Isolation and characterization of a leukocytolytic factor from Aeromonas salmonicida

Graduation date: 1975The purpose of this study was to find what effect temperature\ud and immunization has on the phagocytic activity of fish leukocytes\ud against certain disease causing bacteria. Also proposed was the\ud isolation and characterization of a leukocytolytic factor produced by\ud one of these bacteria, Aeromonas salmonicida.\ud Investigations revealed that temperature had no significant effect\ud on the phagocytic activity of leukocytes from either immunized or\ud nonimmunized rainbow trout toward Aeromonas salmonicida and\ud Vibrio anquillarum. Leukocytes from rainbow trout immunized with\ud killed A. salmonicida vaccine showed a significantly higher phagocytic\ud activity than those from nonimmunized rainbow trout toward A.\ud salmonicida at 12°C and 23°C, but not at 7°C and 18°C. The phagocytic\ud activity of the leukocytes from rainbow trout immunized with\ud killed V. anguillarum vaccine was significantly higher against V.\ud anguillarum at 70, 120, 180, and 230C.\ud A material characterized as a glycoprotein was isolated from\ud the supernatant of a broth culture of A. salmonicida by combinations\ud of ammonium sulfate and ethanol precipitations followed by DEAE\ud chromatography. A virulent strain of A. salmonicida produced more\ud of this substance than an avirulant strain. This material was demonstrated\ud to be cytolytic to rainbow trout leukocytes, and salmon and\ud steelhead embryo cell cultures. An affinity for cell membranes was\ud indicated by a hemagglutinating activity with sheep erythrocytes.\ud Intravenous injection of the material into rainbow trout caused marked\ud leukopenia followed by leukocytosis with an ultimate return of total\ud white cell counts to normal. Simultaneous intraperitoneal injections\ud of the material with one LD₅₀ of A. salmonicida greatly increased\ud the susceptibility of the host to the organism, indicating that this is\ud a virulence factor.\ud The ratio of protein, as assayed by the Lowry method, to\ud hexose, as assayed by the anthrone method, was consistently between\ud 0.35 and 0.45 with small amounts of amino sugars noted. No lipids\ud were detected by the assay methods used. The material was demonstrated\ud to be antigenic and appeared to have a molecular weight of\ud between 100, 000 and 300, 000 as indicated by ultrafiltration methods.\ud Electron micrographs revealed aggregates of oval or egg-shaped\ud particles measuring approximately 40 nm by 25 to 30 nm per particle. It was determined that the leukocytolytic factor is not the endotoxin\ud produced by this bacterium