In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

Abstract

ABSTRACT. Perkinsus marinus, a pathogen of eastern oysters (Crassostrea virginica), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus‐1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue‐black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus‐1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus‐1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell‐free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.

Host-parasite interactions among broadly distributed populations of the eastern oyster Crassostrea virginica and the protozoan Perkinsus marinus

“…Mackin (1962) acknowledged that races differing in virulence may exlst, but lacking any evidence he assumed that differences in virulence were environmentally mediated. The advent of in vitro culture methods for P rnarinus (Gauthier & Vasta 1993, Kleinshuster & Swink 1993, La Peyre et al 1993) enabled us to compare virulence among isolates under controlled laboratory condit~ons. Although we lack information to indicate genetic differences among the isolates, 'transfection' of a common stock of ME oysters and subsequent acclimation of the isolates to in vitro culture should have minimized any environmentally mediated differences among the isolates such as differences in local salinity regimes, nutritive value of oysters, etc.…”

“…No control was run to test the effect of this inoculation procedure on survival since similar procedures have been used in other stud~es with little negative ~m p a c t observed on the oysters (Ray 1954, Valiulis 1973. Gauthier & Vasta 1993, La Peyre et al 1993. Each tank was covered with a fitted lid to prevent crosscontamination.…”

“…marjnus isolate were established from infected oysters using prezoosporangia obtained after incubation of oysters in RFTM (La Peyre & Faisal 1995). Cultures were maintained a s described by La Peyre et al (1993) except that buffers were modified according to Freshney (1987) for maintenance in ambient air rather than a 5' Yn CO2 atmosphere. Because oysters become infected by filtering P marinus from the water (Ray 1954), and heavily infected oysters can harbor more than 10h P marinus cells per gram wet tissue (Choi et al 1989, Gauthier & Fisher 1990, Bushek et al 1994b, it is not likely that a particular oyster would be infected by a single clone.…”

“…is commonly observed histologically in infected oysters) were generated by diluting actively proliferating cultures. Briefly, in vitro cultures of each isolate were subcultured at densities of 4 X 10' cells ml-' in 75 cm2 T-flasks containing 30 m1 JL-ODRP-1 medium (La Peyre et al 1993) buffered for ambient CO,. These were maintained at 27OC.…”

Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus

“…Unique to the genus Perkinsus is that the species in this group are one of the few protozoan parasites that can be propagated in vitro as clonal cultures in defined media and in the absence of host cells, reaching cell densities of 10 7 cells/ml (Gauthier and Vasta, 1993;La Peyre et al, 1993). The availability of culturing methodologies for Perkinsus spp.…”