Basic studies on prevention of experimental salmonellosis (4). Purification of S. enteritidis SPA (author's transl)]

The protective activity against infection (SPA activity) of the aseptic filtrate from cultures of S. enteritidis2547, a high SPA-producing strain, in TSA medium reached a plateau at 4 days of cultivation and remained at essentially the same level over the ensuing 30 days. As progressive bacterial autolysis occurred in the culture on incubation, however, it was decided to use culture fluid harvested after 4 days of incubation at 37°C and sterilized by filtration, for purification of SPA.The filtrate of culture fluid thus obtained contained SPA activity which was stable between PH 5.0 and 9.0 and resistant to heating at 100°C for 30 minutes.One hundred liters of the aseptic culture fluid filtrate was concentrated to one-hundredth of the original volume and purified by TEAE-Cellulose and DEAE-Sephadex A-50 column chromatographies and then by gel filtration on Sepharose 2B.The final purified preparation proved to form single precipitation line by Ouchterlony immunodiffusion with a mouse immune serum to a crude SPA preparation. It also showed a single spot on high voltage paper electrophoresis. Qualitative analyses by color reactions disclosed the prepation to be a polysaccharide free from lipids, proteins and nucleic acids.