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Scrapie associated fibril detection on decomposed and fixed ovine brain material
Research in Veterinary Science 1993, 55, 173-178
Scrapie associated fibril detection on decomposed and fixed ovine brain material M. J. STACK, A. C. SCOTT, S. H. DONE, M. DAWSON, Central Veterinary Laboratory, Woodham
Lane, New Haw, Addlestone, Surrey KT15 3NB
phalopathy (BSE) as a neurological degenerative
Samples of cerebral cortex from eight scrapie affected sheep and two unaffected control sheep were stored for up to nine days at temperatures ranging from 18°C to 29°C. Scrapie associated fibrils (SAF) could be detected in proteinase K treated brain extracts from all the eight scrapie affected animals after five days storage and in six out of the eight after nine days storage. SAF could not be detected in any brain extracts from the two control animals. Formol saline fixed brain material from a further six scrapie affected and two clinically normal sheep, were also subjected to an extraction technique used to detect fibrils. No characteristic SAF were observed in any of these fixed samples. Long filamentous structures were observed in four of the fixed scrapie affected brain extracts and in one of the fixed unaffected control brain extracts.
disease with similarities to scrapie in sheep (Wells et al 1987, Fraser et al 1988, Hope et al 1988, Wilesmith et al 1988) the use of SAF detection for diagnosis has taken a more prominent role within this laboratory, especially where autolysis or physical damage has rendered the brain material as unfit for histological examination. There have also been cases where the only tissue available for examination has been fixed in formol saline. This paper describes the effects of tissue decomposition on SAF detection in extracts from ovine brains and also reports attempts to recover SAF from formol saline fixed ovine brain tissue.
Materials and methods
Animals and tissues
THE demonstration of scrapie associated fibrils (SAF) by transmission electron microscopy (TEM), in brain extracts from clinically affected rodents, was first reported by Merz et al (1981). This has led to the acceptance of SAF detection as an additional diagnostic criterion for natural scrapie (Dawson et al 1987, Gibson et al 1987, Rubenstein et al 1987, Scott et al 1987). Strnctures termed prion rods have also been detected in extracts from scrapie infected brains (Prusiner et al 1983). Although both prion rods and SAF appear to be constituents of a protease resistant, neuronal membrane glycoprotein, termed PrP Sc (Chesebro et al 1985, Oesch et al 1985), there are morphological differences between the two which reflect differences in the extraction treatments used (Hope et al 1988). As the extraction methods used here produce fibrils rather than priori rods the SAF terminology is used. With the emergence of bovine spongiform ence-
All the 14 clinically suspect sheep used for these studies were histopathologically confirmed as scrapie cases and were from different flocks and of various breeds. The four control sheep were from a flock with no recorded history of scrapie and their brains showed no histopathological changes. Brains from eight scrapie cases and two control sheep were used in the decomposition study. From these, five 10 g samples of cerebral cortex were dissected from each brain and put into glass universal containers. These were placed in a cardboard box on an east-facing window ledge and the temperature monitored using a continuous thermograph. One brain sample was removed after 0, two, five, seven and nine days and stored at -70°C before biochemical extraction for electron microscopy using method 1. Brains from the other six scrapie cases and the two remaining control sheep were used in the fixation study. From these, 10 g aliquots of pooled
173
M. J. Stack, A. C. Scott, S. H. Done, M. Dawson
174
Brain tissue 10 g (Method 1) 1 g (Method 2) 10% homogenate in (sol A) 10% N-lauroyl sarcosine pH 7.4 homogenise 30 s (Silverson blender) C
22,000 gx 30 rain (60 Ti) 10 g equiv 22,000 gx 10 rain (TLA100'3) 1 g equiv i
i
S save
P Discard Top up with Sol A
215,000 g x 2 h (60 Ti) 10 g equiv 540,000 g x 20 rain (TLA 100.3) 1 g equiv ] P Resuspend 10% NaCI +1% N-lauryl sarcosine pH 7.2
[
S Discard
C 215,o00 gx 2.5 h (60 Ti) 10 g equiv 540,000 gx 25 rain (TLA 100.3) 1 g equiv
~ [
]
P (10 g equiv)
S Discard
I
I
P (1 g equiv)
Resuspend in 1.5 ml 0.01 M Tris 10% NaCI + 1% sarcosine pH 7.2
i
S Discard
Reeuspend in 1.5 ml 0.01 M Tris +10 pg m1-1 Proteinase-K pH 7.2 Magnetic stirrer 37°C x 1 h , ~
~
I
C 22,000 gx 10 rain (TLA100.3) 10 g or 1 g equiv [
P (10 g equiv)
] S Discard
I P (1 g equiv)
I S Discard
Resuspend in 50 pl deionised H2O and vertex
Resuspend in 1.5 ml 0.01 M Tris +10 p.g/m1-1 Proteinase-K pH 7.2 Magnetic stirrer 37°C x 1 h
NEGATIVE STAIN FOR TEM C 22,000 gx lO rain (T/A 100.3) 10 g equiv I
P Resuspend in 50 pl deionised H20 and vortex
I
S Discard
NEGATIVE STAIN FOR TEM FIG 1 : FLow chart - methods 1 and 2 for SAF extraction
175
Fibril detection in ovine brain
grey matter from the cerebral cortex and cerebellum were dissected from each brain. One gram of fresh brain was stored at -70°C, the remainder was fixed in 10 per cent formol saline and stored at room temperature for a minimum of two weeks. Biochemical extraction for electron microscopy was carried out using method 2.
Biochemical extraction for SAF detection Method 1. This was based on the method of Hilmert and Diringer (1984). Ten grams of brain were homogenised in 10 per cent N-lauroylsarcosine and after differential centrifugation, digested with proteinase K. Centrifuge timings were achieved using a Beckman L8 ultracentrifuge with a 60 Ti fixed angle rotor.
Method 2. This was based on a modification of method 1 (Hope et al 1990), where 1 g of brain was used and shorter centrifuge times achieved using a Beckman benchtop ultracentrifuge with a TEA 100.3 miniature rotor. The methods used are fully described in the form of a flow diagram (Fig 1).
Electron microscopy The final pellet from both extraction methods was resuspended in 50 gl of deionised water. Electron microscope grids were prepared from these suspensions and negatively stained with 2 per cent potassium phosphotungstate, as fully described by Scott et al (1987). Examination of grids was carried out in a Philips 410LS TEM with an accelerating voltage of 80 kV and at magnifications above x 25,000. If no fibrils were observed within a 20 minute observation time the extract was considered negative.
TABLE 1: SAF detection results using method 1 at various decomposition times
Animal 1 2 3 4 5 6 7 8 9 10
Clinical status Affected Affected Affected Affected Affected Affected Affected Affected Unaffected Unaffected
Histology
0
2
Days 5
7
9
+ + + + + + + +
+ + + + + + + + .
+ + + + + + + +
+ + + + + + + +
+ + + + + + +
+ + + + + +
.
.
. .
. .
. .
. .
F i s h e r ' s e x a c t test P = 0 . 0 2 2 P = 0 - 0 6 7 P = 0 . 1 3 3
tion of the neuronal cytoplasm in the dorsal vagal, cuneate or olive nuclei, and on the presence of spongiform change in the grey matter neuropil. The brains of the four control animals showed no spongiform change. The results of the decomposition study are summarised in Table 1. No characteristic SAF were observed in any of the brain extracts from the two control animals. SAF were detected in the brain extracts from all eight sheep affected with scrapie after two days and five days storage at 18°C to 29°C. This was a statistically significant difference (P=0.022) using Fisher's exact test. SAF were also detected in seven of the extracts after seven days (P=0.067) and in six of the extracts after nine days decomposition (P=0-133) (Fig 2).
Histopathology Histological examination was carried out on 5 Bm sections of the medulla of each brain, as previously described (Scott et al 1987).
Results A histological diagnosis of scrapie was confirmed in the 14 brains of the animals showing suspect clinical signs. Histological confirmation was based on finding multiple or single vacuola-
F i G 2: E l e c t r o n m i c r o g r a p h (x 7 8 , 0 0 0 ) s h o w i n g n e g a t i v e l y s t a i n e d S A F r e c o v e r e d f r o m o v i n e brain t i s s u e a f t e r nine d a y s d e c o m p o sition. Barline = 1 0 0 n m
176
M. J. Stack, A. C. Scott, S. H. Done, M. Dawson
TABLE 2: SAF detection results
Animal 1 2 3 4 5 6 7 8
Clinical Status
Histology
Method 2 fresh brain
Affected Affected Affected Affected Affected Affected Unaffected Unaffected
+ + + + + + -
+ + + + + + -
Method 2 fixed brain -
LF -
-
LF LF LF LF -
LF L o n g filaments P=0.036
The results of the fixation study are summarised in Table 2. No characteristic SAF were found in extracts of the fresh brain pools from the two control animals. SAF were detected in extracts of the fresh unfixed brain pools from all six scrapie affected animals during the 20 minute TEM examination (P=0.036). No SAF were detected in the formol saline fixed brain extracts from any of the six scrapie affected sheep, or the two unaffected control sheep. Long filaments (9 to 16 nm in width and 1 to 4 gm in length), which were not observed in extracts from any of the fresh brain tissues, were seen in the fixed brain tissue extracts from four scrapie-affected and one unaffected control sheep (Fig 3).
Discussion The decomposition experiment showed that autolysis of ovine brain material, of up to five days duration, does not affect fibril detection within the temperature and time parameters studied. There were three instances where SAF were not detected in scrapie affected brain extracts. These were from two sheep brains where only two or three fibrils were detected within the 20 minute search time, at two and five days decomposition. The failure to detect SAF may have been due to the sublocalisation of PrP SC within those three brain samples (Scott et al 1990, Stack et al 1991), that the limit of detection of the fibril extraction method had been reached, or that there was an adverse :affect on PrP sC stability due to decomposition beyond five days. A similar study of autolysis in BSE-affected brains also showed variation in the frequency of fibril detection (Scott et al 1992). This study, using ovine brain material, suggests that there may be a limit to how long material can be left to autolyse before it begins tO affect a SAF diagnosis.
FIG 3: Electron micrograph (x 68,000) showing a typical long filament o b s e r v e d in brain extracts from formalised tissue, found in both scrapie affected and unaffected control s h e e p
Diagnosis of scrapie and other spongiform encephalopathies is traditionally carried out on formaldehyde fixed brain tissue using histopathology to identify vacuolation and, in some cases, amyloid plaque formation. PrP sC can also be observed in chemically fixed brain tissue by immunocytochemical staining techniques, using antisera raised against SAF protein (Bendheim et al 1984, McBride et al 1988). The extraction techniques used to recover SAF from fresh unfixed tissue are generally based on obtaining a crude mitochondrial pellet which is subfractionated to obtain synaptosomal and synaptic plasma membrane fractions (Whittaker 1969), or postsynaptic density fractions (Cohen et al 1977). The detergents used in the procedures render any membrane protein non-sedimentable at low centrifugal forces (22,000 g) but sedimentable at higher forces (215,000 g). This sedimentable pellet can be treated with a proteinase enzyme and with extractions from scrapie affected brain the surviving PrP sC is visualised as SAF in the electron microscope. These results show that extraction methods used to visualise the protein as SAF are unsuccessful when applied to 10 per cent formol saline fixed ovine brain material. Using extraction techniques similar to the methods described in this paper,
Fibril detection in ovine brain
Gibbs et al (1985) reported an unsuccessful attempt to isolate SAF from formalin fixed human brain tissue. They were also unable to detect any associated PrP SC using sodium dodecyl sulphatepolyacryamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot. The tissue was derived from a confirmed case of CreutzfeldtJakob disease (CJD), who had been a recipient of suspect human pituitary growth hormone. SAF and PrPsC had already been isolated from fresh-frozen brain tissue taken at autopsy from the same individual. Tintner et al (1986) tried to detect SAF by TEM and PrP sC by SDS-PAGE from embalmed brain tissue derived from an exhumed human. The exhumation was carried out to verify CJD in this individual who was another recipient of suspect human pituitary growth hormone. Neither SAF nor associated PrP SC protein were detectable. Brown et al (1990) suggest that PrP sC may acquire sufficient rigidity from the molecular cross-linking effect of formaldehyde to resist the subsequent destruction of hydrogen bonds by heat. It therefore seems possible that this cross-linking effect may also render the PrP SC resistant to extraction by detergent and proteinase K treatment. Morphologically, the long filaments observed in the scrapie affected and unaffected control extracts from fixed ovine brain material, could not be characterised. They may be subfractions of neurons which have only been partly digested by the proteinase K due to a protective effect of the formaldehyde cross-linking. In conclusion, the diagnosis of scrapie using SAF detection is useful in cases where histopathological confirmation is not possible due to mechanical damage or autolysis of the brain material, but there may be a problem of false negatives, where delays are longer than five days. It is unlikely that extraction methods presently being used to detect SAF or PrP sC will be successful when applied to 10 per cent formol saline fixed brain tissue, as presented for histopathological examination.
Acknowledgements We are indebted to Yvonne Spencer of the Pathology Department, Central Veterinary Laboratory, for her skilled technical assistance and to Robin Sayers of the Epidemiology Department for his statistical advice.
177
References BENDHEIM, P. E., BARRY, R. A., DeARMOND, S. J., STITES, D. P. & PRUSINER, S. B. (1984) Antibodies to a serapie prion protein. Nature 310, 418-422 BROWN, P., LIBERSKI, P. P., WOLFF, A. & GADJUSEK, D. C. (1990) Resistance of scrapie infectivity to steam autoclaving after formaldehyde fixation and limited survival after ashing at 360°C: practical and theoretical implications. Journal of Infectious Diseases 161,467-472 CHESEBRO, B., RACE, R., WEHRLY, K., NISHIO, J., BLOOM, M., LECHNER, D., BERGSTROM, S., ROBBINS, K., MAYER, L., KEITH, J. M., GARON, G. & HAASE, A. (1985) identification of scrapie prion protein specific mRNA in scrapie-infected and uninfect~ ed brain. Nature 315, 331-333 COHEN, R. S., BLOMBERG, F., BERZINS, K. & SIEKEVITZ, P. (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. Journal of Cell Biology 74, 181-203 DAWSON, M., MANSLEY, L. M., HUNTER, A. R., STACK, M. J. & SCOTT, A. C. (1987) Comparison of scrapie associated fibril detection and histology in the diagnosis of natural sheep scrapie. Veterinary Record 121,591 FRASER, H., McCONNELL, I., WELLS, G. A. H. & DAWSON, M. (1988) The transmission of bovine spongiform encephalopathy to
mice. Veterinary Record 123, 472 GIBBS, C. J., JOY, A., HEFFNER, R., FRANKO, M., MIYAZAK2, M., ASHER, D. M., PAR2S2, J. E., BROWN, P. W. & GAJDUSEK, D. C. (1985) Clinical and pathological features and laboratory confirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derived human growth hormone. New England Journal of Medicine 313, 734738 GIBSON, P. H., SOMERVILLE, R. A., FRASER, H., FOSTER, J. D. & KIMBERL2N, R. H. (1987) Scrapie associated fibrils in the diagnosis of scrapie in sheep. Veterinary Record 120, 125-127 HILMERT, H. & DIR2NGER, H. (1984) A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Bioscienee Reports 4, 165-170 HOPE, J., REEK2E, L. J. D. & GIBSON, P. H. (1990) On the pathogenesis of SAF. Proceedings of the I2 symposium international sur les virus non conventionnels du systeme nerveux cenncal (Paris 1986). Eds L. A. Court, D. Dormont, P. Brown, D. T. Kingsbury. pp
536-546 HOPE, J., REEK2E, L. J. D., HUNTER, N., MULTHAUP, G., BEYREUTHER, K., WHITE, H., SCOTT, A. C., STACK, M. J., DAWSON, M. & WELLS, G. A. H. (1988) Fibrils from brains of cows with new cattle disease contain scrapie-associated fibrils Nature
336, 390-392 McBRIDE, P. A., BRUCE, M. E. & FRASER, H.
(1988)
Immunostaining of scrapie cerebral arnyloid plaques with antisera raised to scrapie-associated fibrils (SAF). Neuropathology and
Applied Neurobiology 14, 325-336 MERZ, P. A., SOMERVILLE, R. A., WISNIEWSKI, H. M. & IQBAL, K. (1981) Abnormal fibrils from scrapie-infected brain. Acta Neuropathologica 54, 63-74 OESCH, B., WESTAWAY, D., WALCHLI, M., McKINLEY, M. P., KENT, S. B. H., AEBERSOLD, R., BARRY, R. A., TEMPST, P., TEPLOW, D. B., HOOD, L. E., PRUS2NER, S. B. & WEISSMANN, C. (1985) A cellular gene encodes scrapie PrP 27-30 protein. Cell 40, 735-746 PRUSINER, S. B., McKINLEY, M. P., BOWMAN, K. A., BOLTON, D. C., BENDHEIM, P. E., GROTH, D. F. & GLENNER, G. G. (1983) Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35, 349-358 RUBENSTE2N, R., MERZ, P. A., KASCSAK, R. J., CARP, R. i., SCAL2CI, C. L., FAMA, C. L. & W2SNIEWSK2, H. M. (1987) Detection of serapie-associated fibrils (SAF) and SAF proteins from scrapie affected sheep. Journal of Infectious Diseases 156, 36-46 SCOTT, A. C., DONE, S. H., VENABLES, C. & DAWSON, M. (1987) Detection of scrapie-associated fibrils as an aid to the diagnosis of
natural sheep scrapie. Veterinary Record 120, 280-281 SCOTT, A. C., WELLS, G. A. H., CHAPLIN, M. J. & DAWSON, M.
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(1992) Bovine spongiforrn encephalopathy: detection of fibrils in the central nervous system is not affected by autolysis. Research in Veterinaty Science 52, 332-336 SCOTT, A. C., WELLS, G. A. H., STACK, M. J., WHITE, H. & DAWSON, M. (1990) Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain. Veterinary Microbiology 23, 295-304 STACK, M. J., SCOTT, A. C. DONE, S. H. & DAWSON, M. (1991) Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep. Veterinary Record 128, 539-540 TINTNER, R., BROWN, P., HEDLEY-WHYTE, E. T., RAPPAPORT, E. B., PICCARDO, C. P. & GADJUSEK, D. C. (1986) Neuropathologic verification of Creutzfeldt-Jakob disease in the exhumed American recipient of human pituitary growth hor-
mone: epidemiologic and patbogenetic implications. Neurology 36, 932~936 WELLS, G. A. H., SCOTT, A. C., JOHNSON, C. T., GUNNING, R. F., HANCOCK, R. D., JEFFREY, M., DAWSON, M. & BRADLEY, R. (1987) A novel progressive spongiform encephalopathy in cattle. Veterinary Record 121, 419-420 WHITTAKER, V. P. (1969) Chapter 14. The synaptosome. In Handbook of Neurocheraistry vol 2 Ed A. Lajtha, New York, Plenum Press. pp 327-364 WILESMITH, J. W., WELLS, G. A. H., CRANWELL, M. P. & RYAN, J. B. M. (1988) Bovine spongiform encephalopathy: epideminlogical studies. Veterinary Record I23, 638-644
Received October 12, 1992 Accepted February 12, 1993
Scrapie associated fibril detection on decomposed and fixed ovine brain material M. J. STACK, A. C. SCOTT, S. H. DONE, M. DAWSON, Central Veterinary Laboratory, Woodham
Lane, New Haw, Addlestone, Surrey KT15 3NB
phalopathy (BSE) as a neurological degenerative
Samples of cerebral cortex from eight scrapie affected sheep and two unaffected control sheep were stored for up to nine days at temperatures ranging from 18°C to 29°C. Scrapie associated fibrils (SAF) could be detected in proteinase K treated brain extracts from all the eight scrapie affected animals after five days storage and in six out of the eight after nine days storage. SAF could not be detected in any brain extracts from the two control animals. Formol saline fixed brain material from a further six scrapie affected and two clinically normal sheep, were also subjected to an extraction technique used to detect fibrils. No characteristic SAF were observed in any of these fixed samples. Long filamentous structures were observed in four of the fixed scrapie affected brain extracts and in one of the fixed unaffected control brain extracts.
disease with similarities to scrapie in sheep (Wells et al 1987, Fraser et al 1988, Hope et al 1988, Wilesmith et al 1988) the use of SAF detection for diagnosis has taken a more prominent role within this laboratory, especially where autolysis or physical damage has rendered the brain material as unfit for histological examination. There have also been cases where the only tissue available for examination has been fixed in formol saline. This paper describes the effects of tissue decomposition on SAF detection in extracts from ovine brains and also reports attempts to recover SAF from formol saline fixed ovine brain tissue.
Materials and methods
Animals and tissues
THE demonstration of scrapie associated fibrils (SAF) by transmission electron microscopy (TEM), in brain extracts from clinically affected rodents, was first reported by Merz et al (1981). This has led to the acceptance of SAF detection as an additional diagnostic criterion for natural scrapie (Dawson et al 1987, Gibson et al 1987, Rubenstein et al 1987, Scott et al 1987). Strnctures termed prion rods have also been detected in extracts from scrapie infected brains (Prusiner et al 1983). Although both prion rods and SAF appear to be constituents of a protease resistant, neuronal membrane glycoprotein, termed PrP Sc (Chesebro et al 1985, Oesch et al 1985), there are morphological differences between the two which reflect differences in the extraction treatments used (Hope et al 1988). As the extraction methods used here produce fibrils rather than priori rods the SAF terminology is used. With the emergence of bovine spongiform ence-
All the 14 clinically suspect sheep used for these studies were histopathologically confirmed as scrapie cases and were from different flocks and of various breeds. The four control sheep were from a flock with no recorded history of scrapie and their brains showed no histopathological changes. Brains from eight scrapie cases and two control sheep were used in the decomposition study. From these, five 10 g samples of cerebral cortex were dissected from each brain and put into glass universal containers. These were placed in a cardboard box on an east-facing window ledge and the temperature monitored using a continuous thermograph. One brain sample was removed after 0, two, five, seven and nine days and stored at -70°C before biochemical extraction for electron microscopy using method 1. Brains from the other six scrapie cases and the two remaining control sheep were used in the fixation study. From these, 10 g aliquots of pooled
173
M. J. Stack, A. C. Scott, S. H. Done, M. Dawson
174
Brain tissue 10 g (Method 1) 1 g (Method 2) 10% homogenate in (sol A) 10% N-lauroyl sarcosine pH 7.4 homogenise 30 s (Silverson blender) C
22,000 gx 30 rain (60 Ti) 10 g equiv 22,000 gx 10 rain (TLA100'3) 1 g equiv i
i
S save
P Discard Top up with Sol A
215,000 g x 2 h (60 Ti) 10 g equiv 540,000 g x 20 rain (TLA 100.3) 1 g equiv ] P Resuspend 10% NaCI +1% N-lauryl sarcosine pH 7.2
[
S Discard
C 215,o00 gx 2.5 h (60 Ti) 10 g equiv 540,000 gx 25 rain (TLA 100.3) 1 g equiv
~ [
]
P (10 g equiv)
S Discard
I
I
P (1 g equiv)
Resuspend in 1.5 ml 0.01 M Tris 10% NaCI + 1% sarcosine pH 7.2
i
S Discard
Reeuspend in 1.5 ml 0.01 M Tris +10 pg m1-1 Proteinase-K pH 7.2 Magnetic stirrer 37°C x 1 h , ~
~
I
C 22,000 gx 10 rain (TLA100.3) 10 g or 1 g equiv [
P (10 g equiv)
] S Discard
I P (1 g equiv)
I S Discard
Resuspend in 50 pl deionised H2O and vertex
Resuspend in 1.5 ml 0.01 M Tris +10 p.g/m1-1 Proteinase-K pH 7.2 Magnetic stirrer 37°C x 1 h
NEGATIVE STAIN FOR TEM C 22,000 gx lO rain (T/A 100.3) 10 g equiv I
P Resuspend in 50 pl deionised H20 and vortex
I
S Discard
NEGATIVE STAIN FOR TEM FIG 1 : FLow chart - methods 1 and 2 for SAF extraction
175
Fibril detection in ovine brain
grey matter from the cerebral cortex and cerebellum were dissected from each brain. One gram of fresh brain was stored at -70°C, the remainder was fixed in 10 per cent formol saline and stored at room temperature for a minimum of two weeks. Biochemical extraction for electron microscopy was carried out using method 2.
Biochemical extraction for SAF detection Method 1. This was based on the method of Hilmert and Diringer (1984). Ten grams of brain were homogenised in 10 per cent N-lauroylsarcosine and after differential centrifugation, digested with proteinase K. Centrifuge timings were achieved using a Beckman L8 ultracentrifuge with a 60 Ti fixed angle rotor.
Method 2. This was based on a modification of method 1 (Hope et al 1990), where 1 g of brain was used and shorter centrifuge times achieved using a Beckman benchtop ultracentrifuge with a TEA 100.3 miniature rotor. The methods used are fully described in the form of a flow diagram (Fig 1).
Electron microscopy The final pellet from both extraction methods was resuspended in 50 gl of deionised water. Electron microscope grids were prepared from these suspensions and negatively stained with 2 per cent potassium phosphotungstate, as fully described by Scott et al (1987). Examination of grids was carried out in a Philips 410LS TEM with an accelerating voltage of 80 kV and at magnifications above x 25,000. If no fibrils were observed within a 20 minute observation time the extract was considered negative.
TABLE 1: SAF detection results using method 1 at various decomposition times
Animal 1 2 3 4 5 6 7 8 9 10
Clinical status Affected Affected Affected Affected Affected Affected Affected Affected Unaffected Unaffected
Histology
0
2
Days 5
7
9
+ + + + + + + +
+ + + + + + + + .
+ + + + + + + +
+ + + + + + + +
+ + + + + + +
+ + + + + +
.
.
. .
. .
. .
. .
F i s h e r ' s e x a c t test P = 0 . 0 2 2 P = 0 - 0 6 7 P = 0 . 1 3 3
tion of the neuronal cytoplasm in the dorsal vagal, cuneate or olive nuclei, and on the presence of spongiform change in the grey matter neuropil. The brains of the four control animals showed no spongiform change. The results of the decomposition study are summarised in Table 1. No characteristic SAF were observed in any of the brain extracts from the two control animals. SAF were detected in the brain extracts from all eight sheep affected with scrapie after two days and five days storage at 18°C to 29°C. This was a statistically significant difference (P=0.022) using Fisher's exact test. SAF were also detected in seven of the extracts after seven days (P=0.067) and in six of the extracts after nine days decomposition (P=0-133) (Fig 2).
Histopathology Histological examination was carried out on 5 Bm sections of the medulla of each brain, as previously described (Scott et al 1987).
Results A histological diagnosis of scrapie was confirmed in the 14 brains of the animals showing suspect clinical signs. Histological confirmation was based on finding multiple or single vacuola-
F i G 2: E l e c t r o n m i c r o g r a p h (x 7 8 , 0 0 0 ) s h o w i n g n e g a t i v e l y s t a i n e d S A F r e c o v e r e d f r o m o v i n e brain t i s s u e a f t e r nine d a y s d e c o m p o sition. Barline = 1 0 0 n m
176
M. J. Stack, A. C. Scott, S. H. Done, M. Dawson
TABLE 2: SAF detection results
Animal 1 2 3 4 5 6 7 8
Clinical Status
Histology
Method 2 fresh brain
Affected Affected Affected Affected Affected Affected Unaffected Unaffected
+ + + + + + -
+ + + + + + -
Method 2 fixed brain -
LF -
-
LF LF LF LF -
LF L o n g filaments P=0.036
The results of the fixation study are summarised in Table 2. No characteristic SAF were found in extracts of the fresh brain pools from the two control animals. SAF were detected in extracts of the fresh unfixed brain pools from all six scrapie affected animals during the 20 minute TEM examination (P=0.036). No SAF were detected in the formol saline fixed brain extracts from any of the six scrapie affected sheep, or the two unaffected control sheep. Long filaments (9 to 16 nm in width and 1 to 4 gm in length), which were not observed in extracts from any of the fresh brain tissues, were seen in the fixed brain tissue extracts from four scrapie-affected and one unaffected control sheep (Fig 3).
Discussion The decomposition experiment showed that autolysis of ovine brain material, of up to five days duration, does not affect fibril detection within the temperature and time parameters studied. There were three instances where SAF were not detected in scrapie affected brain extracts. These were from two sheep brains where only two or three fibrils were detected within the 20 minute search time, at two and five days decomposition. The failure to detect SAF may have been due to the sublocalisation of PrP SC within those three brain samples (Scott et al 1990, Stack et al 1991), that the limit of detection of the fibril extraction method had been reached, or that there was an adverse :affect on PrP sC stability due to decomposition beyond five days. A similar study of autolysis in BSE-affected brains also showed variation in the frequency of fibril detection (Scott et al 1992). This study, using ovine brain material, suggests that there may be a limit to how long material can be left to autolyse before it begins tO affect a SAF diagnosis.
FIG 3: Electron micrograph (x 68,000) showing a typical long filament o b s e r v e d in brain extracts from formalised tissue, found in both scrapie affected and unaffected control s h e e p
Diagnosis of scrapie and other spongiform encephalopathies is traditionally carried out on formaldehyde fixed brain tissue using histopathology to identify vacuolation and, in some cases, amyloid plaque formation. PrP sC can also be observed in chemically fixed brain tissue by immunocytochemical staining techniques, using antisera raised against SAF protein (Bendheim et al 1984, McBride et al 1988). The extraction techniques used to recover SAF from fresh unfixed tissue are generally based on obtaining a crude mitochondrial pellet which is subfractionated to obtain synaptosomal and synaptic plasma membrane fractions (Whittaker 1969), or postsynaptic density fractions (Cohen et al 1977). The detergents used in the procedures render any membrane protein non-sedimentable at low centrifugal forces (22,000 g) but sedimentable at higher forces (215,000 g). This sedimentable pellet can be treated with a proteinase enzyme and with extractions from scrapie affected brain the surviving PrP sC is visualised as SAF in the electron microscope. These results show that extraction methods used to visualise the protein as SAF are unsuccessful when applied to 10 per cent formol saline fixed ovine brain material. Using extraction techniques similar to the methods described in this paper,
Fibril detection in ovine brain
Gibbs et al (1985) reported an unsuccessful attempt to isolate SAF from formalin fixed human brain tissue. They were also unable to detect any associated PrP SC using sodium dodecyl sulphatepolyacryamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot. The tissue was derived from a confirmed case of CreutzfeldtJakob disease (CJD), who had been a recipient of suspect human pituitary growth hormone. SAF and PrPsC had already been isolated from fresh-frozen brain tissue taken at autopsy from the same individual. Tintner et al (1986) tried to detect SAF by TEM and PrP sC by SDS-PAGE from embalmed brain tissue derived from an exhumed human. The exhumation was carried out to verify CJD in this individual who was another recipient of suspect human pituitary growth hormone. Neither SAF nor associated PrP SC protein were detectable. Brown et al (1990) suggest that PrP sC may acquire sufficient rigidity from the molecular cross-linking effect of formaldehyde to resist the subsequent destruction of hydrogen bonds by heat. It therefore seems possible that this cross-linking effect may also render the PrP SC resistant to extraction by detergent and proteinase K treatment. Morphologically, the long filaments observed in the scrapie affected and unaffected control extracts from fixed ovine brain material, could not be characterised. They may be subfractions of neurons which have only been partly digested by the proteinase K due to a protective effect of the formaldehyde cross-linking. In conclusion, the diagnosis of scrapie using SAF detection is useful in cases where histopathological confirmation is not possible due to mechanical damage or autolysis of the brain material, but there may be a problem of false negatives, where delays are longer than five days. It is unlikely that extraction methods presently being used to detect SAF or PrP sC will be successful when applied to 10 per cent formol saline fixed brain tissue, as presented for histopathological examination.
Acknowledgements We are indebted to Yvonne Spencer of the Pathology Department, Central Veterinary Laboratory, for her skilled technical assistance and to Robin Sayers of the Epidemiology Department for his statistical advice.
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References BENDHEIM, P. E., BARRY, R. A., DeARMOND, S. J., STITES, D. P. & PRUSINER, S. B. (1984) Antibodies to a serapie prion protein. Nature 310, 418-422 BROWN, P., LIBERSKI, P. P., WOLFF, A. & GADJUSEK, D. C. (1990) Resistance of scrapie infectivity to steam autoclaving after formaldehyde fixation and limited survival after ashing at 360°C: practical and theoretical implications. Journal of Infectious Diseases 161,467-472 CHESEBRO, B., RACE, R., WEHRLY, K., NISHIO, J., BLOOM, M., LECHNER, D., BERGSTROM, S., ROBBINS, K., MAYER, L., KEITH, J. M., GARON, G. & HAASE, A. (1985) identification of scrapie prion protein specific mRNA in scrapie-infected and uninfect~ ed brain. Nature 315, 331-333 COHEN, R. S., BLOMBERG, F., BERZINS, K. & SIEKEVITZ, P. (1977) The structure of postsynaptic densities isolated from dog cerebral cortex. Journal of Cell Biology 74, 181-203 DAWSON, M., MANSLEY, L. M., HUNTER, A. R., STACK, M. J. & SCOTT, A. C. (1987) Comparison of scrapie associated fibril detection and histology in the diagnosis of natural sheep scrapie. Veterinary Record 121,591 FRASER, H., McCONNELL, I., WELLS, G. A. H. & DAWSON, M. (1988) The transmission of bovine spongiform encephalopathy to
mice. Veterinary Record 123, 472 GIBBS, C. J., JOY, A., HEFFNER, R., FRANKO, M., MIYAZAK2, M., ASHER, D. M., PAR2S2, J. E., BROWN, P. W. & GAJDUSEK, D. C. (1985) Clinical and pathological features and laboratory confirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derived human growth hormone. New England Journal of Medicine 313, 734738 GIBSON, P. H., SOMERVILLE, R. A., FRASER, H., FOSTER, J. D. & KIMBERL2N, R. H. (1987) Scrapie associated fibrils in the diagnosis of scrapie in sheep. Veterinary Record 120, 125-127 HILMERT, H. & DIR2NGER, H. (1984) A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Bioscienee Reports 4, 165-170 HOPE, J., REEK2E, L. J. D. & GIBSON, P. H. (1990) On the pathogenesis of SAF. Proceedings of the I2 symposium international sur les virus non conventionnels du systeme nerveux cenncal (Paris 1986). Eds L. A. Court, D. Dormont, P. Brown, D. T. Kingsbury. pp
536-546 HOPE, J., REEK2E, L. J. D., HUNTER, N., MULTHAUP, G., BEYREUTHER, K., WHITE, H., SCOTT, A. C., STACK, M. J., DAWSON, M. & WELLS, G. A. H. (1988) Fibrils from brains of cows with new cattle disease contain scrapie-associated fibrils Nature
336, 390-392 McBRIDE, P. A., BRUCE, M. E. & FRASER, H.
(1988)
Immunostaining of scrapie cerebral arnyloid plaques with antisera raised to scrapie-associated fibrils (SAF). Neuropathology and
Applied Neurobiology 14, 325-336 MERZ, P. A., SOMERVILLE, R. A., WISNIEWSKI, H. M. & IQBAL, K. (1981) Abnormal fibrils from scrapie-infected brain. Acta Neuropathologica 54, 63-74 OESCH, B., WESTAWAY, D., WALCHLI, M., McKINLEY, M. P., KENT, S. B. H., AEBERSOLD, R., BARRY, R. A., TEMPST, P., TEPLOW, D. B., HOOD, L. E., PRUS2NER, S. B. & WEISSMANN, C. (1985) A cellular gene encodes scrapie PrP 27-30 protein. Cell 40, 735-746 PRUSINER, S. B., McKINLEY, M. P., BOWMAN, K. A., BOLTON, D. C., BENDHEIM, P. E., GROTH, D. F. & GLENNER, G. G. (1983) Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35, 349-358 RUBENSTE2N, R., MERZ, P. A., KASCSAK, R. J., CARP, R. i., SCAL2CI, C. L., FAMA, C. L. & W2SNIEWSK2, H. M. (1987) Detection of serapie-associated fibrils (SAF) and SAF proteins from scrapie affected sheep. Journal of Infectious Diseases 156, 36-46 SCOTT, A. C., DONE, S. H., VENABLES, C. & DAWSON, M. (1987) Detection of scrapie-associated fibrils as an aid to the diagnosis of
natural sheep scrapie. Veterinary Record 120, 280-281 SCOTT, A. C., WELLS, G. A. H., CHAPLIN, M. J. & DAWSON, M.
178
M. J. Stack, A. C. Scott, S. H. Done~ M. D a w s o n
(1992) Bovine spongiforrn encephalopathy: detection of fibrils in the central nervous system is not affected by autolysis. Research in Veterinaty Science 52, 332-336 SCOTT, A. C., WELLS, G. A. H., STACK, M. J., WHITE, H. & DAWSON, M. (1990) Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain. Veterinary Microbiology 23, 295-304 STACK, M. J., SCOTT, A. C. DONE, S. H. & DAWSON, M. (1991) Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep. Veterinary Record 128, 539-540 TINTNER, R., BROWN, P., HEDLEY-WHYTE, E. T., RAPPAPORT, E. B., PICCARDO, C. P. & GADJUSEK, D. C. (1986) Neuropathologic verification of Creutzfeldt-Jakob disease in the exhumed American recipient of human pituitary growth hor-
mone: epidemiologic and patbogenetic implications. Neurology 36, 932~936 WELLS, G. A. H., SCOTT, A. C., JOHNSON, C. T., GUNNING, R. F., HANCOCK, R. D., JEFFREY, M., DAWSON, M. & BRADLEY, R. (1987) A novel progressive spongiform encephalopathy in cattle. Veterinary Record 121, 419-420 WHITTAKER, V. P. (1969) Chapter 14. The synaptosome. In Handbook of Neurocheraistry vol 2 Ed A. Lajtha, New York, Plenum Press. pp 327-364 WILESMITH, J. W., WELLS, G. A. H., CRANWELL, M. P. & RYAN, J. B. M. (1988) Bovine spongiform encephalopathy: epideminlogical studies. Veterinary Record I23, 638-644
Received October 12, 1992 Accepted February 12, 1993