(2) are capable of disrupting the fluke in vitro and are not the inactive compounds they were assumed to be previously. They may well contribute to drug action in vivo as well, indicating that drug action is due to the additive effects of several metabolites, rather than being due to a single active metabolite, namely, TCBZSO.

The distribution of Fasciola hepatica and Fasciola gigantica within southern Tanzania - constraints associated with the intermediate host

In East Africa, Fasciola gigantica is generally the causative agent of fasciolosis but there have been reports of F. hepatica in cattle from highland regions of Kenya, Ethiopia, Uganda and Zaire. The topography of the Southern Highlands of Tanzania provides an environment where the climatic conditions exist for the sustenance of lymnaeid species capable of supporting both Fasciola hepatica and F. gigantica. Theoretically this would allow interaction between fasciolid species and the possible creation of hybrids. In this report we present molecular data confirming the existence of the snail, Lymnaea truncatula, at high altitude on the Kitulo Plateau of the Southern Highlands, Tanzania, along with morphometric and molecular data confirming the presence of F. hepatica in the corresponding area. At lower altitudes, where climatic conditions were unfavourable for the existence of L. truncatula, the presence of its sister species L. natalensis was confirmed by molecular data along with its preferred fasciolid parasite, F. gigantica. Analysis based on a 618 bp sequence of the 28S rRNA gene did not reveal the presence of hybrid fasciolids in our fluke samples.

Fasciola hepatica expresses multiple alpha- and beta-tubulin isotypes.

We have identified five α-tubulin and six β-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke α-tubulin and between 65 and 97% for β-tubulin isotypes. Nucleotide sequence identity ranged between 68–77% and 62–80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the α-tubulins and two of the β-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on β-tubulin, three of the fluke isotypes had tyrosine at position 200 of β-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.

Multiple origins of European populations of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), a liver parasite of ruminants ☆

The giant liver fluke, Fascioloides magna, a liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384bp) and nicotinamide dehydrogenase subunit I (nad1; 405bp) were used. Phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European foci of infection (Italy, Czech Republic and Danube floodplain forests involving the territories of Slovakia, Hungary and Croatia) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (eight), while a more substantial level of genetic diversity and a greater number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes, supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities which indicates that introduction of F. magna to the European continent occurred more than once. In the Czech focus of infection, a south-eastern USA origin was revealed. Identical haplotypes, common to parasites from the Czech Republic and from an expanding focus in Danube floodplain forests, implies that the introduction of F. magna to the Danube region came from an already established Czech focus of infection.

Development of an egg hatch assay for the diagnosis of triclabendazole resistance in Fasciola hepatica: proof of concept.

The aim of this study was to develop an Egg Hatch Assay (EHA) test for the detection of triclabendazole (TCBZ) resistance in Fasciola hepatica. A number of fluke isolates were used, of differing sensitivity to TCBZ. Eggs were exposed to solutions of triclabendazole sulphoxide (TCBZ.SO) for 14 days, then triggered to hatch. Egg development was divided into 6 distinct and easily identifiable stages: dead, empty, unembryonated, cell division, eye spot and hatched. The number of eggs reaching those stages was recorded. Initially, the discriminating dose (1% hatch) was determined for the Cullompton isolate, used as TCBZ-susceptible (TCBZ-S) standard. Once this concentration had been resolved, the response of different isolates to this concentration was examined. The hatch rate of the Fairhurst isolate was not significantly different from that of the Cullompton isolate, confirming its TCBZ-S status. The Patagonia isolate has not been exposed to TCBZ in the field and should be TCBZ-S: the results of the EHA supported this. The egg hatch response of the Oberon and Dutch isolates differed significantly from that of the Cullompton isolate; the former isolates are regarded as TCBZ-resistant (TCBZ-R) and the results confirmed this. Another isolate, the Leon isolate, was originally described as being TCBZ-R, but has since been shown to be TCBZ-S. There was no difference in its response to TCBZ.SO in the EHA from the Cullompton (and Fairhurst and Patagonia) isolate(s), further indicating its TCBZ-S status. The impact of TCBZ.SO treatment on the component stages of egg development was determined and revealed differences between the isolates. In conclusion, the results of the study have shown that it is possible to discriminate between TCBZ-S and TCBZ-R isolates of F. hepatica on the basis of the response of their eggs to an EHA and the test could be used to evaluate the TCBZ sensitivity of unknown field isolates.

Effect of the metabolic inhibitor, methimazole on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica.

SUMMARY A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered in the presence of a metabolic inhibitor. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-sensitive isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 microM). Flukes were then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); MTZ+NADPH+TCBZ (15 microg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 microg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater surface disruption to the triclabendazole-susceptible than -resistant isolate. However, co-incubation with MTZ and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own; this was not seen for the TCBZ-susceptible Cullompton isolate. Results of this study support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

Population dynamics of the liver fluke, Fasciola hepatica : the effect of time and spatial separation on the genetic diversity of fluke populations in the Netherlands

An evaluation of the genetic diversity within Fasciola hepatica (liver fluke) may provide an insight into its potential to respond to environmental changes, such as anthelmintic use or climate change. In this study, we determined the mitochondrial DNA haplotypes of > 400 flukes from 29 individual cattle, from 2 farms in the Netherlands, as an exemplar of fasciolosis in a European context. Analysis of this dataset has provided us with a measure of the genetic variation within infrapopulations (individual hosts) and the diversity between infrapopulations within a herd of cattle. Temporal sampling from one farm allowed for the measurement of the stability of genetic variation at a single location, whilst the comparison between the two farms provided information on the variation in relation to distance and previous anthelmintic regimes. We showed that the liver fluke population in this region is predominantly linked to 2 distinct clades. Individual infrapopulations contain a leptokurtic distribution of genetically diverse flukes. The haplotypes present on a farm have been shown to change significantly over a relatively short time-period.