Use of murine bioassay to resolve ovine transmissible spongiform encephalopathy cases showing a bovine spongiform encephalopathy molecular profile

Abstract

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrP(Sc) immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrP(Sc) deposition pattern in wild-type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrP(Sc) deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE.

Figure 1

UK‐1 and UK‐2 LP following primary isolation in RIII mice. Profiles for RIII mice inoculated with UK‐1 (A) and UK‐2 (B) were obtained following quantification of specific vacuolation in nine neuroanatomical gray matter areas. Where less than five clinically and histopathologically positive mice were available, separate profiles representing all haematoxylin and eosin (H&E)‐positive mice (dashed line) and mice that were both clinically and H&E positive (solid line) are plotted alongside representative bovine spongiform encephalopathy (BSE) profiles. Error bars indicate standard error of the mean. Ovine BSE 1 and Ovine BSE 2 represent duplicate bioassays using the same ovine source. The numbers of animals that contributed to each profile are given in parentheses.

Figure 2

PrP^Sc deposition patterns of UK‐1 and UK‐2 following transmission to RIII mice. Representative photographs of PrP^Sc deposition in the hippocampus and cerebellum are shown for bovine spongiform encephalopathy (BSE) (A). UK‐1 gave two distinct patterns termed UK‐1 (1) (B) and UK‐1 (2) (C). Representative PrP^Sc IHC labeling for UK‐2 is shown in (D). Scale bars represent 100 µm.

Figure 3

PrP^Sc deposition patterns of UK‐1 and UK‐2 following transmission to C57BL mice. Representative photographs of PrP^Sc deposition in the hippocampus and cerebellum are shown for BSE (A). UK‐1 and UK‐2 each gave two distinct patterns as shown, termed UK‐1 (1) (B), UK‐1 (2) (C), UK‐2 (1) (D) and UK‐2 (2) (E), respectively. Scale bars represent 100 µm.

Figure 4

PrP^Sc deposition patterns of UK‐1 and UK‐2 following transmission to VM mice. Representative photographs of PrP^Sc deposition in the hippocampus, solitary tract and periaqueductal gray are shown for bovine spongiform encephalopathy (BSE) (A), UK‐1 (B) and UK‐2 (C). Scale bars represent 100 µm.

Figure 5

Western blot analysis in wild‐type mice. Western blot analysis of proteinase‐K‐digested PrP^Sc from UK‐1 and UK‐2 challenged RIII (A) and C57BL (B) mice, detected with Sha31 and 12B2 antibodies. Lanes 1 and 12: molecular mass markers (kDa), Lanes 2 and 11: blank, lane 3: unchallenged proteinase‐K digested mouse brain homogenate, lane 4: mouse challenged with ovine classical scrapie, lanes 5–6 and 8–9: mice challenged with UK‐1 or UK‐2, lane 7: mouse challenged with murine passaged ME7 strain, lane 10: mouse challenged with sheep bovine spongiform encephalopathy (BSE). Exposure time was1 minute for all blots.

Figure 6

LP following sub‐passage of UK‐1 and UK‐2 in wild‐type mice. Lesion profiles (LP) of UK‐1 and UK‐2 at second passage in RIII, C57BL and VM mouse lines are shown plotted alongside reference classical scrapie strains. Where less than five clinically and histopathologically positive mice were available, separate profiles, representing all haematoxylin and eosin (H&E)‐positive mice (dashed line) and mice that were both clinically and H&E positive (solid line) are plotted. Error bars indicate standard error of the mean. The numbers of animals that contributed to each profile and the IP (mean days post inoculation) for each profile are given in parentheses. The 87A in C57BL profile was reproduced from Fraser and Dickinson (23).

Figure 7

UK‐1 and UK‐2 LP following primary isolation in ovine transgenic mice. Lesion profiles (LP) following transmission of UK‐1 and UK‐2 to tg338 mice are shown in (A) and (C), respectively. Profiles represent mice that were both clinically and haematoxylin and eosin (H&E) positive plotted alongside a classical scrapie LP. Error bars indicate standard error of the mean. In (B), UK‐1 profiles were re‐plotted according to the PrP^Sc deposition pattern, denoted P₃₃₈, G₃₃₈ or composite, shown by individual mice.

Figure 8

PrP^Sc deposition patterns of UK‐1 following transmission to tg338 mice. PrP^Sc labeling in tg338 mice showed distinct patterns and deposition types. The characteristic deposition type and labeling within the habenular bodies are also shown. P₃₃₈ (A) was predominantly punctuate and intraneuronal labeling. G₃₃₈ (B) was characterized by granular labeling within the neuropil and distinctive aggregates within the medial habenular bodies. A number of mice showed a mixture of P₃₃₈ and G₃₃₈ (composite) (C). Scales bars represent 100 µm.

Figure 9

Western blot analysis of ovine transgenic mice inoculated with UK‐1. Detection of PrP^Sc in proteinase‐K‐treated brain homogenates by Sha31 and 12B2 monoclonal antibodies. Lane 1: original UK‐1 inoculum, lane 2: sheep bovine spongiform encephalopathy (BSE), lane 3: classical scrapie, lanes 4–8: UK‐1 inoculated tg338 mouse brains exhibiting (with Sha31 mAb) a 19‐kDa profile as referred to the migration of the unglycosylated band (lanes 4–5), a mixed profile (lane 6) or a 21‐kDa profile (lanes 7–8), lane 9: classical scrapie in tg338 mice, lane 10: sheep BSE in tg338 mice.