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Abstracts
Workgroup: Frontiers in Disease Research
Induction of potential pathological states in Perkinsus marinus by exposure to oyster tissue extracts. Modulation of cell morphology and protease/antigen production.
Principal Investigator(s):
Dr. Stephen L. Kaattari, Virginia Institute of Marine Science, kaattari@vims.edu
Co-Investigator(s):
Dr. Kimberly S. Reece, Virginia Institute of Marine Science
Dr. Eugene M. Burreson, Virginia Institute of Marine Science
Dr. Eugene M. Burreson, Virginia Institute of Marine Science
Funding Period: 10/01/01-09/30/03
(Project is in progress)
Objective I: Determine the nature of the induced P. marinus molecules, and develop specific serological and molecular probes for these molecules.
Status: This objective builds upon the results from previous ODRP grant research regarding the alteration in cellular differentiation and expression of potential virulence factors by inclusion of oyster products in the cell culture medium. The isolation of differentially expressed genes by subtractive hybridization using a P. marinus cell line grown in the presence or absence of oyster products is proceeding. We are currently in the process of screening the cDNA libraries, and it is expected that several differentially expressed genes will be identified. In conjunction with the molecular subtractive techniques, subtractive serological probes are also being developed. Briefly, mice are immunized with the extracellular products (ECP) of P. marinus grown in unsupplemented medium, the B-lymphocytes which are reactive to constituents of that ECP are then deleted using cyclophosphamide, and the mouse is re-immunized with ECP from cells previously grown in supplemented medium. This technique is expected to skew the immune response toward those antigens which are only present in the latter immunogen. Initial attempts at the technique were unsuccessful, due to a generally poor immune response of the mice toward the ECP. Effort was directed at enhancement of the immunogenicity of the ECP preparations, and it has now been determined that ECP not only contains intrinsic factors which are immunosuppressive in the mouse, but that an immunosuppressive media component was being co-concentrated with the ECP proteins during processing. Development of a purification and concentration technique has subsequently allowed production of a panel of IgG isotype monoclonal antibodies directed against components of unsupplemented ECP. Concurrently, the subtraction protocol has been refined using a highly immunogenic protein mixture, C. virginica hemolymph. These refinements are now allowing the use of subtractive immunization for detection of upregulated proteins.
Objective II: Examine various C. virginica stocks to determine whether there is an intra-species correlation between relative resistance and the ability to induce protease expression. Status: The original proposal listed several groups of oysters which were to be supplied by the VIMS oyster hatchery. Unfortunately, there were high mortalities in several of the groups, and the remaining groups have remained very small under quarantine conditions. This has necessitated acquisition of several groups of oysters which have been previously deployed in the field. Since this experiment involves comparison of body burdens after experimental infection, we have elected to proceed with this experiment during late winter, when the naturally occurring infection levels will be at their lowest. At that time, we will gradually acclimate the oysters to warmer water and proceed with this phase of the experiment.
Objective III: Homogenates from distinct oyster tissues will elicit varying degrees of serine protease expression and parasite differentiation. Status: Initial results have shown that there are distinct patterns of cell differentiation in cultures supplemented with homogenates of different organs. There has not, however, been consistent induction of the low molecular weight proteases that are postulated to be virulence factors. We are currently in the process of repeating this experiment, using oysters from a different geographic area. We are also supplementing the cultures at a higher protein level. Previous studies have demonstrated a dose-responsive elicitation of the low molecular weight protease when using whole oyster homogenate and oyster hemolymph. The dose range for any single organ is not known, and the initial experimental dose may have been under the critical limit for induction.
Objective IV: Purify, isolate, and identify oyster molecules that induce low molecular weight protease expression in P. marinus. Status: Oyster homogenate and hemolymph are extremely complex and apparently have a tendency to react with sugar moieties present on typical commercial size exclusion chromatographic beads. Initial attempts to broadly separate the components of oyster products into size classes was frustrated by aggregation of the proteins, and their resultant passage through the column in the void volume. Several different buffers have been used, and it has been found that inclusion of magnesium can reduce the amount of aggregation. We are currently attempting size separation using an acrylamide-based matrix, which should reduce the interaction of the oyster proteins with the column. Additionally, we have optimized a fractionation technique using preparative isoelectric focusing for use in the event that size exclusion becomes untenable, or that we need further resolution of a particular size fraction.
Objective V: Determine the effect of culture supplementation on P. marinus infectivity (reversal of in vitro induced attenuation): Status: We currently have 7 groups of P. marinus cells which are being grown in media supplemented with both high and low concentrations of tissue homogenates from oyster strains and species of varying susceptibility to P. marinus infection. After four weeks of culture, these cells will be used to infect C. virginica oysters, and the degree of infection will be determined by enumeration of total parasite body burden four weeks after infection. In addition, we will at that time assess the induction of the low molecular weight proteases by each of these oyster groups and assess correlation between infectivity and ability to upregulate protease expression.
IMPACTS and/or BENEFITS:
Until all of the results can be assessed at the end of the grant period, it is difficult to project the total impact. We have already succeeded in production of antibody probes to P. marinus ECP, which has been an elusive goal in P. marinus research. In addition, the recognition that P. marinus ECP contains intrinsic immunosuppressive factors in the murine model leads to interesting path of investigation as to the role of specific ECP components in the modulation of oyster defenses.
PROJECT PUBLICATIONS:
Earnhart, C. and Kaattari, K. Development of serological techniques to host-induced proteins of the oyster parasite, Perkinsus marinus. Poster. Presented to the International Symposium on Aquatic Animal Health, 2002, New Orleans, LA.
Earnhart, C. and Kaattari, K. Submitted. The murine humoral response to in vitro generated parasite antigens is critically diminished by the Pluronic F-68 block copolymer, a defined media component.
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