PrPres in placental tissue following experimental transmission of atypical scrapie in ARR/ARR sheep is not infectious by Tg338 mouse bioassay

Abstract

Nor98-like atypical scrapie is a sporadic disease that affects the central nervous system of sheep and goats that, in contrast to classical scrapie, is not generally regarded as naturally transmissible. However, infectivity has been demonstrated via bioassay not only of brain tissue but also of certain peripheral nerves, lymphoid tissues, and muscle. This study examines placental tissue, a well characterized route of natural transmission for classical scrapie. Further, this study was conducted in sheep homozygous for the classical scrapie resistant ARR genotype and is the first to characterize the transmission of Nor98-like scrapie between homozygous-ARR sheep. Nor98-like scrapie isolated from a United States ARR/ARR sheep was transmitted to four ARR/ARR ewes via intracerebral inoculation of brain homogenate. These ewes were followed and observed to 8 years of age, remained non-clinical but exhibited progression of infection that was consistent with Nor98-like scrapie, including characteristic patterns of PrPSc accumulation in the brain and a lack of accumulation in peripheral lymphoid tissues as detected by conventional methods. Immunoblots of placental tissues from the infected ewes revealed accumulation of a distinct conformation of PrPres, particularly as the animals aged; however, the placenta showed no infectivity when analyzed via ovinized mouse bioassay. Taken together, these results support a low risk for natural transmission of Nor98-like scrapie in ARR/ARR sheep.

Fig 1. Accumulations of PrP^Sc in the brain of recipient ewes.

(A) Rank scores of PrP^Sc accumulation for each recipient ewe by brain region: G1—caudal medulla at the obex, G2—cerebellar cortex, G3—superior colliculus, G4/5—hypothalamus and medial thalamus, G6—hippocampus, G7—septum, G8—cerebral cortex at level of G4/5, G9—forebrain cortex at level of G7, W1—cerebellar white matter, W2—mesencephalic tegmentum. G1 was plotted as a mean of the rank scores to represent the two brain sections. G6 and G7 were plotted as a mean of the rank scores to include all the gray matter neuroanatomy in the respective section. (B) Punctate to globular extracellular PrP^Sc in spinocerebellar nerve fiber tract. Animal 3917, caudal medulla, IHC. (C) Absent PrP^Sc in the dorsal motor nucleus of the vagus nerve. Animal 3917, caudal medulla, IHC. (D) Patchy but diffuse, coalescing finely granular PrP^Sc in the molecular layer of the cerebellum (solid arrow), and punctate PrP^Sc in the white matter of the folia (solid arrowheads). Rare PrP^Sc in the granular layer (open arrowhead). Animal 3917, cerebellum, IHC. (E) Plaques (dashed ellipses) in the corona radiata with displacement of nerve fibers and mild gliosis (solid arrowheads); H&E. (F) PrP^Sc multicentric aggregates (dashed ellipses) coinciding with the localization of plaques on H&E; IHC. (E, F) Animal 3916, forebrain.

Fig 2. Western blots demonstrating stable experimental transmission of ARR/ARR atypical scrapie to ARR/ARR sheep and Tg338 ovinized mice.

A) Brain homogenate from the original donor sheep (1204–02) shows an anti-PrP western blot profile indicative of atypical scrapie following treatment with proteinase K (PK). Brain homogenates from a sheep positive for classical scrapie (4533), and scrapie-naïve sheep (4509, 4759) are shown for comparison. B) Western blot of brain homogenates from the original atypical scrapie donor (1204–02), recipient ewes (3913, 3914, 3916, 3917), and a scrapie-naïve sheep (4759). C) Western blot of brain homogenates from Tg338 ovinized mice inoculated with brain homogenate from the original atypical scrapie donor (1204–02). Passage 1 (P1) mice were inoculated with brain homogenate from 1204–02 and Passage 2 (P2) mice were inoculated with brain homogenates from P1 mice. D) Western blot of brain homogenates from Tg338 ovinized mice inoculated with brain homogenate from recipient ewes (representative blot from ewe 3916 shown). Western blots labeled with molecular weight markers of 20, 40 kDa.

Fig 3. Accumulation of non-infective PrP^res in the placentas of ARR/ARR ewes infected by atypical scrapie from a US ARR/ARR sheep.

A) PrP^Sc was not detected in cotyledons (Ct.) from infected ewes (3913, 3914, 3916, 3917) by immunohistochemical staining; note red staining in cotyledon from a sheep with classical scrapie shown for comparison. B) Anti-PrP western blot showing accumulation of PrP^res in cotyledons from infected ewes. Brain homogenate (Br.) from ewe 3913 shown for comparison. C) Anti-PrP western blot showing lack of PrP^res in brain homogenates from Tg338 mice inoculated with homogenates of cotyledons containing PrP^res (Ct.–M); inocula were prepared from the same cotyledons shown in (B). Brain homogenate of passage 2 mouse (Br.–M*) from original atypical scrapie donor shown for comparison. Western blots labeled with molecular weight markers of 20, 40 kDa. D,E) The proportion of cotyledons containing PrP^res, detected via western blot, increased with the ages of the inoculated ewes.