This application is in response to the PAR-03-056 (NIA Pilot Research Grant Program) and focuses on the topic number four, Transmissible Spongiform Encephalopathies. Transmissible spongiform encephalopathies, also called prion diseases, are a group of fatal infectious neurodegenerative disorders affecting humans and animals. Although rare diseases, the recent outbreak of Bovine Spongiform Encephalopathy and Chronic Wasting disease and the transmission of the disease from cattle to humans have risen a great concern about a possible epidemic of Creutzfeldt-Jakob disease. This problem is aggravated by the lack of an early and sensitive diagnosis to identify individuals incubating the disease during the pre-symptomatic phase. The infectious agent (termed prion) is composed exclusively by a misfolded version of a normal protein and does not contain any nucleic acid. According to the prion hypothesis, the disease is transmitted by propagation of the misfolding from the disease associated isoform (termed PrPres) to the normal host protein (termed PrPc), which become converted into the pathological form. We have recently described a procedure to induce the conversion of PrPc into PrPres in vitro starting with minute quantities of brain PrPres. This procedure, named Protein Misfolding Cyclic Amplification (PMCA) mimics the process of prion replication in vivo, but at an accelerated speed resulting in an exponential amplification of the initial amount of PrPres. PMCA has tremendous promise to increase detection of prions in tissues and biological fluids during early stages of the disease and thus may be useful for pre-symptomatic diagnosis of prion disease. The major goal of this project is to take advantage of the PMCA technology to attempt developing a highly sensitive and non-invasive diagnosis of prion diseases in humans and animals. In specific aim 1 we will attempt the pre-symptomatic detection of PrPres in the brain of experimentally infected cattle sacrificed at different times after infection. In specific aim 2 we will attempt detection of PrPres in peripheral tissues of humans and animals. Specific aim 3 proposes the development of the conditions for amplification of PrPres from blood of experimental animals in order to reach reproducible detection of prions in blood. The results generated in this project may provide the basis for the development of a novel highly-sensitive pre-mortem and pre-symptomatic diagnosis of prion disease. Such test will have tremendous applications in public health to minimize the risk of further propagation of prion to humans.