Cooperative Fish and Wildlife Research Units Program: Wisconsin Wildlife
Education, Research and Technical Assistance for Managing Our Natural Resources

NSF has funded a portion of our larger-scale proposal to focus our research efforts on adapting, validating and applying a novel, biochemical method to detect the pathogenic prion protein (PrPTSE) associated with chronic wasting disease in environmental samples. The pathogenic prion protein co-purifies with prion infectivity and constitutes the major, if not sole, component of the prion. PrPTSE refers to pathogenic prion protein in general, and PrPCWD to PrPTSE specifically associated with chronic wasting disease (CWD). Our goal will be to detect PrPCWD in environmental samples using the protein misfolding cyclical amplification (PMCA) technique. PMCA ranks among the most important recent developments in prion measurement. PMCA exploits the ability of PrPTSE to seed the conversion of the normal, cellular form of the prion protein (PrPC) to the misfolded isoform in a manner conceptually similar to the polymerase chain reaction (PCR). The amount of PrPTSE is increased by successive cycles of sonication (to disrupt PrPTSE aggregates) and incubation with PrPC from brain homogenate as a substrate. Sensitivity can be increased by replenishing the substrate after a round of PMCA, this method has been recently automated allowing PMCA to be applied to large numbers of samples simultaneously. PMCA has been successfully employed to detect prions in blood, urine and tissues of presymptomatic animals. The sensitivity depends on the number of PMCA rounds; successive rounds of PMCA dramatically increase the level of amplification. The lowest detection limit reported to date for infected brain homogenate is ~26 molecules or 1.3 ag, a thousand-fold more sensitive than the next most sensitive animal bioassay of 5.3 fg. The exquisite sensitivity of PMCA and the recent automation of the method make it a particularly attractive candidate to adapt for application to environmental matrices.