Cytotoxicity and Antiparasitic Effects of Toltrazuril Against Enteromyxum leei in Olive Flounders: In Vitro and In Vivo Analyses.

The parasite Enteromyxum leei causes serious emaciation disease in marine fish species. Toltrazuril exhibits antiparasitic effects; however, the simultaneous impacts of toltrazuril on parasites and hosts are unclear. Here, we evaluate the cytotoxicity of three commercial toltrazuril products (A, B and C) and the treatment efficacy against E. leei infections in olive flounder (Paralichthys olivaceus). Product A was confirmed safe in Hirame natural embryo cells, even at a toltrazuril concentration of 50 μg/mL, and was selected for further studies. In vitro assays revealed that toltrazuril effectively killed E. leei spores at concentrations of 5.0, 7.5 and 10.0 μg/mL. In vivo experiments on olive flounders infected with E. leei showed that oral toltrazuril administration at doses of 10 and 20 mg/kg body weight for 5 days effectively damaged spores in the fish intestine, with E. leei death confirmed by propidium iodide staining. Administering toltrazuril mixed feed (20 mg/kg) to infected olive flounders in a fish-farm setting also proved effective in killing spores present in the intestine. Furthermore, Hoechst-propidium iodide double staining confirmed that toltrazuril was effective in killing E. leei spores both in vitro and in vivo, indicating its potential as a treatment for emaciation diseases in marine fish.