Improved cell culture propagation of Amyloodinium ocellatum

Abstract

A system for cultivating Amyloodinium ocellatum was developed using aggregates of red drum Sciaenops ocellatus dorsal fin cells in suspension. Tomonts harvested from infected fish using density gradient centrifugation in Perroll® were placed in saltwater media (30 ppt) containing antibiotics (penicillin 100 IU ml-1, streptomycin 100 mg ml-1). Microbe-free infective dinospores emerging from the tomonts were used to infect red drum cell aggregates. Dinospores attached and transformed into trophonts on the cell aggregates, developing into normal size (80 μm) trophonts in saltwater media (10 ppt). Trophont infected cell aggregates were placed in increased saltwater concentration (30 ppt) at 48 h incubation to induce the release of trophonts and their encystment as tomonts. Tomonts were separated from the cellular debris by saltwater media washing and Percoll® density gradient centrifugation. Tomonts were then incubated to produce a second generation of dinospores. The parasite was maintained in vitro for 10 complete life cycles with no observed decreased viability using this system.