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Abstract
This study evaluated three experimental infection methods for Enterocytozoon hepatopenaei (EHP) in juvenile whiteleg shrimp (Penaeus vannamei): oral administration of EHP-infected hepatopancreatic tissue, direct injection of infected tissue into the hepatopancreas of healthy shrimp, and cohabitation with EHP-infected shrimp. The results showed that oral administration resulted in the highest infection rate (70%) and parasite load (7.84 × 10⁷ copies/µL of DNA), indicating its potential as a reliable method for establishing experimental EHP infections. Cohabitation resulted in a lower infection rate (30%) and parasite load (1.39 × 10⁷ copies/µL of DNA), but more closely represented the natural transmission route of EHP. In contrast, direct injection into the hepatopancreas caused high acute mortality, likely due to mechanical damage, making it impossible to accurately assess EHP infectivity in juvenile shrimp; thus, this method was unsuitable for establishing an experimental infection model. These findings provide a useful basis for selecting appropriate experimental infection methods for EHP research, particularly in epidemiological studies and the development of preventive strategies.