TWENTY-SECOND ANNUAL EASTERN FISH HEALTH WORKSHOP ROYAL PAVILLION RESORT, ATLANTIC BEACH, NC MARCH 18 - 20, 1997 In Vivo Activation of Nonspecific Cytotoxic Cells with a Phosphatase Inhibitor: Correlation of the Expression of Signalling Phosphoproteins with Innate Resistance to a Bacterial Pathogen. Donald L. Evans, John H. Leary III, Emmett B. Shotts and Liliana Jaso-Friedmann. Department of Medical Microbiology, University of Georgia, Athens, GA. USA In vivo treatment of catfish with the phosphatase inhibitor sodium orthovanadate (vanadate) produced resistance to challenge with the catfish enteric pathogen Edwardsiella ictalurus. The mechanism of innate resistance to fulminant infection was closely associated with nonspecific cytotoxic cell (NCC) activity and with the appearance of "signalling phosphoproteins" in activated NCC. To determine this relationship, catfish were treated (48 h before infection or 0 h) with 25 uM vanadate. Fish were infected by immersion or IP injection. In the absence of vanadate, both infection protocols produced fulminant disease by 6-10 days. Vanadate treatment was associated with 17-100% survival of infected fish. Fish which had vanadate-induced resistance to primary infection were "immune" to secondary challenge with an LD100 dose of Edwardsiella (w/o secondary vanadate treatment). To determine the mechanism(s) of the altered innate resistance, catfish were injected (IP) with Edwardsiella and simultaneously treated with 25 uM vanadate. At 0, 24, 48, 72 and 96 hours post-infection (PI) and vanadate treatment, cytotoxicity and phosphoserine and phosphotyrosine levels were determined. Both cytotoxicity and phosphoprotein levels increased at 48-96 hours PI. This occurred only in vanadate treated fish. Both serine and tyrosine residues on the same NCC proteins were phosphorylated. These were a dimer (20-30 and 50-60 kDa) tentatively identified as a calcium transporting ATPase and 60-70 kDa unidentified protein. Increased phosphoprotein accumulation subsided to background levels by 96 hours P-I and vanadate treatment. These data demonstrated a direct linkage between phosphatase regulation of innate resistance, NCC cytotoxicity and signalling phosphoprotein levels. Return to 22nd Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page