Prions in the environment: From the host to the environment and back again

In the first chapter of this dissertation we examine the binding capacity of prions to soil by a subtractive infectivity bioassay. We quantified the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in chronic wasting disease (CWD)-endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). BASICS indicated SLS bound and removed 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula suspension, effectively preventing disease in the mice. These data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.;In the second chapter of this dissertation we outline efforts to develop a novel detection assay using aptamers, small oligonucleotieds capable of epitope specific labeling. Our objective was to develop this assay to allow for an increased detection limit of prions in soil and tissue samples. In collaboration with InfoScitex we selected for aptamers reactive against PrP RES, candidate aptamers were purified and incubated with positive and negative control samples. PrPRES detection by western blot showed some promise for two prion strains but was difficult to replicate and did not work for CWD. A second method of amplification was tested for samples containing prions that were below the detection threshold of a western blot. Bound aptamers would then be amplified by rtPCR. Data from this method was inconsistent and indicated a non-specific binding to negative elk brain homogenate molecules. Further aptamer selection and research should be pursued with this technique to achieve targeted PrPRES binding.;Enhanced surveillance of CWD in both free-ranging and captive elk and deer herds is an essential aspect of study and management of this disease. Currently, immunohistochemistry (IHC) is the gold standard for CWD diagnosis in cervids. In the third chapter of this dissertation we compare the sensitivity and specificity of IHC to sPMCA. Obex samples from free-ranging Rocky Mountain elk were blindly tested by sPMCA and compared to IHC findings of PrPRES in obex and lymph nodes of the same animals. Hierarchical Bayesian analysis found the sensitivity of sPMCA on obex tissue (95%) was higher than IHC on the same obex tissue (71%). Only when IHC was used on three different tissues did the sensitivity match that of sPMCA. These data are significant for the identification of a previously unrecognized sub-clinical population on the landscape, potentially capable of shedding and transmitting CWD. Additionally, our findings challenge the idea of CWD being an invariably fatal disease, instead suggesting a possibly infectious but subclinical or carrier state may be more common than previously believed.;The transmission ecology and epidemiology of CWD through environmental reservoirs is poorly understood due to the novelty of the agent and the difficulty of prion detection in environmental samples. It is unclear what an infectious dose is in a natural setting, and if a difference in transmission rate between a single or chronic exposure exists. In the fourth chapter of this dissertation we test chronic exposure to CWD in naturally contaminated soils. Our objectives were to investigate the role of indirect transmission by exposing PrP CWD susceptible transgenic mice to PrPCWD contaminated soil, to evaluate a dose response of prions in soil, to estimate average soil ingestion by laboratory mice, and to estimate annual exposure to prions from chronic environmental exposure of prion contaminated soil. This study was the first to successfully use transgenic mice to test soil infectivity from natural sources. We found that chronic exposure was more efficient for CWD transmission than a single concentrated oral dose of CWD-spiked soil. Epidemiology of the studies also suggests that infectivity differences existed between our two sources of naturally contaminated soil, it is unclear if a difference in titer or CWD strain is responsible. Additionally through soil ingestion estimates we calculated an annual ingestion of > 3.6x105 LD 50 infectious units by our mouse model. (Abstract shortened by UMI.).