IN-VITRO CULTURE OF THE EASTERN OYSTER PARASITE PERKINSUS-MARINUS - OPTIMIZATION OF THE METHODOLOGY

We have succeeded in establishing an in vitro continuous culture of th e eastern oyster (Crassostrea virginica) parasite Perkinsus marinus (G authier and Vasta, 1993) and have now characterized a number of variab les, such as temperature, salinity, pH, seeding density, and selected nutritional requirements that affect parasite proliferation. Optimum t emperature, salinity, and pH ranges were 28-32 degrees C, 25-30 ppt, a nd 6.6-6.8, respectively. Proliferation rates during the first 48 hr o f culture were directly related to the size of the inoculum. The addit ion of Ham's F12 nutrient mixture (a serum replacement) to Dulbecco mo dified Eagle's (DME) base medium substantially enhanced proliferation, particularly at a DME:Ham's F12 ratio of 1:2. Five percent fetal bovi ne serum was required for optimal growth, but higher concentrations (1 0-20%) were inhibitory. The presence of oyster plasma enhanced growth only at low concentrations of fetal bovine serum (0-0.1%) and doubled addition of Ham's F12 (DME:Ham's 1:2). After approximately 30 passes o ver a 6-month period, the cultured parasite exhibited morphological an d virulence features that are similar to those from the freshly collec ted specimens. The optimized medium apparently provides an environment comparable to the intracellular conditions of the host since the smal l (approximately 4 mu m) trophozoite stage frequently observed to prol iferate within the oyster hemocyte is maintained. (C) 1995 Academic Pr ess, Inc.