Post on 06-Feb-2018
Inactivation of template-directed misfolding of infectious 1
prion protein by ozone 2
3
Ning Ding1, Norman F. Neumann2,3*, Luke M. Price2, Shannon L. Braithwaite2, Aru 4
Balachandran4, Miodrag Belosevic2,5 and Mohamed Gamal El-Din1* 5
6
1Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta 7
Canada; 2Department of Public Health Sciences, University of Alberta, Edmonton, 8
Alberta Canada; 3Provincial Laboratory for Public Health, Edmonton, Alberta, Canada; 9
4Canadian Food Inspection Agency, Ottawa, Ontario, Canada; 5Department of Biological 10
Sciences, University of Alberta, Edmonton, Alberta, Canada. 11
12
13
14
15
*Corresponding Authors: Dr. Mohamed Gamal El-Din 16
Department of Civil and Environmental Engineering 17
University of Alberta 18
Edmonton, Alberta, T6G 2W2, Canada 19
Tel: 1-780-492-5124 20
E-mail: mgamalel-din@ualberta.ca 21
22
Dr. Norman F. Neumann 23
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.06791-11 AEM Accepts, published online ahead of print on 2 December 2011
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Department of Public Health Sciences 24
University of Alberta 25
Edmonton, Alberta, T6G 2T4, Canada 26
Phone: 1-780-492-8502 27
E-mail: Norman.Neumann@albertahealthservices.ca 28
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Abstract 31
Misfolded prions (PrPSc) are well known for their resistance to conventional decontamination 32
processes. The potential risk of contamination of the water environment, as a result of disposal of 33
specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the 34
water industry for inactivation of microbial contaminants and was tested in this study for its 35
ability to inactivate prions (263K hamster scrapie = PrPSc). Treatment variables included initial 36
ozone dose (7.6–25.7 mg/L), contact time (5 s and 5 min), temperature (4 °C and 20 °C) and pH 37
(pH 4.4, 6.0, 8.0). Exposure of dilute suspensions of the infected 263K hamster brain 38
homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating 39
properties of PrPSc, as measured by the protein misfolding cyclic amplification (PMCA) assay. 40
Highest levels of prion inactivation (≥4 log10) were observed with ozone doses of 13.0 mg/L, at 41
pH 4.4 and 20 °C, resulting in a CT (the product of residual ozone concentration and contact time) 42
value as low as 0.59 mg·L-1 min. A comparison of ozone CT requirements among various 43
pathogens suggests that prions are more susceptible to ozone degradation than some model 44
bacteria and protozoa, suggesting that ozone treatment may be an effective solution for 45
inactivating prions in water and wastewater. 46
47
Keywords: PMCA, Ozone dose, Contact time, Temperature, pH 48
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1. Introduction 50
The normal cellular prion protein (PrPC) is present in most tissues of humans and other 51
animals (15), and is expressed at particularly high levels in neuronal tissues (20, 40). PrPC can 52
transform into a protease resistant, infectious, misfolded pathological isoform (PrPSc) which can 53
subsequently act as an efficient template for conformational misfolding of PrPC. Template 54
directed misfolding of PrPC by PrPSc represents the fundamental mechanism for disease 55
progression in prion-related transmissible spongiform encephalopathies [TSEs] such as scrapie, 56
chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) (28). Moreover, 57
the associated change in conformational structure results in the protein (PrPSc) becoming 58
resistant to most treatment methods used for the inactivation of microbial pathogens (37). 59
Tissues associated with the nervous and immune systems are considered specified risk 60
materials (SRMs) in the food animal production industry. Due to the resistant nature of PrPSc and 61
the potentially high levels of PrPSc present in SRM tissues, the liquid and solid wastes generated 62
from the disposal of SRM pose serious concerns due to the possible release and bioaccumulation 63
of misfolded infectious prion proteins in the environment. PrPSc has been found to be extremely 64
recalcitrant in the environment. Results reported by Brown and Gajdusek (5) demonstrated that PrPSc 65
infectivity remained in soil for 3 years, and both soil and the aqueous extracts from contaminated 66
soil were infectious in animal models (33). Recently, the prion agent of CWD in cervids was 67
detected in one surface water sample from a CWD endemic area in USA (23). These authors 68
suggested that the persistence and accumulation of prions in the environment may promote the 69
transmission of CWD (23).Water and wastewater may act as transport agents for prions from 70
liquid wastes from slaughtering houses, rendering plants, agricultural digesters, and some septic 71
systems (27). PrPSc appears to be resistant to conventional municipal water and wastewater 72
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treatment regimens, such as chlorination (usually ~1 mg/L available Cl2 in water) (38), UV 73
irradiation (11), and mesophilic anaerobic sludge digestion (12). The high resistivity of PrPSc to 74
conventional inactivation in water and wastewater intensifies concerns about prion 75
contamination of the environment, thus effective approaches for prion decontamination in 76
aqueous environment are desirable. 77
Ozone as an advanced oxidation technology is widely used in water and wastewater 78
treatment processes for inactivation of bacteria (44), viruses (19, 39), and protozoa (18). 79
Although certain advanced oxidation processes have been shown to inactivate infectious prions 80
(14, 25, 26, 34, 35), several major knowledge gaps exist, in particular how interactions between 81
oxidant reaction conditions (e.g., temperature, pH, duration of exposure, ozone dose and organic 82
load) affect inactivation levels of prions. Consequently, the derivation of a CT disinfection value 83
for prions has never been reported. The CT value is defined as the product of the residual 84
disinfectant concentration [e.g., mg/L] and the contact time [e.g., minutes] required to achieve a 85
certain level of inactivation of a particular target organism (i.e., CT99 is the CT product required 86
for 99 % inactivation [or 2-log10]). Maintaining and measuring the residual oxidant dose is 87
critical during the disinfection process, since advanced oxidant products are short-lived and 88
consumed rapidly by non-target biomolecules (i.e., first order decay), and a sustained residual 89
dose is required for continuous reaction against more resistant biomolecular structures (e.g., 90
aggregates of PrPSc). The United States Environmental Protection Agency (USEPA) routinely 91
uses a CT concept for characterizing disinfection requirements for microbial pathogens under a 92
given set of reaction conditions (pH and temperature), and for which a comparative assessment 93
of the susceptibility of the pathogens can be made (41, 42). Consequently, the CT value is 94
commonly used as an engineering target for inactivation of pathogens in water matrices, as 95
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regulatory standards in water treatment, and for modeling the inactivation kinetics of 96
physicochemical disinfectants (19, 39). 97
Until recently, a major challenge associated with studying physicochemical inactivation 98
of prions has been the lack of a cost effective, high volume assay for detection and quantification 99
of PrPSc, and therefore animal bioassays remain the gold standard for assessing prion infectivity. 100
However, the protein misfolding cyclic amplification (PMCA) assay has recently emerged as a 101
powerful in vitro tool for detection and quantification for PrPSc (32). Protein misfolding cyclic 102
amplification results in the template directed misfolding of PrPC (naturally present in normal 103
brain homogenates) as a result of seeding a small quantity of infectious PrPSc into the in vitro 104
reaction, thereby mimicking the pathological process of disease progression in vivo (6). The 105
overall amount of amplification obtained in PMCA is contingent on the amount of infectious 106
seed used to initiate template directed misfolding (32), thus allowing for quantification of PrPSc 107
found in the original infectious seed. The PMCA assay has a wide dynamic range of sensitivity 108
(31). This assay considerably reduces the time required for generating results (i.e., 3 days) 109
compared to animal bioassay models (several months to more than a year), and studies 110
examining heat sterilization of PrPSc demonstrated that results obtained by PMCA correlated 111
with animal infectivity (22, 36). 112
The objective of this study was to determine the effectiveness of ozone for inactivating 113
template directed misfolding properties of PrPSc (263K scrapie), as determined by PMCA, under 114
a variety of experimental conditions (ozone dose, contact time, pH, and temperature) and for 115
which CT values for ozone could be derived. 116
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2. Materials and Methods 117
2.1 Buffers 118
Phosphate buffered saline (130 mM sodium chloride, 20 mM potassium chloride, 7 mM 119
sodium phosphate, and 3 mM potassium phosphate in Milli-Q water) was prepared and used to 120
make brain homogenates; and for making ozone stock solution in inactivation experiments. An 121
alternate PBS buffer (0.66x) was used as a component of conversion buffer for PMCA assay. 122
The 0.66x PBS was prepared using PBS tablets (BioBasic Inc., Markham, ON, Canada), 123
dissolving 1 tablet per 150 mL Milli Q water. 124
PMCA conversion buffer was prepared with a final concentration of 0.15 M sodium 125
chloride (Fluka/Sigma-Aldrich, Toronto, Canada), 5 mM EDTA (Gibco Invitrogen Canada Inc., 126
Burlington, ON, Canada), and 1% Triton (MP Biochemicals, Salon, OH, USA) in 0.66x PBS, 127
and adding 1x complete protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada) 128
according to manufacturers’ instructions. 129
130
2.2 Animals 131
Three to six week old female Syrian golden hamsters (Charles River Laboratories 132
International, Inc., Wilmington, MA, USA) were used to prepare infectious brain homogenates 133
(IBH) and normal brain homogenates (NBH), respectively. The hamster handling protocol used 134
in this study adhered to the Canadian Council of Animal Care (CCAC-Canada) guidelines. 135
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136
2.3 Preparation of hamster brain homogenates 137
2.3.1 Infectious prion brain homogenates (IBH) 138
Female Syrian Gold hamsters were either exposed orally (100 µL) or by intraperitoneal 139
injection (50 µL) to an inoculum of 263K scrapie positive brain homogenates at the Canadian 140
Food Inspection Agency (CFIA) Transmissible Spongiform Encephalopathy (TSE) Laboratory 141
in Nepean, Ontario, Canada. Hamsters displaying clinical signs of scrapie, typically 95–110 142
days post inoculation, were euthanized with carbon dioxide and the brains harvested in as short 143
of time as possible. All infected hamsters were confirmed positive by routine diagnosis at the 144
CFIA TSE Laboratory (ELISA and immunohistochemistry). The infectious dose of brain 145
homogenates was subsequently determined to be 109.94 ID50 per gram of brain tissue as 146
confirmed by hamster infectivity end point titration assays. Ten percent IBH samples 147
(weight/volume in 1x PBS) were manually disrupted (15–20 strokes) on ice using a Potter/glass 148
tissue grinder/homogenizer and allowed to stand on ice for 30 min followed by 1 min 149
centrifugation at 1000x g. The clarified 10% IBH supernatant was used as stock solutions for 150
ozone inactivation and PMCA experiments. 151
2.3.2 Normal brain homogenates (NBH) 152
Hamsters were sacrificed by exposure to excess carbon dioxide (dry ice in a kill box), 153
upon confirmed death. Cold 1x PBS with 5 mM EDTA (Gibco Invitrogen Canada Inc., 154
Burlington, ON, Canada) was perfused through the hamster circulatory system with the 155
assistance of a peristaltic pump attached to a syringe with the needle puncture to the left ventricle 156
of the heart. A 10% NBH was prepared by adding 1 g of perfused brain suspended in 8 mL of the 157
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conversion buffer and 1 mL of 15 USP sodium heparin solution (BD Vacutainer, Franklin Lakes, 158
NJ, USA). The sodium heparin solution was originally prepared by the addition of 10 mL of 159
0.66x PBS to a pre-coated 150 USP sodium heparin vacutainer (BD Vacutainer), and 160
subsequently aliquoted and froze at –20 °C. Normal brain samples in the conversion 161
buffer/sodium heparin solution were manually disrupted (15–20 strokes) on ice using a 162
Potter/glass tissue grinder/homogenizer and allowed to stand on ice for 30 min followed by 1 163
min centrifugation at 1000x g. The clarified 10% NBH supernatant was used for 164
PMCA/experimentation. 165
166
2.4 Ozone inactivation of PrPSc 167
2.4.1 Preparation of ozone-demand free reagents and reactors 168
The 1x PBS was used to make ozone stock solution. The pH was adjusted to pH 4.4, 6.0 169
and 8.0 with 1M HCl or 1M NaOH, respectively. The PBS buffer, reaction tubes, pipette tips and 170
magnetic stir bars were made ozone demand-free prior to use. The ozone-demand free (ODF) 171
PBS buffer was prepared by bubbling ozone gas (see ozone gas preparation below) through a 172
glass bottle for 20 min. The bottle was then placed at room temperature with the lid loose until 173
ozone residuals were completely dissipated, as confirmed by Indigo method (1). The reaction 174
tubes, pipette tips and stir bars were soaked in an ozone solution in deionized water (initial 175
concentration > 10 mg/L) overnight, followed by drying at room temperature for 3 days. 176
2.4.2 Ozone inactivation experiment 177
Ozone stock solutions (in 1x PBS at pH 4.4, 6.0, and 8.0) were generated from ultra pure 178
oxygen using an ozone generator (G30, PCI WEDECO). Concentrated ozone stock solutions in 179
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PBS buffer were prepared by bubbling ozone gas through 1 L of PBS buffer chilled at 4 °C, for 180
around 30 min and ozone dose within the stock solution determined using the Indigo method (1). 181
For ozone inactivation experiments, shell vial reaction tubes (Fisher Scientific, Canada) 182
were mounted on top of a stir plate with a Teflon-coated magnetic stir bar in each tube to ensure 183
even mixing. The temperature was controlled by submerging the reaction tubes in ice water (4 °C) 184
or at room temperature (20 °C). Separate reaction tubes were set up to withdraw samples at pre-185
determined reaction times. The experiment was carried out by adding ODF PBS buffer (same pH 186
as the stock solution) and diluted IBH into reaction tubes, followed by ozone stock solution with 187
a final volume of 1 mL. During the reaction, reaction tubes were covered with plastic lids. 188
Samples were withdrawn at pre-determined reaction times for residual ozone concentration 189
determination, immediately followed by addition of 20 µL of 1 M sodium thiosulfate to 190
neutralize residual ozone in the reaction tubes. The ozonated samples were then frozen at –80 °C 191
until PMCA assay. The non-ozonated control samples were treated in the same manner except 192
that ozone was completely neutralized prior to addition of diluted IBH. The residual ozone was 193
determined by Indigo method (1) with a UV-visible spectrophotometer (Biospec Mini 1240, 194
Shimadzu, Japan) immediately after the experiment. Absorbance measurement was performed at 195
600 nm in 1 cm quartz cell. The presence of diluted IBH and the applied sodium thiosulfate in 196
the control samples had negligible effect on Indigo method (data not shown). The absorbance of 197
the control samples was set up as reference for calculation of residual ozone concentration at 198
predetermined reaction time points. 199
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2.5 Protein misfolding cyclic amplification (PMCA) assay 201
The PMCA assay was used to measure inactivation of the templating properties of ozone 202
treated PrPSc samples. Control and ozonated samples were serial diluted 10-fold in 10% NBH 203
prior to the PMCA assay. Subsequently, two replicates of 8 µL aliquots samples of each 10-fold 204
dilution series was mixed with 72 µL of 10% NBH in 200 µL flat cap, thin wall PCR tubes 205
(Axygen,Unison City, CA, USA) by inversion. As a negative control for PMCA, 80 µL of a 10% 206
NBH was also prepared in the thin wall PCR tubes. PCR tubes were randomly placed in a 207
Misonix Sonicator Model 4000MXP (Misonix Inc Farmingdale, NY, USA) with the sonicator 208
microplate cup horn housed within the acoustic enclosure (provided with the instrument) and the 209
water reservoir temperature set to 37 °C. PMCA was performed for 19 h, with 40 s of sonication 210
followed by 29 min and 20 s incubation periods within each cycle (total number of cycle = 38) at 211
a potency of 90%. Samples were subsequently frozen at –80 °C and PrPSc detected by western 212
blot. 213
214
2.6 Proteinase K digestion, SDS-PAGE and western blot 215
Proteinase K (PK) digestion (200 µg PK/mL) was carried out on all PMCA samples and 216
non-PMCA controls (1% IBH in NBH). The digestion was performed at 37 °C for 20 min and 217
stopped with the addition of an equivalent volume of 2x Laemmli buffer [28.5 mL of Laemmli 218
sample buffer (Bio-Rad Laboratories, Mississauga, ON, Canada), 0.6 g SDS (Sigma-Alrich 219
Canada Ltd, Oakville, ON, Canada), and 1.5 mL β-mercapto-ethanol (Sigma-Alrich Canada Ltd, 220
Oakville, ON, Canada)] and incubated at 100 ± 5 °C for 5 min. Twenty five µL of denatured 221
samples were fractionated by SDS-PAGE (Pierce precise 12% precast polyacrylamide gels, 222
Thermo Scientific, Rockford, IL, USA) at 100 V for 1 h, and transferred onto PVDF membrane 223
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(Bio-rad laboratories, Mississauga, ON, Canada) at 20 V overnight on ice. The blots were 224
blocked in 5% skim milk in 1x PBS (Bio-rad laboratories, Mississauga, ON, Canada) with 0.1% 225
Tween 20 (Bio-rad laboratories, Mississauga, ON, Canada) for 1 h at room temperature. The 226
blots were then probed with primary anti-prion protein 3F4 antibody (Millipore, Billerica, MA, 227
USA) at 1:20,000 in 1x PBS (10 mM sodium phosphate and 150 mM sodium chloride) 228
containing 0.1% Tween 20 for 1 h at room temperature, followed by washing three times (10 min 229
each) in 1x PBS-0.1% Tween 20. The conjugated secondary antibody, goat anti-mouse horse-230
radish peroxidase (HRP, Bio-Rad) (1:10,000 in 1x PBS-0.1% Tween 20), was subsequently 231
added to bind to the primary antibody and incubated for 1 h at room temperature, followed by 232
washing three times (10 min each) in 1x PBS-0.1% Tween 20, and washing twice (5 min each) 233
in 1x PBS without Tween 20. Immunoreactive bands were then visualized using ECL reagent 234
and ImageQuant RT ECL Imager (Amersham, GE Life Sciences, Canada). 235
236
2.7 Quantitative analysis of western blot images 237
The blot images of control and ozonated samples were analyzed using ImageQuantTM TL 238
software (GE Health care). The net intensity of each blot was obtained from the software. The 239
signal intensity of all dilutions of ozonated samples, and 2 to 6 log10 dilutions of the non-240
ozonated control samples were normalized as a percentage of the average signal intensity 241
(saturated) of two replicates of 1 log10 dilution of the non-ozonated control samples. The 242
background intensity was subtracted before normalization. Assuming the normalized intensity 243
(as a percentage) vs. dilution fold follows exponential relationship before signal intensity 244
saturation (32), the inactivation of PrPSc by ozonation was calculated according to Eq. 1. 245
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××
=2) Fold(Dilution )Intensity2 Normalized(log
1) Fold(Dilution )Intensity1 Normalized(loglog )(logon Inactivati
10
1010
010 N
N
(1) 246
Where N0 is the concentration of PrPSc template in PMCA non-ozonated control samples, N is 247
the concentration of PrPSc template in PMCA ozonated samples. Normalized Intensity 1= the 248
intensity of the highest dilution of non-ozonated sample divided by the intensity of 1log10 249
dilution of non-ozonated sample; Dilution Fold 1= dilution (in the form of 10n where n is an 250
integer) of the highest diluted lane of non-ozonated control sample, i.e. 104, 105. Normalized 251
Intensity 2 = the intensity of the highest dilution of ozonated sample divided by the intensity of 252
1log10 dilution of non-ozonated sample; Dilution Fold 2 = dilution (in the form of 10n where n is 253
an integer) of the highest diluted lane of ozonated control sample, i.e. 101, 102,103, 104. 254
255
2.8 Estimation of CT values 256
The CT values were estimated for the purpose of gaining the credit of ozone inactivation 257
of PrPSc. Since ozone decomposition follows first order kinetics after initial ozone demand, the 258
CT values at a contact time of 5 min was estimated by the area under the ozone decay curve at 259
the specific time, using Eq. 2. 260
−−== )]'exp(1['
)( 0 tkk
CdttCCT (2) 261
where C0 is the ozone concentration at time 5 s (closest to time zero), k’ is the first order ozone 262
decomposition rate (min-1), t is contact time (min). To be conservative, CT for contact time of 5 s 263
was calculated by multiplying instantaneous ozone concentration by contact time. 264
265
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3. Results 266
3.1 Evaluation of ozone demand 267
Prior to investigating the effect of ozone inactivation on PrPSc, preliminary experiments 268
were carried out to determine the ozone demand associated with inactivation of PrPSc in 269
experimental brain homogenates. At an ozone dose of ~12 mg/L, ozone was completely 270
consumed by a 0.1% IBH suspension within 5 s, while a residual ozone concentration was 271
maintained for up to 5 min when a 0.01% IBH was used (Table 1). For this reason a 0.01% IBH 272
was used for all subsequent ozonation experiments for determining the inactivation efficiency of 273
PrPSc by ozone and for calculation of an ozone CT product for PrPSc. 274
275
3.2 Detection of PrPSc by PMCA assay 276
A 0.1% suspension of IBH represented the lowest concentration of IBH for which PrPRes 277
could be detected by western blot alone (Fig. 1A), indicating that western blot was insufficient as 278
detection tool for characterizing ozone inactivation of PrPSc. Incorporation of PMCA upstream of 279
western blot increased the sensitivity of detection of PrPSc upwards of 6-7 log10 compared to 280
western blot alone (Fig. 1). A 10% IBH sample could be diluted 100 million-fold (108) and still 281
be detected after only a single round of PMCA (38 cycles) (Fig. 1B). This expanded range of 282
sensitivity allowed for detection and quantification of ozone inactivation of PrPSc under the 283
conditions necessary to maintain an ozone residual (i.e., 0.01% IBH suspension), and 284
consequently for derivation of a range of CT values based on approximately 4 orders of 285
magnitude of inactivation (i.e., up to CT99.99 [detection of prion signal between 0.01% IBH and 286
0.000001% IBH using PMCA]). 287
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288
3.3 Ozone inactivation of PrPSc 289
Ozone inactivation experiments were performed under varying initial ozone doses (7.6 290
mg/L to 25.7 mg/L), contact time (5 s and 5 min), pH (4.4, 6.0, and 8.0), and temperatures (4 and 291
20 °C), to assess optimal conditions for inactivating the templating properties of PrPSc. In 292
preliminary low dose ozone reactions (7.6 mg/L) carried out at pH 8.0 and 20 °C, a PrPSc 293
reactive band was observed in control blots (i.e., samples in which ozone was neutralized by 294
sodium thiosulfate) as low as 5 log10 dilution of a 0.01% IBH sample using PMCA (Fig. 2, Panel 295
C). However, after only 5 s of exposure to this ozone dose, pH and temperature, a loss of 1 log10 296
in PrPSc western blot signal intensity was observed (Fig. 2, Panel C), and exposure to ozone at 297
this dose for 5 min resulted in 3 log10 loss in observable PrPSc signal intensity (Fig. 2, Panel C). 298
In fact, exposure of an IBH (0.01%) sample to ozone at any condition tested always resulted in a 299
measureable loss of signal intensity by PMCA compared to controls (Fig. 2). At pH 4.4 and 20 300
°C, both low dose (13.0 mg/L) and high dose ozone (23.5 mg/L) readily inactivated PrPSc, as 301
assessed by the inability of ozone-treated IBH to act as a seeding template for conformational 302
misfolding of PrPC by PMCA (Fig. 2, Panel A). Exposure to ozone for as little as 5 s at low pH 303
(4.4) and 20oC appeared to completely inactivate the templating properties of the PrPSc present in 304
a 0.01% IBH (Fig. 2, Panel A). In this context, PMCA represented a valuable tool for examining 305
ozone inactivation of the templating properties of PrPSc. 306
To generate a more accurate quanititative estimate of ozone inactivation, western blot 307
images obtained from PMCA reactions were analyzed by densitometry (Fig. 3). Densitometric 308
analysis of western blots were normalized against saturated signal intensity of PMCA non-309
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ozonated control samples and the normalized intensity was used to estimate log10 reduction of 310
signal intensity of ozone treated samples, using Eq. 1. (Fig. 3). 311
A summary of the log estimates of the levels of ozone inactivation of PrPSc based on 312
PMCA densitometric analysis of western blots under the various experimental conditions is 313
provided in Table 2. Ozone dose, contact time, pH, and temperature were all shown to affect 314
ozone inactivation of PrPSc. Higher ozone doses and longer contact times resulted in greater 315
PrPSc inactivation at any given pH and temperature. For example, at pH 6.0 and 20 °C, low dose 316
ozone exposure (11.9 mg/L) , resulted in 1.9 log10 inactivation of the templating properties of 317
PrPSc after 5 s, while a higher inactivation of 3.6 log10 was achieved at 5 min exposure (Table 2). 318
At this same pH and temperature, but with a higher ozone dose (20.7 mg/L), greater inactivations 319
were achieved (2.2 log10 and ≥4 log10 after exposure of PrPSc to ozone for 5 s and 5 min, 320
repectively [Table 2]). 321
In addition to ozone dose and contact time dependency, ozone inactivation of PrPSc was 322
pH and temperature dependent. A reaction pH of 4.4 provided greater levels of inactivation of 323
PrPSc compared to a higher pH (6.0 or 8.0) at the same temperature and ozone dose. For example, 324
when the pH of the reaction was increased from 4.4 to 6.0 at 20 °C, low dose ozone (11.9 mg/L) 325
did not completely inactivate the templating properties of the infectious IBH seed after 5 s or 5 326
min contact time (Fig. 2, Panel B). Increasing the ozone dose to 20.7 mg/L at this same pH and 327
temperature inactivated the templating properties of the IBH seed after a 5 min exposure but not 328
after a 5 s exposure (Fig. 2, Panel B). This is in contrast to experiments carried out at pH 4.4 329
where a dose of 13.0 mg/L of ozone completely inactivated the templating properties of the PrPSc 330
in the IBH after only 5 s exposure (Fig 2, Panel A). When the pH of the reaction was further 331
increased to pH 8.0, ozone doses of 11.3 mg/L did not completely inactivate templating 332
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properties of PrPSc in the IBH after 5 min (Fig. 2, Panel C). Greater inactivation of PrPSc was 333
observed at higher temperatures. For example, at pH 4.4, and ozone dose of 13.0 mg/L and 334
contact time of 5 s, PrPSc inactivation increased from 2.8 to ≥4 log10 as temperature was 335
increased from 4 to 20 °C (Table 2). Overall, the ideal reaction conditions for PrPSc inactivation 336
were at pH 4.4 and 20 °C (Table 2). 337
Ozone inactivation CT values for PrPSc under various treatment conditions were 338
generated using Eq. 2 and are presented in Table 2. With a range of initial ozone doses between 339
11.3 and 14.1 mg/L, at 4 °C, a CT between 0.59 and 0.72 mg·L-1 min resulted in 2.8 log10 340
inactivation of PrPSc at pH 4.4, followed by 1.9 log10 at pH 6.0, and 1.1 log10 at pH 8.0. At 20 °C, 341
a CT between 0.36 and 0.66 mg·L–1 min resulted in ≥4 log10 of inactivation at pH 4.4, followed 342
by 2.4 log10 at pH 6.0, and 1.9 log10 at pH 8.0. At CT between 25.5 and 32.2 mg·L-1 min, PrPSc 343
inactivation was ≥4 log10 at pH 4.4 and both temperatures, and at pH 6.0 and 20 °C, while the 344
inactivation was 3.6 and 2.9 log10 at 4 °C, pH 6.0 and 8.0, respectively (Table 2). 345
CT values of PrPSc were compared to other well studied waterborne pathogens (Table 3). 346
In general, PrPSc was found to be less resistant to ozone than some encysted waterborne protozoa 347
(i.e., Cryptosporidium) or spore-forming bacteria (Bacillus subtilis) under similar experimental 348
conditions. For example, inactivation of Cryptosporidium oocysts at pH 6-7 at 5 °C required an 349
ozone CT value of 32 mg.L-1 min for 2 log10 inactivation, whereas PrPRes at pH 6 and 4 °C 350
required 1.33 mg.L-1 min for 2.4 log10 inactivation (i.e., Cryptosporidium was >24 times more 351
resistant to ozone than PrPSc). 352
353
4. Discussion 354
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This study investigated the effectiveness of ozone inactivation for infectious prion protein 355
(263K hamster scrapie, PrPSc) in aqueous solution, and represents the first report to generate 356
ozone inactivation CT values for infectious prion protein (263K hamster scrapie; PrPSc). The 357
current paper sets a base for understanding the conditions affecting ozone inactivation of prion 358
proteins and consequently lays the foundation for modeling the kinetics of ozone inactivation of 359
PrPSc (and other chemical disinfectants). A detailed understanding of the kinetics of ozone 360
inactivation of PrPSc is instrumental for assessing the applicability and efficacy of new or 361
existing technologies for mitigating prion contamination risks in water (i.e., SRM generated 362
wastewater). 363
Our data suggests that PrPSc is highly susceptible to inactivation by ozone. The ozone CT 364
values derived for PrPSc in this study were considerably lower than those described for certain 365
waterborne pathogens (i.e., Cryptosporidium) and spore forming bacteria (i.e., B. subtilis) at 366
comparative temperatures and pHs (Table 3). Although the applied ozone dose in this study was 367
higher than is normally done for ozone applications in drinking water disinfection (due to the 368
high ozone demand of the IBH), CT values provide a normalized approach to characterizing 369
susceptibility of a particular microbial contaminant to ozone in an aqueous matrix. Ozone is used 370
extensively for inactivation of pathogens and the degradation of various noxious chemicals in 371
large-scale municipal drinking water and municipal/industrial wastewater treatment systems (10, 372
24, 29, 30). Application of ozone to these water matrices requires that ozone be; a) of sufficient 373
concentration, in order to satisfy all oxidation (i.e., ozone) demand associated with organic loads 374
(lipids, proteins, carbohydrates, etc.), and b) delivered efficiently under continuous flow in order 375
to meet the large-scale treatment volumes necessary in the water and wastewater treatment 376
industry. At an industrial scale, continuous flow reactors currently enable ozone doses (i.e., 377
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dissolved ozone concentrations) to reach 10 mg/L ozone with treatment capacity >500 megalitres 378
of water per day (i.e., >5000 kg of ozone production per day). It has been recently reported that 379
the use of a continuous flow reactor with an impinging bubble column contactor significantly 380
increases the mass transfer rate of ozone into solution, achieving cumulative ozone doses >300 381
mg/L within 20 min of ozonation of a pulp mill effluent (8), and the mass transfer rate of ozone 382
may be higher by using multi-jets ozone contactors (3). Meanwhile, ozone engineering solutions 383
of this magnitude, for the water treatment industry (specifically for disinfection purposes), are 384
fundamentally based on the USEPA’s CT concept of ozone inactivation against target organisms 385
or chemicals, emphasizing the importance of present manuscript for derivation of a CT value for 386
ozone inactivation of PrPSc. Understanding the resistivity of PrPSc to ozone based on a CT value, 387
and comparing this value to other target microbes, provides critical insights into the practicality 388
of applying existing industrial scale ozonation systems to control PrPSc in wastewater produced 389
by rendering facilities. Our data suggests that PrPSc is extremely susceptible to inactivation by 390
ozone (compared to other microbes). Consequently ozone technology solutions that currently 391
exists in the water and wastewater industries may hold promise for control of prions present in 392
wastewater from SRM rendering facilities. Similarly, the data also suggests that ozone my be 393
extremely valuable for other disinfection purposes, such as sterilization of medical instruments in 394
hospital. 395
Ozone inactivation was shown to be dose, contact time, pH, and temperature dependent. 396
The pH conditions of 4.4, 6.0 and 8.0 were chosen to be representative of moderate acidic, 397
slightly acidic and slightly alkaline conditions for disinfection; higher pH was not selected due to 398
the high ozone decomposition rate at alkaline conditions (16). Temperatures of 4 °C and 20 °C 399
were chosen to represent typical disinfection temperatures associated with temperate climate 400
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conditions. Inactivation of PrPSc was greatest at a pH of 4.4 and lowest at the highest pH (8.0). 401
The pH effect alone does not appear to act directly on the conformational stability of the 402
infectious property of the prion agent, as pH by itself has been shown to have little effect on 403
scrapie infectivity over the range of pH 2–10 (21). Since ozone decomposition has been shown to 404
be more rapid at a higher pH (16), acidic conditions may favor the sustained and direct attack of 405
molecular ozone on the prion protein itself or on biomolecular targets affecting the stability of 406
the misfolded conformer (i.e., lipids) (2). Direct oxidation of protein targets by molecular ozone 407
predominate reactions kinetics at lower pH as indirect oxidative byproducts (i.e., hydroxyl 408
radical generation) comprise a minor component of the oxidative potential at low pH (7). High 409
inactivation rates of ozone at more acidic pHs have also been observed for E. coli (35), norovirus 410
(19) and helminth eggs (43). We are currently addressing what contributions molecular ozone 411
and its oxidative derivatives (OH., H2O2, etc) have on inactivation of PrPSc as a means of 412
characterizing the molecular mechanisms responsible for inactivation. Furthermore, 413
optimization of the experimental methods and approaches to prion inactivation, as described in 414
this manuscript, lays the foundation for a more thorough examination of the kinetics of ozone 415
inactivation of prions. 416
The effect of temperature on the inactivation of microorganisms and the degradation of 417
organic pollutants by ozone has also been investigated by us and others (9, 18). In general, 418
elevated temperatures result in more rapid inactivation of microbial pollutants by ozone, an 419
outcome also observed in our study. Interestingly, temperature has opposing effects on ozone 420
solubility and disinfection rates (4). 421
A recent study by Johnson et al. (14) demonstrated that UV-ozone treatment of hamster-422
adapted transmissible mink encephalopathy prions induced inactivation of PrPSc >5 log10. Ozone 423
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was generated by UV at a wavelength of 185 nm, and then decomposed by UV at another 424
wavelength of 254 nm to produce hydroxyl radicals. However, the UV-ozone system studied by 425
Johnson et al. (2009) produced limited ozone thus generating limited hydroxyl radicals, requiring 426
exposure times up to several weeks, questioning the practicality of using UV-generated ozone as 427
a decontamination/sterilizing approach to prion inactivation. Prion inactivation has also been 428
studied with other advanced oxidation methods, such as copper and hydrogen peroxide (17, 34), 429
iron and hydrogen peroxide (35), photo-Fenton treatment (25), and titanium dioxide photo-430
catalysis (26). Hydrogen peroxide inactivation (100 mmol/L) in the presence of copper (0.5 431
mmol/L) was reported to achieve ≥5.2 log10 inactivation of 263K scrapie prion with a contact 432
time of 2 h at room temperature (34). Increasing the concentration of hydrogen peroxide to 2.2 433
mol/L reduced the contact time to 30 min for the same level of inactivation (17). Hydrogen 434
peroxide inactivation (1.5 mol/L) in the presence of Fe2+ (15.7 mmol/L), heating at 50 °C for 22 435
h was able to obtain approximately 6 log10 reduction of prion infectivity (35). PrPSc could also be 436
degraded by ≥2.4 log10 by photo-Fenton treatment (147 mmol/L H2O2, 8.9 mmol/L Fe3+) after 5 437
h UV-A exposure (25), and be degraded by ≥2 log10 by titanium dioxide photo-catalysis (25 438
mmol/L titanium dioxide and 118 mmol/L H2O2) after 12 h UV-A exposure (26). Due to the low 439
sensitivity of the PrPSc detection methods used in the last two studies, inactivation higher than 440
2.4 log10 was not derived. In these advanced oxidation studies, hydroxyl radicals as a sole 441
component to inactivate PrPSc demonstrated its capabilities, however, prolonged exposure times 442
(from 30 min to 22 h) were essential to continuously generate potent hydroxyl radicals sufficient 443
for the inactivation. In contrast, ozone at a pH of 4.4 and 20 °C in this study caused a very rapid 444
≥4 log10 inactivation of PrPSc after 5 s of exposure, suggesting that ozone treatment might be 445
more efficient for PrPSc inactivation than other advanced oxidants. 446
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447
5. Conclusions 448
PMCA is an extremely sensitive tool for detection and quantification of PrPSc and for 449
measuring ozone inactivation of the template directed misfolding properties of PrPSc. Ozone 450
inactivation of scrapie 263K was shown to be dose, contact time, temperature, and pH dependent. 451
In addition, ozone was found to be extremely effective on inactivation of 263K scrapie, with 452
more than 4 log10 inactivation observed at CT of 0.59 mg·L-1 min at pH 4.4 and 20 °C. The 453
derived ozone CT product for PrPSc was similar to that of poliovirus, and considerably less than 454
encysted protozoan parasites such as Cryptosporidium spp. and Giardia spp. 455
456
Acknowledgements 457
This research was financially supported by the Alberta Prion Research Institute (APRI) 458
and PrioNet Canada through grants provided to MB, NFN, and MGE, and through an NSERC 459
Discovery Accelerator Grant to MGE. 460
461
462
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TABLE 1. Ozone demand of PrPSc at pH 6.0, 4 °C 584
Ozone dose (mg/L)
Concentration of IBH
Contact time Residual ozone
(mg/L)
12.9 0.1% 5 s 0 30 s 0 2 min 0 5 min 0
12.5 0.01% 5 s 8.3 30 s 8.2 2 min 6.0 5 min 5.0
585
586
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TABLE 2. Summary of ozone inactivation of PrPSc under various conditions 587
pH Temperature (°C)
Ozone dose (mg/L)
Contact time CT (mg·L-1 min)
Inactivation log10 (N0/N)
4.4 4 13.7 5 s 0.59 2.8 5 min 31.6 ≥4 25.7 5 s 1.52 ≥4 5 min 67.8 ≥4 20 13.0 5 s 0.59 ≥4 5 min 28.6 ≥4 23.5 5 s 1.17 ≥4 5 min 56.9 ≥4
6.0 4 12.5 5 s 0.69 1.9 5 min 32.2 3.6 20.7 5 s 1.33 2.2 5 min 56.5 ≥4 20 11.9 5 s 0.66 2.4 5 min 26.0 4.4 20.7 5 s 1.15 2.9 5 min 41.5 ≥4
8.0 4 9.4 5 s 0.40 0.2 5 min 14.7 2.4 14.1 5 s 0.72 1.1 5 min 25.5 2.9 20 7.6 5 s 0.02 0.9 5 min NA 2.9a
11.3 5 s 0.36 1.9 5 min 6.51 3.0
a No residual ozone maintained 588
589
590
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TABLE 3. Summary of the efficacies of ozone inactivation of microorganisms in water and 591
PrPSc 592
Microorganisms / PrPSc
pH Temperature
(°C) CT (mg·L-1 min)
Inactivation (log10)
Reference
E. coli 6-7 5 0.02 2 (13) Rotavirus 6-7 5 0.006 – 0.06 2 (13)
Adenovirus 40 7 5 0.01 – 0.02 2 (39) 7 5 0.07 – 0.60 4
Poliovirus 7.2 5 0.60 2 (41) 1.20a 4a 20 0.25 2 0.50 4
Giardia lamblia cysts
7 5 1.30 2 (41) 1.90 3 20 0.48 2 0.72 3
Cryptosporidium parvum oocysts
6-7 5 32 2 (42) 47 3 20 7.8 2 12 3
PrPSc (263K scrapie) 4.4 4 0.59 2.8 This study 20 0.59 ≥4 6.0 4 0.69 1.9 20 0.66 2.4 8.0 4 0.72 1.1 20 0.36 1.9
a extrapolated by applying first order kinetics with a safety factor of 3 593
594
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595
Fig. 1. A comparison between traditional western blot methodologies and PMCA for detection of 596
263K scrapie. Panel (A): western blot of a 10-fold serially diluted IBH (10%) without PMCA. 597
Lanes labeled 10 to 0.001 represent IBH in percentage (%), respectively. Panel (B): western blot 598
of a 10-fold serially diluted IBH (10%) with PMCA. Lanes labeled 1 to 0.00000001 represent 599
IBH in percentage (%), respectively. Lanes labeled as ‘PK-‘ represent 1% IBH not treated with 600
PK. Lane labeled as ‘-‘ represents 10% NBH treated with PK. Lane labeled as ‘+’ represents 1% 601
IBH treated with PK. Molecular weight markers (lane labeled MW) at 50, 37, 25 and 20 kD are 602
indicated. 603
604
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605
Fig. 2. Western blots from 0.01% IBH samples treated with ozone at 20 °C and amplified by 606
PMCA. The images in each panel, from left to right, represent non-ozone treated control samples, 607
samples treated with ozone for 5 s, and samples treated with ozone for 5 min. The applied ozone 608
doses are provided to the left of each panel. Panel A = pH 4.4; Panel B = pH 6.0; and Panel C = 609
pH 8.0. The numbers at the top of each image represent dilutions of 0.01% IBH in log10. Lane 610
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labeled as ‘-’ represents 10% NBH treated with PK. Lane labeled as ‘+’ represents 1% IBH 611
treated with PK. Molecular weight markers (lane labeled MW) at 50, 37, 25 and 20 kD are 612
indicated. 613
614
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615 Fig. 3. Densitometry analysis of western blot images in Panel A and B of Fig. 2. (A) Normalized 616
intensity of images in Panel A, Row 2 (pH 4.4, ozone dose of 23.5 mg/L). (B) Normalized 617
intensity of images in Panel B, Row 2 (pH 6.0, ozone dose of 20.7 mg/L). The bars show the 618
range of two replicates. 619
620
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Table 1. Ozone demand of PrPSc at pH 6.0, 4 °C
Ozone dose (mg/L)
Concentration of IBH Contact time Residual ozone
(mg/L)
12.9 0.1% 5 s 0 30 s 0 2 min 0 5 min 0
12.5 0.01% 5 s 8.3 30 s 8.2 2 min 6.0 5 min 5.0
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Table 2. Summary of ozone inactivation of PrPSc under various conditions
pH Temperature
(°C) Ozone dose
(mg/L) Contact time
CT (mg·L-1 min)
Inactivation log10 (N0/N)
4.4 4 13.7 5 s 0.59 2.8 5 min 31.6 ≥ 4 25.7 5 s 1.52 ≥ 4 5 min 67.8 ≥ 4 20 13.0 5 s 0.59 ≥ 4 5 min 28.6 ≥ 4 23.5 5 s 1.17 ≥ 4 5 min 56.9 ≥ 4
6.0 4 12.5 5 s 0.69 1.9 5 min 32.2 3.6 20.7 5 s 1.33 2.2 5 min 56.5 ≥ 4 20 11.9 5 s 0.66 2.4 5 min 26.0 4.4 20.7 5 s 1.15 2.9 5 min 41.5 ≥ 4
8.0 4 9.4 5 s 0.40 0.2 5 min 14.7 2.4 14.1 5 s 0.72 1.1 5 min 25.5 2.9 20 7.6 5 s 0.02 0.9 5 min NA 2.9a
11.3 5 s 0.36 1.9 5 min 6.51 3.0
a No residual ozone maintained
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TABLE 3. Summary of the efficacies of ozone inactivation of microorganisms in water and PrPSc
Microorganisms / PrPSc
pH Temperature
(°C) CT (mg·L-1 min)
Inactivation (log10)
Reference
E. coli 6-7 5 0.02 2 (10) Rotavirus 6-7 5 0.006 – 0.06 2 (10)
Adenovirus 40 7 5 0.01 – 0.02 2 (31) 7 5 0.07 – 0.60 4
Poliovirus 7.2 5 0.60 2 (32) 1.20a 4a 20 0.25 2 0.50 4
Giardia lamblia cysts
7 5 1.30 2 (32) 1.90 3 20 0.48 2 0.72 3
Cryptosporidium parvum oocysts
6-7 5 32 2 (33) 47 3 20 7.8 2 12 3
PrPSc (263K scrapie) 4.4 4 0.59 2.8 This study 20 0.59 ≥4 6.0 4 0.69 1.9 20 0.66 2.4 8.0 4 0.72 1.1 20 0.36 1.9
a extrapolated by applying first order kinetics with a safety factor of 3
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