Evaluation of Indirect ELISA and Western Blotting for the Diagnosis of Amphistomes Infection in Cattle and Buffaloes

Abstract

Amphistomes are snail-borne trematodes infect rumens and reticulums causing acute and chronic diseases in cattle and buffaloes. The economical losses caused by Amphistomes resulted from drop in milk and meat yield in addition to mortalities. Eighty four serum samples (50 from cattle and 34 from buffaloes, 30 from amphistomes infected and 54 from amphistomes free cattle and buffaloes) collected from Giza and Garbia governourates, Egypt. The collected Amphistomes were morphologically and histologically classified. The identified worms of Paramphistomum and Carmyerius were used for the preparation of somatic antigens separately. The collected 84 serum sample were tested twice by indirect ELISA, firstly with Paramphistomum somatic antigen and secondly with Carmyerius somatic antigen. The antigenic profiles of adult Paramphistomum spp somatic antigen and Carmyerius gergaerius somatic antigen were analysed by (SDS-PAGE). Four male Rabbit were used for the preparation of hyper-immune serum against Paramphistomum and Carmyerius somatic antigens. Nine serum samples (two rabbit hyper immune sera; one for Paramphistomum and the another for Carmyerius , 3 serum sample from Paramphistomum infected cattle and 3 serum samples from Carmyerius infected cattle and one serum sample from non infected cattle as negative control) were blotted and tested twice at the same time on the nitrocellulose membrane by Western blotting techniques., firstly by using Paramphistomum somatic antigen and secondly with Carmyerius antigen. The results of both ELISA and Western blotting were statistically analysed. The sensitivity, specificity and accuracy for ELISA and Western blotting were (74% and 100%),(82.4% and 33.3%) and (79.76% and77.78%) respectively . The antigenic profile of adult Paramphistomum somatic antigen showed 14 distinct protein bands of protein molecular weights ranging from 11.5 to 174 KDa. While Carmyerius somatic antigen showed 13 distinct protein bands ranging from 11.5 to 166KDa. One distinct immunogenic band at 63 KDa was found to react with all sera from infected cattle and buffaloes with Paramphistomum somatic antigen while the same serum samples gave one distinct immunogenic band at 71 KDa with Carmyerius somatic antigen. It is concluded that ELISA is more reliable test for early diagnosis of amphistomiasis. There is a strong cross immune reaction between Paramphistomum and Carmyerius.