Transmissible mink encephalopathy: infectivity of corneal epithelium.

Corneal epithelium from hamsters dying of transmissible mink encephalopathy contained a virus titer of 10-4.8 times the 50 percent lethal dose (10-4.8 LD50) per 0.05 milliliter when assayed as a cell suspension derived directly from the infected animal. After one passage in tissue culture, an equivalent concentration of cells contained only 10-0.8 LD50 per 0.05 milliliter.. It is concluded that corneal tissues are infectious; the infectivity may be mainly associated with free nerve endings. However, the most important immediate inference is that corneas from human beings affected with Creuzfeldt-Jakob disease are likely to be lethal if transplanted to healthy recipients.

Transmissible mink encephalopathy: experimental transmission to the squirrel monkey.

A progressive, fatal spongiform encephalopathy developed in three squirrel monkeys 11 months after inoculation with primate-passaged transmissible mink encephalopathy agent. The clinical symptoms and histopathologic and electron microscopic findings suggest that this naturally occurring disease of mink has been transmitted experimentally to squirrel monkeys.

Susceptibility of mink to sheep scrapie.

A progressive, fatal spongiform polioencephalopathy was induced in mink intracerebrally inoculated with a suspension of brain from a Suffolk sheep with naturally acquired scrapie. The clinical signs and pathological lesions of the experimental disease were indistinguishable from transmissible mink encephalopathy, a disease of undetermined origin that occurs in mink.

Human papovavirus (JC): induction of brain tumors in hamsters.

Eighty-three percent of hamsters inoculated at birth with JC virus, a human papovavirus isolated from brain tissue of a case of progressive multifocal leukoencephalopathy, developed malignant gliomas within 6 months. Three brain tumors have been serially transplanted as subcutaneous tumors. JC virus was isolated from five of seven tumors tested. Cells from four tumors were cultivated in vitro. These cells contained an intranuclear antigen with the characteristics of a T antigen, and this antigen was antigenically related to SV40 T antigen. Although virus was not recovered from extracts of serially cultured tumor cells, JC virus was rescued when one tumor cell line was fused with permissive cells.

Effect of bacterial flora and mouse genotype (euthymic or athymic) on scrapie pathogenesis.

Euthymic and athymic female BALB/c mice, reared under either germfree or defined flora conditions, were used to investigate the pathogenesis of scrapie after intracerebral or intraperitoneal inoculation. Time in days to onset of clinical signs (Stage I), to endstage (Stage II), and the time interval between Stage I and Stage II were compared among groups. In addition, scrapie agent titers in spleen were determined at 28 and 90 days after infection, as were agent titers in spleen and brain at Stage II. Three-way analysis of variance indicated that the bacterial flora, the presence or absence of a thymus, and the route of agent inoculation interact to produce significant differences in the pathogenesis of disease. The three factors in the experimental design also influenced the spleen titers of scrapie infectivity. The variation in scrapie pathogenesis among the groups of mice is likely to be mediated by differences in their reticuloendothelial systems. These differences may alter the agent's adsorption in spleen and/or route of transport from spleen to brain.

Photoreceptor degeneration preceding clinical scrapie encephalopathy in hamsters.

Hamsters experimentally inoculated with scrapie had histologic and ultrastructural changes in their retinas before clinical signs of central nervous system disease became evident at 50 days postinoculation. The retinal changes were limited to the photoreceptors and began with degenerative changes in the inner segments as early as 8 days postinoculation. As the inner segment lesions became more prominent, associated degenerative changes within outer segments were seen (38 days postinoculation). An influx of macrophages was associated with increasing degenerative changes; these cells engulfed cellular debris did not appear to contribute directly to the degenerative process. Retinas having the greatest damage to the inner segment area also had moderate reduction in outer nuclear layer density and outer segment length, and in numbers of outer segments. All of the retinal changes described occurred almost exclusively in the central to mid-region of retina with relative sparing of peripheral retina.

Retinal degeneration during clinical scrapie encephalopathy in hamsters.

Weanling hamsters were inoculated intracerebrally with brain suspensions from normal or scrapie-infected hamsters. A third group of uninoculated animal was fed cuprizone. Histologic and electron microscopic examination of the neural retinas and retinal pigment epithelium was done. At 50 days postinoculation, when scrapie-infected animals began to show clinical signs of encephalopathy, there was a variable degree of photoreceptor degeneration. By the time animals were moribund, at 74 days postinoculation, photoreceptor degeneration was severe, as demonstrated by loss of most outer and inner segments and cell bodies. The outer plexiform, inner nuclear, and inner plexiform layers were reduced in thickness. Some retinal pigment epithelial cells contained lipoidal inclusions. The neural retinas and retinal pigment epithelium of noninfected and cuprizone-treated animals were normal. We have previously shown that the scrapie agent accumulates in the retina; that together with our present work, we conclude that the scrapie agent is the cause of photoreceptor degeneration in experimentally inoculated hamsters.

Ocular effects of scrapie agent in hamsters: preliminary observations.

Scrapie is caused by one of a group of so-called slow viruses responsible for the subacute spongiform encephalopathies. In the present study, young hamsters were inoculated intracerebrally with hamster-adapted scrapie agent. At termination, all inoculated animals showed signs and central nervous system pathology compatible with scrapie infection. The eyes appeared well developed grossly, but histologically the retina and optic nerve were abnormal. There were varying degrees of thinning of the retina, with the photoreceptor layer being most severely affected. Although the ganglion cell layer was not much different from the controls, the optic nerve appeared more cellular than that of the controls.

Bovine spongiform encephalopathy: a new disease of cattle?

Bovine spongiform encephalopathy (BSE) was first recognized in Great Britain in 1985. Most believe that the disease is of recent origin initiated by feeding rendered animal protein from scrapie-infected sheep to cattle, then perpetuated by feeding rendered infected cattle to other cattle. This paper explores an alternative hypothesis that BSE existed in cattle populations in an unrecognized form for a much longer time until amplified by changes in the rendering process that allowed cattle to cattle transmission to occur. This viewpoint is supported by observations that transmissible mink encephalopathy, a disease that first occurred 45 years ago, is likely caused by feeding downer cows to mink, and that the sporadic form of Creutzfeldt-Jakob disease occurs spontaneously with no evidence of natural transmission. This epidemiologic scenario on the origin of BSE has important implications for prevention of the disease in BSE-free countries. Mainly, emphasis needs to put on practices of feeding animal protein to cattle rather than in reducing the prevalence of sheep scrapie. If BSE is already present in the cattle population, the major threat becomes feeding cows to cows.

Immunolocalization of scrapie amyloid in non-congophilic, non-birefringent deposits in golden Syrian hamsters with experimental transmissible mink encephalopathy.

Transmissible mink encephalopathy (TME), a naturally occurring subacute spongiform encephalopathy in commercially ranch-reared mink (Mustela vision), is characterized neuropathologically by spongiform changes in the neuropil, intracytoplasmic neuronal vacuolation and astrocytic hypertrophy and hyperplasia. Amyloid deposits have not been observed in brain tissue sections from animals with natural and experimental TME using conventional histochemical stains such as Congo red. To determine if amyloid deposits be visualized by immunocytochemical techniques, we stained formalin-fixed, formic acid-treated brain tissue sections from several animal species with natural and experimental TME, using a rabbit antiserum directed against scrapie amyloid (PrP27-30). Scrapie amyloid-immunoreactive deposits were found in golden Syrian hamsters experimentally infected with TME, but were absent in mink with natural and experimental TME, as well as in ferrets and squirrel monkeys with experimental TME. The scrapie amyloid-immunoreactive deposits, which were non-congophilic and non-birefringent, were distributed in the subependymal, subpial and perivascular regions of the brain, as in hamsters infected with the 263K strain of scrapie. Ultrastructurally, scrapie amyloid-immunoreactive deposits revealed a collection of degenerating neurites with numerous abnormal mitochondria and degenerating synapses. Amyloid fibrils were not observed. Anti-scrapie amyloid antibodies immunoabsorbed with scrapie amyloid abolished immunostaining. Our data indicate the presence of scrapie amyloid lacking the molecular conformation of amyloid fibrils in hamsters with experimental TME.

Immunohistochemical detection of prion protein in sheep with scrapie.

Prion protein (PrP), which is involved in the pathogenesis of scrapie, occurs in 2 forms. The form extracted from scrapie brain is protease resistant (PrP-res), whereas PrP from normal brain is protease sensitive (PrP-sen). This study examined whether PrP-res could be detected in brains of sheep with scrapie by immunohistochemistry (IHC). A suitable IHC procedure was developed using brain tissue from hamsters that had been inoculated with the transmissible mink encephalopathy agent. Tissue samples were fixed in PLP (periodate, lysine, paraformaldehyde) that contained paraformaldehyde at a concentration of 0.125%. Before application of the IHC technique, tissue sections were deparaffinized and treated with formic acid to simultaneously enhance PrP-res immunoreactivity and degrade PrP-sen. Primary antibody was obtained from a rabbit immunized to PrP-res extracted from brains of mice with experimentally induced scrapie. Brain from 21 sheep with histopathologically confirmed scrapie were examined by IHC. In all 21 brains, PrP-res was widely distributed throughout the brain stem. Staining was particularly intense in neuronal cell bodies and around blood vessels. The IHC technique successfully detected PrP-res in brain samples that had been frozen or that were severely autolyzed before fixation in PLP. Brains from 11 scrapie-suspect sheep that were not considered histologically positive were also examined by IHC. PrP-res was found in 4 of these brains. Sections of brains from 14 clinically normal sheep did not have detectable PrP-res. Results of this study indicate that IHC detection of PrP-res is equivalent, and perhaps superior, to histopathology for the diagnosis of scrapie in sheep. Furthermore, IHC is applicable to tissues that have autolytic changes or processing artifacts that prevent satisfactory histopathologic evaluation for lesions of scrapie.

Experimental infection of cattle with the agents of transmissible mink encephalopathy and scrapie.

Cattle are susceptible to experimental infection with the Stetsonville isolate of the transmissible mink encephalopathy (TME) agent. To determine if they are susceptible to other TME isolates, two groups of calves were inoculated intracerebrally with homogenate of mink brain containing the Hayward isolate or the Blackfoot isolate. For comparison, a third group was inoculated with a brain homogenate from a steer infected with the Stetsonville isolate in its primary cattle passage and a fourth group was inoculated with a pool of brain homogenate from three cattle experimentally infected with a sheep and goat scrapie agent in its primary cattle passage. Clinical signs of neurological disease appeared in each steer of every group between 15 and 25 months after inoculation. An encephalopathy characterized by severe spongiform change and pronounced astrocytosis occurred in the three groups inoculated with the TME agent. In contrast, the neurohistological changes in the steers inoculated with the cattle-passaged scrapie agent were slight and subtle. Analysis of the octapeptide repeat region of the bovine protease-resistant protein (PrP) gene showed that variations in incubation period, clinical signs, and neurohistological changes were unrelated to the homozygous or heterozygous condition of six or six/five octapeptide repeats.

Transmissible mink encephalopathy.

Transmissible mink encephalopathy (TME) is a rare disease of ranch-raised mink caused by exposure to an as yet unidentified contaminated food ingredient in the ration. The clinical and pathological similarities between TME and scrapie, together with the indistinguishable physicochemical characteristics of their transmissible agents, suggest that sheep may be the source of infection. However, experimental testing of oral susceptibility of mink to several different sources of sheep scrapie have been unsuccessful. These results indicate that either the feeding of scrapie-infected sheep tissues to mink is not the cause of TME, or that there exists a strain of sheep scrapie having high mink pathogenicity that remains unknown. Additional sources of sheep scrapie need to be tested in mink, and epidemiological investigations of new incidents of TME need to emphasise obtaining a thorough history of past feeding practices.

Immunochemical characterization of proteins from scrapie-infected hamster brain, using immunoblot analysis.

Preparations of brain plasma membrane from scrapie-infected or noninfected hamsters were extracted with a solvent and were used to inoculate rabbits. Antisera evaluated by immunoblot analysis revealed a protein of 45 kD in scrapie-infected hamster brain that had a greater signal compared with proteins of comparable relative mass in noninfected brain. This 45-kD protein was not increased in scrapie-infected mouse, sheep, or goat brain. Seemingly, the 45-kD protein may be a degradation product of glial fibrillary acidic protein.