Determination of the effects of temperature on viability, metabolic activity and proliferation of two Perkinsus species, and its significance to understanding seasonal cycles of perkinsosis.

The range of water temperatures in which Perkinsus species can survive and proliferate remains ill-defined, particularly at lower temperatures. The in vitro viability, metabolic activity, and proliferation of 3 isolates each of P. marinus and P. olseni trophozoites at 28 degrees C, and at 15 and 4 degrees C, after transfer from 28 degrees C, were compared. Both species showed declines in metabolic activity and proliferation from 28 degrees C to 15 degrees C. At 4 degrees C, both species had viability after 30 days incubation time (P. marinus 49%, P. olseni 58%), but limited metabolic activity and no proliferation. Perkinsus marinus viability was further compared when transferred directly from 28 degrees C, 18 degrees C and progressively from 18 degrees C (0.5 degrees C/day) to 2, 4 and 6 degrees C and maintained for up to 4 months. Viability was highest under progressive transfer (77% and 54% after 30 and 60 days exposure to test temperatures). The decrease in P. marinus viability at the lower temperatures in vitro only partially explains decreasing parasite infection intensities in eastern oysters in the colder months of the year. Moreover, the significant decrease in parasite infection intensities in late winter and early spring, as temperatures increase, is likely due to an active process of elimination by oyster host defences.

Control of Listeria monocytogenes and Salmonella anatum on the surface of smoked salmon coated with calcium alginate coating containing oyster lysozyme and nisin.

This study investigated the antimicrobial effect of oyster lysozyme with or without nisin added to calcium alginate (CaAlg) coated on the surface of smoked salmon against Listeria monocytogenes and Salmonella anatum. L. monocytogenes or S. anatum inoculated smoked salmon samples (1 g) were dipped into CaAlg with either oyster lysozyme (OysL) or hen egg white lysozyme (HEWL), with or without added nisin (N), then stored at 4 degrees C for 35 d. Our results indicated that the effectiveness of oyster lysozyme or hen egg white lysozyme was enhanced when added to calcium alginate coatings. After 35 d at 4 degrees C the growth of L. monocytogenes and S. anatum was suppressed in the range of 2.2 to 2.8 log CFU/g with CaAlgNOysL or CaAlgNHEWL coatings compared to the control nontreated samples. There was no significant difference between oyster lysozyme and hen egg white lysozyme treatments against L. monocytogenes or S. anatum inoculated on the surface of salmon. Calcium alginate coatings containing lysozyme with nisin or without could be used to reduce the growth of L. monocytogenes and S. anatum on the surface of ready-to-eat smoked salmon at refrigerated temperatures.

A new lysozyme from the eastern oyster (Crassostrea virginica) indicates adaptive evolution of i-type lysozymes.

A new lysozyme (cv-lysozyme 2) with a MALDI molecular mass of 12 984.6 Da was purified from crystalline styles and digestive glands of eastern oysters (Crassostrea virginica) and its cDNA sequenced. Quantitative real time RT-PCR detected cv-lysozyme 2 gene expression primarily in digestive gland tissues, and in situ hybridization located cv-lysozyme 2 gene expression in basophil cells of digestive tubules. Cv-lysozyme 2 showed high amino acid sequence similarity to other bivalve mollusk lysozymes, including cv-lysozyme 1, a lysozyme recently purified from C. virginica plasma. Differences between cv-lysozyme 2 and cv-lysozyme 1 molecular characteristics, enzymatic properties, antibacterial activities, distribution in the oyster body and site of gene expression indicate that the main role of cv-lysozyme 2 is in digestion. While showing that a bivalve mollusk employs different lysozymes for different functions, findings in this study suggest adaptive evolution of i type lysozymes for nutrition.

Cryopreservation of heart cells from the eastern oyster.

Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75 degrees C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at -80 degrees C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells fozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45 degrees C. After thawing, atrial cells showed 53+/-5% of the metabolic activity, 84+/-5% of the number, and 92+/-2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25 degrees C yielded the best results. The thawed ventricular cells showed 83+/-5% of the metabolic activity, 91+/-5% of the number, and 96+/-2% of the viability of nonfrozen cells.

Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica.

At present, establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells of the eastern oyster, Crassostrea virginica, to aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin, poly-D-lysine, and two types of uncoated tissue culture plates (Falcon and Corning). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a tetrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24 h and at 5 d (P > 0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P > 0.0001). At 24 h, poly-D-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon substrate, and collagen I performed most poorly. At 5 d, poly-D-lysine and the uncoated Corning substrate induced significantly higher levels of attachment and spreading than did the uncoated Falcons substrate, and collagen I performed most poorly. From these results, poly-D-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 d) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.

Perkinsus marinus extracellular protease modulates survival of Vibrio vulnificus in Eastern oyster (Crassostrea virginica) hemocytes.

The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues.

In vitro interaction of Perkinsus marinus merozoites with eastern and Pacific oyster hemocytes.

This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites.

Acute osmotic tolerance of cultured cells of the oyster pathogen Perkinsus marinus (Apicomplexa:Perkinsida).

Cultured Perkinsus marinus cells were exposed for 24 hr to salinities of 0, 3, 6, 9, 12 and 22 ppt at temperatures of 1, 5, 10, 15 and 28 degrees C in artificial seawater (ASW) and to the same salinities at 28 degrees C in ASW with the osmotic concentration adjusted with sucrose to the equivalent of 22 ppt. At 28 degrees C mortality increased as salinity decreased below 22 ppt. Mortality was greater than 99% at 0 ppt and greater than 90% at 3 ppt. Mortality was 70% at 6 ppt, 43% at 9 ppt and 20% at 12 ppt. Mortality was low (< 5%) and equal to that at 22 ppt in all treatments where osmotic concentration was maintained with sucrose. Mortality occurred rapidly, within 5 min of exposure to experimental conditions. In the region where mortality was most sensitive to salinity changes (6-12 ppt), lower temperature caused an increase in mortality, but the temperature effect was significant only at 9 ppt.