TWENTY-FOURTH ANNUAL EASTERN FISH HEALTH WORKSHOP Royal Pavilion Resort, Atlantic Beach, NC MARCH 9-11, 1999 An Inducible Protease In Flavobacterium columnare: A Possible Role In Virulence? Joel A. Bader1, Somak Vinitnanthrat2, and Phillip H. Klesius1 1United States Department of Agriculture, Agricultural Research Service, Fish Diseases and Parasites Research Laboratory,PO Box 0952, Auburn, AL 36831; 2The Aquatic Animal Health Division of ALPHARMA, Bellevue,WA 98005 Flavobacterium columnare, which causes columnaris disease, is the most important bacterial pathogen in warm water fish culture throughout the world. An effective vaccine to F. columnare is desirable and would help reduce the economic loss caused by this bacterium. An important step in production of such a vaccine is the identification and characterization of virulence factors. Earlier studies have identified bacterial proteases as possible virulence factors. In preliminary studies we have identified and studied one specific protease, the 58 kd protein that is dominant and immunogenic to channel catfish. This protease is always associated with virulence and has specific proteolytic activity. The protein is inactivated in standard formalin-killed bacterin preparations and is not thermodynamically stable. The proteolytic activity was inactivated at 65°C after 15 min. Western blots using F. columnare infected channel catfish serum and whole-cell bacterial lysates heat inactivated for 15, 30, and 45 min at 65°C showed that catfish also only produce antibodies to the protease up to 30 min at 65°C. 45 min at 65°C is the typical temperature for heat kill bacterins and therefore this data suggests that those conditions are inappropriate for bacterin production for catfish. Further, the protease is differentially induced by the bacteria under various environmental conditions. The conditions which stimulated the highest protease production were, incubation temperature of 30°C, pH of 8.5, and high supplemental protein, 2.0 g/L (tryptone). The protease is produced best at the stationary phase of bacterial growth in liquid medium under aerobic incubation conditions and could be observed in cultures grown at up to 37°C. The inducible nature of this protease may explain why bacterial challenges often fail for columnaris and lends support to the hypothesis that the outbreak of columnaris disease is strongly influenced by external environmental factors. These preliminary results show that the 58 kDa protease should be studied further and may be a good candidate for future subunit vaccine development. Studies are currently underway to determine the molecular biology and immunoprotective value of this protease. Return to 24th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page