An immunohistochemical approach to define mechanisms of scrapie pathogenesis

Transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases caused by a variant form of a normal cellular protein (PrPc) that is conformationally altered into a disease-causing form (PrPd). TSEs include scrapie in sheep and goats as well as chronic wasting disease (CWD) in deer, elk, and moose. TSEs pose a significant challenge to study due to their similarity to the normal cellular protein as well as the lack of a typical immune response.;In this study we were the first laboratory to examine the deposition patterns of scrapie and CWD in early infection. First, a novel method of antigen retrieval was developed to be used in prion immunohistochemistry. This antigen retrieval technique allowed staining of sheep immune cells as well as PrP sc within lymph nodes and increased the sensitivity of immunohistochemical techniques used in prion detection. This new technique will enable the use of many monoclonal antibodies with consistent and reliable results and allow the detection of small amounts of PrPsc in lymph nodes of sheep infected with scrapie.;In the next series of experiments, the kinetics of PrPsc deposition in lymph nodes at varying timepoints was studied. PrPsc in the lymph nodes of sheep was observed as early as 15 minutes after injecting the infectious agent near the lymph node in both genetically susceptible sheep and genetically resistant sheep. Accumulation of the scrapie agent was observed for approximately a week before it disappeared from the lymph nodes of resistant animals. All lymph nodes of susceptible animals continued to show accumulation, replication and retention of the infectious agent.;In the final set of experiments the deposition of CWD in lymph nodes of muntjac deer that had been infected with CWD at varying timepoints with elk-derived and white-tailed deer-derived strains of CWD was examined. Deposition of the infectious agent began as early as 24 days post-inoculation and continued throughout the course of infection. The sensitivity of immunohistochemistry and Western Blot techniques for detecting PrPCWD in tissue of infected animals was compared and the two techniques yielded comparable detection thresholds except in very early CWD infection. In early infection immunohistochemistry techniques were superior in detecting the presence of PrPCWD to Western Blot analysis.;Overall, the work presented here has provided novel information regarding the activity and behavior of prions during the acute stages of infection. By tracking the deposition of PrPd when it initially enters the lymph node and germinal centers of infected animals, it may be possible to identify immune cells involved in the pathogenesis of the disease in the future. Furthermore, PrPd infection remains constant throughout the course of disease in genetically susceptible animals and defines a timeline of clearance in genetically resistant animals. This information is vital to gaining a better understanding of an infectious agent that still remains an enigma in many ways, and may someday lead to possible treatment and testing to diagnose prion disease prior to death.