Increasing the in vitro proliferation rate of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis.

Perkinsus mediterraneus is an alveolate parasite first described in Ostrea edulis from the Balearic Islands (Mediterranean Sea, Spain), and little is known about its biology or the disease it causes. Continuous in vitro cultures of P. mediterraneus have recently been established in the protein-deficient culture medium JL-ODRP-2F to facilitate its study. Parasite proliferation rate in vitro however was low, with densities increasing 2- to 6-fold between subcultures at 6-week intervals. To increase the proliferation rate of P. mediterraneus cultures to rates similar to other Perkinsus species, various culture conditions (temperature, osmolality, pH, O(2), and CO(2) concentrations), culture procedures (seeding density and frequency of medium changes), concentrations of medium components, and addition of medium supplements (oyster tissue lysate, oyster plasma, animal sera, growth factors, and hormones) were tested. All treatments were evaluated by measuring parasite densities after 2 weeks of culture. The greatest increase in parasite densities, a 35-fold increase over the cell seeding density and 18 times that of the control (cells without supplementation), occurred in medium supplemented with 1,000 μg/mL of O. edulis tissue lysate. P. mediterraneus proliferation was also significantly increased by oyster tissue lysate concentration as low as 125 μg/mL.