First report of Astragalus polysaccharide nanoemulsion efficacy against Clinostomid metacercariae in Nile Tilapia.

Herein, we present the development of a novel plant-based nanotechnology strategy employing Astragalus polysaccharide nanoemulsion (APS-NE) to combat clinostomid excysted metacercariae, metacercarial parasites of major global public health concern. A survey conducted between January and August 2024 on 200 Nile tilapia (Oreochromis niloticus) from Giza, Egypt, revealed a high prevalence of encysted metacercariae infection (43.50 %; 87/200). Morphological and molecular analyses (targeting the cytochrome oxidase subunit I gene) confirmed the presence of Clinostomum phalacrocoracis and Euclinostomum heterostomum (99-100 % sequence identity). The APS-NE was characterized by transmission electron microscopy and dynamic light scattering, which demonstrated well-dispersed nanoemulsions (average size 75.6 nm; polydispersity index (PDI) 0.04; zeta potential -35 mV), indicating robust colloidal stability. In vitro assays revealed a dose- and time-dependent increase in excysted metacercariae mortality. Specifically, Clinostomum spp. mortality increased from 5 % at 0.01 % concentration after 10 min to 100 % at 1 % within 1 h, with lethal concentration 50 % (LC₅₀) values decreasing from 0.488 % to 0.013 %. Euclinostomum spp. exhibited even greater sensitivity, with LC₅₀ values ranging from 0.396 % to 0.004 %. Statistical analysis confirmed significant differences in mortality across concentrations and timepoints (Chi-square, p < 0.05). Ultrastructural damage, including tegumental rupture and sucker edema, was visualized using scanning electron microscopy. Comet assays revealed significant DNA fragmentation in the treated worms. For example, the percentage of DNA in the tail increased from merely 6 % to approximately 12 % for Clinostomum spp., and from approximately 9 % to over 25 % for Euclinostomum spp. (p ≤ 0.05), comparable to praziquantel. Molecular docking simulations revealed strong binding affinities of APS to parasite mitochondrial enzymes (e.g., Euclinostomum cytochrome c oxidase: -8.59 kcal/mol), supporting the proposed mechanism of action. These findings represent the first in vitro evaluation of APS nanoemulsions against excysted metacercariae of Clinostomum and Euclinostomum, underscoring their promising potential for targeted parasite control. However, comprehensive in vivo and ecotoxicological studies are essential to validate their therapeutic efficacy and establish their feasibility for large-scale implementation.