<<

. 15
( 45 .)



>>

a Black side of cassette
b Scotchbrtte.
c 2X chromatography papers
d Gel
e Transfer membrane.
f 2X chromatography papers.
g Scotchbrtte
h Clear side of cassette
34. Place the cassette into the blottmg tank with the black side next to the negative
(black) electrode.
35 Top up the blottmg tank with blotting buffer, and gently shake the cassette up and
down to dislodge any au bubbles
36 Connect the tank to an appropriate power supply and run at a constant voltage of
30 V overnight
When blotting IS complete, remove the cassette and open. The prestained mark-
37
ers should be vtsrble on the transfer membrane
Mark the furthest posmon traveled of each marker with a pm hole, since the
38.
colors may fade during subsequent mcubatrons

The immunostammg (mcludmg blocking) can be carried out according to
the mstructions supplied with the Auroprobe BL plus and IntenSE BL Kits
obtained from CAMBIO
The primary antibody currently used at CVL 1s lB3, a polyclonal rabbit
antiserum raised against formic-acid-treated SAF protein extracted from the
brains of clinically affected mice infected wrth the ME7 strain of scrapie, as
supplied by The Institute for Animal Health, AFRC/MRC Neuropathogenesis
Unit (Edinburgh). The antibody is used at a dilution of 1:1000. (A negative
rabbit serum is used, at the same dilution, as a negative control.)
Diagnosis of BSE and Scrap/e 101
Detection of scrapre PrP can be achieved by silver stammg, but thts IS not as
successful for BSE because of the lower levels of PrP m the bratn. Ktts for
silver staining can be obtamed commercially (BioRad Laboratories, cat. no.
161-0443).

3.5. Interpretation of Results
3.5.1. Positive Brain-Fig. 4A,B (Lanes Labeled +ve Brain)
1 Treated wtth postttve anttsernm (Fig 4A) For non-PK-treated samples a wade, dif-
fuse region of stained bands is present at sites corresponding to protems of molecular
masses of 33-35 kDa. For PK-treated samples a wade,diffuse region of stamed bands
IS present at sttes correspondmg to protems of molecular masses of 27-30 kDa
2. Treated with negattve anttserum (Fig 4B)
No stained bands can be identified m the discussed regions.

3.5.2. Negative Brain-Fig. 4A,B (Lanes Labeled -ve Brain)
No stamed bands can be identified in the described regions with either the
positive antiserum (Fig. 4A) or the negative antiserum. Note: Bands of
approxtmate molecular masses 19, 22, and 43 kDa occur in blots reacted with
positive and negative antisera (Fig. 4A,B); they occur even when no primary
antibody has been reacted (picture not shown). These bands are at present con-
sidered to be nonspecific.
3.5 3. Inconclusive
A further sample should be prepared from the same or different parts of the
frozen central nervous tissue. Examme as determmed and reclassify.
References
1 Merz, P. A., Rohwer, R. G., Kascsak, R , Wismewskl, H. M., Somerville, R A.,
Gibbs, C J J , et al. (1984) Infection-specific particle from the unconventional
slow virus diseases. Sczence 225,437-440
2. Merz, P. A., Somerville, R. A , Wismewskt, H M., and Iqbal, K. (1981) Abnor-
mal fibrtls from scrapte-infected brain. Acta Neuropathol S4,63-74
3. Prusiner, S B , McKmley, M. P , Bowman, K A , Bolton, D C , Bendheim, P E.,
Groth, D F., et al (1983) Scrapte prtons aggregate to form amyloid-like birefrm-
gent rods. Cell 35,34%358
4. Merz, P. A , Somervtlle, R A., Wtsniewski, H. M , Manuehdis, L., and Manuehdis,
E E (1983) Scrapte-associated fibrils m Creutzfeldt-Jakob disease. Nature 306,
474-476
5 Dawson, M , Mansley, L. M , Hunter, A. R., Stack, M J., and Scott, A C. (1987)
Comparison of scrapte associated tibril detection and histology m the diagnosis of
natural sheep scrapre. Vet Ret 121,591
102 Stack, Keyes, and Scott
6 Gibson, P H , Somervtlle, R A , Fraser, H , Foster, J D , and Ktmberlm, R H (1987)
Scrapie associated fibrtls m the dtagnosts of scrapie m sheep Vet Ret 120, 125127
7 Rubenstem, R., Merz, P A , Kascsak, R J , Carp, R I , Scalict, C L , Fama, C L ,
et al (1987) Detectton of scrapte-associated fibrtls (SAF) and SAF proteins from
scrapie affected sheep J infect Du 156, 3&46
8 Scott, A C , Done, S H , Venables, C , and Dawson, M. (1987) Detection of
scrapie-associated fibrils as an aid to the diagnosis of natural sheep scrapie Vet
Ret 120,280-28 1
9 Hope, J , Reekie, L J D , Hunter, N., Multhaup, G., Beyreuther, K , White, H , et
al (1988) Fibrtls from brains of cows with new cattle disease contam scrapie-
associated protein Nature 336, 39&392.
10 Wells, G. A H. and Scott, A. C (1988) Neuronal vacuolatton and spongtosus, a
novel encephalopathy of adult cattle Neuropathol Appl Neuroblol 14,247
11 Wells, G A H , Scott, A. C , Johnson, C T , Gunning, R F , Hancock, R. D.,
Jeffrey, M , et al (1987) A novel progressive spongtform encephalopathy m cattle
Vet Ret 121,419-420
12 Wells, G A H , Scott, A C , Wilesmtth, J W , Simmons, M M , and Matthews,
D (1994) Correlation between the results of a histopathological exammation and
the detection of abnormal brain fibrils m the dtagnosts of bovine spongtform
encephalopathy Res Vet Scz 56, 346-351
13 Pearson, G R , Wyatt, J M , Gryffed-Jones, T J , Hope, J , Chong, A , Higgins,
R J , et al (1992) Feline spongiform encephalopathy fibrtl and PrP studies Vet
Ret 131,307-3 10
14 Peet, R L and Curran, J M (1992) Spongiform encephalopathy m an imported
cheetah (Actrnonyxjubatus) Aus Vet J 69, 117
1.5 Ktrkwood, J K , Wells, G A H , Cunningham, A A , Jackson, S I, Scott, A C ,
Dawson, M , et al (1992) Scrapte-like encephalopathy in a greater kudu
(Tragelaphus strepsiceros) which had not been fed ruminant-derived protein Vet
Ret 130,365-367
16. Guiroy, D. C., Wtlhams, E. S , Song, K J , Yanagihara, R , and Gajdusek, D C
(1993) Ftbrils m brains of Rocky Mountain elk with chrome wasting disease con-
tam scrapte amyloid. Acta Neuropathol. 86,77-80.
17 Liberski, P P and Brown, P (1993) Scrapte-associated fibrils, m Lzght and Elec-
tron Mzcroscopzc Neuropathology of Slow Vu-us Disorders (Ltberski, P. P , ed ),
CRC, Boca Raton, FL
18. McKinley, M. P., Bolton, D. C., and Prusmer, S B (1983) A protease-resistant
protein IS a structural component of the scrapte prton Cell 35,57-62
19. Bolton, D. C., McKmley, M P , and Prusmer, S B (1982) Identtficatton of a
protein that purifies with the scrapte priori Sczence 218, 1309-l 3 11
20. Advisory Committee on Dangerous Pathogens (1994) Precautions for Work with
Human and Anrmal TSEs HMSO ISBN 0- 1 l-32 1805-2.
21. European Cornmtssion for Agrtculture (1994) Transmzsslble Spongzfkm Enceph-
alopathles Protocols Used at CVL for the Laboratory Dlagnosls and ConjGma-
tton of Bovine Sponglform Encephalopathy and Scraple A Report from the
Diagnosis of BSE and Scrap/e 103
Sclenttfic Veterinary Commzttee European Commtsston, Dtrectorate General for
Agrtculture, Unit for Veterinary Legislatron and Zootechmcs
22 Scott, A C., Wells, G A H , Chaplin, M J., and Dawson, M (1992) Bovme
spongtform encephalopathy. detection of fibrils in the central nervous system IS
not affected by autolysts. Res Vet Scz.32,332-336.
23 Stack, M J , Scott, A C., Done, S H., and Dawson, M (1993) Scraple-associated
fibril detection on decomposedand fixed ovine bran-tmaterial. Res Vet Scr 55,
173-178
Stack, M. J , Scott, A C , Done, S H , and Dawson, M (199 1) Natural scraple
24
detection of fibrtls m extracts from the central nervous system of sheep Vet Ret
128,53%540.
Scott, A C., Wells, G A. H , Chaplm, M J , and Dawson, M (1990) Bovine
25
spongiform encephalopathy detection and quantttatton of fibrtls, fibrtl protein
(PrP) and vacuolatton m brain. Vet Mcroblol 23,295-304
Htmert, H and Dumger, H (1984) A rapid and efficient method to enrich SAF-
26
protein from scrapte brains of hamsters Broscz Rep. 4, 165-I 70
Hope, J , Reekte, L. J D , and Gibson, P. H (1990) On the pathogenesisof SAF,
27
m Unconventional Vu-us Diseasesof the Central Nervous System, Pat-u I986
(Court, L. A., Dormont, D., Brown, P , and Kmgsbury, D T , eds.), Commtssartat
a I™Energre Atomtque, Dtpartement de Protection Samtatre, Service de Documen-
tation Fontenay-aux-Roses Cedex, pp. 536-546
Laemmh, U. K (1970) Cleavage of structural proteins during the assemblyof the
28.
head of bacteriophage T4 Nature 277,68&685
29 Towbm, H., Staehlm, T , and Gordon, J. (1979) Electrophoretrc transfer of pro-
teins from polyacrylamlde gels to nitrocellulose sheets Proc Nat1 Acad Scz
USA 76,435&4354.
Exposure to, and Inactivation
of, the Unconventional Agents that Cause
Transmissible Degenerative Encephalopathies
David M. Taylor


1. Introduction
Some fatal degenerative encephalopathies of mammals form a distmct group
because they are caused by unconventional but uncharacterized transmissible
agents that evoke no classical immune response in the affected host. The animal
diseases are bovme spongtform encephalopathy (BSE), chronic wastmg disease
of elk and mule-deer, feline spongiform encephalopathy (FSE) of the domestic
cat, scrapie in sheep and goats, and transmissible mink encephalopathy (TME).
A number of exotic ruminant and felid species maintamed m, or originating
from, zoological collecttons m the United Kingdom have also developed fatal
encephalopathies that, like FSE, are considered to have been caused by BSE
agent; the affected species are cheetah, eland, gemsbok, kudu, nyala, ocelot, oryx,
and puma. The human diseases are Creutzfeldt-Jakob disease (CJD), fatal famil-
ial Insomnia (FFI), Gerstmann-Straussler-Schemker syndrome (GSS), and kuru.
Although other tissues can be infected, the highest titers of infectivity are
always found within the central nervous system (CNS) during the termmal
stage of disease. It is only within such tissues that any histopathological
changes can be observed, usually as an intraneuronal spongiform degeneration
accompanied by astrocytic hypertrophy with or without discrete extracellular
amyloid plaques. Although spongiform change is common, tt is not a consis-
tent feature of these diseases, and the term transmissible degenerative
encephalopathies (TDE) has been considered to be a more appropriate
alterna-
twe to descriptions such as “transmissible spongiform encephalopathies” (1).
A dominant feature of the TDE is the uncertainty and controversy regarding

From Methods m Molecular Medune Pnon Dfseases
Edlted by H Baker and R M Rldley Humana Press Inc , Totowa, NJ

105
106 Taylor

the nature of then unconventtonal causal agents, which will be addressed else-
where in this volume.
A notable characteristic of TDE agents is their relative resistance to macti-
vation by chemical and physical procedures that are effective with conven-
tional microorgamsms (2,3), and they are capable of prolonged survival m the
general environment owmg to their resistance to desiccation (4), freezing (5),
and ultraviolet u-radiation (6). Scrapie-infected brain has been shown to retam
substantial infectivity after burial for 3 yr (7). Scrapie mfectivity IS considered
to be capable of survival for several years on grazing pastures (8), and CJD
agent has been shown to retam mfecttvity after a 28-mo holdmg period at room
temperature (9).

2. Accidental Transmission of TDE
Among the known instances of accidental transmission of TDE there are
some that are suspected or known to be attributable to failure of decontamma-
non procedures. Thus, CJD has been transmitted from human to human (10, I I),
scrapie from sheep to sheep (22) and probably from sheep to cows, giving rise
to BSE (Z3) In addition, a significant number of human patients have devel-
oped CJD after mJection of growth hormone or gonadotrophm derived from
the pituitary glands of human cadavers, or surgical repair with cadaveric human
dura mater (14) There had been no perceived risk of such occurrences when
these products were first used, but from later studies on the inactivation poten-
tial of the manufacturing processes when challenged with scrapie agent it was
concluded that mfecttvity might survive (25,16) or crosscontammate the manu-
facturing processes (2 7)
The Incidence of scrapie in sheep m some countries is sufficiently high to
conclude that the human consumption of infected tissues has been common-
place, given that scrapie agent is relatively resistant to the temperatures
achieved during cooking by boiling (18) or in the oven (29). Concern that this
might be a causal factor for CJD m humans was not supported by a number of
eptdemiological studies that also dismissed occupational exposure to scrapie
agent as a risk factor for CJD (e g ,20) Although there are no known examples
of transmission of animal TDE to humans, concern regarding this possibthty
has been rekindled because of the occurrence of BSE that may represent the
novel transmission of scrapie to cattle. The concern is that, having breached
the cattle species barrier, the cattle-passaged agent might have acquired an
enhanced capacity to penetrate the human species barrier but the possibihty of
this is considered to be low (21,22). Although not stattstrcally significant, the
occurrence m recent years of CJD m two farmers who tended herds of cattle
affected by BSE has heightened this concern (23,24) As a precautionary mea-
sure, specified bovine offals have been prohibited from use as human food on
Transmissible Degenerative Encephalopathles 107

the basts of what IS known about the levels of mfecttvtty m these organs m
scrapte-infected sheep (25) or m calves infected experimentally with BSE agent
(26) However, recent studies have shown that apart from the CNS none of the
many bovine ttssues tested from natural BSE cases have any demonstrable
mfecttvity by mouse bioassay (27). This contrasts sharply with the sttuatton m
scrapte-affected sheep in which lymphoreticular and other nonneural trssues
can be shown by the same bioassay system to harbor mfectivtty (28).
Although medical products have been manufactured for many years from
sheep tissues sourced from countries where scrapte 1s endemic without any
apparent problems, the emergence of BSE has resulted m stricter gutdelmes
for the use of ruminant ttssues m the manufacture of such products; the prmct-
pal recommendatton IS that raw matertals should be obtained from countries
where both BSE and scrapte are known reliably to be absent (29).
3. Occupational Exposure to TDE Agents
As far as occupattonal exposure to either animal or human TDE agents IS
concerned, the prmctpal groups at risk appear to be research scientists, health-
care workers mvolved m human or veterinary medicine, undertakers, embalm-
ers, farmers, butchers, abattotr workers, and those involved m the rendering
industry The degree of actual exposure to TDE agents for these various groups
will vary constderably given that these agents have not been found generally to
be present in the bloodstream, feces, urine, or secrettons from the host. Conse-
quently, those dealing with intact living hosts are at mmtmal risk of exposure,
whereas the theoretical risk IS increased for those who have to deal with
infected tissues through surgery, postmortem procedures, or as part of an
mdustrtal process, especially tf the tissues are derived from the central nervous
or lymphorettcular systems. Much useful advice for such workers has been
published recently (30), but tt 1simportant to re-emphasize that the only known
examples of humans acquiring CJD accidentally are related to the use of medi-
cal procedures mvolvmg the use of CJD-contammated surgical mstruments,
transplant material, or inJectable therapeutic products. This chapter will mclude
informatton on the currently available practrcal measures for mactivatron of, or
reduction of exposure to, TDE agents with parttcular reference to the laboratory
srtuatton but that can be extrapolated to other occupatronal sttuations where the
level of exposure to TDE agents 1sperceived to be a theoretical occupattonal risk.
4. Inactivation Studies on TDE Agents
4.1. Background
Until the 1980s the information on approprrate methods for decontamma-
non of scrapte-hke agents was piecemeal and sometimes mappropriate because
bioassays were often termmated prematurely, grven the delayed dose-response
108 Taylor

curves that are known to occur frequently after chemical or phystcal treatment
of TDE agents compared with comparable titers of untreated rnfectivity

<<

. 15
( 45 .)



>>