Virulence Factors Associated with the Causative Agent of Coldwater Disease Flavobacter psychrophila
IAAAM 1999
Jason W. Heffernan; Kimberly Bynum; Anita Afzali; Larisa Ford; Katherine Byrne
Department of Animal Sciences, Washington State University
Pullman, WA, USA


Flavobacter psychrophila strains were isolated from healthy and diseased fish, as well as from influent and effluent water from hatcheries around the Pacific Northwest. Isolates were separated initially by morphology then biochemically characterized and compared using attributes previously seen in Coldwater isolates. The prominent phenotypic qualifications that separate these bacteria are its circular, raised, glossy bright yellow colonies, with some strains exhibiting colonies with spreading layers of uneven diameter. Biochemical tests (Indole, Catalase, Dextrose, Sorbitol, Esculin, Lactose, Nitrate, or Mannitol) were used in conjunction with growth conditions and Gram staining to further characterize the bacteria as Flavobacter psychrophila. Isolated strains were then grown up in TYES media and then tested for virulence factors at 4, 10, 14, and 18 days, for the presence of Hemolysin, Protease, S-Layer proteins, and endotoxin. The production of virulence factors paralleled log phases of growth. Hemolysin production was detected by measuring bovine red blood cell hemoglobin release after exposure to extracellular products from Flavobacter psychrophila. Presence of proteases was examined using Azocasein as a substrate. S-Layer proteins were detected following SDS polyacrylamide gel electrophoresis after cell wall lysis and pH adjustment. Bacterial endotoxin lipopolysaccharide presence was detected using a standard curve after disruption of the bacteria's LPS constituents.

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Jason W. Heffernan
Department of Animal Sciences, Washington State University
Pullman, WA, USA

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