Effects of plasma from bivalve mollusk species on the in vitro proliferation of the protistan parasite Perkinsus marinus

Abstract

The in vitro culture of the Eastern oyster parasite Perkinsus marinus has provided a unique opportunity to examine its susceptibility to putative recognition and effector defense mechanisms operative in refractory bivalve species. In this study, we report the effect of supplementing the culture medium with plasma from: (1) uninfected to heavily infected Eastern oysters; (2) oyster species considered to be disease-resistant; and (3) bivalve mollusk species that are naturally exposed to the parasite but show no signs of disease. We also examined in vitro the interaction between hemocytes from Crassostrea virginica and C. gigas and P. marinus trophozoites. Our results revealed a significant decrease (32%) in proliferation of P. marinus in the presence of plasma from heavily infected C. virginica oysters. The inhibitory effects were less pronounced with plasma from moderately infected and uninfected oysters. In contrast, plasma from C. rivularis and C. gigas enhanced P. marinus proliferation. Proliferation was significantly reduced in media supplemented with plasma from Mytilus edulis, Mercenaria mercenaria, and Anadara ovalis. The highest inhibitory activity was apparent in M. edulis, for which 5% plasma-supplemented medium reduced growth by 35% relative to the controls. M. edulis active component(s) was heat-stable, yet pronase-sensitive. The significantly higher uptake of live P. marinus trophozoites by hemocytes from C. virginica, relative to those from C. gigas, suggests a certain level of specificity in the recognition/endocytosis of the parasite by its natural bivalve host species.

Perkinsosis in molluscs: A review

“…The authors stated that anti-P. marinus activity of mussel sera may depend on cytotoxic molecules other than, or in addition to, lysozyme or antimicrobial peptides. Gauthier and Vasta (2002) also showed that plasma from bivalves that are less susceptible to P. marinus infections, Mercenaria mercenaria, Anadara ovalis and, especially, M. edulis, inhibited P. marinus in vitro proliferation more effectively than that of C. virginica.…”

Supplementation of Perkinsus marinus Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity

“…Compared with growth in unsupplemented ODRP-3, P. marinus cells grown in the presence of plasma supplements from Crassostrea gigas, Crassostrea ariakensis, or infected C. virginica oysters show reduced in vitro proliferation (20). Uninfected C. virginica plasma supplementation, however, results in only minimal inhibition of proliferation (20,29). Oyster tissue homogenatesupplemented medium produces marked changes in cell proliferation, morphology, and differentiation, including enlargement of trophozoites and induction of tomont stages, which are rarely seen in unsupplemented ODRP-3 medium but are commonly observed during infection (29).…”

“…In an effort to more closely simulate, in vitro, the milieu to which P. marinus is exposed in vivo, oyster tissue homogenate and plasma from P. marinus-susceptible and -tolerant oyster species have been employed as culture supplements (20,21,29). Compared with growth in unsupplemented ODRP-3, P. marinus cells grown in the presence of plasma supplements from Crassostrea gigas, Crassostrea ariakensis, or infected C. virginica oysters show reduced in vitro proliferation (20). Uninfected C. virginica plasma supplementation, however, results in only minimal inhibition of proliferation (20,29).…”

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

“…Gauthier et al (1995) also found that adding plasma to the commercial medium DME/Ham's F12 with low FBS concentrations (0% and 0.1%) enhanced the parasite proliferation as measured by changes of optical density. Adding plasma to DME/Ham's F12 with higher FBS concentrations (1-20%), however, greatly reduced P. marinus proliferation (Gauthier and Vasta 2002;Gauthier et al 1995) compared with medium with FBS. Results from these past studies on the full effects of plasma are however difficult to interpret because the concentrations of basal medium constituents were not held constant as the concentrations of plasma or FBS were tested.…”

“…For example, Giardia duodenalis, an intestinal protistan of vertebrates is cultured in medium supplemented with bile (Binz et al 1992), and Trypanoplasma borreli, a pathogenic flagellate from the blood of cyprinid fishes, only grows in media containing carp serum (Steinhagen et al 2000). In the case of the genus Perkinsus, the effect of host components (i.e., plasma and tissue lysates) on in vitro proliferation has only been reported for P. marinus (Gauthier and Vasta 2002;MacIntyre et al 2003;Earnhart et al 2004). In contrast to our results with P. mediterraneus, MacIntyre et al (2003 ) found that P. marinus cultured in the chemically defined medium JL-ODRP-3 (La Peyre and Faisal 1997) supplemented with eastern oyster (C. virginica) tissue lysates had lower densities than cultures in unsupplemented JL-ODRP-3, and densities were dependent on tissue lysate concentrations.…”