Transmission of atypical scrapie to homozygous ARQ sheep

Abstract

Two Cheviot ewes homozygous for the A₁₃₆L₁₄₁R₁₅₄Q₁₇₁ (AL₁₄₁RQ) prion protein (PrP) genotype were exposed intracerebrally to brain pools prepared using four field cases of atypical scrapie from the United Kingdom. Animals were clinically normal until the end of the experiment, when they were culled 7 years post-inoculation. Limited accumulation of disease-associated PrP (PrP^Sc) was observed in the cerebellar molecular layer by immunohistochemistry, but not by western blot or enzyme-linked immunosorbent assay. In addition, PrP^Sc was partially localized in astrocytes and microglia, suggesting that these cells have a role in PrP^Sc processing, degradation or both. Our results indicate that atypical scrapie is transmissible to AL₁₄₁RQ sheep, but these animals act as clinically silent carriers with long incubation times.

Fig. 1.

Features of IHC PrP^Sc staining in the cerebellum of a sheep (case 2) with subclinical infection 85 months after inoculation with atypical scrapie. Arrows show accumulation of PrP^Sc in the cerebellar molecular layer. Tissue was stained with the mAb 2G11 and counterstained with hematoxylin. M: molecular layer, G: granule cell layer, WM: medulla of white matter.

Fig. 2.

Dual immunofluorescence labeling with mAbs against PrP^Sc (2G11; a–d, green) and GFAP (a and b) or Iba-1 (c and d). Astrocytes (a and b) and microglia (c and d) are labeled in red. The panels show higher magnification images of the area shown in Fig. 1, obtained from serial sections of the sample. Fine to coarse granular PrP^Sc deposits can be observed principally in the neuropil and associated with the cytoplasmic protrusions (yellow) of astrocytes (a) and microglia (c). Boxed regions in panels (a) and (c) are shown at higher magnification in panels (b) and (d), respectively. PrP^Sc deposits can be discerned within astrocytes and microglia or at the periphery of these cells. The section was counterstained with TO-PRO-3. bv: Blood vessel.

Fig. 3.

Western blotting using mAb P4 assessing the presence of PK-resistant PrP^Sc in the brain of a sheep (case 1) inoculated with atypical scrapie. Compared to the sample from a classical scrapie-affected sheep (lane 11), an additional smaller fragment (~7 kDa) is detected in an atypical scrapie case (lane 9). Lanes 1–2: cerebellum samples from negative sheep [10 mg of tissue equivalent (eq.)]; lanes 3–7: samples from five different areas of the cerebellum from case 1 (10 mg tissue eq.); lane 9: cerebellum sample from an ARQ/ARQ atypical scrapie natural case as a positive control (2 mg tissue eq.); lane 11: brainstem sample from a classical scrapie affected sheep as a positive control (5 μg tissue eq.); lane X: empty lanes. The nonspecific staining band migrates around ~7–9 kDa at bottom of lanes 1–7. Molecular markers are shown on the left (kDa).