Western Blot (WB), immunohistochemistry (IHC), and protein-misfolding cyclic amplification results and incubation periods of Tg[CerPrP] mouse bioassay.

<p>Numerators indicate the number of animals testing positive by a particular assay, while denominators designate the total number tested. PMCA analysis was reserved for mice testing negative by traditional assays. Incubation periods indicate the survival times in days post inoculation +/− one standard deviation. N.A. - not assayed.</p

Spongiform degeneration and PrP^CWD identified by histopathology and immunohistochemistry.

<p>Vacuolated neurons and spongiform degeneration of the neuropil characteristic of a TSE is evident on H&E staining, with the colocalization of PrP^CWD specific immunostaining of florid plaques in the cortices of mice inoculated with positive control inoculum and concentrated urine and saliva from CWD-infected cervids. Negative control mice showed no evidence of spongiform degeneration or PrP^CWD immunostaining. HRP-conjugated BAR-224 was used as a primary antibody. (Measure bar, 50 µm).</p

Western Blot detection of PrP^CWD in urine and saliva-inoculated mice.

<p>Western blotting analysis of control and test mice, demonstrating PrP^CWD in positive control mice (lanes 1 and 2), as well as urine (lanes 3 and 4) and saliva (lanes 5 and 6) inoculated mice. Protease-resistant prions were not detected in negative control mice (lanes 7 and 8). Flanking lanes represent undigested PrP^C.</p

Serial PMCA amplification of PrP^CWD in concentrated deer urine and in the brains of urine-inoculated mice.

<p>A) PrP^CWD was detectable by serial PMCA (sPMCA) in control and urine inocula (lanes 1 and 2, respectively), while PrP^CWD could not be identified in saliva and negative control inocula (lanes 3 and 4, respectively) after 3 rounds of amplification. B) Three rounds of sPMCA also amplified PrP^CWD in the brains of CWD-infected mice, including positive-control inoculated mice and a single mouse inoculated with lyophilized urine (lanes 1 and 3, respectively). PrP^CWD was not amplified in mice inoculated with negative control material (lanes 5 and 6) or in other mice inoculated with either urine (lane 2) or saliva (lane 4) from CWD+ deer. All flanking lanes represent undigested PrP^C.</p

Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease

<div><p>Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrP^d) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrP^d detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrP^d identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF. </p> </div

Immunohistochemistry of white-tailed deer obex.

<p>Immunohistochemistry results from deer #4502 (<b>A</b>), 4129 (<b>B</b>), 144 (<b>C</b>) and 4488 (<b>D</b>). Normalized scores from IHC+ animals ranged from 0.17 (e.g. #4129) to 1.0 (e.g. #4502 and 144). Sham-inoculated deer (e.g. #4488, D) were consistently negative by IHC. </p

Serial PMCAb detection of prion seeding activity in CSF from CWD-positive deer.

<p>Cerebrospinal fluid samples, along with positive and negative controls, were assayed in duplicate for three rounds of sPMCAb, with the total number of positive rounds tallied for each sample. Positive control samples were positive in each duplicate through all rounds (Lanes 2 and 3, for a total of 6/6 positive rounds and a normalized score of 1), while CSF samples varied in their total number of positive rounds. CSF from deer #4502 (Lanes 4 and 5) and #4129 (Lanes 8 and 9) demonstrated amplification in 3/6 rounds of sPMCAb, for a normalized score of 0.5, while CSF from deer 144 was positive in 6/6 rounds (Lanes 6 and 7). Negative controls, including deer #4488 (Lanes 11 and 12) and unspiked brain homogenate (NBH – lanes 12 and 13) remained negative throughout all rounds.</p

RT-QuIC seeded amplification detected by ThT fluorescence in CSF of deer.

<p>A 10^-3 dilution of cervid brain pool (red), as well as 10^-1 dilutions of CSF from deer #4502 (blue) and 144 (orange) showed evidence of prion seeding activity (ThT binding) in 48 hour RT-QuIC experiments. The time to reaching threshold fluorescence (C_t – red horizontal dashed line) varied amongst RT-QuIC positive animals, and correlated with IHC scores. CSF from some CWD-positive deer (e.g. deer #4129), all sham-inoculated deer (e.g. deer #4488), and all untreated controls failed demonstrate seeding activity. </p

Optimization of sPMCAb and RT-QuIC.

<p>Various spike volumes and dilutions were chosen for optimization of each assay for use with cervid CSF. Cerebrospinal fluid from six CWD-positive deer (#106, 110, 133, 144, 346, and 4116) and one CWD-negative deer (#4488) were chosen for optimization. With sPMCAb (A.), spike volumes including 20μl (Lanes 2, 5, 8, 11, 14, 17, and 20), 10μl (Lanes 3, 6, 9, 12, 15 18, and 21), and 1μl (Lanes 4, 7, 10, 13, 16, 19, and 22) were evaluated using three rounds of sPMCAb. For RT-QuIC (B.), dilutions of CSF including 10°, 10^-1, and 10^-2 were evaluated in 48hr experiments. The threshold for positive amplification (C_t) is represented by the horizontal hashed red line. Data encompassing results from IHC, sPMCAb, and RT-QuIC from CSF samples from seven deer (C.) were evaluated to estimate the optimum spike volumes and dilutions of CSF, and revealed that 10μl of whole CSF appeared to be optimal for sPMCAb, while a 10^-1 dilution of CSF had the greatest precision in RT-QuIC analysis. </p

Treatment with heterologous recombinant HaPrP delayed loss of motor function.

<p>The hanging wire assay was used as an objective measure of motor coordination and muscle strength. The graphs show the results of A) mock-treated mice, B) uninfected age matched mice, C) low-dose-treated mice, and D) high-dose-treated mice. E) Shows the mean values for each group. The X-axis shows the day post-infection and the Y-axis shows the time of latency to fall from a wire grid in a 120 second trial. In A-D), individual averages of triplicate trials are shown with error bars showing the standard deviation. Mixed model analysis (which uses the data from all time points to test for differences between groups) finds significant differences between both treatment dose groups and the mock infected group (<i>p</i><0.001 for both tests). Significant differences (p<0.001) were also seen between the three groups (mock, low dose, high dose) and the uninfected age matched mice. Note, one of the low-dose treated animals included in this study (indicated with an asterisk in C) was euthanized due to the development of a skin disease towards the end of the study at 347 dpi.</p