Determination of diagnostic antigens in cattle amphistomiasis using western blotting

ABSTRACT

Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis. Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphistomum cervi [mixed infection], the most common species, were collected from cattle was determined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspection. After their homogenization and centrifugation, somatic antigens were prepared and analyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterologous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subsequent gels staining. Immunoblotting analysis using sera from cattle naturally infected with amphistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed. Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 micro gr/ml and 8 micro gr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demonstrate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cattle naturally infected with mixed amphistomes. Ninety-kDa protein can be specific diagnostic antigen for mixed amphistomiasis in Iran