DISCOVERY OF A PARTICLE UNIQUE TO BRAIN AND SPLEEN SUBCELLULAR-FRACTIONS FROM SCRAPIE-INFECTED MICE

Fresh mouse scrapie brain and frozen mouse scrapie spleen were homogenized, sized by ultrafiltration to a fraction containing particles of 30 nm to 200 nm and separated by zonal centrifugation in sucrose gradients. In both the brain and spleen, the maximum titre of scrapie infectivity banded between densities of 1.125 to 1.200 g/ml; but in spleen a second fraction of scrapie (infectivity was observed between 1.200 to 1.250 g/ml. A unique 30 to 60 nm particle was found in mouse spleen gradient fractions with high scrapie (infectivity titres. This particle was not observed in similar fractions isolated from normal mouse spleen or brain and was rarely found in subcellular fractions of low titre from scrapie-infected tissues. The particle was first observed by negative staining and then confirmed in thin sections after en bloc staining with ruthenium red and potassium permanganate. The previously observed 'smearing' of scrapie (infectivity over broad ranges in sucrose density gradients may now be explained since these particles were often found as aggregates or chains of particles. The significance of these particles in scrapie infectivity remains uncertain at this time, but absence of these structures in normal tissue fractions may provide a promising new morphological approach to the purification of the infective scrapie agent.