Rapid antemortem detection of CWD prions in deer saliva.

Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other) prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA) and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC) to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3%) diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%). In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1%) of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination

In vitro detection of prionemia in TSE-infected cervids and hamsters.

Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans

RT-QuIC analysis of CWD-positive clinically ill white-tailed deer.

<p>Both 1:10 diluted and PTA precipitated saliva show positive results by RT-QuIC. Each line represents an individual experiment with the average of four replicates. Blue and light blue lines represent 1:10 diluted saliva experiments and red and dark red lines represent PTA precipitated counterpart of the same saliva samples. The left axis (blue) denotes ThT fluorescence for 1:10 diluted saliva samples and the right axis (red) denotes ThT fluorescence for PTA saliva samples. Dashed lines represent threshold for a positive sample. Blue for 1:10 diluted saliva experiments and red for PTA saliva experiments. Positive RT-QuIC results were observed for saliva from clinically ill white-tailed deer in 5 of 9 1:10 diluted saliva samples and 8 of 9 PTA precipitated saliva samples. Each saliva sample was from an individual deer.</p

SHrPrP substrate can detect CWD-positive brain over a range of dilutions from 10^-4 to 10^-7.

<p>CWD positive and negative brain was serially diluted and analyzed by RT-QuIC assay. Each line for positive CWD samples represents the average of 4 individual wells from 4 independent experiments.</p

Kaplan-Meier survival curve for mice inoculated with CWD+ saliva.

<p>The same saliva samples from deer assayed in Fig. 1 (#133 and 144) were inoculated into cervidized mice (Tg[CerPrP] 5037) as well as saliva from a sham inoculated deer. 50% of mice were euthanized due to clinical disease after ~500 days.</p

Correlation of RT-QuIC analysis of terminal saliva samples with western blot PrP^Res signal in brains.

<p>A. Cartoon representation of sections analyzed for PrP^Res by western blotting. Seven rostral to caudal sections were analyzed plus the obex/brainstem region. B. (left) RT-QuIC analysis of saliva samples from white-tailed deer. Colors and axis are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074377#pone-0074377-g001" target="_blank">Figure 1</a> (right). Western blot analysis of 8 brain sections including CWD (+) and (-) control samples. PK was added to all samples except one CWD (-) brain sample. Antibody BAR-224-HRP used for detection.</p

PTA precipitation increases sensitivity of the RT-QuIC assay.

<p>A. Each data point shows the ThT fluorescence average of 4 replicate wells loaded with either 2 µl of brain homogenate dilution or PTA pellets from the same brain homogenate equivalents. Lines with triangles represent PTA precipitated samples while smooth lines are non-precipitated counterparts or controls (in the latter case, invisible under the green triangles). The data for spiked PTA precipitated deer saliva (purple triangles) comes from a separate experiment performed under the same experimental conditions. B. End-point dilution analysis of a CWD (+) brain homogenate including 10^-7 to 10^-9 dilutions with or without PTA precipitation. PTA precipitated samples were done in triplicate (purple) and represent the ThT fluorescence average of 4 replicate wells each. Non-precipitated samples (red) represent the ThT fluorescence average of all 12 replicate wells. Normal deer brain homogenates (green) are shown for PTA precipitated (3 overlapping curves of data points representing the average of 4 replicate wells) and non-precipitated (average of 12 replicate wells) samples.</p

RT-QuIC analysis of whole blood collected in various anticoagulants.

<p>Blood was collected from a CWD-infected and CWD-naïve white-tailed deer and preserved in one of three anticoagulants: CPDA, EDTA, or heparin. Serial blood sample dilutions (neat to 10^-6) were assayed by RT-QuIC for 60 hours and ThT fluorescence level above threshold determined positivity. Detection of PrP^C-converting activity for each replicate is shown for blood collected in CPDA (A), EDTA (B), and heparin (C). </p

RT-QuIC comparison of brain and blood samples.

<p>Ten percent (10%) brain homogenates were serially diluted (10^-5 to 10^-8) and assayed by RT-QuIC for 60 hours. Blood samples were diluted to 10^-2 and run in triplicate for 60 hours with ThT fluorescence level above threshold determining positivity. CWD-infected blood diluted 10^-2 is seen to have similar levels of PrP^C converting activity as CWD-positive brain diluted 10^-6 and 10^-7. UN= Uninfected; INF= Infected.</p

RT-QuIC analysis of samples before and after treatment with NAPTA.

<p>Samples were untreated or concentrated using NAPTA, serially diluted (neat to 10^-6), and assayed by RT-QuIC in triplicate for 60 hours. ThT fluorescence level above threshold determined positivity, each replicate is present. (A and C) Limited detection is seen in untreated blood samples. (B and D) Improved detection of PrP^C-converting activity is seen in blood samples precipitated with NAPTA from CWD-infected white-tailed deer. (E) Note increased false-positives in untreated samples from a CWD-naïve white-tailed deer. (F) No PrP^C-converting activity was seen in samples precipitated with NAPTA from a CWD-naïve white-tailed deer.</p