Purification of Piscirickettsia salmonis and partial characterization of antigens.
Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, an economically significant disease affecting the salmon aquaculture industry. As with other rickettsial pathogens, antigenic analysis of P. salmonis has been limited by the inherent difficulties of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diatrizoate sodium (DMDS) density gradient centrifugation to purify P. salmonis grown in chinook salmonis was consistently concentrated in a visible band within the DMDS density gradient at density of 1.15 to 1.16 g ml(-1). Recovery of purified, viable organisms from DMDS density gradients varied from 0.6 to 3%. Preparations of uninfected CHSE-214 cells, CHSE-214 cells infected with P. salmonis, and gradient-purified P. salmonis were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess the degree of purification and to identify P. salmonis-specific proteins. Although gradient-purified P. salmonis preparations were not completely free of host cell material, 8 bacterial proteins were identified. Polyclonal rabbit antiserum was used in an immunoblot of proteins from purified P. salmonis to identify 3 major and 5 minor antigens. The major antigens of 56, 30 and 20 kDa were potential candidates for experimental vaccines and development of novel diagnostic assays.
School of Fisheries, University of Washington, Seattle 98195, USA.
M N
MN
Barnes
M L
ML
Landolt
D B
DB
Powell
J R
JR
Winton
D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis* / Q000737:chemistry; D017756:Aquaculture; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005393:Fish Diseases / Q000382:microbiology*; D015151:Immunoblotting; D008970:Molecular Weight; D012287:Rickettsiaceae / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D012288:Rickettsiaceae Infections / Q000382:microbiology / Q000662:veterinary*; D012474:Salmon
D000942:Antigens, Bacterial; D001426:Bacterial Proteins