In silico and in ovo model evaluation of thymol as an anticryptosporidial agent

Cryptosporidiosis constitutes a serious public health problem, notably in immunocompromised individuals, while effective therapeutic options remain limited. The objective of the present work was to evaluate the anticryptosporidial activity of thymol through in silico and in ovo approaches. Molecular docking assessed thymol interactions with four key Cryptosporidium targets involved in essential metabolic pathways: glycolysis [lactate dehydrogenase (LDH)], protein biosynthesis [lysyl-tRNA synthetase (LysRS)], nucleotide synthesis [inosine monophosphate dehydrogenase (IMPDH)], and calcium-dependent signaling [calcium-dependent protein kinase (CDPK)]. The anticryptosporidial activity of thymol was experimentally validated using the embryonated chicken egg (in ovo) model a cost-effective and ethically sound alternative to mammalian models at non-embryotoxic concentrations (0.025, 0.05, 0.1, 0.2, and 0.4 mg/egg). Docking results showed favorable binding affinities toward all investigated targets, with binding free energies ranging from -6.0 to -6.3 kcal/mol. Evaluation of oocyst counts in the chorioallantoic fluid collected seven days post-inoculation revealed a dose-dependent inhibition of oocyst multiplication. Complete inhibition was achieved at concentrations of 0.1, 0.2, and 0.4 mg/egg compared with untreated controls, without embryonic mortality. To the best of our knowledge, this is the first study employing the in ovo model to evaluate the anticryptosporidial activity of thymol. These findings suggest that thymol could serve as a promising, natural, and economically accessible candidate for the development of novel therapeutic agents against cryptosporidiosis.