Composting?

Prions Inactivation Through
Composting Process
Shanwei Xu & Tim A McAllister
Agriculture and Agri‐Food Canada
Compost Matters in Alberta
February 25 2014
Edmonton Alberta
TSE Disease




Scrapie – Sheep and goats
CWD – Deer and elk
BSE – Cattle
PrPC
CJD – Humans
PrPTSE
Alpha helix
Pleated sheet
2
CWD and BSE in Canada
• 19 BSE cases in Canada
• 17 cases – typical BSE (Feed contamination)
• 2 cases – atypical BSE (Spontaneous mutation)
• 173 CWD cases in Alberta
• Economic losses
1. Cattle industry – $6 billion
2. Cervid industry – $43 million
3
Specified Risk Materials
Tonsils
Dorsal root
ganglia
Skull
Brain
Eyes
Tonsils
Trigeminal ganglia
Dorsal root ganglia
Vertebral column
Spinal cord
Distal ileum
4
Specified Risk Materials

250,000 tonnes SRM in Canada

74,000 tonnes SRM in Alberta

50,000 cervid carcasses from farms and road kills

SRM are rendered, dehydrated and disposed of in
landfills

$300/tonne cost for disposal
5
Methods for Prion Inactivation
Alkaline or thermal hydrolysis
pH 14 and 150-180°C at 4-12 atmospheric
pressure for 1-3 h
Incineration
Prion
Inactivation
Two stages at 850°C and 1000°C for 16 h
Sodium hydroxide
2 N for 1 h
Autoclaving
134°C for 1 h in saturated steam
6
Enzymes Effective Against Prion
Composting?
Enzyme reaction
Enzymes
Source microbes
Keratinase
Bacillus licheniformis
50
8
Alcalase
Bacillus licheniformis
35
7.2
Proteinase K
Tritirachium album
50
7.2
Properase, protease M
Bacillus spp.
60
12
Alkaline proteinase
Streptomyces sp.
60
11
Protease E
Thermus sp.
80
7
Keratinolytic protease
Nocardiopsis sp.
60
10
Temp. (°C)
pH
Susuki et al. (2006)
7
Outline
 Lab-Scale Composting




Biocontainment laboratory level 3
Approx. 40 kg
Composted for 28 days; Two 14 day cycles
SRM; Scrapie 263K, CWD, BSE (WB & PMCA)
 Field-Scale Composting
 Approx. 85,000 kg;
 Composted for 230 days
 16 cattle mortalities; SRM; Scrapie 263K (Bioassay)
8
Laboratory Compost
Lethbridge Research Centre
(AAFC)
Level 3 laboratory
(CFIA)
9
Laboratory Compost - Sampling
1 ml 10% TSE BH
SRM or manure sphere
1 g dry manure sphere
10
Methods for Prion Detection
 Western blot (WB)
 Protein misfolding cyclic amplification (PMCA)
PrPC
PrPTSE growth
PrPTSE
Sonication
Incubation
Colby and Prusiner (2011)
11
Field Compost Layout

All dimensions in cm
12
Baker Retrieval Pyramid
13
Binding to Stainless Steel Beads




440C Stainless Steel; Hamster 263K
Mass: ~0.5 mg; Diameter: 0.5 mm
Surface area: 0.8 mm2; Density: 7.68 g cm-3
Syrian hamster model – IC implant
Clean beads
Beads coated with brain
homogenate
14
Temperature
 Lab-scale compost
 Field-scale compost
70
Ambient
Control
Feather
Temperature °C
60
50
40
30
20
10
0
0
7
14
21
28
46
92
138
184
230
Days of composting
15
SRM Degradation
Dry Matter Degradation
100
Control compost
80
Feather compost
DMD (%)
 Lab-scale
60
40
20
0
7
DMD(%)
 Field-scale
14
21
28
120
P80 DMD%
100
P160 DMD%
80
60
40
20
0
0
7
14
28
56
84
Days of composting
112
230
147
16
Microbial Invasion Brain
Day 7 Brain
Day 14 Brain
17
Bacterial Characterization
Rubrobacter radiotolerans (U65647)
Actinomycetospora succinea (AB514514)
54
Keratinase activity
F-7-3 (JN596831)
99
Saccharomonospora viridis (AB588632)
98
85
Saccharomonospora
Al-Zarban (2002)
C-7-1 (JN596829)
F-14-6 (JN596834)
100
74
80
Actinobacteria
F-7-4 (JN596832)
C-7-2 (JN596830)
Thermobifida fusca (AB562472)
Keratinase activity
F-21-10 (JN596838)
82
Actinomadura
Puhl et al. (2009)
Actinomadura sp. (FN178409)
70
88 F-28-12 (JN596840)
Actinomadura hallensis (AM932278)
F-14-7 (JN596835)
55
Thermoactinomycetaceae bacterium (AB362280)
58
Firmicutes
Thiohalospira alkaliphila (EU169227)
50
C-14-5 (JN596833)
95
C-21-9 (JN596837)
50
51
55 52
C-28-11 (JN596839)
Proteobacteria
Pseudomonas pertucinogena (EF673695)
C-21-8 (JN596836)
50
Enterobacter sp. (HM461153)
F-28-13 (JN596841)
0.1
18
Fungal Characterization
Mortierellales sp. (EF126342)
Trichaptum abietinum (U63475)
F-28-15 (JN596856)
51 F-21-13 (JN596854)
C-21-11 (JN586952)
Lipase activity
Trichaptum
Goud et al. (2009)
C-21-12 (JN596853)
90
C-28-14 (JN596855)
Basidiomycota
F-28-16 (JN596857)
Udeniomyces megalosporus (D31657)
96
Udeniomyces puniceus (DQ836006)
Day0-1 (JN596842)
Day0-2 (JN596843)
Ascomycete sp. (EF469670)
100
Chaetomium sp. (FJ646600)
88 F-7-7 (JN596848)
Proteinase activity
Chaetomium
Breuil and Huang (1994)
F-7-6 (JN596847)
C-14-9 (JN596850)
67
F-14-10 (JN586951)
Ascomycota
C-14-8 (JN596849)
Dothideomycetes sp. (HQ418433)
57
C-7-3 (JN596844)
74
C-7-5 (JN596846)
Cladosporium cladosporioides (EU263604)
62
C-7-4 (JN596845)
0.1
Proteinase activity
Cladosporium
Li and Li (2009)
19
Scrapie 263K Degradation
 Lab-scale compost – WB
% Dilution
(a)
M
10
5
2.5
1.3
0.6
0.3
0.2
0.1
0.04 0.02
30 kDa
2 log10
20 kDa
Day 0
(b)
M
IBH
Day 14
C1 C2
F1 F2
Day 28
C1 C2
F1 F2
Day 2
C
F
30 kDa
20 kDa
20
Scrapie 263K Degradation
 Lab-scale compost – PMCA
Day 0 VS Day 14
NBH
NBH
1:108
1:107
1:106
1:105
1:104
No PK
Day 0
Detected at 6 log
Day 14 Control
Detected at 5 log
NBH
1:108
1:107
1:106
1:105
1:104
No PK
1:108
1:107
1:106
1:105
1:104
PK
No PK
Ladder
1:108
1:107
1:106
1:105
1:104
1:103
1:102
1:101
PK
No PK
Amplification
Control
Day 14 Feather
Detected at 4 log
21
Scrapie 263K Degradation
 Lab-scale compost – PMCA
Day 0 VS Day 28
NBH
NBH
1:108
1:107
1:106
1:105
1:104
No PK
Day 0
Detected at 7 log
Day 28 Control
Detected at 5 log
NBH
1:108
1:107
1:106
1:105
1:104
No PK
1:108
1:107
1:106
1:105
1:104
PK
No PK
Ladder
1:108
1:107
1:106
1:105
1:104
1:103
1:102
1:101
PK
No PK
Amplification
Control
Day 28 Feather
Detected at 5 log
22
Bioassay – Stainless Steel Bead
 Steel Bead IC implant
Syrian hamsters
Dilution
Clinical DPI
(mean ± SD)
No. of sick hamsters/total
no. of hamsters
10-1
107 ± 2
3/3
10-2
119 ± 6
5/5
10-3
109 ± 4
4/4
10-4
133 ± 10
4/4
10-5
196 ± 38
3/4
10-6
179 ± 32
3/5
10-7
>330
0/5
10-8
>330
0/4
Infectivity titre: 106 ID50 / bead = 100 ng brain tissue (Reed and Muench 1938)
23
Scrapie 263K Degradation
 Field-scale compost
Compost Day 0
11/11 clinical disease
at 103 ± 3 dpi
Compost Day 112
2/5 clinical disease
at 123 ± 0 dpi
Compost Day 230
1/5 clinical disease
at 252 ± 0 dpi
24
Summary - Scrapie 263K
Lab-scale compost
Field-scale compost
•
Total 28 days
• Total 230 days
•
≥ 55°C for 3 days
• ≥ 55oC for 75 days
• Feather > Control after 14 days
• Effective to reduce infectivity
• Approx. 2 log after 28 days
• 4.8 log* after 230 days
*Four-parameter logistic regression model (SAS)
25
CWD Degradation
 Lab-scale compost – WB
% Dilution
(a)
M
IBH 10
5
2.5
1.3
0.6
0.3
0.2
0.1 0.05
30 kDa
1-2 log10
20 kDa
Day 0
(b)
M
IBH
Day 14
C1 C2
F1
Day 28
F2
C1 C2
F1 F2
30 kDa
20 kDa
26
CWD Degradation
 Lab-scale compost – PMCA
Day 0 VS Day 14
1:1010
1:109
1:108
1:107
1:106
1:105
No PK
Day 14 Control
Detected at 8 log
Ladder
1:1010
1:109
1:108
1:107
1:106
1:105
No PK
1:1010
1:109
1:108
1:107
1:106
1:105
No PK
NBH
NBH
NBH
1:1010
1:109
1:108
1:107
PK
No PK
Day 0
Detected at 9 log
Amplification
Control
Day 14 Feather
Detected at 8 log
27
CWD Degradation
 Lab-scale compost – PMCA
Day 0 VS Day 28
1:1010
1:109
1:108
1:107
1:106
1:105
No PK
Day 28 Control
Detected at 6 log
Ladder
1:1010
1:109
1:108
1:107
1:106
1:105
No PK
1:1010
1:109
1:108
1:107
1:106
1:105
No PK
NBH
NBH
NBH
1:1010
1:109
1:108
1:107
PK
No PK
Day 0
Detected at 9 log
Amplification
Control
Day 28 Feather
Detected at <5 log
28
BSE Degradation
 Lab-scale compost – WB
% Dilution
(a)
M
IBH 10
5
2.5
1.3
0.6
0.3
30 kDa
1 log10
20 kDa
(b)
Day 0
M
IBH
C1
Day 14
C2 F1 F2
Day 28
C1 C2 F1 F2
30 kDa
20 kDa
29
Future Directions
Composting degrades:
• SRM completely
• Prions at the logarithmic level
• CWD elk at field-scale after 230 days?
• BSE at lab-scale after 25 cycles (=230 days
of field-scale composting)?
30
Acknowledgements
•
Brandon Gilroyed – University of Guelph
•
Tim Reuter – Alberta Agriculture
•
Norman Neumann, Mike Belosevic, Jerry Leonard – University of
Alberta
•
Aru Balachadran, Stefanie Czub, Catherine Graham – Canadian Food
Inspection Agency
•
APRI, AAFC, NSERC
31
Thank you

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