Analysis of Cathepsin-L and Cathepsin-B Gene Expression in Embryonated Eggs of Fasciola gigantica and Effect of Various Protease Inhibitors on Miracidial Hatching

Abstract

Background: Fasciola gigantica is a helminth parasite of veterinary importance and a constraint on the growth and productivity of domestic livestock in the tropical countries of Asia and Africa. Present study aimed at determining the expression of four cysteine protease genes including cathepsin (cat) L-1 D and cat-Bs in the embryonated egg stage of F. gigantica by polymerase chain reaction (PCR). In addition to the analysis of expression of the above genes in the embryonated eggs, inhibition of in vitro hatching of the miracidia from the eggs was carried out using three protease inhibitors viz. serine, cysteine and leucine aminopeptidase inhibitors to understand the possible role of these proteases in the hatching of the miracidia from the eggs. Methods: Cat- L-1D, cat B-1, cat B-2 and cat B-3 genes were amplified by reverse transcriptase and quantitative-PCR, respectively for determining their expression in the embryonated eggs. Inhibition of in vitro hatching of the miracidia from the eggs was carried out by exposing the embryonated eggs to different concentrations of iodoacetamide, phenyl methyl sulphonyl fluoride and bestatin as cysteine, serine and leucine aminopeptidase inhibitors. Result: Results showed that cat L1-D, cat B-1 and cat B-3 genes were expressed at the embryonated egg stage but cat B-2 gene was not expressed at this stage of the parasite. Iodoacetamide and phenyl methyl sulphonyl fluoride (cysteine and serine protease inhibitors) inhibited the miracidial hatching at different concentrations, indicating probable role of cysteine and serine proteases in its hatching process. However, leucine aminopeptoidase does not seem to influence the miracidial hatching from the eggs as bestatin did not inhibit its hatching.