Molecular characterization of different amphistomes infecting ruminants in India

Adult amphistomes collected from buffaloes were identified to the species level following available information. Genomic DNA from the individual flukes belonging to Paramphistomum epiclitum, Gastrothylax crumenifer and Gigantocotyle explanatum was isolated using the commercial kit. PCR amplification of the ITS2 region along with flanking 5.8S and 28S regions (ITS2+ region) was done for all 3 species and subsequently amplicons were cloned and sequenced. Sequence data generated from the cloned products confirmed the presence of 484 continuous bases in each case, out of which only 288 nucleotides encodes for ITS2 region (total length). Primary ITS2 sequence analysis recorded a single nucleotide polymorphism (SNP) at 259^th position in the targeted amphistome species (Bareilly isolates). However, total nucleotide variations recorded with respect to consensus were 9 in case of G. explanatum and 6 each in case of P. epiclitum and G. crumenifer. Study further recorded species wise clustering of different isolates except G. crumenifer (Bareilly isolate). Intra-specific variation recorded in the ITS2 region of P. epiclitum, G. crumenifer and G. explanatum were 0.3 to 0.7%, 0.7 to 2.9% and 2.8 to 5.0%, respectively.