Continuous in Vitro Culture of the Eastern Oyster Parasite Perkinsus marinus

The severe disease caused by the endoparasitic protozoan Perkinsus marinus (Apicomplexa: Class Perkinsea) is a major cause of mortality of the eastern oyster (Crassostrea virginica) along the Gulf of Mexico and Atlantic coasts and no natural resistance against this parasite appears to have developed so far. This has resulted in a drastic reduction of existing populations, has critically impaired oyster production, and has strengthened the need for a detailed understanding of the cellular and molecular aspects of this disease in order to accomplish its eradication as an ultimate goal. Although we are currently developing new methods for the purification of large numbers of the freshly isolated throphozoites from oyster tissues, we decided to attempt the in vitro continuous propagation of P. marinus. In vitro cultures of other Apicomplexa parasitic protozoa, such as Plasmodium and Toxoplasma, have been accomplished in host cell lines. However, because no oyster cell lines have been developed so far, a host cell-free culture system was the only remaining option for establishing a continuous parasite culture. Our preliminary attempts to cultivate P. marinus trophozoites included the use of a considerable variety of media and media combinations, including Dulbecco's modified Eagle's (DME), RPMI 1640, Wolf's amphibian and Schneider's Drosophila (GIBCO 172) media, and one formulation currently being tested for the establishment of an invertebrate cell line.