Sheep-passaged bovine spongiform encephalopathy agent exhibits altered pathobiological properties in bovine-PrP transgenic mice

Abstract

Sheep can be experimentally infected with bovine spongiform encephalopathy (BSE), and the ensuing disease is similar to scrapie in terms of pathogenesis and clinical signs. BSE infection in sheep is an animal and human health concern. In this study, the transmission in BoPrP-Tg110 mice of prions from BSE-infected sheep was examined and compared to the transmission of original cattle BSE in cattle and sheep scrapie prions. Our results indicate no transmission barrier for sheep BSE prions to infect BoPrP-Tg110 mice, but the course of the disease is accelerated compared to the effects of the original BSE isolate. The shortened incubation period of sheep BSE in the model was conserved in subsequent passage in BoPrP-Tg110 mice, indicating that it is not related to infectious titer differences. Biochemical signature, lesion profile, and PrP(Sc) deposition pattern of both cattle and sheep BSE were similar. In contrast, all three sheep scrapie isolates tested showed an evident transmission barrier and further adaptation in subsequent passage. Taken together, those data indicate that BSE agent can be altered by crossing a species barrier, raising concerns about the virulence of this new prion towards other species, including humans. The BoPrP-Tg110 mouse bioassay should be considered as a valuable tool for discriminating scrapie and BSE in sheep.

FIG. 1.

Overview of the inoculations conducted in BoPrP-Tg110 mice. Survival times (as days post inoculation) ± standard error of the mean and the percentage of animals scored PrP^res positive are indicated.

FIG. 2.

Histopathological alterations in BoPrP-Tg110 mice inoculated with BSE, BSE/sheep, or scrapie. (A) Vacuolation profiles in BSE-, BSE/sheep-, and SC-UCD-99-inoculated animals. Lesion scoring is undertaken for seven areas of the brain (cerebral cortex, cerebellum, corpus striatum, thalamus/hypothalamus, hippocampus, mesencephalon, and brainstem at the obex). Bars represent means ± standard deviation. (B) Images of vacuolation in the cerebellum and striatum of inoculated mice. The main differences are an increase in the number and size of vacuoles in these areas of animals inoculated with scrapie compared to those in either the BSE- or BSE/sheep-inoculated animals.

FIG. 3.

Immunohistochemical detection of PrP^res in BoPrP-Tg110 mice inoculated with BSE, BSE/sheep, and scrapie. (A) Deposition scores for the different types of PrP^res deposits (IN, intraneuronal; IG, intraglial; GA, glial associated deposits; NR, neuropil deposits; VS, vascular; EP, ependimary deposits; Plaque-like, amyloid plaque-like deposits) in the brain of BoPrP-Tg110 mice inoculated with BSE/sheep, BSE, or SC-UCD-99; second passages of brain material from these inoculated mice are shown in panel B. (C) Illustrations of the immunohistochemical detection of PrP^res in the brain stem and hippocampus of inoculated animals. Plaque-like deposits appear in the brain stem and hippocampus of BSE- and BSE/sheep inoculated animals, but not in the hippocampus of the SC-UCD-99-inoculated animals. PrP deposition in the brain stem of SC-UCD-99-inoculated animals is observed as intraneuron staining.

FIG. 4.

Electrophoretic profiles and antibody labeling of PrP^res detected with the Sha31 (A), 12B2 (B), and 9A2 (C) MAbs. Lanes show the original BSE₅ inoculum (lane 1), BSE₅-infected BoPrP-Tg110 mice (lane 2), second-passage BSE₅ in BoPrP-Tg110 mice (lane 3), BSE/sheep (lane 4), BSE/sheep in BoPrP-Tg110 mice (lane 5), second-passage BSE/sheep in BoPrP-Tg110 mice (lane 6), scrapie field isolate SC-UCD-99 (lane7), SC-UCD-99 in BoPrP-Tg110 mice (lane 8), second-passage SC-UCD-99 in BoPrP-Tg110 mice (lane 9), scrapie field isolate SC-Langlade (lane 10), and scrapie field isolate SC-N662-97 (lane 11). Panels A, B, and C were loaded with the same quantities of extracted PrP^res from each sample. MW, molecular mass given in kilodaltons. (D) PrP^res fragment yields are represented according to MAb reactivity. Arrows indicate proteinase K cleavage points, as predicted by the PeptideCuter program of the EXPASY server (http://us.expasy.org/). Numbers reflect sheep/cattle PrP sequence positions. Continuous lines represent the main PrP^res fragments, and broken lines represent PrP^res fragments found in minor proportions.

FIG. 5.

Triangular plot of the glycosyl fractions of PrP^res after proteinase K digestion and Western blotting using the Sha31 antibody. The data shown are the means of six or more measurements obtained from density scans in two or more different Western blots. To interpret the plot, read the values for the diglycosyl, monoglycosyl, and aglycosyl fractions along the left, base, and right axes of the triangle, respectively. For each point, the sum of the three values is 100. As an example, the values for the individual points marked by arrows are 64, 24, and 12% for the diglycosyl, monoglycosyl, and aglycosyl fractions, respectively.

FIG. 6.

Differential proteinase K (PK) resistance ELISA for PrP^Sc from the inoculum (A) and from the BoPrP-Tg110-inoculated mice (B).