Inactivation of template-directed misfolding of infectious...

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

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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

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*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: [email protected] 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: [email protected] 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

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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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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

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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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References 463

1. APHA. 2005. Standard methods for the examination of water and wastewater. 21 ed., 464

Washington, D. C. 465

2. Appel, T. R., M. Wolff, F. von Rheinbaben, M. Heinzel, and D. Riesner. 2001. Heat 466

stability of prion rods and recombinant prion protein in water, lipid and lipid-water 467

mixtures. J. Gen. Virol. 82:465-473. 468

3. Baawain, M. S., M. G. El-Din, D. W. Smith, and A. Mazzei. 2011. Hydrodynamic 469

characterization and mass transfer analysis of an in-line multi-jets ozone contactor. 470

Ozone-Sci. Eng. 33:1-14. 471

4. Bablon, G., W. D. Bellamy, G. Billen, M. Bourbigot, F. B. Daniel, F. Erb, C. 472

Gomella, G. Gordon, P. Hartemann, J. Joret, W. R. Knocke, B. Langlais, A. 473

Laplanche, B. Legube, J. B. Lykins, G. Martin, N. Martin, A. Montiel, M. F. Morin, 474

R. S. Miltner, D. Perrine, M. Prevost, D. A. Reckhow, P. Servais, P. C. Singer, O. J. 475

Sproul, and C. Ventresque. 1991. Practical application of ozone: principles and case 476

studies. In Ozone in Water Treatment: Application and Engineering (Edited by Langlais 477

B., Reckhow, D.A. and Brink D.R.), Lewis, Chelsea, Mich. 478

5. Brown, P., and D. C. Gajdusek. 1991. Survival of scrapie virus after 3 years internment. 479

Lancet 337:269-270. 480

6. Castilla, J., P. Saa, C. Hetz, and C. Soto. 2005. In vitro generation of infectious scrapie 481

prions. Cell 121:195-206. 482

7. Chu, W., and C. W. Ma. 2000. Quantitative prediction of direct and indirect dye 483

ozonation kinetics. Water Res. 34:3153-3160. 484


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

24

8. El-Din, M. G., and D. W. Smith. 2001. Maximizing the enhanced ozone oxidation of 485

kraft pulp mill effluents in an impinging-jet bubble column. Ozone-Sci. Eng. 23:479-493. 486

9. El-Din, M. G., D. W. Smith, F. Al Momani, and W. X. Wang. 2006. Oxidation of resin 487

and fatty acids by ozone: Kinetics and toxicity study. Water Res. 40:392-400. 488

10. Gunukula, R. V. B., and M. E. Tittlebaum. 2001. Industrial wastewater treatment by an 489

advanced oxidation process. J. Environ. Sci. Health Part A-Toxic/Hazard. Subst. Environ. 490

Eng. 36:307-320. 491

11. Hartley, E. G. 1967. Action of disinfectants on experimental mouse scrapie. Nature 492

213:1135-1135. 493

12. Hinckley, G. T., C. J. Johnson, K. H. Jacobson, C. Bartholomay, K. D. McMahon, D. 494

McKenzie, J. M. Aiken, and J. A. Pedersen. 2008. Persistence of pathogenic prion 495

protein during simulated wastewater treatment processes. Environ. Sci. Technol. 496

42:5254-5259. 497

13. Hoff, J. C. 1986. Inactivation of microbial agents by chemical disinfectants. Water 498

Engineering Research Laboratory, USEPA. 499

14. Johnson, C., P. Gilbert, D. McKenzie, J. Pedersen, and J. Aiken. 2009. Ultraviolet-500

ozone treatment reduces levels of disease-associated prion protein and prion infectivity. 501

BMC Res. Notes 2:121-125. 502

15. Krasemann, S., M. Neumann, M. Geissen, W. Bodemer, F. J. Kaup, W. Schulz-503

Schaeffer, N. Morel, A. Aguzzi, and M. Glatzel. 2010. Preclinical deposition of 504

pathological prion protein in muscle of experimentally infected primates. PLoS One 5:7. 505

16. Ku, Y., W. J. Su, and Y. S. Shen. 1996. Decomposition kinetics of ozone in aqueous 506

solution. Ind. Eng. Chem. Res. 35:3369-3374. 507


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

25

17. Lehmann, S., M. Pastore, C. Rogez-Kreuz, M. Richard, M. Belondrade, G. Rauwel, 508

F. Durand, R. Yousfi, J. Criquelion, P. Clayette, and A. Perret-Liaudet. 2009. New 509

hospital disinfection processes for both conventional and prion infectious agents 510

compatible with thermosensitive medical equipment. J. Hosp. Infect. 72:342-350. 511

18. Li, H., L. L. Gyurek, G. R. Finch, D. W. Smith, and M. Belosevic. 2001. Effect of 512

temperature on ozone inactivation of Cryptosporidium parvum in oxidant demand-free 513

phosphate buffer. J. Environ. Eng.-ASCE 127:456-467. 514

19. Lim, M. Y., J. M. Kim, J. E. Lee, and G. Ko. 2010. Characterization of ozone 515

disinfection of murine norovirus. Appl. Environ. Microbiol. 76:1120-1124. 516

20. Maddison, B. C., G. C. Whitelam, and K. C. Gough. 2007. Cellular prion protein in 517

ovine milk. Biochem. Biophys. Res. Commun. 353:195-199. 518

21. Mould, D. L., A. M. Dawson, and W. Smith. 1965. Scrapie in mice. Stability of agent 519

to various suspending media pH and solvent extraction. Res. Vet. Sci. 6:151-154. 520

22. Murayama, Y., M. Yoshioka, H. Horii, M. Takata, T. Yokoyama, T. Sudo, K. Sato, 521

M. Shinagawa, and S. Mohri. 2006. Protein misfolding cyclic amplification as a rapid 522

test for assessment of prion inactivation. Biochem. Bioph. Res. Commun. 348:758-762. 523

23. Nichols, T. A., B. Pulford, A. C. Wyckoff, C. Meyerett, B. Michel, K. Gertig, E. A. 524

Hoover, J. E. Jewell, G. C. Telling, and M. D. Zabel. 2009. Detection of protease-525

resistant cervid prion protein in water from a CWD-endemic area. Prion 3:171-183. 526

24. Oneby, M. A., C. O. Bromley, J. H. Borchardt, and D. S. Harrison. 2010. Ozone 527

Treatment of Secondary Effluent at US Municipal Wastewater Treatment Plants. Ozone-528

Sci. Eng. 32:43-55. 529


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

26

25. Paspaltsis, I., C. Berberidou, I. Poulios, and T. Sklaviadis. 2009. Photocatalytic 530

degradation of prions using the photo-Fenton reagent. J. Hosp. Infect. 71:149-156. 531

26. Paspaltsis, I., K. Kotta, R. Lagoudaki, N. Grigoriadis, I. Poulios, and T. Sklaviadis. 532

2006. Titanium dioxide photocatalytic inactivation of prions. J. Gen. Virol. 87:3125-3130. 533

27. Pedersen, J. A., K. D. McMahon, and C. H. Benson. 2006. Prions: novel pathogens of 534

environmental concern? J. Environ. Eng.-ASCE 132:967-969. 535

28. Prusiner, S. B., M. Scott, D. Foster, K. M. Pan, D. Groth, C. Mirenda, M. Torchia, S. 536

L. Yang, D. Serban, G. A. Carlson, P. C. Hoppe, D. Westaway, and S. J. Dearmond. 537

1990. Transgenetic studies implicate interactions between homologous PrP isoforms in 538

scrapie prion replication. Cell 63:673-686. 539

29. Rice, R. G. 1981. Ozone treatment of industrial wastewater. Noyes Data Corp., Park 540

Ridge, NJ. 541

30. Rice, R. G., C. M. Robson, G. W. Miller, and A. G. Hill. 1981. Uses of ozone in 542

drinking-water treatment. J. Am. Water Work Assoc. 73:44-57. 543

31. Saa, P., J. Castilla, and C. Soto. 2006. Ultra-efficient replication of infectious prions by 544

automated protein misfolding cyclic amplification. J. Biol. Chem. 281:35245-35252. 545

32. Saborio, G. P., B. Permanne, and C. Soto. 2001. Sensitive detection of pathological 546

prion protein by cyclic amplification of protein misfolding. Nature 411:810-813. 547

33. Seidel, B., A. Thomzig, A. Buschmann, M. H. Groschup, R. Peters, M. Beekes, and 548

K. Terytze. 2007. Scrapie agent (Strain 263K) can transmit disease via the oral route 549

after persistence in soil over years. PLoS One 2:8. 550


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

27

34. Solassol, J., M. Pastore, C. Crozet, V. Perrier, and S. Lehmann. 2006. A novel 551

copper--Hydrogen peroxide formulation for prion decontamination. J. Infect. Dis. 552

194:865-869. 553

35. Suyama, K., M. Yoshioka, M. Akagawa, Y. Murayama, H. Horii, M. Takata, T. 554

Yokoyama, and S. Mohri. 2007. Assessment of prion inactivation by Fenton reaction 555

using protein misfolding cyclic amplification and Bioassay. Biosci. Biotechnol. Biochem. 556

71:2069-2071. 557

36. Suyama, K., M. Yoshioka, M. Akagawa, Y. Murayama, H. Horii, M. Takata, T. 558

Yokoyama, and S. Mohri. 2007. Prion inactivation by the Maillard reaction. Biochem. 559

Bioph. Res. Commun. 356:245-248. 560

37. Taylor, D. M. 2000. Inactivation of transmissible degenerative encephalopathy agents: a 561

review. The Vet. J. 159:10-17. 562

38. Taylor, D. M., H. Fraser, I. McConnell, D. A. Brown, K. L. Brown, K. A. Lamza, 563

and G. R. A. Smith. 1994. Decontamination studies with the agents of bovine 564

spongiform encephalopathy and scrapie. Arch. Virol. 139:313-326. 565

39. Thurston-Enriquez, J. A., C. N. Haas, J. Jacangelo, and C. P. Gerba. 2005. 566

Inactivation of enteric adenovirus and feline calicivirus by ozone. Water Res. 39:3650-567

3656. 568

40. Tichopad, A., M. W. Pfaffl, and A. Didier. 2003. Tissue-specific expression pattern of 569

bovine prion gene: quantification using real-time RT-PCR. Mol. Cell. Probes 17:5-10. 570

41. USEPA. 1991. Guidance manual for compliance with the filtration and disinfection 571

requirements for public water systems using surface water sources. U.S. Environmental 572

Protection Agency, Washington, D.C. 573


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

28

42. USEPA. 2006. National Primary Drinking Water Regulations: Long Term 2 Enhanced 574

Surface Water Treatment, Final rule ed. U.S. Environmental Protection Agency, 575

Washington D.C. 576

43. Velasquez, M., J. L. Martinez, I. Monje-Ramirez, and M. N. Rojas-Valencia. 2004. 577

Destruction of helminth (Ascaris suum) eggs by ozone. Ozone-Sci. Eng. 26:359-366. 578

44. Zuma, F., J. Lin, and S. B. Jonnalagadda. 2009. Ozone-initiated disinfection kinetics 579

of Escherichia coli in water. J. Environ. Sci. Health Part A 44:48-56. 580

581

582

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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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Inactivation of template-directed misfolding of infectious...

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