|
|
|
Abstracts
Workgroup: Frontiers in Disease Research
Interactions of Crassostrea virginica hemocytes with the putative etiological agent of juvenile oyster disease (JOD).
Principal Investigator(s):
Boettcher, Katherine, Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, ME, boettche@maine.edu
Funding Period: 10-01-01 to 9-30-03
The goal of this research is to understand Crassostrea virginica immunocompetence against the putative agent of juvenile oyster disease. The bacterium, Roseimarina crassostreae (gen. nov., sp. nov.; Boettcher in review IJSEM), colonizes the external body tissues of juvenile oysters. Once established, the bacteria induce signs such as conchiolin deposition, emaciation, and (shortly thereafter) mortality rates that may exceed 90% of annual production. As invertebrates, oysters rely primarily on hemocyte-mediated immunity. Thus, the focus of this project is to determine the contribution of host, pathogen, and environmental factors to the process of hemocyte-mediated killing of the JOD-bacterium. This work is still in progress, and a brief summary of results to date follows:
We are developing a 96-well microplate assay (using guidelines from a previously used protocol) to evaluate the ability of hemocytes to kill target bacteria. Briefly, a tetrazolium dye compound is used as an indicator of bacterial viability after incubations of bacteria with hemocytes extracted from oysters. Respiring cells reduce the dye to produce a formazan product that has a maximal absorbance at 490nm. The intensity of absorbance is determined for suspensions of bacteria alone, hemocytes alone, and bacteria in the presence of hemocytes. A killing index (KI; the percentage of bacteria killed) is then calculated from these values to yield a quantitative measure of cellular immunocompetence.
In experiments using Vibrio parahaemolyticus as the target bacterium (an organism known to be sensitive to hemocyte killing), the following parameters have been optimized: (i) hemocyte collection and washing conditions, (ii) elimination of non-target cells in hemolymph samples, (iii) concentrations of bacterial cells and hemocytes, (ii) bacteria:hemocyte ratios, (iii) killing time and temperature, (iv) growout time and temperature, (v) formazan product development time and temperature.
Preliminary results indicate that the JOD-associated Roseobacter is less susceptible than V. parahaemolyticus to killing by oyster hemocytes.
Table 1: Susceptibility of V. parahaemolyticus and the JOD-associated Roseobacter to hemocyte-mediated killing. KI indicates relative survival of bacterial cells (mean of four replicate wells).
V.Parahemolyticus:
Roseobacter:
Initial cell conc
KI
Initial cell conc
KI
1x107
10.17
1x107
10.87
4x106
47.79
4x106
22.08
2x106
65.98
2x106
32.77
1x106
95.96
1x106
39.80
We are also evaluating and comparing this dye-reduction assay with a plate count assay for optimal quantification and reproducibility of trials. The best method will be employed for (i) investigating the effects of temperature, salinity, oyster age and size, and bacterial strain differences on killing of Roseimarina by C. virginica hemocytes, (ii) comparing the anti-Roseimarina activities of hemocytes taken from other species of bivalves with that of C. virginica, and (ii) directly visualizing the bacterial-hemocyte interactions via epifluorescence and electron microscopy.
IMPACTS and/or BENEFITS:
This project is not yet completed. However, a basic understanding of host/pathogen interactions is expected to lead to improved grow-out methods by oyster growers. Specifically, we will have identified host and environmental parameters that result in optimal killing of the JOD-bacteria by oyster hemocytes.
PROJECT PUBLICATIONS:
None to date- project still in progress.
![[Maryland Sea Grant]](../mdsg_logo.gif){kind=logo}
![[NOAA]](../noaa_logo.gif){kind=logo}
![[Virginia Sea Grant]](../vasg_logo.gif){kind=logo}