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progress m our understanding of prron disease.
Acknowledgments
The development of reliable PrP immunostammg was supported by the Medical
Research Council (SPG9119619) and the CJD surveillance proJect is supported by
the Department of Health. The skilled assistanceof L. McArdle, C. Barrre, D.
Diagnosis of Human Prion Disease 81

Nrcolson, P. Hayward, and I. Goodbrand is gratefully acknowledged. K
Sutherland generated the computer images of PrPCJD mununolocaIization m the
spinal cord and R. Melrose prepared the manuscript. I am Indebted to R. Wtll for
helpful discussion and this work was done in collaboratton with J. W. Ironside

References
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10 Lantos, P L , McGill, I S , Janota, I., Doey, L. J., Collmge, J., Bruce, M , et al
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11. Hashtmoto, K., Mannen, T , and Nobuyuki, N. (1992) Immunohistochemtcal study
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the CNS in patients with Creutzfeldt-Jakob disease Am J Pathol. 140, 1285-l 294
13 Hayward, P A R , Bell, J. E , and Ironside, J W (1994) Priori protem tmmuno-
cytochemtstry the development of reliable protocols for the mvestigation of
Creutzfeldt-Jakob disease Neuropathol Appl Neurobzol 20,375-383
82 Bell

14 Brown, P , GaJdusek, D. C , Gibbs, C J , and Asher, D. M (1985) Potential epr-
demrc of Creutzfeldt-Jakob disease from human growth hormone therapy N Engl
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15 Prckermg-Brown, S , Mann, D M A., Owen, F , Ironsrde, J W , de Stlva, R ,
Roberts, D A., et al (1995) Allehc variations m apoltpoprotem E and prton pro-
tein genotype related to plaque formation and age of onset m sporadic Creutzfeldt-
Jakob disease Neuroscl Lett 187, 127-129.
16 Tatershr, J and Kttamoto, T (1995) Inherited prron diseases and transmission to
rodents. Brarn Path01 5, 53-59.
17 Btllette de Vtllemeur, T , Gelott, A , Deslys, J P., Dormont, D , Duyckaerts, C , Jardm,
L , et al (1994) Iatrogemc Creutzfeldt-Jakob disease m three growth hormone recrpr-
ents. a neuropathologtcal study. Neuropathol Appl Neuroblol 20, 11 l-l 17
18 Fraser, H (1993) Drverslty in the neuropathology of scrapre-like disease m am-
mals Br Med Bull 49,792-809
19 Wells, G A H , Wrlesmrth, J W , and McGrll, I S (1992) Bovine spongrform
encephalopathy Bram Path01 1,69-78.
20 Prusmer, S B and DeArmond, S J (1991) Molecular biology and pathology of
scrapte and the prron diseases of humans. Brazn Path01 1,297-309
21 DeArmond, S L (1993) Overview of the transmtsstble spongrform encepha-
lopathtes prton protein disorders Br Med Bull 49,725-737
22 Baker, H F. and Rtdley, R M (1992) The genetics and transmtsstbility of human
spongtform encephalopathy Neurodegeneratzon 1,3-16
23 Serban, D , Taraboulos, A , DeArmond, S J , and Prusmer, S. B (1990) Rapid
detectron of Creutzfeldt-Jakob disease and scrapre prron protems J Neuropathol
Exp Neurol 49,290
24 Krtamoto, T , Muramoto, T , Mohrr, S , Doh-Ura, K., and Tatershr, J (1991)
Abnormal tsoform of prron protein accumulates m folhcular dendrttrc cells m
mace with Creutzfeldt-Jakob disease J Vwol 65, 6292-6295.
25. Tatetsht, J. and Kttamoto, T (1993) Developments m diagnosis for prton drs-
eases Br &fed Bull 49,971-979
26 Klatzo, I , GaJdusek, D. C., and Zigas, V (1959) Pathology of kuru. Lab Invest
8,799-847
27 Goodbrand, I A , Ironstde, J. W , Ntcolson, D., and Bell, J. E (1995) Prron pro-
tem accumulatton m the spinal cords of patients with sporadic and growth hor-
mone associated Cruetzfeldt-Jakob disease Neuroscz Lett 183, 127-130
28 Gambettt, P , Parcht, P , Petersen, R B , Chen, S. G , and Lugarest, E. (1995)
Fatal famrhal msomma and famtltal Creutzfeldt-Jakob disease: clmtcal, patho-
logical and molecular features Brazn Path01 5,43-5 1
29 Ghetto, B , Taglravmr, F , Masters, C L , Beyreuther, K , Graccone, G , Verga, L ,
et al. (1989) Gerstmann-Straussler-Schemker dtsease II. Neurotibrtllary tangles and
plaques with PrP amylotd coexist m an affected family Neurology 39, 1453-146 1
30 de Silva, R., Ironstde, J W., McCardle, L., Esmonde, T , Bell, J E , Will, R G., et
al (1994) Neuropathologtcal phenotype and “prton protein” genotype correlation
m sporadic Creutzfeldt-Jakob disease. Neuroscl Lett 179, 5CL52
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31 Turner, C , Bell, J E , and Ironside, J W (1993) Localisation of microglia m
CNS amyloid plaques: an nnmunocytochemical and confocal mtcroscoptc study
J Pathol. 170,401.
32 Mryazono, M , Kttamoto, T , Iwaki, T., and Tatetshi, J (1992) Colocahzation of
prton protein and A4/(3 protein m the same amylotd plaques m pattents wtth
Gerstmann-Straussler syndrome Acta Neuropathol 83,333-339
33 Martinez-Lage, J. F , Poza, M , Sola, J , Tortosa, J G., Brown, P , Cervenakova,
L., et al (1994) Accidental transmission of Creutzfeldt-Jakob disease by dural
cadaveric grafts J Neural Neurosurg Psychlat 57, 109 l-l 094.
34 Will, R G and Matthews, W B. (1982) Evidence for case-to-case transmisston
of Creutzfeldt-Jakob disease. J Neurol Neurosurg. Psychtat 45, 235-238
35 Gibbs, C. J , Joy, A , Heffner, R., Franko, M., Miyazakt, M., Asher, D , et al
( 1985) Clinical and pathological features and laboratory confirmatton of Creutzfeldt-
Jakob disease m a recipient of pttuttary-derived human growth hormone N Engl
J Med 313,734-738
36 Weller, R O., Steart, P V., and Powell-Jackson, J D (1986) Pathology of
Creutzfeldt-Jakob disease associated with pituitary-derived human growth hor-
mone admmistration Neuropathol Appl Neuroblol 12, I 17-129
37 Medori, R., Tritschler, J H., LeBlanc, A , Vrllare, F., Manetto, V , Ghen, H Y , et
al. (1992) Fatal famihal msomma, a prton disease with a mutation at codon 178 of
the priori protein gene. N Engl J Med 7,444-449
38 Collmge, J , Owen, F , Poulter, M , Leach, M., Crow, T J , Rossor, M. N , et al
(1990) Prton dementia without characteristic pathology. Lancet 336, 7-9
39 Tomhnson, B. E. (1992) Agemg and the dementias, in Greenjeld™s Neuropathology, 5th
ed. (Adams, J H and Duchen, L W., eds.), Edward Arnold, London, pp 1284-1410
40. Tateishi, J , Kitamoto, T., Doh-ura, K., Boellaard, J. W., and Peiffer, J (1992)
Creutzfeldt-Jakob disease with amyloid angiopathy diagnosis by immunologic
analysts and transmisston experiments. Acta Neuropathol 83,559-563
41 Gray, F., Chretien, F , Cesaro, P., Chatelam, J., Beaudry, P , Laplanche, J L , et
al (1994) Creutzfeldt-Jakob disease and cerebral amylotd angtopathy Acta
Neuropathol 88, IO&11 I
42 Laszlo, L., Lowe, J., Self, T., Kenward, N., Landon, M., McBride, T., et al. (1992)
Lysosomes as key organelles in the pathogenesis of prion encephalopathies J
Path01 166,333-341.
43 Askanas, V., Bilak, M., Engel, W. K., Alvarez, R. B., Tome, F., and Leclerc, A.
(1993) Priori protein is abnormally accumulated in mcluston-body myosms
Neuroreport 5,25-28
44. Westaway, D., DeArmond, S. J., Cayetano-Canlas, J., Groth, D., Foster, D., Yang, S , et
al (1994) Degeneratton of skeletal muscle, peripheral nerves and the central nervous
system m transgemc mice overexpressmg wild-type pnon protems. Cell 76, 117-l 29
45. Sutherland, K., Barrte, C , and Ironside, J W. (1994) Automatic quanttficatton of amyloid
plaque formation m human spongtform encephalopathy. Neurodegenerahon 3,293-300
46. Advisory Committee on Dangerous Pathogens (1994) Precautzons for Work wzth
Human and Animal Transmrsslble Sponglform Encephalopathzes HMSO, London.
5
The Diagnosis
of Bovine Spongiform Encephalopathy
and Scrapie by the Detection of Fibrils
and the Abnormal Protein lsoform
Michael J. Stack, Paula Keyes, and Anthony C. Scott

1. Introduction
Traditionally, confirmation of Transmissible Sponglform Encephalopathy
(TSE) disease m humans or animals IS by conventional hght microscopy of
stained tissue sectlons prepared from specific sites of formalm-fixed tissue after
embedding m paraffin wax. This 1s the statutory method of confirmation of
Bovine Spongiform Encephalphathy (BSE) in the Umted Kingdom and It IS
also used in other Member States of the European Community. However, two
additional diagnostic criteria for TSE, the detection of disease-specific fibrlls
by transmission electron microscopy and the detection of the main constituent of
the fibnls, an abnormal protein by immunoblottmg, have also been reported.
The former IS the current method of confirmation for the statutory diagnosis of
scraple in the United Kingdom.
The purpose of this chapter IS to provide a clear accountof easyto use and effec-
tive protocols, mcludmg reagent preparation, interpretation of results, and equip-
ment required, for the extraction and detection of fibrils and the abnormal protein
from central nervous tissueof clinically suspectcasesof BSE in cattle or scrapie m
sheep.The protocols also can be applied to other species(such as goats,moufflon,
captive wild Bovidue, domestic cats,or wild Felidae) m which TSE is suspected,
but experience of the reliability of the methods m such speciesIS lessextenstve
7.7. Electron Microscopy
Disease-specific structures, called Scraple-Associated Fib& (SAF), were
first reported in brain extracts from affected mice and hamsters experimentally
From Methods IR Molecular Me&one Prlon Dmeases
Ed&d by H Baker and R M Rldley Humana Press Inc , Totowa. NJ

85
86 Stack, Keyes, and Scott

infected with scraple by the use of negative contrast Transmission Electron
Microscopy (TEM) (1,2) Rod-like structures, designated “pnon rods,” were
also reported m extracts from scraple-infected hamster brains (3). Further
studies also showed flbrlls to be present m the human TSE diseases;
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Schemker syndrome, and
kuru (4). The role of SAF detection as a diagnostic aid for natural infections
of scraple m sheep has been well established (5-S) demonstrating a close
correlation (90-l 00%) between fibrll detection and histopathologlcal diag-
nosis. Flbrlls closely resembling those detected from natural sheep scrapte-
affected brains have also been extracted from the brains of BSE-affected
cattle (9-12). Similar flbrlls have also been reported m a domestic cat (13),
a captive cheetah (24), a greater kudu antelope (IS), and m Rocky Moun-
tam elk (I 6)
Although blochemlcally similar, electron microscopy of SAF and prlon rods
does show morphological differences m fibnl size, onentatlon, and clarity of
substructure. It has been suggested that these morphological differences are not
only owing to the differences in preparation method used but may be affected
by the centrifugal sedimentation rate of the species of the brain tissue tested,
or by the strain of scraple agent used for transmission mto laboratory rodents
(I 7) For convenience the term “scrapie-associated fibnls” or SAF 1s used
throughout this chapter, irrespective of the species from which they are
derived.
7.2. Immunoblotting
Biochemically, the main constituent of SAF and prion rods 1s a protease-
resistant neuronal membrane glycoprotem, which is disease specific, and is an
abnormal lsoform of a host protein. The normal lsoform 1s found m a number
of tissues, including the central nervous system (CNS), but 1s dlstmgulshable
from the abnormal lsoform because the latter forms disease-specific fibrlls and
1s relatively resistant to protease digestion
Immunoblotting 1s used to detect the purified, protease-resistant, abnormal
protein by its molecular weight and reaction with specific antibodies. At
present, the test only can be regarded as consistently effective when applied to
CNS tissue where the abnormal tsoform accumulates in particularly large quan-
tities. Further technical developments may allow the reliable and consistent
detection of much smaller amounts of the protease-resistant protein In
lymphoretlcular or other more accessible tissues.
Several different terminologies for the normal and abnormal lsoforms of
the protein are used m the scientific literature Treatment of infected brain
extracts with Sarkosyl (a detergent), followed by centrifugatlon and protemase
K (PK) digestion, can be used to separate the two protein isoforms that have
87
Diagnosis of BSE and Scrap/e

been termed “priori protein” (18) or “protease-resistant protein” (PrP) (19)
The normal cellular protein tsoform (PrPc) 1s soluble and susceptible to
proteinase K digestton, whereas the abnormal scrapie isoform (PrPsc) IS sedi-
mentable and reststant, but treatment with detergents other than Sarkosyl, fol-
lowed by centrifugation, may separate the PrP isoforms differently For
example, PrPC may be found m either or both the sedimentable or soluble frac-
tions, depending on the expertmental conditions The type of protease used is
also an important variable m distmguishing between the normal and abnormal
isoforms of PrP.
1.3. Safety
There would appear to be ltttle risk to humans from the pathogens causing
animal TSE under normal condttions, but there may be risks associated with
very high exposure vta parenteral routes. Therefore, the gutdmg safety prm-
ctple is to mnumize or confine the practices that may result in high exposure of
the pathogens to humans. It should also be remembered that samples sent m for
testmg might contain conventional pathogens, some of which may cause dis-
ease m humans.
There are strtct codes of practice for workmg with TSE-Infected material,
wtth several documents used as references. The safety sttuatton IS complex,
with different hazard levels being assigned according to the species being stud-
ied, the tissue locale, and the type of procedures being carrred out The safety
precautions presently recommended are formulated by the Advisory Commit-
tee on Dangerous Pathogens (ACDP) (20) and there are some published guide-
lines by the European Commtssion of Agriculture (21) Safety IS an important
issue and each laboratory should formulate its own guidelines based on the
most recent credible evtdence and guidance publications from the ACDP. It is
particularly important to recognize that the procedures employed in the proto-
cols described in this chapter will serve to concentrate infecttvity as purtfica-
tton proceeds.
Ideally all potentially hazardous procedures associated with possible opera-
tor exposure via parenteral routes (i.e., homogemzation), should be carried out
in a Class 1 Safety Cabinet (BS 5726 or equivalent) with latex gloves worn at
all times. The chemicals marked Care: Toxic in the reagent lists are hazardous
to health. Local risk assessments should be carried out and appropriate safety
precautions should be taken throughout. All glassware, gloves, plasttcware, and
instruments used m the followmg protocols must be decontaminated before
disposal, or reuse, by one of three procedures:
1 Porous load autoclavmg at a holding temperature of 134-138°C for 18 mm,
2 Soaking overnight at 20°C in sodium hypochlorite to provide a final concentra-
tion of 2% (20,000 ppm) of available chlorine,
88 Stack, Keyes, and Scott
3 Soaking overmght at 20°C m sodmm hydroxide to provrde a final concentratron
of 2N NaOH

2. Materials
2.1. Tissue Collection
For adequate dtagnosis of TSE there generally would be a prtortty to collect
a portion of ttssue from the medulla brain region (dissected at the obex) to

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