Studies on the oyster pathogen Perkinsus marinus (Apicomplexa): Interactions with host defenses of Crassostrea virginica and Crassostrea gigas, and in vitro propagation

The disease caused by the protozoan Perkinsus marinus has been a major source of mortality in the eastern oyster, Crassostrea virginica. Variations in susceptibility to P. marinus infection among eastern oysters collected from the Chesapeake Bay and Gulf of Mexico, as well as between eastern and Pacific (Crassostrea gigas) oysters were determined. Since oyster host defense may play a role in determining susceptibility to pathogen infection, cellular and humoral defense activities of the oyster and their interactions with P. marinus were investigated. Procedures also had to be established to isolate, purify, and propagate in vitro, P. marinus. Eastern oysters from all sites were found to be highly susceptible to the pathogen. Cellular and humoral activities were significantly affected by heavy intensity of P. marinus infection. Prevalence and intensity of P. marinus infection were lower in Pacific oysters than in eastern oysters. Pacific oysters may offer a less favorable environment for the development of P. marinus compared to eastern oysters for at least two possible reasons: the elevated cellular and humoral activities may degrade the parasite more effectively, and lower plasma protein levels may limit parasite growth. Incubation of merozoites with hemocytes of eastern and Pacific oysters in vitro suggested that limited intracellular killing of P. marinus occurred but that killing was not mediated by oxygen metabolites. Perkinsus marinus was successfully propagated in vitro in a culture medium containing most of the known constituents of cell-free oyster hemolymph. Cultures of the parasite were initiated from heart fragments of infected oysters. The cultured protozoan was similar in morphology to P. marinus, enlarged in fluid thioglycollate medium, reacted with polyclonal antibodies raised against hypnospores and was infective. Continuous cultures of P. marinus could also be initiated from hypnospores. Two types of division, progressive cleavage and successive bipartition of the mother cell protoplast, were observed