Intra-host mathematical model of chronic wasting disease dynamics in deer (<i>Odocoileus</i>)

<p>Bioassays of native cervid hosts have established the presence of infectious chronic wasting disease (CWD) prions in saliva, blood, urine, and feces of clinically diseased and pre-clinical infected deer. The intra-host trafficking of prions from the time of initial infection to shedding has been less well defined. We created a discrete-time compartmentalized model to simulate the misfolding catalysis, trafficking, and shedding of infectious prions throughout the organ systems of CWD-infected cervids. Using parameter values derived from experimental infections of North American deer (<i>Odocoileus spp</i>.), the exponential-based model predicts prion deposition over time with: 1) nervous tissues containing the highest deposition of prions at 20 months post-infection and 2) excreted fluids containing low levels of prions throughout infection with the highest numbers of prions predicted to be shed in saliva and feces (as high as 10 lethal doses (1.34 × 10²⁹ prions) in 11–15 months). These findings are comparable to prion deposition described in literature as assayed by conventional and ultrasensitive amplification assays. The comparison of our model to published data suggests that highly sensitive assays (sPMCA, RT-QuIC, and bioassay) are appropriate for early prion detection in bodily fluids and secretions. The model provides a view of intra-host prion catalysis leading to pre-clinical shedding and provides a framework for continued development of antemortem diagnostic methods.</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

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 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

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

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

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

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

Comparison of PrP^C expression among tissue types.

<p>We used one-way ANOVA and Tukey’s Multiple Comparison post-test to analyze differences between PrP^C expression levels in types of tissue (based on ELISA results.) * indicates p<0.05, ** p<0.01, *** p<0.0001. <i>Lower alimentary tissues</i> include: duodenum, jejunum, ileum, cecum, colon and rectum. <i>Ruminant stomachs</i> include: rumen, reticulum, omasum and abomasum. <i>Oropharyngeal lymph nodes</i> include: tonsil, retropharyngeal lymph node, mandibular lymph node and parotid lymph node. <i>Lower GI lymph nodes</i> includes ileocecocolic lymph node and mesenteric lymph node. As indicated by the asterisks, there are statistically significant differences between the following groups: lower alimentary tissues > all lymph nodes; lower alimentary tissues > spleen; ruminant stomachs > spleen; salivary glands > spleen; salivary glands > oropharyngeal lymph nodes. We plotted means and standard error of the mean.</p

Representative IHC results.

<p>We have displayed 4 tissues that represent the most common PrP^C staining patterns from our IHC experiments. All four tissues are stained with an anti-PrP antibody or an isotype control antibody. Images are 200X; scale bar represents 50μm. Descriptions of the staining patterns are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183927#pone.0183927.t001" target="_blank">Table 1</a>.</p