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 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 detection or PrP^CWD of neural and lymphoid tissues of deer exposed orally to urine and feces of CWD-positive deer.

<p>For each tissue, the number of sPMCA rounds producing a positive result was tabulated for three independent experiments of three rounds each. Samples appearing as positive in round 2 continued to be positive in round 3, and thus over the course of three experiments, were positive a total of 6 out of 9 rounds (e.g. obex samples from deer 134 and 150). Samples becoming positive in the first round were succeeded by positivity in rounds 2 and 3, and thus were positive in 9 out of 9 rounds (e.g. tonsil and RLN samples from deer 122 and 113). As would be expected, the greatest number of positive results was observed in the positive control tissues, with some variance among tissues and between animals. Deer orally exposed to urine and feces demonstrated PrP^CWD amplification almost exclusively from the obex; the terminal tonsil collection from a single animal also was positive. By contrast, negative control tissues failed to amplify PrP^CWD. RLN: retropharyngeal lymph node; MedLN: mediastinal lymph node; MesLN: mesenteric lymph node; ILN: ileocecocolic lymph node; IMLT: Intermediolateral spinal cord tract; SU: sample unavailable.</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

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

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 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