Immunomodulatory Effects And Efficacy Of A DNA Vaccine For Aquatic Mycobacteriosis     David J. Pasnik and Stephen A. Smith   Aquatic Medicine Laboratory, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA      DNA vaccine research has demonstrated that plasmid-based vaccines have multiple advantages over killed, attenuated, or subunit vaccines.  DNA vaccines have also been shown to stimulate a strong Th1/cell-mediated immune response.  Because of this, immunogenic vaccines have been made for intracellular pathogens, such as infectious hematopoietic necrosis virus and bacterial kidney disease.  Another intracellular pathogen of fish is Mycobacterium marinum, which causes a chronic progressive disease that leads to systemic infections and ultimately death.  It is an economically important bacterial disease of virtually all species of freshwater and marine fish.  However, an effective vaccine against mycobacteriosis has not been developed.  Because of its advantages and potential to induce a protective Th1 response, a DNA vaccine was chosen as an appropriate construct for aquatic mycobacteriosis.  A vaccine was assembled by cloning the M. marinum 85A gene in the pcDNA 3.1 eukaryotic expression vector (Invitrogen, San Diego, CA).  In the resulting pCMV-85A construct, the 85A gene is expressed under a cytomegalovirus (CMV) promoter after injection into fish tissue.  Hybrid striped bass (Morone saxatilis x M. chrysops), a proven susceptible species, were used as a model to test the vaccine.  Intramuscular or intraperitoneal vaccinations of the construct were administered on days 0 and 14 at doses of 0.01, 0.25 or 0.50 µg, along with two control groups injected with an empty plasmid, pCMV-0, or saline alone.  Every two weeks, blood was drawn from the caudal vessels of the fish to evaluate the humoral and cell-mediated responses by ELISA, lymphoproliferative assay, and cytokine assay.  Then on day 70, the fish were challenged with M. marinum.  Routine histological examination was performed and splenic bacterial counts evaluated to determine the relative degree of susceptibility among the different vaccination groups.