Background: Identification of liver flukes, Fasciola hepatica, and Fasciola gigantica by morphometric parameters is not always reliable due to the overlapping measurements. This study aimed to characterize the liver flukes of animals from different parts of Iran by the genetic markers, ITS1, and COXI. Methods: We collected flukes from infected livestock in six provinces of Iran from Sep to Nov 2016. The flukes were identified by amplification of a 680 bp sequence of ITS1 locus followed by a restriction fragment polymorphism (RFLP) assay. The genetic diversity among isolates was evaluated by amplification and sequencing of a 493 bp fragment of the COXI gene. Results: We obtained 38 specimens from Khuzestan, 22 from Tehran, 10 from Isfahan, 10 from Mazandaran, 4 from Kurdistan, and 3 from Ardabil provinces. PCR-RFLP analysis revealed two patterns, representing F. hepatica, and F. gigantica. Fifty specimens from cattle and sheep exhibited F. hepatica pattern and 37 from the cattle, sheep, buffalo, and goat that of F. gigantica. The phylogeny based on COXI revealed two distinct clades separating F. hepatica from F. gigantica. In our phylogeny, the Iranian F. gigantica isolates showed a distinct separation from the African flukes, while grouped with the East Asia specimens demonstrating a common ancestor. The F. hepatica isolates clustered with the flukes from different parts of the world, including East Asia, Europe, and South America. Conclusion: The present study revealed a substantial genetic difference between F. gigantica populations of Asia and Africa, while F. hepatica isolates from different parts of the world shared high similarities.

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Background: Efforts to find a reliable non-molecular means of identification has been the main purpose of the current work that always is persuaded by researchers interested in the field of parasitology. Methods: Adult fasciolids were obtained from the slaughterhouses in different parts of Iran in 2017, and investigated using the classical old fashion morphological appearances of the worms implementing a camera lucida equipped microscope. Histological procedure was subsequently performed for almost the entire collected adult worms followed by Hematoxylin and Eosin (H&E) staining technique. DNA extraction and RFLP-PCR technique were carried out for the entire fasciolid liver flukes. To attain more comparable morphological conclusions, Scanning Electron Micrographs were also implemented for two molecularly identified fasciolids. Results: Based on spine morphology observed in worm’s tissue sections two types of tegumental spines, “pointed” and “molar” shapes have been identified addressing to distinguish F. hepatica and F. gigantica species respectively. The present identification has been also supported by Molecular analysis using RFLP-PCR technique. Conclusion: There are some hidden morphological characters implemented in species identification for certain helminths. Meanwhile, the emergence of computer image analysis system (CIAS) on the scene of taxonomy, has revolutionized the accuracy of measurement in morphology by employing detailed parameters that have not been regarded before. The current study has illustrated the tegumental spines of two Fasciola species in tissue sections which has not been enough considered in helminthological publications so far.