Genotypic and phenotypic analysis of Fasciola isolates

ABSTRACT

To identify the fasciolid species by morphometric and molecular methods in Zanjan, northwest of Iran. Adult Fasciola worms [n=584] were obtained from cattle and sheep in Zanjan slaughterhouse in 2007. Living flukes were washed, then worms' images were taken by 3CCD camera and finally apical zone of each worm was obtained. Morphometric values such as body length, body width, body area, body perimeter and the distance between ventral sucker and posterior end of body were obtained using AutoCAD image analysis software. Total gDNA was extracted from individual flukes by modified phenol-chloroform method. PCR amplification of ITS2 fragment was performed the isolated DNA samples and the amplicons were consequently subjected to RFLP assay and nucleotide sequencing to distinguish between fasciolid species. Mean of morphometric values in flukes from sheep was greater than those of cattle. Accordingly, the identified species included 31% F. hepatica-like, 7% F. gigantica-like and 62% intermediate forms. However, ITS2 fragment of 535 amplified specimens, showed no variation at the species-specific nucleotide sites 230, 340 and 341. The amplified fragment composed of partial 5.8S sequence [62bp], the complete ITS2 sequence [361bp] and partial 28S sequence [34bp]. The nucleotide contents of ITS2 region were 69 A, 116 T, 81 C and 95 G and the average G+C content was approximately 49%. Comparing of ITS2 sequences with the BLAST GenBank database, also confirmed that all specimens were F. hepatica. A simple and rapid PCR-RFLP assay can be used for distinguishing between these species