Article pubs.acs.org/est

Biodegradation of Prions in Compost Shanwei Xu,†,¶ Tim Reuter,‡,¶ Brandon H. Gilroyed,§,¶ Gordon B. Mitchell,∥ Luke M. Price,⊥ Sandor Dudas,# Shannon L. Braithwaite,⊥ Catherine Graham,# Stefanie Czub,# Jerry J. Leonard,▽ Aru Balachandran,∥ Norman F. Neumann,⊥ Miodrag Belosevic,○ and Tim A. McAllister*,† †

Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta T1J 4B1, Canada Alberta Agriculture and Rural Development, Lethbridge, Alberta T1J 4V6, Canada § School of Environmental Sciences, University of Guelph Ridgetown Campus, Ridgetown, Ontario N0P 2C0, Canada ∥ National and OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, Ontario K2H 8P9, Canada ⊥ School of Public Health, University of Alberta, Edmonton, Alberta T6G 1C9, Canada # Canadian and OIE Reference Laboratories for BSE, Canadian Food Inspection Agency Lethbridge Laboratory, Lethbridge, Alberta T1J 3Z4, Canada ▽ Department of Agriculture, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada ○ Department of Biological Science, University of Alberta, Edmonton, Alberta T6G 2E9, Canada ‡

S Supporting Information *

ABSTRACT: Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP263K), chronic waste disease (PrPCWD), and bovine spongiform encephalopathy (PrPBSE) in lab-scale composters and PrP263K in field-scale compost piles. Western blotting (WB) indicated that PrP263K, PrPCWD, and PrPBSE were reduced by at least 2 log10, 1−2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP263K and 3 log10 in PrPCWD. Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP263K and PrPCWD. For field-scale composting, stainless steel beads coated with PrP263K were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP263K infectivity. Our findings show that composting reduces PrPTSE, resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

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INTRODUCTION

endemic within Alberta and Saskatchewan in Canada and 19 states across the United States.6 As of May 2014, 70 domestic cervid herds have been confirmed to be infected with CWD in Canada,7 and 211 cases of CWD have been confirmed in wild cervids within the province of Alberta.8 In the United States, 408 CWD cases have been confirmed in Illinois9 and prevalence is approaching 50% of the mule deer in Wyoming.10 Therefore, mortalities in farmed herds and wild cervids killed in

Transmissible spongiform encephalopathies (TSE) are a group of fatal neurodegenerative diseases including scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt−Jackob disease (CJD) in humans. The cause of TSE is the sequential conformational changes of normal prion protein (PrPC) into misfolded and infectious prion proteins (PrPTSE).1 To date, 23 cases of BSE have been confirmed in North America, 19 in Canada and four in the United States.2,3 Economic losses as a result of BSE in farmed cattle are estimated at over $11 billion in Canada and the United States.4,5 Chronic wasting disease is even more prevalent and © 2014 American Chemical Society

Received: Revised: Accepted: Published: 6909

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Sources of Tissues and PrPTSE. Three- to six-week-old female Syrian golden hamsters (Charles River Laboratories International Inc., Washington, USA) infected with PrP263K were euthanized with carbon dioxide, and the brains were harvested as a source of scrapie prions. Infectious cervid and bovine brain tissues from elk and cattle positive for CWD and classical BSE, respectively, were obtained from Canadian National laboratory collections. Brain tissues from noninfected hamsters, elk, and cattle served as controls in studies with PrP263K, PrPCWD, and PrPBSE, respectively. The infectious titer of hamster 263K brain tissues in Syrian golden hamsters was estimated at 109.94 50% infectious doses (ID50) g−1 as described by Ding et al.28 The titers of CWD brain tissues were 107 ID50 g−1 and 107.2 ID50 g−1 in Tg(CerPrP-E226)5037+/− and Tg(CerPrP-M132)1536+/− mice, respectively,29 while PrPBSE was not titrated. All brain tissues were homogenized (1 g + 9 mL) in phosphate buffered saline using the MediFASTH homogenizer (Consul AR, Villeneuve, Switzerland) to yield a 10% brain homogenate (BH). Lab-scale Composting Experiment. Passively aerated lab-scale composters were used as described by Xu et al.30 Infectious prions, i.e., PrP263K, PrPCWD, and PrPBSE, were composted in a cattle manure−wood shavings matrix with or without chicken feathers from a commercial abattoir. The control compost mixture consisted of 72% (w w−1; dry-weight basis) cattle manure and 28% wood shavings, while 5% chicken feathers were substituted for manure in the feather treatment.31 The physicochemical properties of the ingredients are described in Table S1 (see Supporting Information). Composting of PrPTSE was conducted in a level 3 containment laboratory at the CFIA in Lethbridge, Alberta. Identical matrices without PrPTSE were prepared and composted out of containment with samples collected for measurement of physicochemical parameters during composting. Experiments in and out of containment were started simultaneously with three replicate composters per treatment outside and two replicate composters in containment. PrPTSE Sampling Procedures. PrPTSE was introduced into compost by inoculating dried manure spheres (1.0 ± 0.1 g; dryweight basis) with 1 mL of a 10% BH solution containing the appropriate PrPTSE. Subsequently, each inoculated manure sphere with PrPTSE was sealed in a nylon bag (53 μm pore size; ANKOM Technology, Macedon, USA). Two replicate nylon bags were then placed in a larger polyester mesh bag (5 mm pore size) along with 200 g of mixed compost matrix. Each mesh bag contained manure spheres that were inoculated with only one type of PrPTSE. As each composter was filled, triplicate PrP263K and duplicate PrPCWD and PrPBSE mesh bags were placed 30 cm below the surface of the compost matrix, resulting in a total of seven bags in each composter. To determine if PrPTSE could still be detected after a short duration of composting, one PrP263K bag was collected after 2 days. After 14 days, three mesh bags containing PrP263K, PrPCWD, and PrPBSE were randomly collected. After collection, each composter was emptied, and contents were then mixed with water to return compost to its original moisture level prior to a second composting cycle. As the composters were refilled, the remaining PrP263K, PrPCWD, and PrPBSE mesh bags were placed in each composter at the same depth as the first cycle. After 28 days, all mesh bags were collected, and temperature, moisture, and pH of the compost were measured at the same depth at which the mesh bags were implanted.

vehicular collisions generate significant quantities of carrion requiring disposal.11−13 In Canada, the identification of BSE in 2003 led the Canadian Food Inspection Agency (CFIA) to impose an enhanced feed ban in 2007 to prevent the introduction of specified risk materials (SRM, i.e., skull, brain, trigeminal ganglia, eyes, palatine tonsils, spinal cord, and dorsal root ganglia of cattle 30 months or older, as well as the distal ileum from cattle of all ages) into the feed and food chain. If SRM are not removed, the entire mortality is designated SRM. A similar regulation was passed in the United States in 2008.14 It is estimated that more than 250 000 tonnes of SRM are generated in Canada annually15 with its proper disposal being critical to the efforts of regulatory agencies to prevent the spread of TSE. In North America, the majority of SRM are rendered, dehydrated, and disposed of in landfills,16 a process that does not inactivate all PrPTSE. Disposal of SRM in a manner that inactivates PrPTSE is challenging owing to the recalcitrant nature of this infectious agent.17 Current disposal practices approved by CFIA for PrPTSE infected material include two stage incineration at 850 and 1000 °C, 1 h disinfection with 2 N sodium hydroxide, autoclaving in saturated steam at 134 °C for a period of 60 min, thermal hydrolysis at 180 °C under 12 atmospheric pressure for no less than 40 min, and alkaline hydrolysis at 150 °C and 4 atmospheric pressure with 15% sodium hydroxide or 19% potassium hydroxide (w w−1) for at least 180 min. All of these methods are costly due to the large volumes of SRM that must be processed and the level of energy these systems require to inactivate PrPTSE. Composting is an aerobic decomposition process whereby organic matter including proteins is degraded by the actions of mesophilic and thermophilic bacteria and fungi. Previous research has shown that some proteinases produced by Streptomyces, Thermus, Bacillus, and Tritirachium can degrade PrPTSE.18−20 Members of these genera have been isolated from compost,21,22 and an array of proteases are present in compost.23,24 Compost is highly alkaline (pH 8−10), and temperatures in the core of compost piles commonly exceed 55 °C for weeks or months,25 conditions ideal for protein denaturation. Exposure to proteases under these conditions for weeks or months raises the possibility that the microbial consortia in compost may have the capacity to degrade PrPTSE. If composting is to be adopted as a reliable method of SRM disposal, inactivation of PrPTSE during composting needs to be confirmed. Huang et al.26 reported that ovine scrapie prions (PrPSc) declined to levels undetectable by Western blotting (WB) after 108 days of composting. However, PrPTSE in compost was not quantified by more sensitive assays such as protein misfolding cyclic amplification (PMCA) nor was the impact of composting on the infectivity of PrPTSE investigated. Therefore, the objectives of this study were (1) to assess the fate of scrapie PrP263K, PrPCWD, and PrPBSE during composting in lab-scale composters; (2) to use PMCA to further investigate the degree of PrP263K and PrPCWD degradation during composting; and finally (3) to assess the impact of field-scale composting on the infectivity of PrP263K in a hamster bioassay.

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EXPERIMENTAL SECTION Ethics Statement. All animal work was performed in accordance with Canadian Council on Animal Care guidelines.27 The protocol (ACC 10−09) was approved by the accredited Animal Care Committee at the CFIA, Ottawa Laboratory-Fallowfield. 6910

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et al.41 An amplification control of 10% infectious BH and PrPTSE samples were serially diluted 10-fold in 10% noninfectious BH, respectively. Subsequently, 80 μL of each 10-fold dilution series and a negative control (10% noninfectious BH) were placed in a Misonix model Q700 sonicator (Misonix Inc., Farmingdale, USA). Samples of PrP263K were incubated at 37 °C and subject to a single round of amplification with 37 cycles of 40 s sonication followed by 29 min and 20 s incubation at a potency of 35%. For PrPCWD, three rounds of PMCA were performed under the same conditions as PrP263K except at a potency of 40% with each new round using a 1:10 dilution of amplified materials from the previous round in 10% noninfectious TgElk BH. All PMCA products and non-PMCA controls (10% noninfectious and infectious BH) were then PK digested and analyzed by WB as described by Ding et al.41 with mAb 3F4 and mAb 6H4 (1:20 000; Prionics) used for detection of PrP263K and PrPCWD, respectively. Field-scale Composting Experiment. Duplicate fieldscale biosecure composters were built at the AAFC Research Centre in Lethbridge, Alberta.25 Briefly, compost was contained in a straw bale bunker and sealed within plastic sheeting to ensure biocontainment (see Figure S1, Supporting Information). Approximately 60 cm of cattle manure was overlaid onto a 40 cm base of straw and 16 mature cattle carcasses that had died of natural causes were placed in each structure and covered with 100 cm of manure. Each structure was 25 × 5 × 2.4 m and contained approximately 85 000 kg (wet-weight basis) of compost. Bioassays of PrPTSE derived from compost present a significant challenge, as the susceptibility of lab animals to infection may be influenced by the formation of complexes between prions and organic molecules within compost. Zobeley et al.42 and Flechsig et al.43 demonstrated that stainless steel has the ability to bind prions, which can result in the transmission of TSE via surgical instruments. To overcome these challenges, we bound prions to stainless steel beads prior to their implantation in compost. Specifically, stainless steel beads (440C stainless steel; 0.5 mm diameter; grade 0025; Hoover Precision Products, Cumming, USA) were coated with hamster PrP263K and placed in composters. Beads were incubated in 2mL microcentrifuge tubes with 10% infectious 263K BH at room temperature for 1 h. After incubation, BH was decanted and beads were washed five times for 1 min with 1 mL of PBS to remove unbound material. Subsequently, six beads were sealed in nylon bags (53 μm pore size; ANKOM Technology) and placed within Baker retrieval pyramids44 along with a compost matrix. Baker retrieval pyramids were suspended in each composter at a depth of 100 cm and removed after 14, 56, 112, and 230 days. Thermal couples connected to a data logger (Onset Computer, Bourne, USA) were placed in each retrieval pyramid to measure temperature. Compost temperatures at depths of 40 and 160 cm were also measured at multiple locations as previously described.25 A total of six pyramids were retrieved from each composter. Composted beads were then collected for use in a Syrian hamster bioassay. Hamster Bioassay. Three- to six-week-old female Syrian golden hamsters (Charles River Laboratories International Inc.) were used to detect infectivity associated with stainless steel beads in a manner similar to that for stainless steel wires.45 To accurately calculate log10 reductions of PrP263K infectivity by composting, it was necessary to establish the relationship between the dose and time to disease for the hamster 263K bead implantation model. Therefore, stainless steel beads

Composters out of containment were managed using identical matrices except that manure spheres were not inoculated with PrPTSE. Compost samples were collected from each composter at day 14 after mixing and moistening and from each mesh bag for physicochemical analyses. Compost temperature and oxygen concentration at the depth of mesh bags in each composter, and moisture, bulk density, total carbon (TC), total nitrogen (TN), pH, electrical conductivity (EC), and mineral N (NH4+ and NO2− + NO3−) were measured as previously described.30 PrPTSE Extraction and Western Blotting. Infectious prions are highly hydrophobic and aggregate into plaque-like complexes that exhibit a high affinity for soil and sludge solids.32−34 Among detergents, SDS is an effective means to release soil- and sludge-bound PrPTSE into solution for subsequent WB detection.35,36 A modification of the procedure of Xu et al.31 was used to extract PrPTSE from manure spheres. Manure spheres inoculated with PrPTSE and composted for 0, 2, 14, and 28 days were extracted using sodium dodecyl sulfate (SDS), digested by proteinase K (PK), and then precipitated by sodium phosphotungstic acid (PTA) for WB analysis. To enhance prion extraction efficiency, 4.5 mL of a 1% aqueous solution of SDS was added to each manure sphere during extraction. Subsequently, 500 μL of the supernatant was removed for PK digestion (25 μg mL−1 of PK) at 37 °C for 1 h and precipitated with PTA (4%, w v−1, in 170 mM MgCl2, pH 7.4) at a final concentration of 0.3% (w v−1). The precipitated pellet was resuspended in 200 μL of 1 × Laemmli’s sampling buffer and heated to 100 °C for 5 min prior to WB as described by Xu et al.31 except that mAb 3F4 (1:20 000 dilution; Human PrP109−112; Millipore, Billerica, USA) was used for the detection of PrP263K and mAb 6H4 (1:5000 dilution; Ovine PrP148−157; Prionics, Zurich, Switzerland) for the detection of PrPCWD and PrPBSE. For the positive control, 10% PrPTSE BH was PK digested as described above prior to WB. For the negative control, manure inoculated with water was extracted, PK digested, and PTA precipitated. To ensure PrPTSE was extractable from compost over time, manure spheres without PrPTSE were composted for 14 and 28 days. These composted manure spheres were inoculated with 1 mL of 10% PrPTSE BH and extracted to assess the recovery and detection of PrPCWD and PrPBSE by WB and PrP263K by PMCA. Sensitivity of Western Blotting. To determine the sensitivity of WB for PrPTSE in manure, 10% PrP263K, PrPCWD, and PrPBSE BH were initially diluted in an equal volume of 10% noninfectious hamster, elk, and cattle BH, respectively. A series of 5%, 2.5%, 1.25%, 0.625%, 0.32%, 0.16%, 0.08%, 0.04%, 0.02%, and 0.01% dilutions were generated for each type of PrPTSE. The 10% to 0.01% dilutions of TSE BH were then inoculated onto manure spheres (1.0 ± 0.1 g; dry-weight basis) and extracted for WB analysis. Protein Misfolding Cyclic Amplification (PMCA) Assay. In vitro methods such as PMCA are ultrasensitive, increasing sensitivity over WB by 9 log1037 and bioassay by 1−3 log10 depending on the strain of PrPTSE.38,39 Recently, Nagaoka et al.40 developed a PMCA method for the detection of ovine PrPSc in soil, demonstrating 103−104 increased sensitivity over a SDS extraction method followed by WB. To increase the limit of detection, PMCA was used to measure PrP263K and PrPCWD extracted from manure spheres before and after composting. Prior to PMCA, 10% noninfectious hamster and TgElk BH solutions containing sodium heparin were prepared as per Ding 6911

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Figure 1. Western blotting (WB) of (A) PrP263K, (B) PrPCWD, and (C) PrPBSE extracted from manure spheres collected after 0, 2, 14, and 28 days of lab-scale composting. M, reference marker; PC (positive control), 10% infectious brain homogenate was PK digested as a positive control; C, control compost; F, feather compost; C1−C2, duplicate control compost; F1−F2, duplicate feather compost; MS_D14 and MS_D28, manure spheres without PrPTSE inoculation were composted for 14 and 28 days and then collected and directly inoculated with PrPCWD or PrPBSE, followed by extraction and WB analysis. All extractable PrP263K, PrPCWD, and PrPBSE samples were proteinase K digested and precipitated using phosphotungstic acid prior to WB.

coated with 10-fold serially diluted infectious PrP263K BH in PBS were prepared. Individual beads from 10−1 to 10−8 dilutions were intracranially implanted into anesthetized hamsters, with four to five individuals per group. Inoculated hamsters were monitored for neurological disease and euthanized when they displayed clinical signs or were asymptomatic after >300 days post inoculation (dpi). Disease transmission was confirmed by immunohistochemical staining of brain sections using an automated immunostainer (Ventana Medical Systems, Tucson, USA) and the monoclonal antibody SAF84 (Cayman Chemical, Ann Arbor, USA) and the infectious titer of PrP263K estimated.46 A wide range of toxic biomolecules and infectious pathogens produced in the compost may also affect animal health during bioassays. Therefore, composted beads that were not coated with PrP263K were washed five times with 1000 μL of sterile Milli-Q water for 1 min with shaking at 350 rpm. Subsequently, washed and nonwashed beads were tested for microbial contamination on 5% blood agar plates (PB75; DALYNN, Calgary, Canada) for 72 h. No microbial colonies formed with washed beads, suggesting that this procedure precluded microbial contamination. To determine if composting reduced infectivity, groups of five to six hamsters were intracranially inoculated with washed noncomposted or composted beads at each time point that they were removed from the composters. Negative control beads which were composted but not coated with PrP263K were also included in bioassays. Hamsters were euthanized, and PrP263K was assessed by immunohistochemistry as described above.

Statistical Analysis. Changes in the physicochemical compost parameters during lab-scale composting were analyzed using the mixed procedure of SAS (version 9.2; SAS Institute Inc., Cary, USA) with time treated as a repeated measure, composter as an experimental unit, and mesh bag as a replicate in the model. Main effects were considered to be statistically significant at P < 0.05. The log10 reductions of prion infectivity by composting were calculated from the clinical dpi data of animal bioassay based on a standard curve of four-parameter logistic regression model (i.e., clinical dpi = d + (a − d)/(1 + c*exp(b*log10 dilution)) using the nonlinear regression procedure of SAS (version 9.2; SAS Institute Inc.). Physicohemical parameters for the field-scale composting experiment were analyzed as described previously and reported in Xu et al.25

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RESULTS Lab- and Field-scale Compost Properties. Temperature curves did not differ between control and feather compost in lab-scale composters (see Figure S2, Supporting Information). Composts heated rapidly, with temperature peaking at 68 °C in the control and 66 °C in the feather compost after 3 days (see Table S2, Supporting Information). Subsequently, temperatures steadily declined but remained above 55 °C for 2 days. In the second cycle, temperatures peaked at 59 and 56 °C on day 15 in the control and feather compost, respectively, but were ≥55 °C for only 1 day. In field-scale composters, temperatures at the depth of prion coated beads peaked at 61 °C after 31 days in both structures (see Table S2, Supporting Information) and 6912

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Figure 2. Western blotting of PrP263K (A and B) and PrPCWD (C and D) extracted from noncomposted and composted manure spheres collected at days 0, 14, and 28 days from lab-scale composters after protein misfolding cyclic amplification (PMCA). The left panel contains amplification controls, i.e., infectious brain homogenate (BH), verifying PMCA amplification was occurring. PK, 10% infectious BH digested with proteinase K; no PK, 10% noninfectious BH not digested with proteinase K; NBH, 10% noninfectious BH digested with proteinase K; control, control compost; feather, feather compost. Molecular weight markers at 50, 37, 25, and 20 kDa are indicated.

were ≥55 °C for 71 and 79 days in structures 1 and 2, respectively. Temperature, pH, moisture, and chemical properties of compost were similar in and out of containment (see Figure S2 and Table S3, Supporting Information). Upon completion, TN and NH4−N were higher (P < 0.05), and TC and the C/N ratio were lower (P < 0.05) in the feather than the control compost. Field-scale composters lost an average of 13 305 kg of dry matter, while lab-scale composters lost 3.7 kg of dry matter after two composting cycles (see Table S2, Supporting Information). Losses averaged 44.0% for TC and 31.6% for TN in field-scale composters but were lower in lab-scale composters, averaging 27.8% for TC and 19.2% for TN after 28 days. Inactivation of Prions Measured by Western Blotting. All three forms of PrPTSE in manure spheres were detected by WB after SDS extraction, with the limits of detection estimated at 2 log10 for PrP263K, 1−2 log10 for PrPCWD, and 1 log10 for PrPBSE (see Figure S3, Supporting Information). The reactivity of PrPTSE observed by WB before and after composting was not influenced by nonspecific binding of antiPrPTSE antibodies, as no signals were observed when isotype

control antibodies were examined. Prior to composting, PrP263K, PrPCWD, and PrPBSE were all detected after extraction from manure (Figure 1). After each composting cycle, decreasing levels of PrPTSE were detected for all prions. Signal intensity of PrP263K was slightly reduced after 2 days of composting (Figure 1A), clearly reduced by day 14 in the control compost and undetectable in the feather compost. After 28 days, PrP263K was not detected in either control or feather compost. After 14 days, a similar reduction in PrPCWD was observed in the control compost and was undetectable in the feather compost (Figure 1B) and both composts after 28 days. After 14 days, signal intensities of PrPBSE were still apparent in one replicate control compost but absent in the other and in all feather compost (Figure 1C). After 28 days, PrPBSE was no longer detectable in either compost type. Changes in the chemical and physical properties of compost overtime may have influenced the extraction efficiency of PrPTSE. However, PrPTSE added directly to 14 or 28 day-old compost was detectable at a level similar to that in fresh manure by WB for PrPCWD and PrPBSE (Figures 1B and C) and by PMCA for PrP263K (see Figure S4, Support Information). This offers evidence that irreversible binding of PrPTSE to humic acids or other organics in compost was not responsible for the 6913

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Table 1. Inactivation of Scrapie PrP263K Bound to Stainless Steel Beads during 230 Days of Field-scale Composting

temporal decline in WB signal during composting. Our WB results suggest at least a 2, 1−2, and 1 log10 reductions in PrP263K, PrPCWD, and PrPBSE, respectively, after 28 days of composting. Inactivation of Prions Measured by PMCA. A 10% infectious 263K BH was detectable at dilutions up to 107 after a single round of PMCA, whereas PrPCWD was detectable at 109 after three rounds of PMCA (Figure 2). Components in manure or compost coisolated during prion extraction inhibited PMCA detection of PrP263K and PrPCWD after one round of amplification by 1−2 log10 (data not shown). This inhibition was overcome by further dilutions to ≥3 log10, enabling PrP263K and PrPCWD to be detected in both manure and compost (Figure 2). As detected by PMCA, PrP263K declined by 1 log10 in the control and by 2 log10 in feather compost after 14 days (Figure 2A). After 28 days, PrP263K declined by 2 log10 in all composters (Figure 2B), suggesting that PrP263K degradation was limited during the second compost cycle. After 14 days, PrPCWD declined by 3 log10 in feather compost after 28 days (Figure 2D). Similar results of inactivation of PrP263K and PrPCWD as measured by PMCA were observed in the replicate lab-scale composters (see Figure S5, Supporting Information). Inactivation of Prions Measured by Bioassay. Infectivity of bead-bound PrP263K was titrated in Syrian hamsters using a series of 10-fold dilutions of infectious BH (see Table S4, Support Information). All hamsters implanted with PrP263K beads at 10−1−10−4 developed TSE with an increase in the duration to clinical disease from 107 ± 2 to 133 ± 10 dpi. At 10−5, 75% of the hamsters succumbed to disease, exhibiting clinical signs at 196 ± 38 dpi. Only 60% of hamsters developed TSE at 10−6, with no hamsters developing clinical symptoms at 10−7 and 10−8 after 330 dpi. These results indicated that the bioassay remained sensitive to bead-bound dilutions up to 10−6 with an average clinical dpi of ∼100−200 days. An infectious titer of bead-bound PrP263K was estimated as 106.0 ID50 per bead as per Reed and Muench.46 To determine the relationship between disease onset and log dilution, Cox47 and four-parameter logistic28 regression models were fitted to data from the titration assay. As the fourparameter logistic model fit better (R2 = 0.97) than the Cox model (R2 = 0.92), it was used in subsequent calculations. All hamsters displayed clinical signs of disease by 103 ± 3 dpi in control beads that were not composted (Table 1). Composting for 14 and 56 days resulted in 2.6 and 4.8 log10 reduction in the infectivity of PrP263K-bound beads, respectively (Table 1). A further reduction in infectivity was not observed in PrP263K beads that were composted for 112 days. However, only one of five hamsters developed clinical TSE from PrP263K beads that were composted for 230 days, suggesting at least a 4.8 log10 reduction of infectivity (Table 1).

Syrian hamsters clinical dpia (mean ± SEM) negative control noncompost day 0 compost day 14b compost day 56b compost day 112b compost day 230b

total number of scrapie positive hamsters/total number of hamsters

infectivity reduction (log10)

>400

0/6

103 ± 3

11/11

133

1/3c

≥2.6

257 ± 39

3/4c

≥4.8

123, 123

2/3c

≥2.2

252

1/5c

≥4.8

a

Clinical dpi: days post inoculation (dpi) when hamsters displayed clinical transmissible spongiform encephalopathy disease signs. bDue to the hamster losses from the illness unrelated to scrapie, clinical dpi data from the hamsters inoculated with the composted beads collected from compost structures 1 and 2 were combined for the calculations. c These groups contained animals that were euthanized prior to 300 dpi (a range from 108 to 253 dpi) due to illness unrelated to scrapie as verified by immunohistochemistry and were not included in the calculations. The remaining hamsters had no indication of scrapie after >300 dpi and were scrapie negative, as verified by immunohistochemistry after euthanasia.

studies. Critical to this process was to confirm that these composting systems contained the microbial communities with the potential to degrade SRM and PrPTSE.22,25,49 Our manure sphere-bound PrPTSE model simulates a natural scenario in which PrPTSE were allowed to interact directly with components in compost. Saunders et al.50 found that there was no difference in the enzymatic degradation of soil-bound and unbound PrPCWD, but not hamster-adapted PrPTME. However, as the components in compost differ from soil, there is the possibility that the affinity of PrPTSE for matrix components changed during composting,31 a factor that could have influenced PrPTSE degradation. Our study further confirmed that SDS effectively extracts PrPSc, PrPCWD, and PrPBSE from manure and compost for subsequent detection. More importantly, detection of PrPTSE was not impacted by changes in the compost matrix over time (Figure 1). However, low recovery of PrPTSE from environmental samples such as wastewater sludge36 and soils51,52 and the limited sensitivity of immunoblottting47 can be an issue. Further analysis using PMCA enabled detection of PrP263K of ∼7 log10 and PrPCWD of ∼9 log10, ∼105−107 times higher than WB. This ultrasensitive method further validated the 2 log10 reduction in PrP263K and 3 log10 reduction in PrPCWD after 28 days of lab-scale composting. Previous research has shown that binding of PrPTSE to soil alters infectivity, decreasing intracerebral53 and increasing oral infectivity.54 However, PrP263K clearly remained infective when bound to stainless steel beads in our study. Infectious prions on stainless steel surgical instruments have been implicated in nosocomial infections,55 and stainless steel wires are used in prion bioassays.38,56,57 We estimate that ∼110 ng of hamster brain tissue bound to the surface of each bead, an amount less than ∼250 ng of transgenic bovine mice brain tissue estimated to bind a 4 mm × 0.25 mm stainless steel wire.47 However, even with less infectious tissue, Syrian hamsters developed TSE

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DISCUSSION Our research team has been involved in the refining of research tools, such as Western blotting (WB),31 protein misfolding cyclic amplification (PMCA),41 and immunoquantitative PCR (iq-PCR),48 to assess the feasibility of using composting to inactivate prions. Lab- and field-scale composting systems25,30 along with suitable sampling methods30,44 for immobilizing and isolating of prions from compost laid the foundation for present 6914

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within a period of 110 dpi at an estimated detection level of ∼6 log10. Microbial activity in both lab- and field-scale composters was considerable as reflected by temperature profiles, but limited biomass in lab-scale composters resulted in thermophilic temperature (≥55 °C) for only 3 days after two composting cycles over 28 days. This contrasts with field-scale composters where temperatures ≥55 °C were recorded for 75 out of 230 days of composting. These differences in microbial activity were also reflected in the proposed degree of inactivation of PrP263K with a 2 log10 reduction in lab-scale composters and a 4.8 log10 reduction in field-scale composters. Differences in microbial activities between these models likely account for the reduced destruction of PrPTSE under lab- vs field-scale composting. The possibility exists that PrPTSE may have migrated from manure spheres or been cleaved off the stainless steel beads without being destroyed in this study. Several factors could explain why this is unlikely for the decline in PrPTSE in compost. First, the mobility of prions is limited in environmental samples such as soil52 and waste materials, including composted yard waste and municipal solid waste.58 The detachment of PrPTSE from these environmental samples has not been observed even under stringent chaotropic agents, nonionic detergents, or extreme pH.59,60 Furthermore, PrPTSE remains attached to stainless steel surgical instruments and can cause infection even after extensive decontamination, including sonication,61 chemical reagents,62 enzymatic disinfectants,63 and steam autoclaving at 134 °C.56 The possible migration of PrPTSE in our lab-scale compost was analyzed by WB, but no PrPTSE signals were detected in compost samples collected within the vicinity of nylon bags. Moreover, hamsters developed TSE disease in the earliest and final composted materials, demonstrating that prions bound to the beads remained infective throughout the composting process. These critical points provide support for enzymatic inactivation of PrPTSE during composting. Chicken feathers are predominantly composed of β-keratin (90% dry matter),64 which shares some structural similarities with PrPTSE, as both are rich in β-sheets.1,65 Keratinases with the capacity to degrade feathers may also degrade PrPTSE.22,66 Bohacz and Korniłłowicz-Kowalska67,68 reported that enrichment of a composting matrix with feathers enhanced protease activity in compost and promoted the growth of specialized keratinolytic fungi with the capacity to degrade feathers. Xu et al.31 also found that mixing feathers with cattle manure increased the degradation of SRM in compost. Consequently, enrichment for keratinolytic microorganisms in compost through inclusion of feathers may be a means of promoting the degradation of PrPTSE. In lab-scale composters, higher (P < 0.05) TN and NH4+-N contents were observed with feathers, but temperature profiles were similar between both compost types. Indeed, there was evidence that the degradation of PrP263K and PrPCWD was enhanced during lab-scale composting with feathers as indicated by lower signal intensities with both WB and PMCA. These observations do suggest that enrichment for keratinolytic microbial populations enhances the degradation of PrPTSE in compost. Further characterization of the microbial or proteolytic enzyme profiles within feather compost may yield insight into mechanisms whereby feathers promote PrPTSE degradation in compost. Investigation of prion inactivation based on rodent-passaged prion strains may not be representative of PrPCWD or PrPBSE. After exposure to SDS, PrPBSE was 10- and 106-fold more

resistant to inactivation than PrPCJD and hamster-adapted PrPSc, respectively, as assessed by infectivity titration in transgenic mice.47 Moreover, recent research by Breyer et al.69 indicated that PrPCWD is less resistant to proteolysis by proteinase K than ovine PrPSc. Our lab-scale study ranked degradation of PrPTSE as PrPCWD (3 log10) > PrP263K (2 log10) > PrPBSE (1 log10). Several other factors might also account for differences in degradation among prion strains: (1) varying concentrations of each type of PrPTSE in the infectious brain tissues used, (2) structural differences among strains, and (3) differing affinities for antibodies used in detection. However, the more recalcitrant nature of PrPBSE adds to the significance of our findings, demonstrating that 28 days of composting might result in a 1 log10 or 90% reduction in PrPBSE. Furthermore, previous research showed that the reduction of prion infectivity using various disinfection procedures including advanced ozone, SDS, sodium hydroxide, and steam sterilization as evaluated by PMCA is similar to that of animal bioassays.28,70 This suggests that 28 days of composting could reduce PrPCWD infectivity ∼3 log10 as indicated by PMCA. Consequently, as a logical extrapolation, an equally extensive inactivation of PrPCWD could be expected during field-scale composting. However, the reductions of PrPCWD and PrPBSE infectivity in compost requires further validation in field-scale systems. To our knowledge, this is the first study describing the inactivation of scrapie PrP263K infectivity and degradation of PrPCWD and PrPBSE as result of composting. However, the requirement for complete inactivation of PrPTSE in order for composting to be recommended as a method of SRM disposal is unrealistic. In practice, compost piles are affected by a number of internal and external factors including the heterogeneous nature of animal tissues and other matrix components25 and spatially dissimilar microbial communities within compost.71 Consequently, it is possible that infective PrPTSE would remain in compost where conditions for microbial activity were suboptimal. This likely explains the differences in the reduction of PrPBSE observed between replicate lab composters on day 14. However, considering that composting of prions delayed the onset and reduced the occurrence of disease in intracranially implanted hamsters, the chances of compost acting as a vector for prions is likely infinitesimal. Even if a fraction of prions remained infectious after composting, they would be greatly diluted by the compost matrix. According to Comer and Huntly,72 a BSE-infected cow could contain approximately 41 500 ID50. Assuming that the inactivations of PrP263K and PrPBSE are similar in field-scale composters, the prions remaining in the final compost could be equivalent to one ID50 unit diluted in ∼5600 kg (wet-weight basis) of final compost. Further dispersion of finished compost onto the land makes it highly unlikely that animals would come into contact with sufficient prions to develop TSE as they would be exposed to one ID50 unit every 110 m2 within 5 cm depth of top soil based on compost application guidelines in the United States.73 Setting zero tolerance for standard decontamination procedures for infectious pathogens has proven to be a criterion that is virtually impossible to meet.74 The European Food Safety Agency75 has considered decontamination procedures to be effective if they result in a 5 log10 reduction of nonspore-forming bacteria and nonthermoresistant viruses, a 3 log10 reduction in thermoresistant viruses, and 3 log10 reduction in parasites. With these considerations, composting of SRM with subsequent landfill or land application of compost 6915

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Manhattan, KS, 2005. www.ksre.ksu.edu/bookstore/pubs/MF2678. pdf. (6) Thomsen, B. V.; Schneider, D. A.; O’Rourke, K. I.; Gidlewski, T.; McLane, J.; Allen, R. W.; McIsaac, A. A.; Mitchell, G. B.; Keane, D. P.; Spraker, T. R.; Balachandran, A. Diagnostic accuracy of rectal mucosa biopsy testing for chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds in North America: Effect of age, sex, polymorphism at PRNP codon 96, and disease progression. J. Vet. Diagn. Invest. 2012, 24 (5), 878−887. (7) Canadian Food Inspection Agency Website. Herds infected with Chronic Wasting Disease in Canada since 1996. www.inspection.gc.ca/ animals/terrestrial-animals/diseases/reportable/cwd/herds-infectedsince-1996/eng/1330183608172/1330187238506. (8) Alberta Environment and Sustainable Resource Development Website. Chronic wasting disease (CWD) surveillance update: May 14, 2014. http://esrd.alberta.ca/fish-wildlife/wildlife-diseases/chronicwasting-disease/cwd-updates/default.aspx. (9) Illinois Department of Natural Resources Website. Chronic wasting disease. www.dnr.illinois.gov/programs/cwd/Pages/default. aspx. (10) Saunders, S. E.; Bartelt-Hunt, S. L.; Bartz, J. C. Occurrence, transmission, and zoonotic potential of chronic wasting disease. Emerging Infect. Dis. 2012, 18, 369−376. (11) Haigh, J.; Berezowski, J.; Woodbury, M. R. A cross-sectional study of the causes of morbidity and mortality in farmed white-tailed deer. Can. Vet. J. 2005, 46, 507−512. (12) Huijser, M. P.; Duffield, J. W.; Clevenger, A. P.; Ament, R. J.; McGowen, P. T. Cost-benefit analyses of mitigation measures aimed at reducing collisions with large ungulates in the United States and Canada: a decision support tool. Ecol. Sci. 2009, 14 (2), 15. (13) Cornell Waste Management Institute Website. Pathogen Analysis of New York State Department of Transportation RoadKilled Deer Carcass Compost Facilities. http://cwmi.css.cornell.edu/ tirc.htm. (14) Substances prohibited from use in animal food or feed. Final Rule 21 CFR Part 589, 2008. http://www.gpo.gov/fdsys/pkg/FR2008-04-25/html/08-1180.htm. (15) Gilroyed, B. H.; Reuter, T.; Chu, A.; Hao, X.; Xu, W.; McAllister, T. A. Anaerobic digestion of specified risk materials with cattle manure for biogas production. Bioresour. Technol. 2010, 101 (15), 5780−5785. (16) Carcass Disposal: A Comprehensive Review; National Agriculture Biosecurity Centre Consortium, Kansas State University: Manhattan, KS, 2004. https://krex.k-state.edu/dspace/handle/2097/662. (17) Taylor, D. M. Inactivation of transmissible degenerative encephalopathy agent: A review. Vet. J. 2000, 159, 10−17. (18) Langeveld, J. P.; Wang, J. J.; Van de Weil, D. F.; Shih, G. C.; Garssen, G. J.; Bossers, A.; Shih, J. C. Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. J. Infect. Dis. 2003, 188, 1782−1789. (19) Hui, Z.; Doi, H.; Kanouchi, H.; Matsuura, Y.; Mohri, S.; Nonomura, Y.; Oka, T. Alkaline serine protease produced by Streptomyces sp. degrades PrP(Sc). Biochem. Biophys. Res. Commun. 2004, 321, 45−50. (20) McLeod, A. H.; Murdoch, H.; Dickinson, J.; Dennis, M. J.; Hall, G. A.; Buswell, C. M.; Carr, J.; Taylor, D. M.; Sutton, J. M.; Raven, N. D. Proteolytic inactivation of the bovine spongiform encephalopathy agent. Biochem. Biophys. Res. Commun. 2004, 317, 1165−1170. (21) Ryckeboer, J.; Mergaert, J.; Vaes, K.; Klammer, S.; De Clercq, D.; Coosemans, J.; Insam, H.; Swings, J. A survey of bacteria and fungi occurring during composting and self-heating processes. Ann. Microbiol. 2003, 53 (4), 349−410. (22) Puhl, A. A.; Selinger, L. B.; McAllister, T. A.; Inglis, G. D. Actinomadura keratinilytica sp. nov., a keratin-degrading actinobacterium isolated from bovine manure compost. Int. J. Syst. Evol. Microbiol. 2009, 59, 828−834. (23) Tiquia, S. M. Evolution of extracellular enzyme activities during manure composting. J. Appl. Microbiol. 2002, 92 (4), 764−775.

to areas of low livestock density could be a viable method of SRM disposal. Currently, composting has been approved in several U.S. states as a method for routine or emergency disposal of SRM, while others have not established regulations.74 The Canadian government76 issues temporary permits for the limited use of composting for disposal of SRM, while Australia, New Zealand, and other countries with intensive farming communities are reinvestigating this practice.77 Composting of SRM has been estimated to cost in the range of $120−180 U.S. per tonne, a price that is 2−3 times lower than other disposal methods including landfills, incineration, and alkaline hydrolysis.78 Therefore, the findings herein demonstrate a promising perspective to consider composting as an alternative practice for disposal of potentially PrPTSE-contaminated SRM.

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ASSOCIATED CONTENT

S Supporting Information *

Physicochemical characteristics; comparison of compost properties; physicochemical changes; titration assays; crosssection of a biosecure field-scale composting structure; temperature during composting; and Western blotting (WB) of (A) PrP263K, (B) PrPCWD, and (C) PrPBSE. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (403) 317-2240. Fax: (403) 317-2182. E-mail: tim. [email protected]. Author Contributions ¶

Joint first authors, contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was conducted with the funding from the Prion Inactivation and Environment project, by the Alberta Prion Research Institute to M.B., N.F.N., A.B., and T.A.M., the PrioNet Canada to M.B. and N.F.N., and the Specified Risk Material Disposal Program of Agriculture and Agri-Food Canada to T.A.M. The authors thank F. Van Herk, B. Baker, R. Clark, T. Entz, W. Smart, A. Staskevicius, and P. Shaffer for their technical assistance.

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