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The curve depicts densities of PrP^sc bands in the <i>Scrapie Associated Fibrils</i> (<i>S.A.F.</i>)<i>/O.I.E. Immunoblot</i> plotted against PrP^sc content in the respective tissue homogenates.

<p>Numbers on the right of each density data point correspond to the numbers in the blot photo (inset). <i>R</i>² = 0.999.</p

A performance summary of both rapid and confirmatory tests.

<p>White bars represent the test's detection limitations, as established by the manufacturer. Dotted bars represent our determined theoretical limit of detection for each test, where the corresponding positive tissue dilution yielded a result no different from true negative tissue. Confirmatory tests are above the dashed line. Rapid-tests are below the dashed line.</p

The curves depict densities of PrP^sc bands (diglycosylated (Digly), monoglycosylated (Monogly), unglycosylated (Ungly)) in the <i>Bio-Rad</i>® <i>TeSeE</i> ™ Western Blot.

<p>(inset) and detection curves for digly-, monogly-, and unglycosylated bands of PrP^sc. Numbers on the right of each density data point correspond to the numbers in the blot photo (inset). Each label (Digly, Monogly, Ungly) in the inset photo appears to the left of the band it describes. <i>R</i>²: Digly = 0.9858; Monogly = 0.9659; Ungly = 0.9857.</p

<i>IDEXX HerdCheck</i>™ <i>BSE EIA</i> OD curve plotted against PrP^sc content in the respective tissue homogenates.

<p>Inset text and arrows indicates detection limitations. <i>R</i>² = 0.9980.</p

The curves depict densities of PrP^sc bands (diglycosylated (Digly), monoglycosylated (Monogly), unglycosylated (Ungly)) in the Prionics®-<i>Check WESTERN</i>™ western blot.

<p>Numbers on the right of each density data point correspond to the numbers in the blot photo (inset). Only odd numbered data points appear in the blot photo. Each label (Digly, Monogly, Ungly) in the inset photo appears to the left of the band it describes. Inset text and arrows indicate detection limitations. <i>R</i>²: Digly = 0.9870; Monogly = 0.9841; Ungly = 0.9635.</p

NaPTA precipitation and substrate replacement enable detection of CWD seeding activity in feces.

<p><b>(a)</b> Fecal homogenates (10% w/v) of non-infected elk or mule deer were subjected to NaPTA precipitation and 10fold concentration (lower panel) or not (upper panel). These samples were either used undiluted or in dilutions as indicated for seeding RT-QuIC reactions with mouse rPrP as a substrate. The y-axes show relative ThT fluorescence units, the x-axes depict the reaction time. <b>(b)</b> Fecal homogenates of two individual elk which were orally infected with CWD were subjected to NaPTA precipitation and 10fold concentration. RT-QuIC reactions with mouse rPrP as a substrate were run for 50 hours (upper panel), or for 75 hours (lower panel). For the latter, the reaction was stopped after 25 hours, and 90% of the reaction volume was removed and replaced by freshly prepared RT-QuIC mix containing rPrP substrate and ThT. Then the RT-QuIC assay was continued.</p

Mouse rPrP is a more efficient substrate for amplification of CWD seeding activity.

<p>Serial dilutions of CWD-positive <b>(a)</b> or negative <b>(b)</b> elk brain homogenates were used as seeds for RT-QuIC reactions. As a substrate, either mouse (left panel) or deer rPrP (right panel) were used. Y-axes indicate the relative ThT fluorescence units, x-axes show the reaction time. A threshold to determine positive reactions was calculated by using the average baseline fluorescence plus 5 standard deviations, which is indicated as a solid line at app. 50,000 RFU. <b>(c)</b> The time to reach the threshold (y-axis) was determined for each individual reaction. Dilutions of 2x10^-2 to 2x10^-5 of reactions with either mouse or deer rPrP were included, and the averages of the time to reach the threshold are shown. Bars represent standard deviations. For each dilution mouse and deer rPrP were compared and the difference between the two groups was statistically evaluated using unpaired student’s t-test (GraphPad Prism software; ** = p-value < 0.01; ns = not significant).</p

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

<div><p>Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrP^Sc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive <i>in vitro</i> prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.</p></div

Summary of RT-QuIC results.

<p>Summary of RT-QuIC results.</p