Unveiling Clinostomum spp. In farmed nile tilapia from upper Egypt: morphological clues and molecular evidence.

Abstract

Clinostomum species (spp.) are significant fish-borne zoonotic helminths worldwide, causing yellow grub disease which diminishes the quality, marketability, and productivity of infected fish stocks. Despite their veterinary and public health relevance, detailed molecular investigations of Clinostomum spp. infecting Nile tilapia in Egypt remain limited, especially in Upper Egypt. A total of 400 Nile tilapias were collected from fish farms in Sohag Governorate, Upper Egypt, and initially screened for encysted metacercariae through visual inspection and subsequent light microscopic examination. Recovered parasites were fixed, stained, and identified based on detailed morphological features. For robust molecular confirmation, high-quality genomic DNA was successfully extracted from metacercariae derived from 40 heavily infected representative samples. Targeting of the internal transcribed spacer 2 (ITS2) regions was performed using polymerase chain reaction (PCR) with specific primers. The resulting sequences were examined using BLAST searches and maximum likelihood phylogenetic reconstruction to confirm species identity, and the validated sequences were subsequently deposited in GenBank™. In this study, microscopic examination revealed parasites exhibiting characteristic morphological features of Clinostomum spp., which were detected in 35.0% (140/400; 95% CI: 30.33-39.67) of the examined Nile tilapia (Oreochromis niloticus). The parasite was detected most frequently in tilapia weighing 50-200 g, at a rate of 44.8%, suggesting increased susceptibility among smaller fish. Molecular analyses confirmed the presence of Clinostomum spp. in all positive specimens. The validated sequence (GenBank accession number PQ330909) confirmed through phylogenetic reconstruction identified the encysted metacercariae as Clinostomum phalacrocoracis, showing strong genetic similarity to isolates previously reported from other regions. Overall, these results demonstrate a high prevalence of Clinostomum phalacrocoracis infection in Nile tilapia populations in Upper Egypt, with smaller fish showing the highest frequency. Molecular analyses provide definitive confirmation of Clinostomum phalacrocoracis, complementing morphological identification and offering a comprehensive characterization of the metacercariae. This study also situates the findings within the broader context of previous reports on Nile tilapia in Egypt, providing an updated overview of the parasite's distribution and genetic relationships. Collectively, the evidence underscores the need for enhanced monitoring and management strategies to protect fish health and mitigate potential zoonotic risks in regional aquaculture systems.