Western blot detection of PrP^CWD in mouse CNS tissues.

<p>Except for a single mouse (mouse Tg 2-B, lane 4), all mice inoculated with tissues from deer #134 and 150 succumbed to prion disease (lanes 3–7), as did mice inoculated with CWD+ deer #106 (lanes 1 and 2). Mice inoculated with tissues from sham-inoculated deer showed no evidence of PrP^CWD by western blot (lanes 8 and 9).</p

Serial PMCA amplification of CWD prions in WB and IHC negative deer.

<p>Conventionally negative tissues from deer orally exposed to urine and feces from CWD+ sources (Deer #'s 111, 124, 134, 141, and 150, lanes 4–8) amplified PrP^CWD after 2–3 rounds of PMCA, as did positive control tissues from deer #106 (lane 1). Tissue samples from two sham-inoculated deer (#103 and 123, lanes 2 and 3) and two untreated <i>tg5037</i> mice (lanes 9 and 10) failed to amplify PrP^CWD in three rounds of sPMCA.</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

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

<i>Tg5037</i> mouse bioassay results.

<p>Kaplan-Meyer curve demonstrating prolonged incubation periods in mice inoculated with tissues from deer #134 and 150 as compared to mice inoculated with tissues from deer testing positive by conventional assays.</p

Summary of immunohistochemistry (IHC), western blot (WB), and serial PMCA results and <i>Tg5037</i> mouse bioassay of combined obex/RLN homogenates.

<p>Mean incubation periods with standard deviations in parentheses; numerators indicate number of animals testing positive over total number tested. For PMCA, only those animals testing negative by IHC and WB were assayed. N/A: not assayed.</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

Spongiform degeneration and PrP^CWD in the hippocampus of inoculated mice.

<p>Vacuolated neurons and spongiform degeneration of the neuropil characteristic of TSE demonstrated by H&E staining and co-localization of PrP^CWD florid plaques in the hippocampus of mice inoculated with tissues from urine and feces exposed and positive control deer. Brains of mice inoculated with tissues from sham-inoculated deer showed no evidence of spongiform degeneration or PrP^CWD immunostaining. Anti-prion polyclonal antibody R-505 was used as the primary antibody. (Measure bar, 50 µm)</p

Serial PMCA detection of CWD prions in inoculated mice.

<p>Brain from a single <i>tg5037</i> mouse (mouse Tg 2-B) was WB and IHC negative yet amplified PrP^CWD after three rounds of sPMCA (lane 2), as did a positive control mouse (lane 1). Mice inoculated with tissues from sham-inoculated deer failed to amplify PrP^CWD (lanes 3 and 4).</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