Lesion profiling at primary isolation in RIII mice is insufficient in distinguishing BSE from classical scrapie

Abstract

Primary isolation of bovine spongiform encephalopathy (BSE) in RIII mice generates a lesion profile believed to be reproducible and distinct from that produced by classical scrapie. This profile, which is characterized by peaks at gray matter areas 1, 4 and 7 (dorsal medulla, hypothalamus and septal nuclei), is used to diagnose BSE on primary isolation. The aim of this study was to investigate whether the BSE agent could be present in sheep diagnosed with classical scrapie, using lesion profiles in RIII mice as a discriminatory method. Sixty-two positive scrapie field cases were collected from individual farms between 1996 and 1999 and bioassayed in RIII mice. Fifty-five of these isolates transmitted successfully to at least one mouse. Of the 31 that produced adequate data to allow lesion profile analysis, 10 showed a consistent profile with peaks at brain areas 1, 4 and 7. All inocula for this subgroup were derived from sheep of genotype ARQ/ARQ. While the 1-4-7-scrapie profile exhibited similarities to BSE in RIII mice at primary isolation, it was distinguishable based on histopathology, immunohistochemistry and cluster analysis. We conclude that caution should be taken to distinguish this profile from BSE and that additional parameters should be considered to reach a final diagnosis.

Figure 1

Glycoform ratio chart showing the ovine and bovine BSE samples with a higher percentage of diglycosylated PrP^Sc compared with the ovine scrapie controls and inocula. Abbreviation: BSE = bovine spongiform encephalopathy.

Figure 2

Average lesion profiles in RIII mice of 31 inocula with ≥5 mice per inoculum. A. 10 scrapie sources, all derived from ARQ/ARQ sheep, gave a 1‐4‐7 profile (1‐4‐7‐scrapie). B. 5 scrapie sources gave alternative 1‐4‐7 profiles (1‐4‐7‐subgroup 2). C. 16 scrapie sources gave non‐1‐4‐7 profiles (non‐1‐4‐7‐subgroup). Profiles were obtained following quantitation of specific vacuolation in nine neuroanatomic gray‐matter areas: G1, dorsal medulla nuclei; G2, cerebellar cortex of the folia including the granular layer, adjacent to the fourth ventricle; G3, cortex of the superior colliculus; G4, hypothalamus; G5, thalamus; G6, hippocampus; G7, septal nuclei of the paraterminal body; G8, cerebral cortex (at the level of G4 and G5); and G9, cerebral cortex (at the level of G7).

Figure 3

The 1‐4‐7‐scrapie profile is not consistent with BSE. A. Average lesion profiles for 1‐4‐7‐scrapie isolates plotted against 10 BSE isolates (four bovine, six ovine passaged) for comparison. B. Tree diagram after cluster analysis (Statistica 7.0 statistical software, StatSoft Inc.) of the vacuolation scores in selected neuroanatomic brain areas to show that scrapie and BSE form two distinct groups as measured by Euclidean distance. Abbreviation: BSE = bovine spongiform encephalopathy.

Figure 4

Assessment of disease specific vacuolation by H&E staining revealed differences between BSE and 1‐4‐7‐scrapie pathology. Representative photographs are shown of (A) vacuolation of the granular layer of the cerebellum adjacent to the fourth ventricle (indicated by arrows), (B) the cochlear nuclei (indicated by arrows and shown at higher magnification), (C) the ventral midbrain and (D) the trigeminal nucleus (indicated by arrows). Scales bars represent 500 µm (main photographs) and 200 µm (inset photographs). Abbreviation: BSE = bovine spongiform encephalopathy.

Figure 5

Assessment of abnormal PrP deposition by immunohistochemistry confirmed histopathological findings. Representative photographs are shown of (A) PrP^Sc deposition in the hippocampus, targeted to the molecular layer of the dentate gyrus (MolDG) for 1‐4‐7‐scrapie but to the hippocampal fissure (hif) or at CA2/hif end for BSE isolates, (B) the cerebral cortex: plaques are a consistent feature of 1‐4‐7‐scrapie but not BSE. Scale bars represent 500 µm (main photographs) and 100 µm (inset photographs). Abbreviation: BSE = bovine spongiform encephalopathy.

Figure 6

Survival curves of individual 1‐4‐7‐scrapie and BSE sources (five or more clinically and H&E‐positive mice per inoculum) are shown. Statistical comparison of the mean incubation period between groups was performed using the two‐tailed t‐test, where significance was accepted at P < 0.01 or P < 0.05. Abbreviations: BSE = bovine spongiform encephalopathy; H&E = hematoxylin and eosin.