BSE can propagate in sheep co-infected or pre-infected with scrapie

Abstract

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrP^Sc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrP^Sc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrP^Sc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

Figure 1

Incubation periods of individual VRQ/ARQ, ARQ/ARQ and VRQ/VRQ sheep. Sheep were inoculated with SSBP/1 scrapie, BSE or mixtures (A,B) or were infected with natural scrapie and then superinfected with BSE (C). The codon 141 PRNP genotype is indicated: LL₁₄₁ (grey fill); LF₁₄₁ (black fill); FF₁₄₁ (no fill). Circles are clinical and pathology positive sheep, triangles are non-TSE animals. In A and B, lines represent SSBP/1, BSE, S/B, S + B3m dating from SSBP/1 inoculation (S), and S + B3m dating from BSE inoculation (B). In C, lines represent natural scrapie (NS) and NS superinfected with BSE at 8 months of age (NS + B8m) or 21 months of age (NS + B21m). For natural scrapie incubation periods are calculated from birth (exposure to scrapie).

Figure 2

PrP^Sc phenotype in sheep brain. Brain homogenates were digested with PK and analysed on western blots probed with 6H4 (A) or P4 (B). Representative blots are shown. Approximate molecular weight (MW) indicated at 30 kDa and 20 kDa. Individual sheep references (e.g. Z107 or 70 × 71 formats) are indicated. A relatively low MW for the unglycosylated PrP band with 6H4 staining or a relatively low P4 signal (a ratio of 6H4/P4 signal of > 2) indicated the PrP^Sc had a BSE phenotype; strong P4 staining (a ratio of 6H4/P4 signal of 2 or less) and a relatively high MW unglycosylated band indicated a scrapie phenotype. These criteria and strain phenotypes are indicated. Shaded samples indicate sheep inoculated/infected with a single strain, all of these samples conformed to established molecular phenotypes of BSE or scrapie PrP^Sc and so provided within-blot controls for these phenotypes. Where samples were subjected to repeat analysis, the assay gave consistent results (examples shown for Z107, 70 × 71 and Z13).

Figure 3

sPMCA amplification of BSE prions in sheep brain and LRS tissues. Following amplification of samples by sPMCA, PK-resistant PrP^Sc was detected with either SHa31 (top panels) or P4 (lower panels). Representative blots are shown. Br brain, Sp spleen, Ln PSLN tissue. Scrapie (S+) and BSE (B+) brain controls were also analysed on each blot. Z37 and Z43 were ARQ/ARQ sheep inoculated with BSE only; Z97 was an ARQ/ARQ sheep inoculated with S/B; Z90, Z115 and Z73 were ARQ/ARQ sheep inoculated with S + B3m; Z94 was a VRQ/ARQ sheep inoculated with SSBP/1 only; Z65 and Z48 were VRQ/ARQ sheep inoculated with BSE only; sheep 75 × 44 had natural scrapie and was superinfected, NS + B8m. Molecular weight markers (M) are shown at 20, 30 and 40 kDa and positions are also indicated on each blot.

Figure 4

Discriminatory immunohistochemistry of sheep brain and tonsil from a mixed infection sheep with scrapie signal in brain and BSE in periphery. Sections of medulla [lateral cuneate nucleus; (A, B); × 20] and tonsil [(C,D); × 10) from VRQ/ARQ sheep Z115 were stained with antibody R145 in (A) and (C); or 12B2 in (B) and (D). Results show labelling of intraneuronal PrP^d with both R145 and 12B2 in brain (A,B), whereas in tonsil (C,D) labelling of tingible body macrophages within germinal centre was seen only with R145, the 12B2 reaction being negative.