The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie.

Despite intensive studies on sheep scrapie, a number of questions remain unanswered, such as the natural mode of transmission and the amount of infectivity which accumulates in edible tissues at different stages of scrapie infection. Studies using the mouse model proved to be useful for recognizing scrapie strain diversity, but the low sensitivity of mice to some natural scrapie isolates hampered further investigations. To investigate the sensitivity of bank voles (Myodes glareolus) to scrapie, we performed end-point titrations from two unrelated scrapie sources. Similar titres [10(5.5) ID50 U g(-1) and 10(5.8) ID50 U g(-1), both intracerebrally (i.c.)] were obtained, showing that voles can detect infectivity up to 3-4 orders of magnitude lower when compared with laboratory mice. We further investigated the relationships between PrPSc molecular characteristics, strain and prion titre in the brain and tonsil of the same scrapie-affected sheep. We found that protease-resistant PrPSc fragments (PrPres) from brain and tonsil had different molecular features, but induced identical disease phenotypes in voles. The infectivity titre of the tonsil estimated by incubation time assay was 10(4.8) i.c. ID50 U g(-1), i.e. fivefold less than the brain. This compared well with the relative PrPres content, which was 8.8-fold less in tonsil than in brain. Our results suggest that brain and tonsil harboured the same prion strain showing different glycoprofiles in relation to the different cellular/tissue types in which it replicated, and that a PrPSc-based estimate of scrapie infectivity in sheep tissues could be achieved by combining sensitive PrPres detection methods and bioassay in voles.

Comparative studies on the thermostability of five strains of transmissible-spongiform-encephalopathy agent.

The causal infectious agents of TSEs (transmissible spongiform encephalopathies or prion diseases) are renowned for their resistance to complete inactivation. Survival of TSE infectivity after autoclaving potentially compromises many procedures where TSE infectivity may be present, including surgical instrument sterilization. In the present study, the heat inactivation properties of five different TSE agents were tested in a variety of experiments by exposing them to a range of heat inactivation conditions. Although TSE infectivity was reduced after heating to 200 degrees C in a hot air oven, substantial amounts of infectivity remained. Unlike wet heat inactivation, no TSE strain-dependent differences were observed in the reduction in the amounts of infectivity produced by dry heat inactivation. However, the incubation periods of mice infected with one dry heated TSE strain, ME7, were substantially prolonged, whereas there was little or no effect for two other TSE models. Varying autoclaving conditions for three TSE strains between 132 and 138 degrees C, and times of exposure between 30 and 120 min, had little or no effect on the recovery of TSE infectivity. The results illustrate the limitations of TSE agent inactivation using heat-based methods. The results support the hypothesis that the structures of TSE agents are stabilized during heat-inactivation procedures, rendering them much more refractory to inactivation. This may occur through dehydration of the causal agents, specifically through the removal of the water of solvation from agent structures and hence stabilize interactions between prion protein and TSE agent-specific ligands.

Thermostability of mouse-passaged BSE and scrapie is independent of host PrP genotype: implications for the nature of the causal agents.

Five experimentally maintained strains of scrapie and BSE agents have been passaged in two PrP genotypes of mice. Brain macerates were autoclaved at 126 degrees C and the levels of surviving infectivity were measured by titration. There was a large difference in the survival properties of transmissible spongiform encephalopathy (TSE) infectivity between TSE strains. PrP genotype had little effect. Phenotypic properties of the TSE strains were not affected with the exception that with one strain (ME7), incubation periods of the heated sample were longer than the controls given equivalent doses. It is concluded that PrP is probably not responsible for differences in thermostability between strains. More likely, a host-independent molecule which differs in covalent structure between strains accounts for these properties.