PURIFICATION OF PISCIRICKETTSIA-SALMONIS AND PARTIAL CHARACTERIZATIONOF ANTIGENS

Piscirickettsia salmonis is the etiological agent of salmonid ricketts ial septicemia, an economically significant disease affecting the salm on aquaculture industry. As with other rickettsial pathogens, antigeni c analysis of P. salmonis has been limited by the inherent difficultie s of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diat rizoate sodium (DMDS) density gradient centrifugation to purify P, sal monis grown in chinook salmon embryo (CHSE-214) cells. Plaque assay ti ters and total protein assays confirmed that viable P, salmonis was co nsistently concentrated in a visible band within the DMDS density grad ient at a density of 1.15 to 1.16 g ml(-1). Recovery of purified, viab le organisms from DMDS density gradients varied from 0.6 to 3 %. Prepa rations 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 degr ee of purification and to identify P, salmonis-specific proteins. Alth ough gradient-purified P. salmonis preparations were not completely fr ee of host cell material, 8 bacterial proteins were identified.. Polyc lonal rabbit antiserum was used in an immunoblot of proteins from puri fied P. salmonis to identify 3 major and 5 minor antigens. The major a ntigens of 56, 30 and 20 kDa were potential candidates for experimenta l vaccines and development of novel diagnostic assays.