Karen A. Bowman

Scrapie prions aggregate to form amyloid-like birefringent rods

A large scale purification protocol employing zonal rotor centrifugation has been developed for scrapie prions. The extensively purified fractions derived using this protocol contained only one major protein, designated PrP, and rod-shaped particles. The rods measured 10 to 20 nm in diameter and 100 to 200 nm in length by negative staining; no other particles were consistently observed. SDS denaturation caused the rods to disappear, prion infectivity to diminish, and PrP to become sensitive to protease digestion. Arrays of prion rods ultrastructurally resembled purified amyloid and showed green birefringence by polarization microscopy after staining with Congo red dye. The rods appear to represent a polymeric form of the scrapie prion; each rod may contain as many as 1,000 PrP molecules. Our findings raise the possibility that the amyloid plaques observed in transmissible, degenerative neurological diseases might consist of prions.

Replication of scrapie prions in hamster eyes precedes retinal degeneration.

Progressive degeneration of outer retinal structures occurs in hamsters with scrapie. In order to determine the relationship between histopathologic changes and replication of the scrapie agent, hamsters were inoculated intracerebrally with approximately 10(7) ID50 units. Animals sacrificed at 50 days after inoculation showed no signs of neurologic dysfunction, but had high titers of the scrapie agent or prions in both neural and nonneural portions of the eye. Prion titers in retina were greater than 10(7) ID50 units/ml of 10% (w/v) homogenate and equal to those found in optic nerve and brain. No histopathologic changes were seen by light microscopy in any ocular structure. At 70 days after inoculation, neurologic dysfunction was profound. The titers of the scrapie agent in brain, lens, retinal pigment epithelium, cornea, retina, and optic nerve were not significantly changed compared to those found at 50 days; however, retinal degeneration was severe. No morphologic changes were observed in cornea, pigment epithelium or optic nerve. These findings show that scrapie prion replication to maximal levels precedes the onset of degenerative changes in retina. Furthermore, the retina is preferentially susceptible to the degeneration induced by the scrapie agent while the other ocular structures containing significant levels of prions seem to escape injury.

Immunological studies of scrapie infection

Abstract Scrapie is a show, degenerative nervous system disease of sheep and goats. No obvious inflammatory or immunological response occurs despite early replication of the srapie agent in the spleen. The extend earlier investigations (Garlin et al. 1978a, b) on the immunorcactivity of lymphocytes during scrapie infection, purified populations of T and B cells were prepared from BALB/c mice. No consistent differences in the response to mitogens of T and B cells from scrapie-infected and control mice were observed. An assessment of T-dependent B cell function was obtained using a hemolytic plaque-forming cell assay, but again no differences between scrapie-infected and control splenocytes could be detected. To study the possible influence of B cell maturation on scrapie infection, incubation periods were measured in CBA/N mice, bearing an X-linked deficiency of mature B cells. Scrapie incubation periods were identical in affected males and unaffected females, indicating that B cell maturation does not influence the course of scrapie. Using highly enriched preparations of the scrapie agent, we attempted to raise antibodies in rabbits using an extensive immunization protocol. Scrapie-specific antibodies could not be detected using either indirect immunofluorescence or enzyme-linked immunosorbent assay (ELISA). Some antisera reacted more strongly with scrapie than with control preparations; however, these differences disappeared when the antisera were

Replication of the scrapie agent in hamster brain precedes neuronal vacuolation.

The scrapie agent causes a degenerative neurological disorder in sheep and goats after a prolonged incubation period. Hamsters inoculated intracerebrally with 107 ID50 units of the scrapie agent develop clinical signs of neurological dysfunction 60–65 days later. The titers of scrapie agent in selected regions of the central nervous system (CNS) of hamsters were determined prior to the onset of clinical illness. At 48 days after inoculation, the cerebrum, cerebellum, brain stem, and spinal cord contained 9.3, 9.1, 9.3, and 8.6 log ID50 units/g of tissue, respectively. Sections from the cerebrum showed minimal vacuolation without any astrogliosis. The spinal cord and cerebellum revealed no lesions. At 71 days after inoculation, when clinical signs of scrapie were prominent, another group of hamsters was evaluated. The mean titers of the agent in the same CNS regions were virtually unchanged, but severe vacuolation and moderate astrogliosis were present in the cerebral cortex. A moderate degree of vacuolation and astrogliosis were observed in the cerebellum, brain stem, and spinal cord. These studies indicate that replication of the scrapie agent in the hamster is uniform throughout the CNS and precedes the development of pathological changes.

Toward Development of Assays for Scrapie-Specific Antibodies

At least 40 attempts have been made to detect antibodies directed against the scrapie agent (Table 1). All have failed. More than 10 were by neutralization (1–6), seven by precipitation in gel or tubes (3,7–9), eight by complement fixation (3,5,7), and six by direct or indirect immunofluorescence procedures (3,6,8). Passive hemagglutination, passive hemolysis, passive anaphylaxis, immunoconglutinin (7), and anti-nuclear (10) and anti-cardiolipin (7) antibody tests were also employed. Antisera from goats, sheep, mice, rabbits, roosters, monkeys and humans have been tested.

Prions: Methods for Assay, Purification, and Characterization

Publisher Summary This chapter reviews methods for bioassay and purification as well as molecular and morphological characterization of the scrapie prion. The unusual molecular properties of the scrapie agent seem to distinguish it from both viruses and viroids and have led to the introduction of the term prion for the infectious particle. Procedures that hydrolyze or modify proteins produced a diminution of scrapie prion infectivity; in contrast, procedures that hydrolyze or modify nucleic acids do not alter the infectivity. At present, the only methods for measuring scrapie prion infectivity remain the incubation time interval assay and the end-point titration. Both methods are extremely slow because they require waiting for the onset of clinical neurological dysfunction, following a prolonged incubation period. An end-point titration for the scrapie prion is performed by serially diluting a sample at 10-fold increments. Each dilution is typically inoculated intracerebrally into four to six animals and the waiting process ensues. As the highest dilutions at which scrapie develops are the only observations of interest, 10–12 months must be allowed to pass before the titration may be scored if mice are used.

Scrapie Prions and Degenerative Diseases

Almost two decades ago, a flurry of excitement surrounded studies on slow infectious agents. It was a time when an eight-year search for the cause of kuru was drawing to a close.1–3 Kuru had been classified as a degenerative neurologic disease of unknown origin that was confined to the Fore people and their neighboring tribes in the eastern highlands of Papua New Guinea. Almost every etiologic possibility for degenerative neurologic diseases had been considered with respect to kuru, but none had been identified.4 Based on pathologic and clinical similarities between kuru and scrapie, a neurologic disease of sheep, Hadlow suggested in 1959 that kuru might be caused by a slow infectious agent. Three years later, studies were begun by Gajdusek, Gibbs, and Alpers to test this hypothesis. In 1965, chimpanzees inoculated with brain tissue specimens from patients dying of kuru developed a kuru-like illness.2