NR ARWO
AU Taylor,D.M.; et al.
TI Deactivation of the scrapie agent by simulated rendering methods
QU unbekannt
PT unpublished results
VT
ANNEX B
PHASE 2 EXPERIMENT
DEACTIVATION OF THE SCRAPIE AGENT BY SIMULATED RENDERING METHODS
1. Introduction
1.1. Bovine spongiform enceplialopathy (BSE) is thought to have been caused by ihe inclusion of scrapie-infected meat and bone meal in cattle rations. A significant factor in the expansion of the epidemic is thought to have resulted from the production and feeding of meat and bone meal from the carcases of sub-clinical cases of BSE. Scrapie-like agents are known to be relatively resistant to inactivation by heat treatments that are effective for conventional micro-organisms, but the ability of rendering systems to inactivate the BSE agent had not been examined. This study was designed to investigate, on a pilot scale, the effect on the BSE and Scrapie agents of the various time/temperature regimes used to render animal material within the EU, together wifh some procedures not used generally but which, it was thought, might be expected to be effective.
1.2. Surveys of rendering practices in the UK and other EU member states showed that although a relatively small range of appliances were used, they were operated in many different ways. Because it was not practicable to test every method in precise detail, the first objective was to test the minimum and average conditions employed in each generic process.
1.3. The second objective was to test processes which were not in general use but which were thought likely to inactivate the BSE and scrapie agents, and which might be introduced to enable "safe" products to be manufactured. These involved the exposure of raw materials or meat and bone meal (MBM) to steam above atmospheric pressure.
1.4. The collection of brains from cattle with clinical signs of BSE was completed in November 1990, and processing of this material was completed by March 1991. Mouse inoculation work began shortly thereafter and was completed by September 1991.
1.6. The collection of brains from sheep with clinical signs of scrapie was completed by August 1992. Rendering of this material was completed by 31 September, and solvent extraction on 23 June 1993. Mouse inoculation work began shortly thereafter and is essentially complete.
1.7. Collection of scrapie-infected material in other EU member states proved difficult despite strenuous and varied efforts on the part of the project co-ordinator and the GB authorities. The planned third phase of the experiment was therefore abandoned.
2. Materials and methods
2.1. Description of equipment design and usage
2.1.1. With a few exceptions, the equipment and its mode of use were as described for the phase 1 (BSE) experiment already described at Annex A. The single exception involved the use of an AB 20 twin screw press (Atlas; Balerup, Denmark) instead of the Stord twin screw press used in phase I (BSE) of the experiment. The design and function of the AB 20 press was essentially the same as the Stord press.
2.1. CoIlection and preparation of spiked niaterial
2.1.1. Brains from 2,867 sheep infected with scrapie were collected by MAFF Veterinary Investigation Centres between October 1990 and August 1992.
2.1.2. Brains were collected only if the examining veterinarian considered that the animal exhibited clinical signs of scrapie. All 2,867 brains were placed in individual polythene bags and held at < -20 ° prior to use. To facilitate maceration during the preparation of the brain pool, their temperature was increased to + 4 ° immediately beforehand.
2.1.3. To facilitate maceration during the preparation of the brain pool, the brains were removed from cold storage and allowed to reach approximately 4°. To ensure the homogeneity of the pool, the brains were unwrapped and placed randomly in a presterilised stainless steel bin, and then processed through a mincer fitted with a 10 mm extrusion plate to produce six equal aliquots in sterilised plastic dustbins. These aliquots were mixed further by re-mincing, and were again collected as six equal lots. From these, appropriate amounts were weighed out into double polythene bags for the individual experiments.
2.1.4. Approximately l0g of each aliquot was collected and pooled to provide a sample for measuring infectivity titre by bioassay in mice (Table B2). All aliquots were held at <-20° until required.
2.2. Raw material selection and mixing
2.2.1. For each process the raw material mixtures were prepared on the day of the experiment. The equipment used and the method of mixing were the same as for the BSE study except that only pig bone and intestines were used so as to exclude any possibility of contamination with the BSE agent from the study. The mixtures were processed through a mincer using extrusion plate sizes as appropriate (Table A.1). To maximise the homogeneity of the mixtures, aliquots of bone, intestine and brain were fed sequentially into the mincer in proportion to their inclusion level until all had been processed. The mixtures used in these pilot scale studies were typical of those rendered to produce feed grade tallow and MBM.
2.3. Experimental rendering procedures
2.3.1. The range of processes tested and the rationale adopted towards their use was the same as for the BSE study. The instrumentation used to record times, temperatures, pressure etc were as described previously as were the procedures for the collection and preparation of tallow, greaves and meat and bone meal.
2.3.2. Protocol B: Batch processing
Raw material was processed at atmospheric pressure in a cooker (Iwel; London, UK). This had a steam-jacket and a rotating central shaft with agitator paddles that was also steam-heated. At the start of the process the temperature of the raw material was 40°; at the end of the process (150min) the temperature achieved was 114°
2.3.3. Protocols C, D, E and F: Continuous processing with natural fat content
These protocols were designed to simulate the conditions for full-scale continuous rendering without added tallow at minimum (C and D) and average temperatures (E and F). The raw materials were processed at atmospheric pressure in a model 1 VA Rotadisc drier (Stord International; Bergen, Norway). The temperatures attained during the various cycles are shown in Table B1.
2.3.4. Protocols G and H: Continuous processing with high fat content
These protocols were designed to simulate the average and minimal conditions for full-scale continuous rendering at atmospheric pressure with added tallow (1:1) pre-heated to 100°. The basic design of the cooker was similar to that of the Rotadisc drier except that a sliding valve arrangement permitted vacuum to be applied optionally from a pump. The temperatures attained during the various cycles are shown in Table B1.
2.3.5. Protocols I and J: Continuous vacuum processing with high fat content
The raw material was loaded into the Atlas cooker in which the added tallow had been pre-heated to 65°. Vacuum was applied in two distinct stages to simulate minimal conditions for the production-scale method. The temperatures attained during the various cycles are shown in Table B1.
2.3.6. Protocols K and L: Continuous wet rendering
These protocols were designed to simulate the average and minimal conditions for full-scale rendering by the low temperature wet method. The temperatures attained during the various cycles are shown in Table B1.
2.3.7. Protocol M: Continuous wet rendering
The purpose of this procedure was to produce starting material for solvent rendering protocols. The temperatures attained during the various cycles are shown in Table B1.
2.3.8. Protocols Q, R and S: Pressure cooking
All of these procedures were conducted in the Iwel cooker in which the raw materials were heated initially to 100°. As steam was generated, air was displaced by venting for 10 min. The venting valve was .then closed and the chamber pressure was permitted to increase to the appropriate pressure. Holding times were started when the required temperature was reached on the surface of the raw materials. Exposure times included 10 min to permit heat to penetrate into the particles. The process conditions for these protocols are shown in Table B1.
4. Procedures for decontamination and prevention of cross contamination
4.1. Procedures for chemical decontamination were based upon what was known for the scrapie agent.
4.2. Where possible, sodium hypochlorite solution yielding 20,000 ppm available chlorine was used, and was prepared each day from a stock 14-15% solution (Hay's Chemicals Ltd, Leeds). Where the use of hypochloriie was impractical, 2M sodium hydroxide was utilised, and was prepared daily from a stock 32-35% solution (Hay's Chemicals Ltd, Leeds).
4.3. The experiments were conducted within three separate locations on a single site. Movement of materials between these locations was accompanied by decontamination of containers. Personnel also changed their protective clothing except for wellington boots which were scrubbed with hypochlorite. Where there was a possibility of spillage of infected material, floors and working surfaces were covered with disposable polythene sheeting. In all respects, the procedures for decontamination and prevention of cross contamination were those followed for the BSE experiment.
5. Bacteriological screening
5.1. Although scrupulous measures were adopted to prevent cross-contamination of the experimental samples with BSE agent, it was recognised that the starting materials would be contaminated with conventional microorganisms. Bacteriological screening of the MBM samples was carried out by Q Laboratories Ltd, Preston, UK, the brain macerate and tallow samples were screened at the Neuropathogenesis Unit.
6. Bioassay of BSE infectivity
6.1. Weanling RIII/FaDk-ro mice were used for bioassay because they had been shown to have shorter incubation periods with BSE agent than other mouse genotypes; they were injected by the combined intracerebral (0.02 ml) and intraperitoneal (0.5 ml) routes.
6.2. To assay its infectivity titre, the pooled scrapie brain macerate was homogenised in sterile antibiotic solution containing 5,000 U of penicillin and 5,000 µg streptomycin per ml (Northumbria Biologicals Ltd., Cramlington) in a Griffiths tube to give a 10^-1 w/v preparation. Further tenfold dilutions (v/v) were made in antibiotic solution through to 10^-4. Each dilution was injected into weanling RIII mice.
6.3. Each sample of MBM was homogenised in sterile distilled water to give a 30% (w/v) homogenate using a Virtishear homogeniser (Virtis Co., USA); homogeniser probes were decontaminated between each usage by porous-load autoclaving at 136° for eighteen minutes, followed by washing and re-sterilisation by autoclaving. The homogenates were centrifuged at 500g for l0 min; the supernates were injected into groups of weanling RIII mice. For those samples assayed quantitatively, aliquots of the supernates were also diluted 1:10 in sterile physiological saline. Further serial tenfold dilutions were made as appropriate (Table B2).
6.4. After injection, the mice were observed and scored for the development of clinical neurological disease according to a protocol developed for scrapie in mice. Brains from all mice were immersion-fixed in 10% formol saline. Sections stained with haematoxylin and eosin were prepared from five coronal sections chosen to represent the whole brain, and examined microscopically for the presence of spongiform encephalopathy.
7. Results
7.1. The brain pool macerate contained moderate numbers of Bacillus spp., Micrococcus kristinae and Staphylococcus saprophyticus.
7.2. Table B2 shows that the infectivity titre of the scrapie-infected brain-pool macerate was 10^4.5 LD50/g. Since the concentration of this macerate added to the raw materials for rendering was 10%, the infectivity titre of the spiked starting material was 10^3.5 LD50/g. Because the weight of the solid material remaining after rendering is 30% of that of the Starting material, the maximum titre in the resulting MBM would be 10 ^3.5 LD50/0.3g (or per ml of 30% suspension of MBM).
7.3. Table B3 shows the estimated titre of infectivity surviving in MBM produced by protocols B, D, F, H, J, L and M at 24 January 1996. Titrations have not been conducted on MBM samples C, E, G, 1, K, N, 0 and P. As of 24 January 1996, no infectivity has been detected in MBM samples from protocols Q, R, S, T and U.
8. Discussion
8.1. At this time (24 January 1996), the results indicate that only the batch pressure rendering Systems simulated in Protocols Q - U achieve total inactivation of the scrapie agent as judged by the starting titre in the brain pool and the indicator systems employed. Other simulations appear to achieve a reduction in infectivity varying from 80 - 2000 fold.
8.2. Full and final analysis of Phase 2 (scrapie) of the experiment must await the completion of the remaining bioassays for protocols 3 - P, T and U, later in 1996. However, it is thought unlikely that any more results of significance will emerge. The results are presently being considered by the EU Scientific Veterinary Committee.
----------------------------------------------------------------------------
Table 18: Summary of experimental protocols using scrapie infected raw material
PARTICLE TIME
PROCESS CODE DIAMETER END TEMPERATURE ° (MINUTES)
(MM)
----------------------------------------------------------------------------
Planned Achieved
SCRAPIE BRAIN TITRATION A* homogenate N/A N/A N/A
----------------------------------------------------------------------------
BATCH ATMOSPHERIC B* 150 120 114 150
(NATURAL FAT)
----------------------------------------------------------------------------
CONTINUOUS ATMOSPHERIC C* 30 100-125 102 50
(NATURAL FAT) D* 30 125 121 125
E* 30 100 - 140 100 50
F* 30 140 138 125
----------------------------------------------------------------------------
CONTINUOUS ATMOSPHERIC G* 30 140 134 30
(HIGH FAT) H* 30 140 138 120
----------------------------------------------------------------------------
CONTINUOUS VACUUM I* 10 125 117 10
(HIGH FAT) J* 10 125 126 40
----------------------------------------------------------------------------
CONTINUOUS WET K* 20 100-120 103 120
RENDERING L* 20 120 120 240
(NATURAL FAT) M* 20 70 72 240
----------------------------------------------------------------------------
SOLVENT EXTRACTION OF N* 20 80 80 10
GREAVES FROM PROTOCOL M
----------------------------------------------------------------------------
SOLVENT EXTRACTION OF O* 20 100 100 30
GREAVES FROM PROTOCOL
M PLUS DRY HEAT
----------------------------------------------------------------------------
SOLVENT EXTRACTION OF P* 20 100 100 50
GREAVES FROM PROTOCOL
M PLUS WET& DRY HEAT
----------------------------------------------------------------------------
BATCH PRESSURE - RAW Q 50 133 134 30
MATERIAL(NATURAL FAT) R 30 136 136 28
S 30 145 145 28
----------------------------------------------------------------------------
BATCH PRESSURE - MEAL T 2.2 136 136 20
(NATURAL FAT) U 2.2 145 145 20
----------------------------------------------------------------------------
N/A Not applicable * scrapie positive (confirmed by neurohistopathology)
----------------------------------------------------------------------------
Table 2B: Titration in mice of the infectivity in pooled scrapie infected brain tissue
DILUTION GROUP NUMBER AFFECTED/ INFECTIVITY TITRE/G
_______________ NUMBER INJECTED OF TISSUE
10^-1 23/23
10^-2 23/23
10^-3 12/13 >10^4.5 LD50
10^-4 2/11
----------------------------------------------------------------------------
Table 3B: Summary of results of experimental protocols using scrapie infected raw material
NO. MICE TITRE TIME TO END
PROCESS CODE MATERIAL DILUTION WITH PER ML OF EXPT.
SCRAPIE/NO (LD50) (DAYS)
INJECTED
----------------------------------------------------------------------------
BATCH ATMOSPHERIC B 30% MBM NEAT 22/23 COMPLETE
(NATURAL FAT) 10^-1 13/18 10^1.5 "
___________________ 10^-2 1/12 _______ "
----------------------------------------------------------------------------
CONTINUOUS ATMOSPHERIC C 30% MBM NEAT 16/16 COMPLETE
(NATURAL FAT)
D 30% MBM NEAT 5/12 27
10^-1 5/5 10^1.4 "
10^-2 1/13 "
E 30% MBM NEAT 17/18 COMPLETE
F 30% MBM NEAT 9/11 COMPLETE
10^-1 3/14 10^0.8 "
10^-2 Negative "
----------------------------------------------------------------------------
CONTINUOUS ATMOSPHERIC G 30% MBM NEAT 1/12 COMPLETE
(HIGH FAT)
H* 15% MBM NEAT 6/11 COMPLETE
10^-1 3/17 10^0.8
10^-2 Negative
----------------------------------------------------------------------------
CONTINUOUS VACUUM I 30% MBM NEAT 7/7 COMPLETE
(HIGH FAT) J* l5% MBM NEAT 8/10 75
10^-1 8/16 10^1.6 "
10^-2 1/5 "
----------------------------------------------------------------------------
CONTINUOUS WET K 15% MBM NEAT 16/18 131
RENDERING L 30% MBM NEAT 9/17 228
(NATURAL FAT) 10^-1 2/18 10^0.5 "
10^-2 1/17 "
M 30% MBM NEAT 2/11 264
10^-1 1/22 10^0.01 "
10^-2 Negative "
----------------------------------------------------------------------------
SOLVENT EXTRACTION OF N 30% MBM NEAT 8/15 156
GREAVES FROM PROTOCOL M
----------------------------------------------------------------------------
SOLVENT EXTRACTION OF O 30% MBM NEAT 13/19 143
GREAVES FROM PROTOCOL
M PLUS DRY HEAT
----------------------------------------------------------------------------
* These samples WCTC So absorbative that they had to be prepared as 15% homogenates to provide sufficient fluid for injection
^ Adjusted to correct for 30% suspension
----------------------------------------------------------------------------
TABLE 3B (Cont): Summary of results of experimental protocols using scrapie infected raw material
----------------------------------------------------------------------------
SOLVENT EXTRACTION OF P* 30% MBM NEAT 3/13 149
GREAVES FROM PROTOCOL
M PLUS WET & DRY HEAT
----------------------------------------------------------------------------
BATCH PRESSURE - RAW Q 30% MBM NEAT NEGATIVE COMPLETE
MATERIAL (NATURAL FAT)
R 30% MBM NEAT NEGATIVE COMPLETE
S 30% MBM NEAT NEGATIVE COMPLETE
----------------------------------------------------------------------------
BATCH PRESSURE- MEAL T 30% MBM NEAT NEGATIVE 187
(NATURAL FAT) U 30% MBM NEAT NEGATIVE 180
----------------------------------------------------------------------------
* These samples were so absorbative that they had to be prepared as 15% homogenates to provide sufficient fluid for injection
^ Adjusted to correct for 30% suspension
The status of all mice described as scrapie infected has been confirmed by neurohistopathological examination
SP englisch
OR Prion-Krankheiten 8