Multigene typing and phylogenetic analysis of Fasciola from endemic foci in Iran.

Fasciolosis is a public health problem originally transmitted from livestock. Although molecular analysis of the nuclear and mitochondrial genes allow discrimination between the two known species of Fasciola, F. hepatica and F. gigantica, these markers do not permit the detection of hybrid forms. On the other hand, molecular analysis of the pepck and pold genes from Fasciola do permit the detection of hybrid isolates and this study has not yet been performed on specimens from human and domesticated animals in Iran. Therefore, the aim of this study was to molecularly analyze Fasciola isolates using both conventional and the two new genetic markers mentioned above. Fifty-three adult Fasciola worms were isolated from the livers of 28 domesticated animals, including sheep, cattle and goat. Moreover, an adult worm was obtained from the bile duct of an infected woman during endoscopic retrograde cholangio-pancratography (ERCP). Targeted fragments from the ITS-1, NDI and COX1 genes were amplified using specific primers. PCR products were sequenced and genetically analyzed using MEGA v.7 and DnaSP software. Additionally, the pepck and pold regions were amplified and analyzed using multiplex PCR and RLFP-PCR, respectively. Multiple alignment of sequenced fragments showed highest similarity among the ITS-1 sequences isolated from all four hosts in comparison to the other genes. Furthermore, sequence diversity across the COX1 was higher than the NDI and ITS-1. Diversity among sequences isolated from cattle was higher than those from sheep and goat in all three genes. Tajima's D and Fu's Fs were negative and statistically significant for all the genes except ITS-1. Phylogenetic trees showed that the human F. hepatica isolate was closer to sheep isolates. The results of the pepck and pold analyses showed that all isolates were F. hepatica and there were no hybrid forms among samples. The molecular analyses corroborated this finding.
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