Development of recombinant cathepsin protease based diagnostic assay for Fasciola gigantica infection in domestic ruminants

Abstract

ABSTRACT newlinePresent studies were conducted for evaluating the diagnostic potential of four recombinant newlineantigens and native cathepsin-L protein of the helminth parasite Fasciola gigantica in buffaloes newlineand also for determining the diagnostic potential of native cathepsin-L and recombinant cat L1-D newlinein human fasciolosis. F.gigantica recombinant proteins cat L1-D, cat B-1, cat B-2 and cat B-3 were newlineexpressed in prokaryotic system and used as the target diagnostic antigens in the present study. newlineTotal RNA was isolated from the liver fluke and single stranded cDNA was synthesized. The newlinecDNA coding for cat L1-D, cat B-1, cat B-2 and cat B-3 proteins were PCR amplified with gene newlinespecific primers and cloned in TA cloning vectors for their sequence confirmation. The newlinerecombinant proteins cat L1-D, cat B-1, cat B-2 and cat B-3 were expressed in pPROEXHT-b newlineexpression vector and purified by Ni-NTA affinity chromatography. These recombinant proteins newlineresolved at ~36 kDa, ~37 kDa, ~38 kDa and ~37 kDa, respectively in SDS-PAGE. The antigenicity newlineof the recombinant cat L1-D protein was checked by western blot using Fasciola infected and newlineuninfected buffalo sera wherein the Fasciola infected sera reacted with cat L1-D antigen. Native newlinecathepsin protease was also purified by two step alcoholic fractionation from the adult flukes and newlineevaluated for diagnosis ofFasciola infection in buffaloes and human patients. newlineThe diagnostic potential of recombinant cat L1-D antigen and native cat-L protease in the newlinedetection of fasciolosis in buffaloes was studied in IgG-ELISA. A total number of 325 buffaloes newlinewere screened for anti-Fasciola IgG antibodies with the above antigens. The recombinant cat L1- newlineD antigen showed positive reactivity with 101/122 necropsy positive animals. But 21 of the 122 newlinenecropsy confirmed positive animals were negative in cat L1-D ELISA leading to the sensitivity newlineof 82.8%. However, IgG-ELISA with this antigen depicted 30/203 (14.8%) necropsy negative newlineanimals for Fasciola as sero-positive, thereby showing specificity of 85.2%. With nat