Exposure of RML scrapie agent to a sodium percarbonate-based product and
sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate
infectivity
BMC Veterinary Research 2013, 9:8 doi:10.1186/1746-6148-9-8 Jodi D Smith
(jodi.smith@ars.usda.gov) Eric M Nicholson (eric.nicholson@ars.usda.gov) Gregory
H Foster (ghfoster@mac.com) Justin J Greenlee (justin.greenlee@ars.usda.gov)
ISSN 1746-6148
Article type Research article Submission date 18 September 2012 Acceptance
date 8 January 2013 Publication date 11 January 2013 Article URL http://www.biomedcentral.com/1746-6148/9/8
BMC Veterinary Research
© 2013 Smith et al.
Exposure of RML scrapie agent to a sodium percarbonate-based product and
sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate
infectivity
Jodi D Smith1 Email: jodi.smith@ars.usda.gov Eric M Nicholson1 Email:
eric.nicholson@ars.usda.gov Gregory H Foster1 Email: ghfoster@mac.com Justin J
Greenlee1* * Corresponding author Email: justin.greenlee@ars.usda.gov
1 Virus and Prion Research Unit, National Animal Disease Center, USDA,
Agricultural Research Service, 1920 Dayton Ave, Ames, IA 50010, USA
Abstract
Background
Prions, the causative agents of the transmissible spongiform
encephalopathies, are notoriously difficult to inactivate. Current
decontamination recommendations by the World Health Organization include
prolonged exposure to 1 N sodium hydroxide or > 20,000 ppm sodium
hypochlorite, or autoclaving. For decontamination of large stainless steel
surfaces and equipment as in abattoirs, for example, these methods are harsh or
unsuitable. The current study was designed to evaluate the effectiveness of a
commercial product containing sodium percarbonate to inactivate prions. Samples
of mouse brain infected with a mouse-adapted strain of the scrapie agent (RML)
were exposed to a sodium percarbonate-based product (SPC-P). Treated samples
were evaluated for abnormal prion protein (PrPSc)- immunoreactivity by western
blot analysis, and residual infectivity by mouse bioassay.
Results
Exposure to a 21% solution of SPC-P or a solution containing either 2.1% or
21% SPC-P in combination with sodium dodecyl sulfate (SDS) resulted in increased
proteinase K sensitivity of PrPSc. Limited reductions in infectivity were
observed depending on treatment condition. A marginal effect on infectivity was
observed with SPC-P alone, but an approximate 2–3 log10 reduction was observed
with the addition of SDS, though exposure to SDS alone resulted in an
approximate 2 log10 reduction.
Conclusions
This study demonstrates that exposure of a mouse-adapted scrapie strain to
SPC-P does not eliminate infectivity, but does render PrPSc protease
sensitive.
Keywords Inactivation, Prion, Scrapie, Sodium dodecyl sulfate, Sodium
percarbonate
SNIP...
A major finding of this study was the increased sensitivity of PrPSc to PK
by the SPC-based product without (SPC-PH only) or with SDS at room temperature,
as judged by immunoblotting after exposure of the samples to limited
proteolysis. Based on the loss of detectable PrPSc immunoreactivity after
incubation at pH 11, it appears this effect may be largely pH-dependent. It is
well established that prion infectivity is reduced under extremely basic
conditions, such as exposure to NaOH (pH 12–14) [19-21]. While the pH generated
by SPC-P is lower at 11, it appears to be a favorable characteristic of the
compound with regard to PrPSc protease sensitivity. However, a solely
pH-dependent effect does not explain why SPC-PL treatment alone (pH 11) did not
yield similar WB results. One possible explanation is that a lower concentration
of the product may have contained diminished buffering capacity resulting in a
drop in pH as treatment proceeded, but serial pH evaluation of treated brain
homogenate at 30, 90, and 180 min revealed that the pH remained above 10.7.
Although treatment with the SPC product did render PrPSc sensitive to digestion
by proteinase K, it did not eliminate infectivity. Recent studies examining
prion infectivity in infected tissue and cell cultures have also demonstrated
loss of detectable PrPSc on western blot, but residual infectivity [22,23]. Our
results support the inference that biochemical analysis alone is insufficient
for determination of prion infectivity. The observed PrPSc/infectivity mismatch
in this study and in others warrants a number of considerations including WB
sensitivity, epitope disruption by inactivation treatments, and alternative
infectious agents to PrPSc, such as PK-sensitive forms of PrP or viruses. It is
possible the amount of residual PrPSc in our treated samples was below the
detection limit of our WB (0.025 mg equivalents of brain tissue for this
particular inoculum [24]), or it may be that a true dissociation of PrPSc and
TSE infectivity exists supporting the actuality of alternative infectious agents
to PrPSc [25]. A recent study has demonstrated poor correlation between
infectivity and WB results for sheep scrapie and sheep BSE [26] in line with
observations that PK-sensitive PrP particles are associated with disease
[27,28].
The bioassay results we present indicate that exposure to the selected
SPC-based product alone or in combination with 2.5% SDS is not a viable option
for the inactivation of prions. No decrease in infectivity was observed using
the SPC-PL solution alone, and a modest 1 log10 reduction was achieved with the
SPC-PH solution. However, recent investigations have demonstrated differential
susceptibility of distinct prion strains to the same inactivation procedure
[29]; therefore, we are currently investigating the efficacy of these treatment
conditions in an ovine scrapie model. It should also be acknowledged that
chemical treatment of the scrapie agent has been shown to delay the
dose–response relationship [30,31] resulting in prolonged incubation times
without a change in calculable titer. It is possible our results could be
reflecting this phenomenon, but without bioassay data from serial dilutions of
treated brain homogenate this cannot be definitively determined. Some caution
may therefore be warranted when interpreting these results. The addition of 2.5%
SDS to the SPC-P solutions resulted in a 2–3 log10 reduction in infectivity, but
exposure to SDS alone resulted in an approximate 2 log10 reduction. This
suggests much of the observed combinatorial effect was due to SDS. Prior studies
using SDS have demonstrated minimal effects on CJD infectivity [16], but up to a
3 log10 reduction on scrapie infectivity [17]. Exposure of hamster-adapted Sc237
scrapie to room temperature SDS at pH values of ≤4.5 or ≥10 resulted in
increased PK sensitivity of PrPSc, and exposure to acidic SDS resulted in
decreased infectivity [11]. Since SDS at room temperature is an effective
denaturant at a pH ≥10, this could have contributed to the loss of detectable
PrPScimmunoreactivity we observed after proteolysis in samples treated with
SPC-P and SDS. There was also enhanced reduction in infectivity with the
combination of SPC-PL and SDS. This may be indicative of an enhanced effect of
SDS under basic conditions or a two-step mechanism whereby denaturation of PrPSc
by the relatively high pH of the solution and/or SDS is followed by exposure of
sites sensitive to oxidative damage. Alternatively, the two treatment components
could be acting on different PrPSc fractions in the inoculum resulting in an
additive effect since the combination of SPC-PL and SDS was roughly equivalent
to slightly greater than the sum of the effects of each individual component.
The combination of SPC-PH and SDS did not provide an equivalent or better
increase in survival time than the combination of SPC-PL and SDS. While we are
confident in this result, we cannot definitively explain this observation.
Perhaps disease in this group was exacerbated by oxidative damage induced by the
introduction of treated brain samples containing a greater concentration of
sodium percarbonate. Oxidative stress, whether a cause or consequence of disease
progression, is considered an important contributor to prion neuropathology
[32-34]. It is also possible that the SPC-P solution at higher concentration may
somehow be interfering with the denaturing action of SDS. SDS action may be
enhanced when combined with lower concentrations of SPC-P for longer exposure
times, but restricted by higher concentrations, perhaps via chemical
modification of SDS binding sites on the protein.
Oxidizing agents have been used with variable success in prion inactivation
studies. Exposure of prions to halogens such as sodium hypochlorite at ≥ 20,000
ppm is an accepted means of decontamination [8], but chlorine dioxide is much
less effective at inactivating hamster-adapted 263 K scrapie [35]. Peroxygens
such as liquid hydrogen peroxide [13,35,36] and peracetic acid [37] also promote
limited inactivation. However, recent studies using vaporized hydrogen peroxide
to decontaminate stainless steel surfaces have demonstrated significant
reductions in infectivity for hamster-adapted 263 K scrapie and mouse-adapted
BSE [13,15]. A protective effect from oxidation by peracetic acid has been
demonstrated with the ME7 scrapie agent and attributed to prion aggregation
[37]. Peracetic acid at 2% was effective at inactivating the ME7 scrapie agent
in intact brain tissue, but not homogenized tissue. Samples in the current study
were homogenized, which may have imparted a degree of protection from oxidation
and contributed to the ineffectiveness of SPC-P alone at decreasing infectivity.
We propose that the addition of SDS would have decreased aggregation of cell
membranes to which infectivity is bound, thus enhancing the activity of SPC-P
and perhaps contributing to the increased survival observed with the
combination.
Conclusions
This study demonstrates that exposure of the RML scrapie agent to an
SPC-containing product alone or in combination with SDS does not eliminate prion
infectivity, but does render PrPSc sensitive to proteinase K. Because of this,
it is interesting to consider the potential viability of a combination of SPC
and SDS, even at relatively low concentrations and mild temperatures,
concomitant with or followed by a protease for prion decontamination. Also,
because the SPC product we used contains additional proprietary ingredients, we
cannot rule-out contributions to increased PK-sensitivity or increased survival
by other components of the product. Studies in our laboratory are currently
underway examining exposure of prions to chemical grade SPC with or without SDS
followed by exposure to a protease.
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Comments on technical aspects of the risk assessment were then submitted to
FSIS.
Comments were received from Food and Water Watch, Food Animal Concerns
Trust (FACT), Farm Sanctuary, R-CALF USA, Linda A Detwiler, and Terry S.
Singeltary.
This document provides itemized replies to the public comments received on
the 2005 updated Harvard BSE risk assessment. Please bear the following points
in mind:
Owens, Julie
From: Terry S. Singeltary Sr. [flounder9@verizon.net]
Sent: Monday, July 24, 2006 1:09 PM
To: FSIS RegulationsComments
Subject: [Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine
Spongiform Encephalopathy (BSE)
Page 1 of 98
FSIS, USDA, REPLY TO SINGELTARY
U.S.A. 50 STATE BSE MAD COW CONFERENCE CALL Jan. 9, 2001
2012 atypical L-type BSE BASE California reports
Saturday, August 4, 2012
*** Final Feed Investigation Summary - California BSE Case - July 2012
SUMMARY REPORT CALIFORNIA BOVINE SPONGIFORM ENCEPHALOPATHY CASE
INVESTIGATION JULY 2012
Summary Report BSE 2012
Executive Summary
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Update from APHIS Regarding Release of the Final Report on the BSE
Epidemiological Investigation
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atypical Nor-98 Scrapie has spread from coast to coast in the USA 2012
NIAA Annual Conference April 11-14, 2011San Antonio, Texas
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SCRAPIE AND ATYPICAL SCRAPIE TRANSMISSION STUDIES A REVIEW 2010
Wednesday, February 16, 2011
IN CONFIDENCE
SCRAPIE TRANSMISSION TO CHIMPANZEES IN CONFIDENCE
Tuesday, November 02, 2010
IN CONFIDENCE
The information contained herein should not be disseminated further except
on the basis of "NEED TO KNOW".
BSE - ATYPICAL LESION DISTRIBUTION (RBSE 92-21367) statutory (obex only)
diagnostic criteria CVL 1992
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Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion
disease, Iatrogenic, what if ?
Proposal ID: 29403
TSS
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