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Linguatula serrata is a cosmopolitan parasite whose intermediate hosts are cattle, goats, sheep, and other ruminants. The adult form is found in the nasal airways, frontal sinuses, and tympanic cavity of canines and felines, and it produces hemorrhages and breathing difficulties. To elucidate if L. serrata produces enzymes that are capable of degrading tissues from the intermediate host, proteolytic activities in larval products were studied. Using the zymography technique, one major protease was detected in parasite in vitro-released products with an approximate molecular weight of 75 kDa. This enzyme was inhibited with phenylmethylsulfonyl fluoride, suggesting that it is a serine protease, which was also shown to degrade type I collagen. The serine protease exhibited maximal activity at alkaline pH and temperatures varying from 37 to 45 degrees C. To gather evidence about the physiological roles of the enzyme, further biochemical and functional studies are suggested.

Fasciola hepatica secretes proteolytic enzymes during liver invasion. The present study examined the effects of the cysteine protease inhibitor Ep-475 on sheep considering the following variables: serum levels of aspartate aminotransferase, L-lactate dehydrogenase, and gamma-glutamyltransferase, liver fluke fecundity, egg viability, parasite burden, and size. Twenty-four male sheep were randomly allocated in four groups of six animals each as follows: group A was infected with F. hepatica metacercariae and treated with 50 mg/kg of Ep-475, group B was infected and untreated, group C was uninfected and treated, and group D was uninfected and untreated. All animals were euthanized 10 weeks after the experimental infection. Serum activities of enzymes in infected animals were significantly lower in Ep-475-treated sheep than in untreated controls, although liver damage was produced. No significant reduction in total worm burden was observed between treated and untreated sheep. However, there was a significant difference on the average size, structure development, ova counts, and egg viability of liver flukes from these two groups. Results showed that Ep-475 reduces liver damage due to fasciolosis and induces an impairment of liver fluke growth and fecundity. These effects pinpoint liver fluke proteases as potential targets for pharmacological intervention.

Using the film in situ zymography (FIZ) technique, it has been demonstrated that N-[N-(L: -3-trans-carboxyoxirane-2-carbonyl)-L: -leucyl]-agmatine (E-64) inhibits Fasciola hepatica proteolytic activity in vivo. The aim of this study was to establish the dose-response relationship of the inhibition of proteolysis as assessed by FIZ with E-64 at different dosages in a murine model of fasciolosis. Maximum effective inhibition of proteolysis was achieved at 50 mg/kg/day (87%). Mice treated with this dose survived for a mean of 10.92 days more than untreated controls, and their ova counts and egg viability were significantly (P<0.05) lower than this latter group. These results indicate that intraperitoneal administration of E-64 not only inhibited liver proteolytic activity in a dose-dependent manner but also produced anti-fecundity and anti-embryonation effects, delaying the progression of fasciolosis. Yet, residual proteolysis may suggest that other E-64-non-sensitive enzymes are involved, or that E-64-enzyme chemical interactions are only capable of a partial agonistic-like effect.