In the present study, the carbohydrate structures associated with Fasciola gigantica adult worm were identified by indirect hemagglutination inhibition test. Glucose was found to be the main monosaccharide associated with the fluke. According to indirect hemagglutination inhibition results, purification of glycoprotein fractions from worm crude extract was carried out by affinity chromatography immobilized glucose agarose gel and Con-A lectin columns. The isolated glycoprotein fractions, FI and FII, were characterized by SDS-PAGE which revealed one band in FI of 26 kDa and another one band of 19.5 kDa in FII compared with 12 bands associated with whole worm extract. Both fractions were also characterized by isoelectric focusing technique which proved that both bands were acidic in nature with pIs 6.4 and 6.5 respectively. The comparative diagnostic evaluation of the two isolated glycoprotein fractions and crude extract of experimental fasciolosis in rabbits by ELISA revealed that FII was more potent in the diagnosis during prepatent (first week post infection) and patent periods (10 weeks post infection) than FI and crude extract. Moreover, infected rabbit sera at ten weeks post infection identified both bands; 26 and 19.5 kDa in western blot analysis confirming its immunodiagnostic activities which was proved previously by ELISA. FII proved potency in diagnosis of fasciolosis in 200 buffalo serum samples of different ages and sexes using ELISA which recorded 95 % positive and 5 % negative samples. Moreover, the detailed structural analyses of the most potent fraction, F11, using mass spectrum was made and elucidated chemical structure; O-glycan [Ser-(Arg-Ser-Arg-Ser-GlucNAc)19-GlucNAc]. The present result introduces GlucNAc rich fraction of F .gigantica that can be used successfully in the diagnosis of acute and chronic fasciolosis.
Keywords: Affinity column chromatography; Diagnosis; ELISA; Fasciola gigantica; GlucNAc.