Molecular characterization of Fasciola hepatica isolates by RAPD-PCR and ribosomal ITS1 sequencing

ABSTRACT

Understanding genetic structure and status of genetic variation of the Fasciola hepatica populations has important implications for epidemiology and effective control of fasciolosis. The aim of the present study was to genetically characterize F. hepatica isolates from different hosts, using sequence analysis of ribosomal ITS1 and RAPD-PCR. Fifty three adult F. hepaticas were isolated from naturally infected cattle, sheep, buffalo and goat from two regions in Iran. Genomic DNA was extracted from 70% ethanol preserved flukes. RAPD-PCR with a set of arbitrary primers [UBC90 and R151] was used to estimate genetic variation within the species. Ribosomal ITS1 region of the isolates was amplified, using primers specifically designed for this study. Ten samples [4 sheep, 2 cattle, 3 buffaloes and one goat isolate] were sequenced at ITS1 and analyzed, using DNASIS and ClustalW softwares. F. hepatica ITS1 region was amplified successfully for all samples and a band of 470 bp was shown in all cases. Different isolates did not show any significant genetic variations in rDNA-ITS1 as all the sequences showed to be 100% identical. RAPD results of 52 samples, in particular those with UBC90, showed different patterns within F. hepatica isolates of each host. RAPD data for this primer showed three different patterns for each of sheep and cattle isolates and two patterns in buffalo isolates. All the 14 cattle isolates come up with an identical pattern, using primer R151. The study showed the variability of F. hepatica isolates in Iran, using RAPD markers. No intraspecies variation was seen in the Iranian F. hepatica isolates at ITS1 rRNA gene, indicating highly conserved nature of this region