Further characterisation of transmissible spongiform encephalopathy phenotypes after inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

Abstract

Background: The infectious agent responsible for the bovine spongiform encephalopathy (BSE) epidemic in Great Britain is a transmissible spongiform encephalopathy (TSE) strain with uniform properties but the origin of this strain remains unknown. Based on the hypothesis that classical BSE may have been caused by a TSE strain present in sheep, cattle were inoculated intracerebrally with two different pools of brains from scrapie-affected sheep sourced prior to and during the BSE epidemic to investigate resulting disease phenotypes and characterise their causal agents by transmission to rodents.

Results: As reported in 2006, intracerebral inoculation of cattle with pre-1975 and post-1990 scrapie brain pools produced two distinct disease phenotypes, which were unlike classical BSE. Subsequent to that report none of the remaining cattle, culled at 10 years post inoculation, developed a TSE. Retrospective Western immunoblot examination of the brains from TSE cases inoculated with the pre-1975 scrapie pool revealed a molecular profile similar to L-type BSE. The inoculation of transgenic mice expressing the bovine, ovine, porcine, murine or human prion protein gene and bank voles with brains from scrapie-affected cattle did not detect classical or atypical BSE strains but identified two previously characterised scrapie strains of sheep.

Conclusions: Characterisation of the causal agents of disease resulting from exposure of cattle to naturally occurring scrapie agents sourced in Great Britain did not reveal evidence of classical or atypical BSE, but did identify two distinct previously recognised strains of scrapie. Although scrapie was still recognizable upon cattle passage there were irreconcilable discrepancies between the results of biological strain typing approaches and molecular profiling methods, suggesting that the latter may not be appropriate for the identification and differentiation of atypical, particularly L-type, BSE agents from cattle experimentally infected with a potential mixture of classical scrapie strains from sheep sources.

Fig. 1

Discriminatory Western immunoblot of brain samples from cattle inoculated with the pre-1975 and post-1990 scrapie brain pools. Lanes 1–9 cattle inoculated intracerebrally with the pre-1975 scrapie pool: P75-1, P75-2, P75-3, P75-4, P75-5, P75-6, P75-7, P75-8 and P-75-9. Lanes 10–16 cattle inoculated intracerebrally with the post-1990 scrapie pool: P90-2, P90-3, P90-1, P90-4, P90-5, P90-6 and P90-7. Lanes L, H, C, O controls: L-type BSE, H-type BSE, classical BSE, ovine scrapie. Lanes M molecular mass marker. Animal P90-4, sample lane 13, was an outlier with a lower molecular mass of the unglycosylated band with mAbs Sha31 and SAF84 compared to the other samples previously tested with mAb 6H4. The sample of the other outlier P90-5 (determined previously by testing caudal medulla), sample lane 14, consisted here of rostral medulla and gave a molecular profile similar to the others of the group as observed in the original blot when both brain samples were tested [2]. The lane numbers of those cattle that provided the inocula for bank voles and mice are underlined.

Fig. 2

PK susceptibility ratio for the mild and stringent digestion conditions of samples from the pre-1975 scrapie pool compared to BSE. Controls comprise a UK classical BSE (C-type) sample (two analyses of the same sample and mean) and a UK L-type BSE sample (two analyses of the same sample and mean). Detection with mAbs Sha31 and 6H4. The case that provided the inoculum for bank voles and mice is underlined.

Fig. 3

Scattergram showing the relative glycosylation quantity analysis of brain material from cattle inoculated with the pre-1975 and post-1990 scrapie pools compared to BSE and ovine scrapie controls. Detection with mAb Sha31. Controls comprised an ovine scrapie case, a classical BSE case (two analyses of the same sample each) and L-type and H-type BSE cases from the UK. The black circle indicates all 16 samples from the intracerebrally inoculated cattle clustering together around the L-type BSE control (blue square). The cases that provided the inocula for bank voles and mice are underlined.

Fig. 4

Western immunoblot of bovine brain samples used for transmission in bank voles and comparison with BASE and scrapie. (1) P75-7, (2) molecular mass marker, (3) P90-4, (4) BASE, (5) classical scrapie. Detection with mAb SAF84.

Fig. 5

Western immunoblot and antibody signal ratio of vole brain samples after primary passage of bovine scrapie and comparison with vole-adapted classical scrapie and BSE. WB analysis of PrP^res in voles infected with P75-7 (primary passage) compared with a representative sample of P90-4 (primary passage), vole-adapted classical scrapie and BSE. Replica blots were developed with mAbs SAF84 (upper panel) and 12B2 (lower panel) to compare the antibody reactivity with each sample similar to the discriminative WB used for samples from small ruminants. The graph on the bottom shows the SAF84/12B2 signal ratio relative to the scrapie control. Values higher than 1 denote more C-terminal PK-cleavage and consequential loss of the epitope of 12B2.

Fig. 6

Lesion profiles of vole-adapted bovine scrapie at second and third passages and comparisons with other isolates. a Lesion profiles of vole-adapted P75-7 at second and third passages in voles in comparison with the two ‘sub-strains’ (17 and 18K) of vole-adapted CH1641. b Lesion profiles of vole-adapted P90-4 at second and third passages in voles in comparison with two natural scrapie isolates from sheep (SS-UK6 and SCR6). Scored neuroanatomical areas: 1 medulla, 2 cerebellum, 3 superior colliculus, 4 hypothalamus, 5 thalamus, 6 hippocampus, 7 septum, 8 retrosplenial and adjacent motor cortex, 9 cingulate and adjacent motor cortex.

Fig. 7

Western immunoblot profiles of tg110 mice inoculated with original scrapie brain pools and bovine scrapie sources P90-1 and P75-7. Lanes on top blot. 1 and 14 Molecular mass marker (kDa). 2 Post-1990 scrapie brain pool. 3 Post-1990 scrapie brain pool in Tg110 mice (first passage). 4 Post-1990 scrapie brain pool in Tg110 mice (second passage). 5 P90-1. 6 P90-1 in Tg110 mice (first passage). 7 Pre-1975 scrapie brain pool. 8 Pre-1975 scrapie brain pool in Tg110 mice (first passage). 9 Pre-1975 scrapie brain pool in Tg110 mice (second passage). 10 P75-7. 11 P75-7 in Tg110 mice (first passage). 12 Negative control (brain from non-inoculated Tg110 mice). 13 Positive control (brain from tg110 mice inoculated with classical BSE: case VLA-PG817/00). Antibody: Sha31. The molecular profile of the original post-1990 scrapie brain pool is maintained after passage in Tg110 mice (lanes 2–4) but does not resemble the profile of P90-1 or the mice inoculated with P90-1 brain (lanes 5, 6). By contrast, the profile of the original pre-1975 brain pool (lane 7) differed from the profile obtained from inoculated Tg110 mice (lanes 8, 9), P75-7 (lane 10) and the mice inoculated with P75-7 brain, which were all similar. The profiles obtained in individual mice after inoculation with the steers’ brains were identical within each inoculation group (bottom blot).