Clay Components in Soil Dictate Environmental Stability and Bioavailability of Cervid Prions in Mice.

Chronic wasting disease (CWD) affects cervids and is the only known prion disease to affect free-ranging wildlife populations. CWD spread continues unabated, and exact mechanisms of its seemingly facile spread among deer and elk across landscapes in North America remain elusive. Here we confirm that naturally contaminated soil contains infectious CWD prions that can be transmitted to susceptible model organisms. We show that smectite clay content of soil potentiates prion binding capacity of different soil types from CWD endemic and non-endemic areas, likely contributing to environmental stability of bound prions. The smectite clay montmorillonite (Mte) increased prion retention and bioavailability in vivo. Trafficking experiments in live animals fed bound and unbound prions showed that mice retained significantly more Mte-bound than unbound prions. Mte promoted rapid uptake of prions from the stomach to the intestines via enterocytes and M cells, and then to macrophages and eventually CD21+ B cells in Peyer's patches and spleens. These results confirm clay components in soil as an important vector in CWD transmission at both environmental and organismal levels.

Estimating prion adsorption capacity of soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS).

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200 × g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our 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.

Detection of PrPCWD in feces from naturally exposed Rocky Mountain elk (Cervus elaphus nelsoni) using protein misfolding cyclic amplification.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrP(CWD)) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrP(CWD) in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2 × 10(7) dilution of PrP(CWD) (a 10% infected brain homogenate diluted 1.2 × 10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrP(CWD)/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrP(CWD) was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrP(CWD) concentrations. We used this PMCA scoring system to detect PrP(CWD) and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrP(CWD) IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrP(CWD) in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrP(CWD)/g of feces. These data show for the first time PrP(CWD) in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.