1. Presence of “Mad Cow Disease” (Bovine Spongiform Encephalopathy) and Natural Scrapie in Ovine Transgenic Mice (TgOvPrP4) Christina Hill Department of Biological Sciences, York College of Pennsylvania Project Summary Bovine Spongiform Encephalopathy (BSE) and Natural Scrapie are prion diseases that result from the mis-folding of proteins in the brain. One mis-folded protein can cause other proteins to mis-fold creating florid plaques that can affect motor function and decrease lifespan. Prion diseases can be transferred to animal species that are not naturally susceptible to these diseases through gene transfer. Research shows that brain plaque isolates from animals infected with prion diseases can be collected and inserted into the brains of transgenic mice. In the proposed experiment, 10 (OvTgPrP4) transgenic mice will be injected with normal saline, 10 with BSE isolates and 10 with Natural Scrapie isolates. Inoculated transgenic mice are observed for changes in motor function each week for course of their lifespan. After expiration, the mice can be dissected and their brain removed. The removed brains would be paraffin-embedded for microtome sectioning. These sections will be stained using Immunohistochemistry to look for the presence of florid plaques associated with the respective disease. Isolates from each mouse brain will also be tested for infection using a Sandwich ELISA test. Infection in all mice inoculated with the respective diseases should be found, as well as a decrease in motor dysfunction and lifespan. Effective transmission of prion diseases to species without natural susceptibility can allow for more effective medical models to be developed and increased knowledge of these diseases. Introduction o Protein folding directly affects the structure and function of proteins. o During the protein folding process, proteins can misfold. These misfolded proteins are called prions. o Prions can not be corrected or refolded correctly; they are stuck in their misfolded structure. One misfolded protein, or prion, can coax other proteins to also misfold causing masses of prions to collect. Plaques form from these masses of prions which have collected in the brain and spinal cord. o Diseases such as Bovine Spongiform Encephalopathy (BSE) or “Mad Cow Disease” are prion based. o The plaques that form in disease such as BSE affect the central nervous system of the animal infected causing motor dysfunctions and decreased life span. o Some prion based diseases such as Natural Scrapie which affects sheep dramatically decrease the life span of infected individuals. Review of Literature Transmissible spongiform encephalopathies affect both humans and animals causing progressive neurodegeneration that is fatal (Sejvar et al, 2008). These diseases are caused by prion proteins, or misfolded proteins that interact with normal proteins and cause them to mis-fold (Sejvar et al, 2008). Little is known about the mechanism responsible for the infectivity of the prion (Sejvar et al, 2008). Ovine transgenic mice (TgOvPrP4) have been successfully inoculated with both BSE and Natural Scrapie Isolates (Baron et al, 2008). Differentiation of agents in BSE and Natural Scrapie have been poorly documented in transgenic mice (Cordier et al, 2006). Objectives To successfully inoculate ovine transgenic mice with isolates To record changes in motor function in infected mice To successfully remove brain and spinal cord for further examination To successfully find florid plaques through Immunohistochemistry staining To see infection in mice through ELISA testing Research Design & Methods Expected Results Prion infection in 20 mice inoculated with isolates Slight decrease in motor function as a result of natural aging in 10 saline injected mice Decreased motor function and lifespan in 20 mice inoculated with isolates Quick decrease in motor function and lifespan in 10 mice inoculated with Natural Scrapie isolates Successful use of a small medical model to further investigate the mechanism of prion diseases Literature Cited Baron, T., Bencsik, A., Vulin, j., Biacabe, A-G., Morignat, E., Verchere, J., Betemps, D. 2008. A C-terminal protease-resistant prion fragment distinguished ovine “CH1641-Like” scrapie from bovine classical and L -type BSE in ovine transgenic mice. PLoS Pathogens. 4:1-10. Sejvar, J., Schonberger, L., Belay, E. 2008. Transmissible spongiform encephalopathies. Journal of American Veterinary Medical Association 233:1705-1712. Cordier, C., Benscik, A., Phillippe, S., Betempes, D., Ronzon, F., Calavas, D., Crozet, C., Baron, T. 2006. Transmission and characterization of bovine spongiform encephalopathy sources in two ovine transgenic mouse lines (TgOvPrP4 and TgOvPrP59). Journal of General Virology 87:3763-3771. Acknowledgements I would like to thank Dr. Jeffery Thompson for mentoring this project. I would also like to thank Dr. Deborah Ricker for her support. Introduction Cont. o Prion diseases such as Mad Cow Disease can be transferred to transgenic animals outside of the naturally infected species through injection. Transmitting prion diseases to smaller animals allows for smaller medical models to be used, allowing for easier characterization and testing of the disease. Test Motor Function of mice with Accelerating Rota-rod and Balance Beam Test Dissect brains and spinal cord from diseased mice Obtain 30 TgOvPrP4 Ovine Transgenic Mice Inject 10 mice with saline Inject 10 mice with BSE isolates Inject 10 mice with Natural Scrapie isolates Embed Brains in Paraffin Wax and Complete Microtome sectioning Stain Sections using Immunohistochemistry Complete ELISA on mice brains Observe Differences in Florid Plaques on Stained slides and Infection in ELISA test Between 20 infected mice

2. Presence of “Mad Cow” and Natural Scrapie in Ovine Transgenic Mice (TgOvPrP4) Christina Hill Department of Biological Sciences, York College of Pennsylvania Project Summary Transmissble spongiform encephalies Introduction Review of Literature Objectives To successfully inoculate ovine transgenic mice with isolates Research Design & Methods Expected Results

3. Presence of “Mad Cow” and Natural Scrapie in Ovine Transgenic Mice (TgOvPrP4) Christina Hill Department of Biological Sciences, York College of Pennsylvania Project Summary Transmissble spongiform encephalies Introduction Review of Literature Objectives To successfully inoculate ovine transgenic mice with isolates Research Design & Methods Expected Results