TWENTY-FIFTH ANNUAL EASTERN FISH HEALTH WORKSHOP MARCH 10-13, 2000   Immunogenicity, Stability And Optimal Production Conditions For A 60 Kilodalton Protease Of Flavobacterium columnare     Joel A. Bader   U.S. Department of Agriculture,  Agriculture Research Service, Aquatic Animal Health Research Laboratory, Auburn, AL 36831-0952                 A predominant Flavobacterium columnare protein was studied in the pathogenic AAHRL 1 strain.  The immunogenicity, thermal and pH stability and optimal production conditions were determined for this protein, as was specific cellular activity.  The apparent molecular weights for whole-cell lysate proteins were described after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D densitometric analysis.  Thirty four proteins were observed from 200 to 6.7 kda.  Using an arbitrary 0.1 optical density cut-off, 10 dominant proteins were observed.  These proteins define three protein complexes; Complex 1 at 165, complex II at 134 to 115, and complex III, at 100 to 47.6.  Complex III the immunodominant complex, contained the four immunodominant proteins, 100, 88, 66, and 60.  The 60 kda protein is the most dominant of all the immunodominant proteins and is produced in the greatest amount by the bacterium in vitro.  The 60 kda protein was immunogenic in western blots using sera from channel catfish, Ictalurus punctatus, naturally infected with columnaris disease.  The immunogenicity of this protein was abolished by heating the whole-cell lysate for 30 min at 65°C.  The 60 kda protein exhibited gelatinase and caseinase activities in substrate imbedded SDS-PAGE gels in sonicated lystates and extracellular fluid.  This protease was heat labile after 30 min at 65°C and pH labile above 8.5.  Using densitometric analysis protease production was optimized.  The following conditions were optimal for in vitro production of the 60 kda protease: incubation in a modified Hsu-Shotts media (4 g/L typtone) at 28°C for 24 h, at pH 8.0.     Return to 25th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page