Figure 4. Analysis of FPE lymphoid tissues from subclinical CWD-infected WTD by RT-QuIC.

(a) Representative IHC of FPE lymphoid tissues from subclinical, CWD-infected WTD. Tissues collected at 4 MPI have increased density of PrP^CWD staining when compared to 2 MPI tissues reflecting a greater prion accumulation over the disease course. The low-level of PrP^CWD staining at 2 MPI reflects the early course of disease. RT-QuIC detected amyloid seeding activity in FPE samples that are IHC negative (1148 Retro LN). All IHC images are 200× magnification, measurement bars represent 50 μ m. (b) RT-QuIC of subclinical WTD retropharyngeal lymph nodes. All retropharyngeal lymph nodes from subclinical animals displayed significant amyloid seeding activity (means analyzed by one sample t-test; **p <  0.01, ***p <  0.001) when compared with respective negative control tissues. Lymph nodes collected at 4 MPI had higher RT-QuIC amyloid formation rates than tissues collected at 2 MPI which is consistent with greater PrP^CWD amyloid seeding activity. Each point represents the mean and SEM derived from eight replicates and in two separate experiments. All FPE samples are assayed at a 10⁻¹ dilution. (c) RT-QuIC of subclinical WTD tonsils. All tonsils from subclinical animals displayed significant amyloid seeding activity (means analyzed by one sample t-test; ***p <  0.001) when compared with respective negative control tissues. Similar to the retropharyngeal lymph nodes in (b), tonsils collected at 4 MPI had higher RT-QuIC amyloid formation rates than tissues collected at 2 MPI which is consistent with greater PrP^CWD amyloid seeding activity. Each point represents the mean and SEM derived from eight replicates and in two separate experiments. All FPE samples are assayed at a 10⁻¹ dilution.