Fasciola hepatica: irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke.

Irradiation has been successful in the attenuation of infective stage parasites for use as vaccines against a number of parasites including Fasciola spp. The mechanisms of action of irradiation-attenuated vaccines, however, are not clearly understood. In this study, we examined the effect of 3, 10, and 40 krad of gamma-irradiation on the expression of carbohydrates and cathepsin-B by newly excysted juvenile Fasciola hepatica (NEJ). Following irradiation of metacercariae, the expression of concanavalin A (ConA)-specific sugars was decreased on the surface of NEJ and the expression of wheat germ agglutinin (WGA)-specific sugars was increased in the gut and reduced on the surface of NEJ. Cathepsin proteases are a major component of liver fluke excretory/secretory material (ES) and can cleave host immunoglobulin (Ig). Cathepsin-B protease was localized in nonirradiated NEJ to the gut lumen and to secretory granules within the gut epithelia. Irradiation of fluke with 3, 10, and 40 krad of gamma-rays significantly reduced the tissue expression of cathepsin-B at 8 hr postirradiation in an apparently dose-dependent manner. After a further 24 hr culture tissue expression of cathepsin-B was significantly reduced in 10- and 40-krad-irradiated NEJ. Protease activity of ES samples collected over a 24-hr period from irradiated and nonirradiated NEJ cultured in vitro were tested using a rabbit Ig cleavage assay. The proteolytic activity of ES from 10- and 40-krad-irradiated NEJ was reduced during the initial 6 hr in culture and between 12 and 24 hr when compared to ES from nonirradiated controls. Biosynthetic labeling experiments using [35S]methionine and [35S]cysteine indicated that ES material was actively synthesised during 48 hr in vitro culture. Therefore, from this study, we conclude that gamma-irradiation of NEJ alters expression of cathepsin-B protease and WGA- and ConA-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host by irradiation-attenuated metacercariae of Fasciola spp.

Characterisation of a novel Kunitz-type molecule from the trematode Fasciola hepatica.

A low molecular mass monomeric protein termed Fh-KTM (Fasciola hepatica Kunitz-type molecule) was isolated from the trematode Fasciola hepatica. Fh-KTM is a single polypeptide of 58 amino acids and a Mr of 6751. The complete amino acid sequence of Fh-KTM was determined and revealed significant similarity to the Kunitz-type (BPTI) family of proteinase inhibitors. Several polymorphisms were observed suggesting that more than one Fh-KTM molecule may be expressed by this parasite. Modified proline residues were shown to occur at all four positions in this protein as 3-hydroxy derivatives. This is the first report of 3-hydroxyproline residues in a Kunitz-type molecule. Indirect immunofluorescence and immunogold labelling revealed that Fh-KTM is an abundant molecule within the parasite localised to the gut, the parenchymal tissue and the tegument of adult F. hepatica. Serine protease inhibition assays revealed that Fh-KTM exhibited little or no inhibition against chymotrypsin, kallikrein, urokinase or key serine proteases of the blood coagulation pathways. However, Fh-KTM was able to inhibit trypsin even though the P1 reactive amino acid of Fh-KTM was a leucine residue.

Vaccination of sheep with purified cysteine proteinases of Fasciola hepatica decreases worm fecundity.

There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infection. A cysteine proteinase complex identified in the regurgitant of adult F. hepatica was examined in this context. The thiol-cathepsin-related proteinases of M(r) 28,000 were purified and tested in vaccine trials of sheep infected with liver fluke. Ten animals were immunised with the purified proteinases and developed antibodies to the cysteine proteinases prior to challenge with F. hepatica metacercariae. Infection appeared to cause a boost in antibody response by Week 4 into infection, and antibody levels were generally sustained throughout infection. The cysteine proteinases are not novel antigens, since low-level antibody titres were also detected in nonimmunised controls by late infection. On completion of the trial, there was no difference in worm burden between the two groups. However, faecal egg counts and therefore worm fecundity were significantly decreased.