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The gene for the protective antigen of E. rhusiopathiae will be inserted into a eukaryotic vector both for the production of a DNA vaccine and for large scale production of the recombinant protein (in vitro). The DNA vaccine will be tested in cetacean cell lines for its ability to transfect the cells. The recombinant protein will be used to improve the existing ELISA for determining the antibody titers in cetacean serum samples to E. rhusiopathiae.

The work described in this report is based on discussion with Dr. Linda Chrisey following the catastrophic loss of reagents and samples as a consequence of the effects of Hurricane Katrina. The work is a full departure from the previously approved and funded research, which for both practical reasons (related to loss of reagents as a result of the hurricane), and for programmatic reasons (reflecting changes in personnel and reduced interest in the previously approved research aimed at development and evaluation of a DNA vaccine for E. rhusiopathiae). The research represents the most feasible, as well as the most useful, expenditure of funds and research effort, since it capitalizes on research techniques and products developed in previous years of funding. Objective 1: Use of hybridoma tissue culture batch reactors to produce monoclonal antibodies from existing hybridomas specific for Tursiops truncatus immunoglobulin isotypes IgG (combined reactivity for IgGl and IgG2) and IgA in quantities adequate to conduct quantitative assays of serum samples of interest. Objective 2: Purification of T. truncatus IgGl and IgG2 using Protein G and thiophilic affinity columns Objective 3: Purification of T. truncatus IgM using Sephacryl S-300HR Objective 4: Production of monoclonal antibodies specific for T. truncatus gamma 1, gamma2, and mu heavy chains Objective 5: Production of polyclonal antiserum against T. truncatus IgA, IgM, and IgG (combined) Objective 6: Statistical analysis of samples assayed by this laboratory.