Chronic wasting disease: A model for prion transmission via saliva and urine

Chronic wasting disease (CWD) of cervids is a prion disease distinguished by its high level of transmissibility, wherein bodily fluids and excretions are thought to play an important role. Typical of all prion diseases, CWD is characterized by the forced conversion of the normal prion protein (PrP C) into a misfolded isoform (PrPCWD), which has been shown to accumulate primarily in tissues of the lymphoid and nervous systems, though has also been found in other peripheral tissues including elements of the cardiovascular, musculoskeletal, and urogenital systems.;In this dissertation, two approaches are used to identify infectious CWD prions and PrPCWD in the bodily fluids and tissues of CWD-exposed white-tailed deer: a novel bioassay system using a transgenic mouse line expressing the cervid PrP protein (Tg[CerPrP] mice), and a recently developed prion amplification assay known as serial protein misfolding cyclic amplification (sPMCA).;In initial experiments, concentrated urine and saliva samples from terminal CWD+ white-tailed deer, suspected of harboring infectious CWD prions, was assessed by Tg[CerPrP] bioassay and sPMCA. Authentic prion infectivity was detected in urine and saliva using both detection systems in the case of urine, though only mouse bioassay successfully demonstrated CWD prions in saliva. The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrPCWD levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg[CerPrP] mice. These findings helped to extend the understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.;Based on the identification of CWD prions in saliva ("prionsialia") and urine ("prionuria"), I next sought to determine whether deer previously exposed orally to urine and feces from CWD+ sources, while conventional test-negative, may actually be harboring very low level CWD infection not evident in the 19 month observation period in initial cervid bioassay studies. A selection of tissues, including those of the lymphoreticular and both central and peripheral nervous systems were fully examined, initially using Tg[CerPrP] bioassay to demonstrate true infectivity, and secondarily with sPMCA. Positive controls consisted of issues from CWD+ deer exposed orally to saliva; negative control tissue sets were collected from deer exposed orally and intracranially to CWD-negative brain. PrPCWD was detected in the tissues of orally exposed deer by both sPMCA and Tg[CerPrP] mouse bioassay; each assay revealed very low levels of CWD prions previously undetectable by western blot, ELISA, or IHC. Serial PMCA analysis of individual tissues identified that obex alone was positive in urine/feces exposed deer. PrPCWD was amplified from both LRS and neural tissues of positive control deer but not from the same tissues of negative control deer. Detection of subclinical infection in deer orally exposed to urine and feces (1) suggests that a prolonged subclinical state can exist such that observation periods in excess of two years may be needed to detect CWD infection, and (2) illustrates the sensitive and specific application of sPMCA in the diagnosis of low-level prion infection.;Despite the confirmation of infectious prions in urine and saliva, along with conventional test-negative deer exposed to urine and feces, the manner in which infectivity is transferred to these excreta is unknown. To address this, I went on to apply sPMCA to tissues associated with production and excretion of urine and saliva in an effort to seek proximal sources of prion shedding. I blindly analyzed oropharyngeal and urogenital tissues, reproducibly demonstrating PrPCWD in each tissue examined in 3 rounds of sPMCA; whereas blood samples from the same animals and concurrent negative controls remained negative. Tissue distribution was affected by route of inoculation and CNS burden. The identification of PrPCWD in bodily fluids and conventional-test negative tissues -- in the absence of detection by conventional methods -- may indicate the presence of protease-sensitive infectious prions in excretory tissues not revealed by assays employing PK digestion or other means to remove PrPC reactivity. (Abstract shortened by UMI.)...