Stable In Vitro Cultivation of the Myxozoan Enteromyxum leei for over 12 Months: Integration of a Physical Aggregation Technique for Long-Term Antiparasitic Interventions.

Emaciation disease, primarily caused by the myxosporean parasite Enteromyxum leei, poses a significant economic threat to the olive flounder (Paralichthys olivaceus) aquaculture industry. Research on E. leei has been severely restricted by its status as an unculturable obligate parasite and its extreme sensitivity to cryopreservation. In this study, we developed a standardized in vitro culture system that achieved stable maintenance of E. leei for over 12 months for the first time. To overcome the challenges of primary isolation, a physical aggregation technique was implemented, which effectively concentrated the parasites while reducing bacterial contamination by 99.1%, as validated by qPCR. The established culture, maintained in minimum essential medium supplemented with 30% FBS at 25 °C, exhibited a consistent 20-day asexual proliferative cycle consisting of six distinct developmental stages. Morphological stability and genetic identity were rigorously monitored, showing no phenotypic drift or genetic divergence throughout the 12-month period. Our results demonstrate that the entire proliferative phase of E. leei can be recapitulated in vitro without host intervention. Given that cryopreservation remains non-viable, this continuous culture system provides a critical, unique platform for high-throughput drug screening and myxozoan developmental biology, offering a robust tool to mitigate emaciation syndrome in marine fish farms.