A Consistent PCR-RFLP Assay Based on ITS-2 Ribosomal DNA for Differentiation of Fasciola Species

Fascioliasis is a zoonotic parasitic disease caused by liver fluke species of Fasciola hepatica and Fasciola gigantica. Differentiation of these two species, based on their morphological characteristics, is difficult. The current study aimed to use PCR-RFLP assay to distinguish between F. hepatica and F. gigantica , based on profiles of RFLP, produced by effect of endonucleases on ITS2 of the ribosomal DNA genes from these two species. Adult Fasciola spp. were isolated from bile duct of naturally infected animals. The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene in the isolates. By running the sequences of the samples in NEBcutter, suitable restriction enzymes (MspI and KpnI) were selected. Eight F. gigantica and eighteen F. hepatica samples were evaluated. While RFLP pattern with MspI produced a profile by which it was difficult to differentiate these two species, KpnI along with MspI, produced a consistent pattern of a 231, 212 and 93 bp fragments in F. hepatica. This pattern was not seen in F. gigantica. Findings of this study demonstrated that RFLP with KpnI and MspI produce a suitable pattern which simply differentiates F. hepatica from F. gigantica.