Characterization of Bovine Spongiform Encephalopathy and Scrapie Strains/Isolates by Immunochemical Analysis of PrPSc.

In the past two decades, thoroughly standardized mouse incubation time and brain lesion profile scoring assays have been developed to discriminate between prion strains. However, in these mouse infection experiments, large numbers of animals (about 20 mice/line) from three different highly inbred mouse lines (C57Bl, VM95, RIII), plus their intercrosses, need to be infected, and their brain tissues subsequently examined (1-8). Although results obtained are highly reliable, the effort and time needed for conducting these experiments are considerable. Therefore, alternative criteria and techniques have been developed to characterize transmissible spongiform encephalopathy (TSE) agents. Prion infections are accompanied by the accumulation of an abnormal isoform (designated PrPSc) of normal host-encoded prion protein (PrPC). Both isoforms have the same amino acid sequence and molecular mass, but differ significantly in their three-dimensional structure and biochemical characteristics. The three-dimensional structure of PrPC is characterized by a high ?-helical content (9); all or part of it undergoes a posttranslational modification to ß-sheet in PrPSc (10-12). Although PrPC (33-35 kDa) is completely hydrolyzed by protease treatment, PrPSc is partially resistant to proteinase K(PK) as 62 N-terminal amino acids are cleaved, leaving a core fragment of approx 141 amino acids (27-30 kDa) unhydrolyzed (13).

Protease-resistant prion protein in brain and lymphoid organs of sheep within a naturally scrapie-infected flock.

The hallmark of transmissible spongiform encephalopathies (TSE), such as scrapie in sheep, is the accumulation in tissues of an insoluble and protease resistant form (PrPres) of the cellular prion protein. In this study, we evaluated whether the diversity in both the clinical pattern and the PrP genotypes of scrapied sheep from the same flock was connected with different levels and/or glycoform patterns of the PrPres in the brain and lymphoid organs of the animals. Whereas the PrPres levels in spleen, lymph nodes and tonsils from sheep of different PrP genotypes and clinical status appeared comparable, they were highly variable in brain, particularly in the brain stem and the cerebellum. PrPres was only detected in sheep bearing at least one VRQ allele, including three asymptomatic sheep and the highest PrPres load was found in the cerebellum of VRQ/VRQ animals. All together, levels of PrPres in brain did not necessarily correlate with the severity of the clinical disease but might depend on the PrP genotype of the animals. Different brain regions from a given sheep displayed a similar glycopattern of PrPres, whereas the apparent molecular sizes of the unglycosylated and diglycosylated forms of the protein differed between brain and lymphoid tissues. We did not find any notifiable differences in the glycopattern of PrPres in brain from sheep of different PrP genotypes or different clinical status and this PrPres glycotype was also similar to that found in brain from four cattle BSE.

Characterization of BSE and scrapie strains/isolates.

Following the BSE epidemic in cattle and the emergence of a variant form of Creutzfeldt-Jakob disease in humans, the question was raised whether BSE has been transmitted to small ruminants by the inadvertent feeding of infectious meat and bone meal. Such infections could easily be concealed in countries where scrapie is endemic. To address this issue by immuno-chemically analyzing the PrP(Sc) fragments, we have developed two lines of research. Firstly we have focused on the development of criteria for the differential characterization of experimental BSE and scrapie strains/isolates in rodents. To date, three criteria have been identified: quantification of the relative banding intensities of PrP(Sc) glycotypes using a photoimaging technique; the non-uniform kinetic of proteinase K degradation of PrP(Sc); and differences in the molecular masses of their non-glycosylated PrP(Sc) fragments after PK cleavage in immunoblot. The second line of research focused on the implementation of the criteria described above to representative samples from scrapie diseased Irish sheep. Using these three criteria, no evidence was found for the presence of a BSE infection in these animals. However, the final conclusion must take into account the results of mouse incubation time and mouse lesion profile data which are currently being generated.