J, Comp.

Path.

1992 Vol.

106, 361-381

Comparative Ultrastructural Neuropathology Naturally Occurring Bovine Spongiform Encephalopathy and Experimentally Induced and Creutzfeldt-Jakob Disease

of Scrapie

P. P. Liberski*f, R. Yanagihara*, G. A. H. Wellst, C. J. Gibbs, Jr* and D. C. Gajdusek* *Laboratory of Central .Nervous System Studies, National Institute of .Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, U.S.A., 7 Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, .New Haw, Weybridge, Surrey, U.K. and z Electron Microscopy Laboratory, Department of Oncology, Medical Academy tddi, Lddi, Poland

Summary We report the ultrastructural neuropathology of bovine spongiform encephalopathy (BSE), a recently described slow virus disease first recognized in Friesian/Holstein cattle, and compare it to that of experimental scrapie and Creutzfeldt-Jakob disease. The spongiform change, which was most pronounced in the central grey matter of the midbrain, consisted of membrane-bound vacuoles within neuronal processes, containing curled membrane fragments, secondary chambers and vesicles. Axons and dendrites accumulated whorls of neurofilaments and other subcellular organelles, such as mitochondria and dense bodies, which were entrapped within the filamentous masses. Other neurites accumulated electron-dense bodies, and still others electron-lucent cisterns and branching tubules. Membrane-bound neuronal inclusions, composed of tubules measuring 10 nm in diameter, were found in axonal terminals. Tubulovesieular structures were loosely packed and were occasionally surrounded by a common membrane, a finding previously described only in natural scrapie in sheep. Except for the intraneuronal inclusions, all of the ultrastructural features of BSE resembled those found in scrapie and Creutzfeldt-Jakoh disease.

Introduction Bovine spongiform encephalopathy (BSE), a disease recently discovered among Friesian/Holstein cattle in England (Wells, Scott, Johnson, Gunning, Hancock, Jeffrey, Dawson and Bradley, 1987; Scott, Aldridge, Holmes, Milne and Collins, 1988), is characterized clinically by apprehension, hyperaesthesia, fear and aggressive behaviour, exaggerated startle response to external stimuli and into-ordination of gait with hypermetria leading to falling (Wells et al., 1987). Typically, the disease has an insidious onset and is relentlessly progressive. Neuropathological findings include symmetrical spongiform change in Address for correspondence: Bethesda, MD 20892, U.S.A. 002 lb9975/92/040361+

Dr

2 I $03.00/O

R. Yanagihara,

National

Institutes

of Health,

Bldg.

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36, Rm.

5B-21,

Press Limited

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the neuropil with intraneuronal vacuoles and astrocytosis. Scrapie-associated fibrils (SAF), composed of prion protein, have been isolated from brains of cattle with BSE (Wells et al., 1987; Hope, Reekie, Hunter, Multhaup, Beyreuther, White, Scott, Stack, Dawson and Wells, 1989; Scott, Wells, Stack, White and Dawson, 1990). Brain homogenates from BSE-affected cattle have produced a scrapie-like syndrome with characteristic neuropathology in mice 292 to 342 days following intracerebral inoculation (Fraser, McConnell, Wells and Dawson, 1988) and 15 to 18 months after oral ingestion (Barlow and Middleton, 1990). Furthermore, BSE has been experimentally transmitted to cattle (Dawson, Wells and Parker, 1990). All available data indicate that BSE is a new disease within the subacute spongiform virus encephalopathies (Gajdusek, 1977), which include kuru (Gajdusek, Gibbs and Alpers, 1966), Creutzfeldt-Jakob disease (CJD) (Gibbs, Gajdusek, Asher, Alpers, Beck, Daniel and Matthews, 1968) and Gerstmann-Straussler-Scheinker syndrome (Masters, Gajdusek and Gibbs, 1981) in man, scrapie in sheep and goats (Dickinson, 1976), transmissible mink encephalopathy in ranch-reared mink (Marsh and Hanson, 1979) and chronic wasting disease in captive mule deer and elk (Williams and Young, 1980, 1982). We report, for the first time, the ultrastructural neuropathology of BSE and compare it with that of experimentally transmitted scrapie and CJD. Spongiform change, astrocytic gliosis, neuroaxonal dystrophy and membrane-bound tubular inclusions comprised the ultrastructural neuropathology ofBSE. These electron microscopical findings provide further compelling evidence that BSE is a spongiform virus encephalopathy. Materials

and

Methods

Twenty sampIesof meduila (obex at the IeveI of the solitary tract nucleus and the spinal tract nucleus of the trigeminal nerve), central grey matter of the midbrain and frontal cortex at the gyrus marginalis were obtained several minutes post-mortem from a 6-year-old, BSE-affected Friesian/Holstein cow. The animal developed clinical signs of BSE consisting of behavioural change marked by hypersensitivity to touch and sound, and excessive ear movement and teeth grinding. The disease progressed rapidly with weight loss and wasting and the animal was killed 3 weeks after the onset of disease. Tissues were fixed by immersion for approximately 3 h in 3 per cent glutaraldehyde freshly prepared in 0.2 M phosphate buffer, then transferred to the same buffer before trans-shipment from the United Kingdom to Bethesda, Maryland, U.S.A. Brain tissues from a normal cow were processed in an identical manner. In addition, for comparative ultrastructural studies, 10 NIH Swiss mice and 10 golden Syrian hamsters terminally ill with the Fujisaki strain of CJD virus (Tateishi, Ohta, Koga, Sato and Kuroiwa, 1978; Liberski, Yanagihara, Asher, Gibbs and Gajdusek, 1989b; Liberski, Yanagihara, Gibbs and Gajdusek, 1989e) and the 263K strain of scrapie virus (Kimberlin and Walker, 1977; Liberski, Asher, Yanagihara, Gibbs and Gajdusek, 1989d), respectively, were fixed by intracardiac perfusion with 1 per cent paraformaldehyde and 1.5 per cent glutaraldehyde prepared in either phosphate or cacodylate buffer (pH 7.4). All specimens were postfixed in 1 per cent osmium tetroxide for 2 h, dehydrated through a graded series of ethanol and propylene oxide and embedded in Embed (Electron Microscopy Sciences, Fort Washington, PA, U.S.A.). Ultrathin sections, stained with lead citrate and uranyl acetate, were examined with either a Hitachi 11E or a Philips 300 transmission electron microscope at 75 kV or 60 kV, respectively.

363

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Results Routine neuropathological examination by light microscopy revealed mild changes in the ventral grey matter, moderate changes in the solitary tract nucleus and severe lesions in the spinal tract nucleus of the BSE-affected cow. Ultrastructurally, the pathological findings in the BSE-affected cow resembled those in scrapie-infected hamsters and CJD-infected mice. However, quantitative differences were found in the cerebral cortex (at the level of gyrus marginalis), medulla and central grey matter of the midbrain in the BSEaffected cow (Table 1). The ultrastructural findings in the normal bovine brain were unremarkable, except for mild astrocytosis (when compared with brains from normal rodents) and the presence of lysosomal-like neuronal inclusions (see below). Numerous membrane-bound intracellular vacuoles were observed (Figs IA and B), predominantly in dendrites and rarely within myelinated fibres (Fig. 1B). Some vacuoles, primarily those in the medulla, were extremely large and several neuronal processes appeared to contribute to their formation. Vacuoles contained abundant curled membranes (Fig. 1C), vesicles, secondary chambers and amorphous material. Intranuclear vacuoles were occasionally seen in cerebral cortical neurones, but we did not consider them to be pathological since such vacuoles have been found in both CJD virus- and sham-inoculated mice (Liberski et al., 1989b). Vacuoles within both myelinated and unmyelinated neuronal processes were seen in scrapie-affected hamsters and CJD-affected mice (Fig. 1D). Vacuoles in myelinated fibres were seen within the axoplasm, predominantly in CJD-affected mice, or appeared to result from myelin splitting either at the intraperiod or major dense lines. In scrapie-affected hamsters, the majority of vacuoles were within unmyelinated neuronal processes. Many dendrites and axons, particularly those of larger diameter with thicker myelin sheaths, accumulated masses of interwoven neurofilaments (Fig. 2A), and subcellular organelles, such as mitochondria and electron-dense bodies (vide infra), were occasionally entrapped within these filamentous masses(Figs 2B and C). Some dystrophic neurites were filled completely with

Ultrastructural

Pathological

neuropathology

of three

Table 1 anatomical encephalopathy

changes*

Frontal

Vacuoles in unmyelinated neuronal Vacuoles in myelinated neuronal Astrocytosis Branching tubules Tubular inclusions Tubulovesicular structures Dystrophic neurites containing: Neurofilaments Dense bodies

processes processes

* The

graded

pathological

regions

changes

were

as: - , none;

bovine

spongiform

Midbrain

Brainstem

++ +++ + +

+++ +++ + ++

++ + +++ + + +

+++ +++

+++ +++

++ +++

+ , mild;

cortex

of a cow with

+ + , moderate;

+ + + , marked.

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BSE

Fig. 1.

and

Scrapie

365

process in central Spongiform change in (A-C) BSE and (D) CJD. (A) V acuole in unmyelinated grey matter of the midbrain and (B) vacuole in myelinated process in the brainstem of BSEaffected cow. Whorled membrane fragments (arrowheads) within vacuoles in (C) frontal cortex of BSE-affected cow brain and (D) parietal cortex at the junction of corpus callasum of CJD-affected mouse. Note two dystrophic neurites (stars). Bar= 1 pm.

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BSE and Scrapie

Fig. 2.

367

Dystrophic neurites (stars) in (A-C) BSE and (D) CJD. Dystrophic neurites containing (A, B) microtubules, neurofilaments, mitochondria (m) and (C) dense bodies (d) in cerebral cortex of BSE-affected cow and (D) in patietal cortex at the junction of corpus callosum of CJD-affected mouse. Bar = I Wm.

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normal-appearing mitochondria. A few dense bodies were frequently observed in synaptic terminals and were entrapped by synaptic vesicles. Neuroaxonal dystrophy was also prominent in scrapie-affected hamsters (Fig. 2D) and CJDaffected mice. However, while dystrophic neurites accumulated neurofilamentous masses in BSE, dense bodies and mitochondria predominated in rodents experimentally infected with CJD and scrapie. Also, dystrophic neurites in the BSE-affected cow brain were much larger than those observed in brains of CJD- and scrapie-affected rodents, though this may simply reflect the larger diameter of bovine axons. Occasional distended processes contained a network of branching and intercalating cisterns and tubules (Fig. 3), with entrapped mitochondria, vesicles, larger cisterns and Golgi apparatus. Cisterns and tubules, resembling those found in human neuroaxonal dystrophy (Seitelberger, 197 1; Jellinger, 1973), were less commonly found in hamsters infected with scrapie. In addition to large numbers of lipofuscin accumulations, found also in normal adult bovine brains, two types of neuronal inclusions were observed. The first type, which was also encountered in normal bovine brain tissues but in smaller numbers, was located in neuronal perikarya and appeared to be lysosomal in origin (Fig. 4). The second type of inclusion, which was found only in BSE-affected brain tissues, was located mainly in neuronal processes and was composed of a dense network of tubules (measuring approximately 10 nm in diameter) and circular profiles within a structureless matrix (Fig. 5), surrounded by a common membrane. The circular profiles were suggestive of tubular structures on cross-section. Occasionally, the subsurface cistern was involved in processes which also contained synaptic vesicles, mitochondria, dense bodies and electron-lucent cisterns. Neither of these types of inclusions were ever observed in the rodent models. Numerous hypertrophic astrocytes (Fig. 6A), not infrequently binucleated and containing abundant glial filaments, accompanied the neuronal degeneration (Liberski, 1986b, 198713). Tightjunctions were occasionally observed (Fig. 6B). Astrocytic processes formed a complicated network, filling spaces between other cells and processes. In the medulla, glial bundles were observed among myelinated axons while, in the grey matter, astrocytic processes without glial filaments frequently formed concentric arrays around degenerating neuronal processes. A glial reaction, composed of hypertrophic astrocytes and proliferation of glial bundles, also dominated the picture in both scrapie-affected hamsters and CJD-affected mice (Fig. 6C), but astrocytes digesting myelin fragments were observed primarily in CJD-affected mice, probably reflecting the much greater involvement of myelinated axons in this model. Tubulovesicular structures were observed in neuronal processes (Fig. 7A)? frequently mixed with synaptic vesicles and rarely surrounded by a common membrane (Fig. 7B). These structures were also observed in both rodent models, but, unlike the densely packed tubulovesicular structures in CJDaffected mice, those in BSE were loosely arranged, as in scrapie-affected hamsters (Fig. 7C). Membrane-bound tubulovesicular structures, however, were not seen in the rodent models.

BSE

and

369

Scrapie

Fig. 3.

Neuronal processes containing branching tubules (arrows) and electron-lucent central grey matter of the midbrain of a BSE-affected cow. Bar =0.5 pm.

cisterns

in the

Fig. 4.

Lamellar lysosomal intraneuronal inclusions (arrowheads) in brainstem of BSE-affected Similar inclusions were also encountered in brain tissues of a normal cow. Bar= 0.5 pm.

cow.

370

Fig. 5.

P. P. Liberski

Membrane-bound of a BSE-affected

et al.

inclusion (star) containing tubules, measuring cow. SV = synaptic vesicles. Bar = 0.1 pm.

10 nm in diameter,

in b&stem

Discussion The ultrastructural neuropathology of BSE consisted of spongiform vacuoles in neuronal processes, a florid astrocytic reaction with proliferation of glial bundles, neuroaxonal dystrophy and tubulovesicular structures. In addition, intraneuronal inclusions, not reported in either scrapie or CJD, were found. Spongiform Change Spongiform change is regarded as the most disease-specific and even pathognomanic finding of the subacute spongiform virus encephalopathies (Masters and Gajdusek, 1982; Liberski and Alwasiak, 1983; Liberski et al., 1989b,d). However, in natural scrapie in sheep (Beck and Daniel, 1988) and in a few CJD cases (DeReuck, DeCoster, Vaander Ecken and Daneels, 1975; Kim, Lath and Manuelidis, 1988), spongiform change is limited or conspicuously absent, and in transmissible mink encephalopathy passaged in mink of the Chediak-Higashi genotype, spongiform change is not observed at all (Marsh, Sipe, Morse and Hanson, 1976). Although spongiform change was easily detectable in the BSE-affected cow, the degree of spongiosis was less than that found in mice infected with the Fujisaki strain of CJD virus and was more comparable to that found in hamsters infected with the 263K strain of scrapie virus. The cellular elements responsible for vacuole formation have not been

BSE and Scrapie

Fig. 6.

371

Astrocytic reaction in (A, B) BSE and (C) CJD. (A) Hypertrophic astrocyte and (B) tight junction between two astrocytic processes (arrowheads) in central grey matter of the midbrain of BSEaffected cow. (C) Astrocytic processes contaiting abundant glial filaments (stars) in parietal cortex at the junction of corpus callosum of CJD-affected mouse. Bar= 1 pm.

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precisely identified, but growing evidence indicates that vacuoles originate predominantly, if not exclusively, from dendrites and rarely from axons (Lampert, Earle, Gibbs and Gajdusek, 1969; Lampert, Gajdusek and Gibbs, 1971a, 1975; Bignami and Parry, 1972a; Hirano, Ghatak, Johnson, Partnow and Gomori, 1972; Roy, Gupta and Sethi, 1972; Manuelidis and Manuelidis, 1979; Liberski, 1987c; Liberski et al., 1989b,d). Earlier reports that both neurones and astrocytes or astrocytes alone contribute to vacuole formation reflect suboptimal fixation of brain tissue (Gray, 1985), which is particularly evident in human post-mortem tissue (Martin and Vial, 1964; Gonatas, Terry and Weiss, 1965; Kidd, 1967; Foncin, 1967; Bignami and Forno, 1970; Chandler, 1968, 1973; Ribadeau-Dumas and Escourolle, 1974; Lamar, Gustaffson, Krasovich and Hinsman, 1974; Landis, Williams and Masters, 1981; Manolidis and Balojannis, 1983; Kim and Manuelidis, 1983a,b; Machado, 1986; Miyakawa, Katsuragi, Koga and Moriyama, 1986). Furthermore, we could not substantiate the reports of others (Lampert et al., 1969, 197la; Manuelidis and Manuelidis, 1979; Kim and Manuelidis, 1983a,b, 1986) that local clearings of neuronal and astrocytic cytoplasm, with decreased number of subcellular organelles, were responsible for the early stages of vacuole formation. We regard these changes as fixation artifacts since they are not observed in well-fixed tissues (Liberski, 1987c; Liberski et al., 1989b,d) and microtubules, which are particularly unstable in suboptimal fixation, are only rarely seen. Two types of vacuoles were observed. The first type corresponded to dendrites and predominated almost exclusively in the BSE-affected cow and in hamsters infected with the 263K strain of scrapie (Liberski et al., 1989d). The

373

BSE and Scrapie

Fig.

7.

Tubulovesicular membrane

bound

structures in (B).

(arrowheads) Bar=0.5 pm.

in (A, B) BSE

and

(C)

scrapie.

Note

that

structures

are

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et al.

other type, occurring within myelinated axons, were observed infrequently in BSE and commonly in mice infected with the Fujisaki strain of CJD (Liberski et al., 198913; Liberski, Yanagihara, Wells, Gibbs and Gajdusek, manuscript in preparation) and some other CJD and scrapie isolates (Field and Raine, 1964; Tateishi, Sato and Ohta, 1983). They corresponded to myelin ballooning brought about by splitting at the intraperiod or the major dense lines (Liberski et al., 1989e). Spongiform change occurred predominantly in dendrites or in myelinated axons with different virus strains and in different brain regions, but both types of vacuoles could always be found. The viruses of subacute spongiform virus encephalopathies are intimately associated with membranes (Gibbons and Hunter, 1967). Curled membrane fragments may be the ultrastructural correlates of prion protein 33-35, a membrane protein closely associated with infectivity (Bazan, Fletterick, McKinley and Prusiner, 1987). Thus, the old hypothesis that membranes are the primary target of the virus (Lampert, Hooks, Gibbs and Gajdusek, 197 lb) is worthy of re-evaluation by molecular studies. Neuroaxonal Dystrophy Neuroaxonal dystrophy has been previously documented in experimental kuru CJD (Gonatas et al., 1965; (Lampert et al., 1969), natural and experimental Hirano et al., 1972; Roy et al., 1972; Ribadeau-Dumas and Escourolle, 1974; DeReuck et al., 1975; Manuelidis and Manuelidis, 1979; Kim and Manuelidis, 1983a,b, 1986; Landis et al., 198 1; Manolidis and Balojannis, 1983; Machado,

BSE and Scrapie

375

1986; Liberski, Papierz and Alwasiak, 1987) and natural and experimental scrapie (Chandler, 1968, 1973; Bignami and Parry, 197213;Lamar et al., 1974; Liberski, 1986a, 1987a,c; Liberski, Yanagihara, Gibbs and Gajdusek, 1989a; Liberski, Yanagihara, Gibbs and Gajdusek, 1989c; Liberski et al., 1989b,d). Similarly, neuroaxonal dystrophy was an important feature of the ultrastructural pathology of BSE. However, in BSE, dystrophic neurites primarily contained whorls of neurofilaments rather than dense bodies, which were typically found in CJD and scrapie (Liberski et al., 1989a,b,d). Dystrophic neurites containing dense bodies are most consistent with reactive axonal enlargements as defined by Lampert (1967, 197 1)) who used the term “dystrophic neurites” for structures filled with branching tubules and proliferating plasma membranes. But both subcategories of spheroids are only the extremes of a spectrum (Lampert, 1967; Jellinger, 1973; Raine and Cross, 1989), and both types can be found in subacute spongiform virus encephalopathies (Liberski, 1986a, 1987a; Gibson and Liberski, 1987; Liberski et al., 1989a,b,c). In longitudinal ultrastructural studies of mice infected with the Fujisaki strain of CJD virus and the ME7, 22C and 79A strains of “scrapie and in hamsters infected with the 263K strain of scrapie, neuroaxonal dystrophy was associated with the disease itself, either preceding or following other neuropathological changes such as vacuolation, rather than with aging (Gibson and Liberski, 1987; Liberski et al., 1989a,b,c,e). Neuroaxonal dystrophy may be an ultrastructural marker for neuronal depopulation (Masters and Gajdusek, 1982). Neuroaxonal dystrophy, which also forms a component of neuritic (amyloid) plaques (Klatzo, Gajdusek and Zigas, 1959; Neumann, Gajdusek and Zigas, 1964; Wisniewski, Bruce and Fraser, 1975; Bruce and Fraser, 1975, 1982; Hirano et al., 1972; Horoupian, Powers and Schaumburg, 1972; Wisniewski and Terry, 1973; Bruce, Dickinson and Fraser, 1976; Masters et al., 1981; Fukatsu, Gibbs and Gajdusek, 1984; Gibson, 1985, 1986; Carp, Moretz, Natelli and Dickinson, 1987; McBride, Bruce and Fraser, 1988; Kitamoto, Tateishi and Sato, 1988; Pearlman, Towfighi, Pezeshkpour, Tenser and Turel, 1988; Tatesihi, Kitamoto, Hashiguchi and Shii, 1988), is probably caused by an impairment of slow axoplasmic transport, as shown by the similarity between dystrophic neurites produced experimentally by interference of axonal transport and those observed in the subacute spongiform virus encephalopathies (Lampert, 1971; Griffin, Price, Engel and Drachman, 1977; Spencer and Griffin, 1982; Droz, Chretien, Souyri and Patey, 1982; Dustin and Flament-Durand, 1982). Many types of neurodegenerative disorders may result from an interference of slow axonal transport (Gajdusek, 1985; Liberski et al., 1989b,c,d,e) and BSE is another disorder which fits this picture. Branching

Tubules

and Cisterns,

.Neuronal

Inclusions

and Tubulovesicular

Structures

Branching tubules and cisterns and neuronal inclusions probably reflect the neurodegenerative nature of BSE and the limited repertoire of the central nervous system to degenerate. Various forms of tubules and cisterns, strikingly

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similar to those found in human neuroaxonal dystrophy, have been found in natural and experimental scrapie (Field and Narang, 1972; Narang, 1973; Liberski et al., 1989d). Membrane-bound inclusions composed of tubules and vesicles, measuring 10 nm in diameter, vaguely resemble the “type d” inclusions found in rats experimentally infected with scrapie (Field and Narang, 1972). Their nature is completely unknown. Tubulovesicular structures have been demonstrated in nearly all models of subacute spongiform virus encephalopathies (David-Ferreira, David-Ferreira, Gibbs and Morris, 1968; Bignami and Parry, 197 1; Lampert et al., 1971a; Field and Narang, 1972; Narang, 1973, 1974; Lamar et al., 1974; Baringer, Wong, Klassen and Prusiner, 1979; Narang, Chandler and Anger, 1980; Narang, Asher, Pomeroy and Gajdusek, 1987; Liberski, Yanagihara, Gibbs and Gajdusek, 1988, 1990; Gibson and Doughty, 1989). Previous failures to detect them can be attributed to sampling problems (Baringer et aE., 1979; Kim and Manuelidis, 1983a,b, 1986). Alternatively, virus infectivity titre may need to be sufficiently high for these structures to be detectable (Gibson and Doughty, 1989). The last conclusion is substantiated by our longitudinal ultrastructural studies in mice infected with the Fujisaki strain of CJD virus and in hamsters infected with the 263K strain of scrapie virus, where we found a positive correlation between the number of involved processes and infectivity titre (Liberski et at., 1990). However, the composition and biological significance of tubulovesicular structures are unknown. Whether or not they represent the pathological products of disease or the infectious agents themselves remains to be determined. Suffice it to say that since membrane-bound aggregates of tubulovesicular structures have been previously reported only in natural scrapie (Bignami and Parry, 1971; Bignami, 1974), the occurrence of such aggregates in BSE supports the notion that BSE may have been transmitted to cattle via scrapie-contaminated feed. Acknowledgments Dr P. P. Liberski is the recipient of a fellowship from the Fogarty International Center. MS Petra Friedrich, MS Leokadia Romanska, Mr Ryszard Kurczewski, Mr Kunio Nagashima and MS Elzbieta Naganska are acknowledged for skilful technical assistance. References Baringer, J. R., Wong, J., Klassen, T. and Prusiner, S. B. (1979). Further observations on the neuropathology of experimental scrapie in mouse and hamsters. In Slow Transmissible Diseases of the JVervous System, Vol. 2. S. B. Prusiner and W. J. Hadlow, Eds, Academic Press, New York, pp. 111-121. Barlow, R. M. and Middleton, D. J. (1990). Dietary transmission of bovine spongiform encephalopathy to mice. Veterinary Record, 126, 11 1-I 12. Bazan, J, F., Fletterick, R. I., McKinley, M. P. and Prusiner, S. B. (1987). Predicted secondary structure and membrane topology of the scrapie prion protein. Protein Engineering, 1, 12.5 135. Beck, E. and Daniel, P. M. (1988). Neuropathology of transmissible spongiform encephalopathy. In Prions. .Novel Infectious Pathogens causing Scrapie and CreutdeldtJakob Disease. S. B. Prusiner and M. P. McKinley, Eds, Academic Press, New York, pp. 331-385.

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Bignami, A. (1974). The ultrastructure of status spongiosus and neuronai vacuolation in Jakob-Creutzfeldt disease and other transmissible spongiform encephalopathies. In Neurology. Proceedings of the X International Congress of Neurology, Barcelona, Spain, September S-15,1973. A. Subirana, J. M. Espadaler and E. H. Burrows, Eds, Excerpta Medica-American Elsevier Publishing Co., Inc., Amsterdam-New York, pp. 330-338. Bignami, A. and Forno, L. S. (1970). Status spongiosus in Jakob-Creutzfeldt disease. Electron microscopic study of a cortical biopsy. Brain, 93, 89-94. Bignami, A. and Parry, H. B. (1971). Aggregations of 35-nanometer particles associated with neuronal cytopathic changes in natural scrapie. Science, 171, 389-

390. Bignami, A. and Parry, H. B. (1972a). Electron microscopic studies of the brain of sheep with natural scrapie. I. The fine structure of neuronal vacuolation. Brain, 95, 3 19-326. Bignami, A. and Parry, H. B. (1972b). Electron microscopic studies of the brain of sheep with natural scrapie. II. The small nerve processes in neuronal degeneration. Brain, 95, 487494. Bruce, M. E. and Fraser, H. (1975). Amyloid plaques in the brains of mice infected with scrapie: morphological variations and staining properties. Neuropathology and Applied Neurobiology, 1, 189-202. Bruce, M. E. and Fraser, H. (1982). Effect of age on cerebral amyloid plaques in murine scrapie. Neuropathology and Applied Neurobiology, 8, 7 l-74. Bruce, M. E., Dickinson, A. G. and Fraser, H. (1976). Cerebral amyloidosis in scrapie in the mouse: effect of agent strain and mouse genotype. Neuropathology and Applied JVeurobiology, 2, 47 l-478. Carp, R. I., Moretz, R. C., Natelli, M. and Dickinson, A. G. (1987). Genetic control of scrapie: incubation period and plaque formation in I mice. j’ournal of General Virology, 68, 401-407. Chandler, R. L. (1968). Ultrastructural pathology of scrapie in the mouse: an electron microscopic study of spinal cord and cerebellar areas. British Journal of Experimental Pathology, 49, 52-59. Chandler, R. L. (1973). Ultrastructural observations on scrapie in gerbils. Research in Veterinary Science, 15, 322-328. David-Ferreira, J. F., David-Ferreira, K. L., Gibbs, C. J., Jr and Morris, J. A. (1968). Scrapie in mice: ultrastructural observations in the cerebral cortex. Proceedings of the Society for Experimental Biology and Medicine, 127, 313-320. Dawson, M., Wells, G. A. H. and Parker, B. N. J. (1990). Preliminary evidence of the experimental transmissibility of bovine spongiform encephalopathy to cattle. Veterinary Record, 126, 112-l 13. DeReuck, J., DeCoster, W., Vaander Ecken, H. and Daneels, P. (1975). CreutzfeldtJakob disease: a comparative light microscopic, histochemical and electronmicroscopic study. European Neurology, 13, 154-I 66. Dickinson, A. G. (1976). Scrapie in sheep and goats. In Slow Virus Diseases of Animals and Man. R. H. Kimberlin, Ed., North Holland Publishing Company, New York, Heidelberg, Berlin, pp. 1-14. Droz, B., Chretien, M., Souyri, F. and Patey, G. (1982). Axoplasmic transport in the experimental neuropathy induced by acrylamide. In Axoplasmic Transport in Physiology and Pathology. D. G. Weiss and A. Gorio, Eds, Springer Verlag, Berlin, Heidelberg, pp. 104-108. Dustin, P. and Flament-Durand, J. (1982). Disturbances of axoplasmic transport in Alzheimer’s disease. In Axoplasmic Transport in Physiology and Pathology. D. G. Weiss and A. Gorio, Eds, Springer Verlag, Berlin, Heidelberg, pp. 131-136. Field, E. J. and Raine, C. S. (1964). An electron microscopic study of scrapie in the mouse. Acta Neuropathologica (Berlin), 4, 200-2 11. Field, E. J. and Narang, H. K. ( 1972). An electron-microscopic study of scrapie in the rat: further observations on “inclusion bodies” and virus-like particles. Journal of the Neurological Sciences, 17, 347-364.

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