Evaluation of the effects of controlled autolysis on the immunodetection of PrPSc by immunoblotting and immunohistochemistry from natural cases of scrapie and bse

Abstract

Seventeen clinically suspect scrapie sheep, and twelve suspected bse -affected cattle were confirmed using routine histopathological examination by the detection of characteristic spongiform change in the medulla brain region taken at the level of the obex. Three sheep and four cows acquired as controls showed no spongiform change. Five aliquots of brain tissue from each of four brain regions were taken (cerebellum, medulla, frontal cerebral cortex and occipital cerebral cortex) from each of the 36 animals. One aliquot was frozen at −70°C, the others were subjected to one of four autolysis regimes at 3 or 7 days at 25°C or 37°C. All samples were tested by Western immunoblotting for detection of PrPSc using the Prionics – Check test (Prionics AG, Zurich, Switzerland). Further samples of medulla from 15 suspect scrapie cases, 10 healthy sheep, 13 suspect bse cows and 5 healthy cows, were taken adjacent to the obex, and subjected to autolysis at 37°C for 6, 12, 24 and 48 hours before being fixed in 10 per cent formal saline and subsequently examined by a routine immunohistochemical technique for detection of PrPSc protein. The abnormal protein could not be detected in any of the control animals by either technique. PrPSc could be detected by Western immunoblotting in at least one brain area from all the positive animals after autolysis for 7 days at 37°C. The protein could be detected by immunohistochemistry in all cases which were positive by histopathological examination using all autolysis conditions. From the results of this study it is concluded that autolysis does not significantly compromise the diagnosis of scrapie or bse by either of these diagnostic methods.