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(3,16,31-37).
Although earlier experiments had shown that acetylethyleneimme (18), ethanol
(I 9), and ethylene oxide (38) were ineffective, it was during the 1980s that com-
prehensive studies were conducted in the Umted Kingdom and the Umted States
m an effort to establish decontaminatton standards that could be used reliably for
the agents of CJD and other TDE. During these studies further substances were
found to be meffective; these included chlorine dioxide (36), hydrogen peroxide
(39), peracettc acid (40), phenohc disinfectants (36,41), potassmm permangan-
ate (41), and urea (36). However, in the United States, tt was concluded that
mactivation could be achieved by exposure for 1 h to 1M sodium hydroxide or
gravity-displacement autoclaving at 132°C for 1 h (42) In the United Kingdom,
the preferred options have been the use of sodium hypochlorite solution con-
tammg 20,000 ppm available chlorine for 1 h (41) or porous-load autoclavmg
at 134-138°C for 18 min (43). These regimens have been adopted largely as
mternational standards but, as will be dtscussed, more recent studies have cast
doubt on the efficacy of all but the hypochlorite decontammation procedure
4.2. Sodium Hydroxide
The adoption m the United States of a 1-h exposure to 1M sodium hydroxide
as a recommended procedure for mactrvating CJD agent resulted from the report
that 10% homogenates of guinea ptg or hamster bram infected with CJD agent or
the 263 K strain of scrapie agent, respectively, were macttvated by this proce-
dure (36) However, m the same report it was acknowledged that pre-existing
data had demonstrated the survival of scrapie mfectrvity after exposure to sodium
hydroxide. More recently, survtval of the 263 K and ME7 strains of scrapie agent
has been recorded following exposure to even 2M sodium hydroxide for up to
2 h, the residual titer of 263 K bemg 10 4 2 ID,,/g bram (3). These data appear
superficially to be at gross variance with those reported m the previous study
(36) but they were obtained from samples that were undiluted prior to injection
through careful admstment to neutral pH. In contrast, the samples m the earlier
study required to be diluted before mjectton to eliminate problems of acute
toxictty to the recipient ammals, resultmg m a reduction m the sensitivity of
the bioassay. The general conclusion regarding mactivation of TDE agents by
sodium hydroxide 1s that although substantial reductions m mfectivity titers
can be achieved, the procedure cannot be relied on for complete mactivation.
4.3. Sodium Hypochlorite and Sodium Dichloroisocyanurate
From a study involving the use of the 22A and 139A strains of mouse-
passaged scrapte agent, it was concluded that a 1-h exposure to a sodmm hypo-
109
Transmwible Degenerative Encephalopathies

chlorate solution contammg 20,000 ppm available chlorme 1s an effective
decontamination procedure for TDE agents (4Z), which was later confirmed
for BSE agent (3). However, the expectation that soluttons of sodium
dichloroisocyanurate contammg the same levels of available chlorme would
be equally effective was not realized; under the same experimental conditions
used to test the effectiveness of sodium hypochlorite solutions, the sodium
dichloroisocyanurate soluttons failed to release thetr available chlorine content
freely and were thus less effective (3).
4.4. Gravity-Displacement Autoclaving
The endorsement m the United States of gravity-displacement autoclavmg
at 132°C for 1 h as an effective method for inactivating CJD agent resulted
from the report that 10% homogenates of guinea pig or hamster bram infected,
respectively, wtth the K Fu isolate of CJD agent and the 263 K strain of scrapie
agent were mactrvated by this procedure (36). Unfortunately, subsequent data
obtained using the 263 K strain of scrapie agent mdicate that this procedure is
not entirely reliable (44,4.5). One proposed solution to the problem of questton-
able autoclavmg standards, based on studies wtth CJD agent, 1sto treat contami-
nated materials for 1 h with 1M sodium hydroxide before gravity-displacement
autoclaving at 121°C for 30 min (46). However, there may be a problem if
autoclaves are used routmely for this purpose because of possible progresstve
degradative effects on the autoclave chamber
4.5. Porous-Load Autoclaving
In the United Kingdom, porous-load autoclavmg at 134-138°C for 18 min
has been recommended for mactivatron of CJD agent and, by inference, other
TDE agents. This was based on data that showed that 50 mg macerates of
mouse brain infected with the 22A or 139A strains of scrapte agent were mac-
tivated by porous-load autoclaving at 136°C for 4 min (41). The security of
this procedure appeared to be endorsed by the finding that intact mouse brain
infected with 22A, weighing approx 375 mg, was inactivated by porous-load
autoclavmg at 134OC for 18 mm (471, especrally since 22A is more thermo-
stable than other strains of scrapie agent (19,41). However, more recent studies
using comparable weights of cow brain infected with BSE agent, hamster brain
infected with the 263 K strain of scrapie agent, and mouse brain infected with
the ME7 strain of scrapte agent have produced confhctmg results that suggest
that this standard may not be secure(3). The most obvious difference between the
most recent study and previous ones 1sthat the brain samples m the most recent
study were autoclaved as relatively large ahquots of brain tissue macerates
rather than intact tissue that resulted m some smearing (and probably drying)
of tissue on the surfaces of the glass containers. It has been reported previously
110 Taylor

that scrapte-infected brain tissue dried onto glass surfaces 1s more difficult to
mactivate than salme homogenates (48), whtch may be the explanation for
these apparently anomalous findings that currently are being mvestigated fur-
ther. However, ttssue smearing and dehydration on glass surfaces occurs com-
monly with laboratory equipment that requires TDE decontammation by
autoclavmg, and it may be more realistic to ensure that such conditions prevail
m future autoclave mactivation studies. Despite the theoretical imphcations of
these tindmgs, we have not observed any anomalous experimental data that
might be attributable to cross-contammation; such occurrences would be evi-
dent because of the phenotypic characteristics of BSE and different scrapie
agents that are observed m mice of parttcular genotypes Nevertheless, we have
increased our autoclavmg sterilization standard for scrapie-like agents to 136°C
for 1 h, pendmg the outcome of further studies The risk of iatrogenic transmis-
sion of human TDE through survival of infectivity on surgical instruments that
have been autoclaved has been largely excluded for many years m the United
Kingdom. Because the Department of Health recognized the difficulty of estab-
hshmg secure autoclavmg standards, it has recommended that instruments used
m neurosurgery or ophthalmology on patients with known or suspected CJD
should be destroyed rather than try to decontaminate them (49); more recently,
this recommendation has been extended to mclude newer categories of patients
at risk of developing CJD, such as reciptents of cadavertc human growth hor-
mone or dura mater (50).
4.6. Rendering
Epidemiological studies have indicated that the emergence of BSE in the
United Kingdom during the mid- 1980s was associated with the practice of feed-
mg cattle with diets contammg rummant-derived protem that is manufactured
mainly from abatton waste by the rendermg industry (13). The cooking proce-
dures used by the rendermg industry enable the fat content of animal tissues to
be fluidtzed and collected as commercial tallow, the residual sohds are milled
to produce meat and bone meal that is the source of the ruminant-derived pro-
tem that had been fed to cattle until the UK ban m 1988. It has been hypoth-
esized that scrapie mfectivity m ovme tissues survived rendering procedures at
sufficiently high titer to represent an effective oral challenge for cattle, with a
likely initial exposure at the begmnmg of the 1980s (13). Although the feeding
of meat and bone meal has been commonplace for a much longer period, it was
recognized that there had been a rapid reduction m the use of solvent extraction
as an adjunct to rendermg procedures at a time that coincided with the first
putative enhancement of exposure of cattle to scrapie agent (13). Tradmonally,
solvent extraction had been applied to the solid endproducts yielded by render-
mg procedures to enhance the yield of tallow and to produce low-fat meat and
111
Transmisstble Degenerative Encephatopathies

bone meal that at one time attracted premium prtces. Apart from the exposure
of the raw materials to heated solvents, such as hexane, benzene, petroleum,
and trtchloroethane, the solvent extraction process also Involved exposure of
the processed solrds to dry heat and steam at approx 100°C to evaporate residual
solvent It has been suggested that these procedures may have produced suffi-
cient additional macttvation of scrapte agent to produce meat and bone meal
wtth infecttvity levels that were insufficient to represent an effective dietary
challenge for cattle (23). There ts, however, an absence of any mformation for
the mactivation potential of any of the solvents used, and the scrapte agent 1s
known to be relatively thermostable (51). Experiments are in progress to deter-
mme the effectiveness of such procedures but results are not yet available.
It has also been recognized that none of the experimental conditions under
whtch the thermostability of TDE agents has been studied previously are
directly relevant to rendering procedures (51) Consequently, mdustrtal ptlot-
scale facsimiles of rendering practtces used within the European Community
have been spiked with BSE or scrapte agent (52) The scrapte-spiked experi-
ments are still m progress but the BSE-spiked study is now complete. This has
demonstrated that, even though the spike level was low, BSE mfectivtty was
detectable m meat and bone meal produced by two types of process. These
were. a procedure that involved exposure of the raw materials to heating for
50 mm at atmosphertc pressure wtth the final temperature reaching 112 or
122™C; and processmg raw materials under vacuum with added preheated
tallow for 10 or 40 mm, and the final temperatures reaching 120 or 121“C,
respectively. In view of these findmgs, the mmtmal acceptable temperatures
for rendering have been revised within the European Commumty (53).
Because these types of rendering systemshad been mtroduced mcreasmgly
m the United Kingdom during the 197Os,this m itself does not offer an expla-
nation for the emergence of BSE m the 1980s given that the average age of
cattle developing clmtcal symptoms 1s4-5 yr. However, tf the solvent extrac-
tion process had provided addttional mactivatton as has been postulated, the
rapid decline in the use of this process m the United Kingdom during the late
1970s and early 1980s may have been the key additive factor that permuted
sheep scrapie to be transmitted to cattle. The experiments that are currently m
progress relating to the macttvatton potential of solvent extraction procedures
should clarify this situatton.
5. Working Practice in the Laboratory
Until 1993 CJD had been observed in 24 mdividuals who had been health-
care workers of varrous types, mcludmg a pathologtst and two technicians who
had worked m neurohtstopathology laboratones, but there was no evident asso-
ciation between their developmentof CJD and any occupational exposure to
Taylor
112

CJD agent (54) Furthermore, there was histortcally an interval of 40 yr between
the recogmtton of CJD as a chnrcal entity and the suspicion that the disease
might be transmissible. Even though CJD 1s a rare disease, bram tissue from
CJD-Infected individuals must have been handled worldwide without signA-
cant precautions during this pet-rod by pathologists and laboratory personnel
but without any apparent increased incidence of the disease m such mdividu-
als. Nevertheless, the accidental transmission of CJD to human recipients of
CJD-contaminated human growth hormone by mtramuscular mJection demon-
strates that occupationally acquired disease through trauma is a posstbihty
Survrval of mfecttvny m brain tissue after exposure to formalm has been
described for natural scrapie m sheep (Z2) and for experimental scrapie m ham-
sters (55) and mice (2) The same is true for the agents of BSE (.56), CJD (57),
and TME, which is known to survive for at least 6 yr (58). When hamster brain
contammg 1Or0 * ID,dg of the 263 K strain of scrapie agent was fixed in form01
salme for 48 h, only 1.5 logs of mfecttvity were lost (55); even after full htsto-
logical processmg the titer loss was only 2.8 logs (59). Glutaraldehyde treat-
ment is also known to permit survival of CJD (60) and scrapie mfecttvity (Z9)
Consequently, the handling of fixed CJD-infected tissues m the htstopathology
laboratory has been viewed as a potentially risky activity, and a number of
procedures have been recommended to reduce this risk. One suggestion has
been to fix such tissues m form01 salme containing sodium hypochlorite (61),
although, as discussed above, high concentrations of sodium hypochlorite
macttvate TDE agents, there has been no vahdation of its effectiveness when
combmed with formalm The addition of phenol to form01 salme has also been
suggested (62-64) but the basis of this proposal was flawed (65), and
phenohzed formalm subsequently was shown to be not only meffectrve (66)
but also to produce poor fixation (66,67). Sections stained with hematoxylm
and eosm, prepared from scrapie-infected formol-fixed bram tissue that was
autoclaved at 134°C for 18 mm, retained sufficient integrity to permit quantita-
tive scoring of spongtform encephalopathy (68); tt has been suggested that
autoclaving at 126°C for 30 mm (69) or 132°C for 60 mm (70) could be used to
mactivate CJD mfectlvity m formol-fixed brain. However, mouse or hamster-
passaged scrapie agent m formol-fixed bram has been shown to survive porous-
load autoclavmg at 134°C for 18 mm (47) or gravity-displacement autoclavmg
at 134°C for 30 mm (55), with titer losses of <2 logs. The only procedure that
has been shown to result m significant losses of mfectivtty titer m formol-fixed
tissues, without loss of microscopic morphology, IS a 1-h exposure to concen-
trated formic acid (66). In that study the level of mfectivrty m hamster brain
infected with the 263 K strain of scrapte agent was reduced from 1O™O 2 ID,,/g to
10™ 3 IDSO/g; with human brain infected with CJD agent the original titer of
lo8 5 IDSO/g was reduced to 10 2 3 IDso/g. However, m another study where
Transmissible Degenerative Encephalopathies
mouse brain infected with the 301V stram of BSE agent was fixed using
paraformaldehyde-lysme-perrodate, a necessary prerequisite for the subsequent
tmmunocytochemtcal investigation that is an important aspect of TDE mvestr-
gation, the degree of inacttvatlon by formic acid was calculated to be two logs
less than that achieved with formol-fixed 263 K-infected hamster brain, despite
the equivalent levels of infectivtty of the two agents (72). Thts suggests either
that infected tissues fixed with paraformaldehyde-lysine-periodate are less
amenable to the inactivating effect of formic acid than those fixed with forma-
lin or there is a fundamental difference in the susceptibility of the 263 K agent
compared with 301V; alternatively, both factors may contribute to this obser-
vation. Although further studies are in progress to clarify this situation tt is
evident that there IS no known decontaminatton procedure that can guarantee
the complete absence of infectivity in TDE-infected tissues that have been pro-
cessed by htstopathologrcal procedures. Clearly, autoclavmg of htstologrcal
waste is inappropriate for mactivatmg scrapie-like agents, and reliance must
be placed on mcineratron.
Precautions in the handling of TDE agents in other types of laboratory are
somewhat different from those m the histopathology laboratory. For example,
drsruptron of neural tissues by homogemzation has the potential to release many
more infectious airborne particles than from section-cutting in the histo-
pathology laboratory, especially if the latter tissues were treated with formtc
acrd In the brochemistry laboratory there is also the capabrhty, through partial
purification procedures, to produce samples that contam infectivity titers higher
than those found m naturally infected tissues. Apart from general good labora-
tory practice, the prmclpal recommendatton when handling TDE agents under
such conditions IS to use mtcrobiological safety cabinets. However, what must
be borne in mmd is the resistance of TDE agents to inactrvation by formalm,
which IS the customary fumigant for routine decontamination of safety cabi-
nets The main objective, therefore, is to adopt working procedures that mmt-
mize the potential for contammatton of the cabinet; these include measures
such as the use of disposable covering materials on the work surface, and the
prevention of aerosol drspersron, e.g., by retaining cotton-wool plugs m glass
tissue homogenizers during sample disruption (and for some time thereafter, if
possible). Regardless of these types of precautions, tt would be naive to con-
sider that they would guarantee complete freedom from contaminatton of the
internal surfaces of safety cabinets. Although contammation at this sort of level
IS unlikely to represent any significant risk to the operator, given that such
work should always involve the wearing of disposable gloves and laboratory
coats, the potentral for crosscontammation from different TDE sources m labo-
ratory experiments has to be considered. This can be addressed by adopting a
routine of washing the internal surfaces with a solutton of sodium hypochlorlte
114 Taylor

containing 20,000 ppm available chlorine; however, a compromise has to be
struck between the perceived necessary frequency of such a decontammatton
procedure and its potential progressive degradative effect on exposed surfaces
Class II safety cabmets are suitable for this type of work, and are popular gen-
erally because they combme satisfactory degrees of product and personnel pro-
tection under conditions that are not too restrictive for the operator. However,
the classtcal design of such cabmets has been such that contammatton of the
internal plenum and air-propellmg units IS likely. Although this IS not prob-
lematic for conventional microorgamsms that can be macttvated by formalm
fumigation that penetrates these areas, there is a problem obviously with TDE
agents. Although such TDE contammation does not represent any stgmficant
risk to the operator or the work activity, there is the problem of how to achieve
decontamination before engineers are permitted to carry out repairs or servic-
mg This IS because of the inaccessibthty of the plenum to manual hypochlorite
decontamination, and additionally the potential degradation effects of hypo-
chlorite on the air-propellmg units. An improvement m this sttuation has been
achieved by the manufacture of class II safety cabmets with filters positioned
immediately below the working surface, which means that contammation of

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