Fish‐borne zoonotic trematodes (FBZT) are extremely important zoonotically and can infect humans via the consumption of poorly cooked fish containing active metacercariae. In this context, the present study aimed to update the epidemiological information of FBZT among Nile tilapia (Oreochromis niloticus) collected from Giza and Fayoum Governorates. Concerning the molecular characterization and phylogenetic analysis of adult flukes from experimentally infected pigeons and histopathological alterations of both larval and adult trematode flukes. Oreochromis niloticus were found to be infected with different encysted metacercaria (EMC); Prohemistomum, Haplorchis and Pygidiopsis species from wild caught in Giza and farmed fish in Fayoum with the total mean of prevalence that 81.89 ± 6.48, 18.03 ± 2.9 and 34.64 ± 3.42 respectively. Three recovered flukes from experimentally infected domestic pigeons (Columba livia domestica); Prohemistomum vivax, Haplorchis pumilio and Pygidiopsis genata in their small intestine. First molecular characterization and phylogenetic analysis of heterophyid flukes; P. genata and H. pumilio. The sequences obtained in this study were registered in the GenBank with accession numbers (MT672308.1 and MT707975.1) respectively. Moreover, constructing a phylogeny and phylogenetic relationships between two heterophyid species was performed through analytic study of the second internal transcribed spacer nuclear ribosomal genes (rDNA‐ITS2). Phylogenetic analysis of P. genata and H. pumilio showed 99.42% nucleotide similarity with that sequence from Israel (AY245710) and 99.71% from Vietnam (EU826636.1). In addition, histopathological alterations of EMC and adult flukes induced necrosis of fish muscle bundles and a severe inflammatory response with muscular necrosis in intestinal tract of infected pigeons.
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