Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep

Abstract

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.

FIG. 1.

Pattern of vacuolation observed in brains of 129/Ola wild-type mice (a) and Bov6 mice (b) following inoculation with the cattle BSE brainstem pool and experimental sheep BSE inoculum 1 and inoculum 2. A profile was produced from nine gray matter areas (1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septum; 8, cerebral cortex; 9, forebrain cerebral cortex) and three white matter areas (1*, cerebellar white matter; 2*, midbrain white matter; 3*, cerebral peduncle). Average scores were taken from a minimum of six mice per group and plotted against brain area ± standard errors of the means.

FIG. 2.

Limited PrP^Sc accumulation in the thalamus of one HuMM mouse 706 days postinoculation with experimental sheep BSE (inoculum 1). Panel b is a higher-magnification image of the boxed area in panel a. (b) PrP^Sc deposition appears to be restricted to the thalamus. Images obtained after staining with anti-PrP antibody 6H4 and counterstained with hematoxylin. Magnification is as shown.

FIG. 3.

Variation in pattern and location of PrP^Sc accumulation in the brain of HuMM mice infected with experimental sheep BSE (inoculum 2). (a) Punctate depositions in the cochlear nucleus are similar to those of BSE targeting in wild-type mice. Perineuronal (b) and intraneuronal (c) PrP depositions are seen in the midbrain and areas of the thalamus. (d) Hematoxylin and eosin stain of a large mature florid plaque located in the hippocampus. Punctate (e) and linear (f) PrP deposition in the thalamic region. Images a to c, e, and f were obtained after samples were stained with anti-PrP antibody 6H4 and counterstained with hematoxylin. Magnification is as shown.

FIG. 4.

Comparative analysis of serial sections through the lateral geniculate nucleus (thalamus) of an uninoculated aged (750 days) HuMM mouse and a HuMM mouse infected with experimental sheep BSE (inoculum 2). A HuMM mouse infected with sheep BSE (inoculum 2) shows astro- and microgliosis (b and f) visible when stained with anti-GFAP and anti-Iba1 (respectively). Several amyloid plaques are clearly visible, fluorescing green with thioflavin-S (h) and being stained with anti-PrP antibody 6H4 (d). (a, c, e, g) Sections from a control aged HuMM mouse show mild astro- and microgliosis and the absence of PrP deposits or amyloid plaques. Sections used for immunohistochemical analysis were counterstained with hematoxylin. Magnification, ×20.

FIG. 5.

Comparative Western blot (WB) analysis of the proteinase K-resistant fragment (PrP^Sc) of the prion protein. Discrimination between BSE and natural scrapie is achieved using two monoclonal antibodies, 6H4 (a) and 12B2 (b). Lane 1, 4.2 mg equivalent of brain material (mgE) of natural scrapie isolate from the NPU flock. Lane 2, 20 mg equivalent of inocula NPU J2501, Cheviot sheep experimentally infected via the oral route with cattle BSE. Lanes 3 and 5, 1.2 and 1.5 mg equivalent of 129/Ola mice infected with a natural scrapie isolate. Lane 4, 64 mg equivalent of HuMM transgenic mouse infected with experimental sheep BSE (inoculum 2). Lanes 6 and 7, 2.8 and 0.88 mg equivalent of 129/Ola and Bov6 (respectively) infected with experimental sheep BSE (inoculum 2). Lane 8, 0.6 mg equivalent of ME7/SV control. Molecular markers (M) of the standards are indicated on either side of the panels (in kDa).