Differentiation of amphistome species of cattle in Kerala by polymerase chain reaction - restriction fragment length polymorphism

Abstract

The present study evaluated the PCR-RFLP of ITS-2+genes for species differentiation of three major amphistomes namely Gastrothylax crumenifer, Fischoederius cobboldi and Paramphistomum spp. Molecular analysis using PCR yielded amplicons of 515 bp for each species. The nucleotide differences among the sequences of the three species of amphistomes at different positions were further used for designing suitable RFLP. The amphistome parasites were distinguished taking into account the differences in the recognition sequences of TspRI on the ITS-2+ region by PCR-RFLP. The enzyme cleaved F.cobboldi at two recognition sites, CAGTG and CACTG and yielded 331, 93 and 91 bp products. Gastrothylax crumenifer had one recognition site CAGTG and resulted in 331 bp and 184 bp fragments. Since no recognition sequence was found in Paramphistomum spp., there was no cleavage. It is concluded that PCRRFLP was a promising molecular tool for species identification of amphistomes.