TWENTY-FIFTH ANNUAL EASTERN FISH HEALTH WORKSHOP MARCH 10-13, 2000   Advances in Fish Lymphocyte In Vitro Assays     Rachael L. Roper1, J. Drennan1, R. Beecroft3, D. Machander1, W. Kay1,2, and  J. Thornton1   1Microtek International, Saanichton, British Columbia, 2 University of Victoria and 3Immuno-Precise Antibodies, Victoria, British Columbia, Canada     Our laboratory has been exploring immune responses in fish, particularly with regard to in vitro analysis.  Piscine lymphocytes can be isolated by density centrifugation from peripheral blood, spleen, and the anterior portion of the head kidney.  We have optimized in vitro culture systems using classical  B and T cell mitogens.  For example, there has been much debate regarding the source of serum used for piscine culture; autologous, pooled fish sera or bovine sources.   We have found that bovine serum can support both B and T lymphocyte proliferative responses very well, however it is crucial to pre-screen lots intended for use with fish lymphocytes.  Some lots of bovine serum provide the required growth factors while other batches do not, possibly explaining the contradictory literature.   We have also developed the analysis of specific in vitro immune responses to pathogenic bacteria (Aeromonas salmonicida, Piscirickettsia salmonis) and viral antigens (Infectious Hematopoetic Necrosis Virus, Infectious Pancreatic Necrosis Virus), as well as allo-specific antigens.   We have used these assays to screen vaccine candidates, to evaluate antigens for polyclonal and antigen specific stimulation in primed fish, and also to identify what purified antigens serve as targets of the immune response.  We have been employing the assays to explore strategies for enhancing immune responses and to study the effects of antigen dose, solubility of immunogen, adjuvants and immune enhancing peptides.  In addition, we have developed two monoclonal antibodies, 2C7 and 3D1, that recognize trout and salmon immunoglobulin respectively.  These antibodies are functional in ELISA, FACS, and Western blotting.  These newly developed tools allow us to compare humoral and cellular responses of fish.      Return to 25th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page