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From: TSS (216-119-136-29.ipset16.wt.net)
Subject: Re: THE SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEE OPEN MEETING 11th February 2003
Date: February 11, 2003 at 12:33 pm PST
In Reply to: Re: THE SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEE OPEN MEETING 11th February 2003 posted by TSS on February 11, 2003 at 12:28 pm:
WORKSHOP ON TSEs IN THE
ENVIRONMENT
30 APRIL 2002
NOBEL HOUSE, DEFRA
INTRODUCTION................................................................................................................................1
BACKGROUND..................................................................................................................................1
ANIMAL WASTE AND BY PRODUCTS..........................................................................................2
RENDERING PLANTS........................................................................................................................3
• Rendering condensate .............................................................................................................4
• Greaves/MBM .........................................................................................................................5
• Tallow.....................................................................................................................................5
• Sludge.....................................................................................................................................5
ABATTOIR PRACTICES ...................................................................................................................5
DEFRA FUNDED RESEARCH INTO ENVIRONMENTAL PERSISTENCE OF TSE
INFECTIVITY.....................................................................................................................................6
POTENTIAL RESEARCH AREAS....................................................................................................7
MANAGEMENT OF BURIAL SITES AND PERSISTENCE OF TSES ............................................................7
DETECTION OF PRION PROTEIN............................................................................................................7
DETECTION OF INFECTIVITY IN SOIL MATRIX .......................................................................................8
SOIL TYPE AND PERSISTENCE OF INFECTIVITY .....................................................................................8
AGRICULTURAL PRACTICES/MANAGEMENT AND PERSISTENCE OF INFECTIVITY...................................8
PARTITIONING OF PROTEIN/INFECTIVITY IN SOLID/LIQUID FRACTIONS .................................................9
RISK ASSESSMENTS ............................................................................................................................9
HORIZONTAL TRANSMISSION ..............................................................................................................9
ANNEX 1 ............................................................................................................................................10
ATTENDEES .....................................................................................................................................10
1
Introduction
A meeting was held to identify what research is required to address possible
contamination of the soil and water environment resulting from present and
past farming and agro-industrial practices. The attendees included experts in
soil science, TSE research, risk assessment and relevant government
departments and agencies. Potential research areas were suggested by the
attendees. These are given in italics. These areas will be prioritised by DEFRA
against each other and other research priorities. Those of the most relevance to
DEFRA policy and those that will give meaningful results, within a realistic
timeframe and cost will be included in future research calls.
Background
(Robert Somerville)
Possible sources of environmental contamination were summarised as follows
• Burial or disposal of TSE infected carcasses
• Placentae
• Faeces and urine from infected animals (or carriers)
• Insects, carrion and other vectors
• MBM and other animal products, sometimes used as fertiliser (or spread on
land as a means of disposal)
• Liquid and solid waste fragments from abattoirs, renderers, central
sterilisation units, operating theatres etc
• Incineration – ash
Evidence of transmission through the environmental route was summarised as:
• Icelandic studies have suggested environmental contamination as a route of
scrapie transmission.(Palsson, 1979; Sigurdarson, 1954)
• Chronic Wasting Disease (CWD) in the USA. A high level of lateral
transmission is considered to occur amongst mule deer and elk populations
• BSE in cattle – Epidemiological studies have indicated that horizontal
transmission has not had a big impact on the epidemic. Despite this,
horizontal transmission has not been experimentally validated. Horizontal
transmission may potentially account for the BSE cases observed following
the ban in 1996 on feeding ruminants with animal derived proteins.
However, other factors including non-compliance with the feed ban, feed
imports or sporadic BSE may also form plausible explanations for these
observations
2
The following unknowns with regard to the persistence, movement and
infectivity of TSEs in the environment are:
• Communities of soil microrganisms and animals involved in carcass
degradation
• Susceptibility to proteolytic degradation
• Adsorption to and entrapment in soil and organic/mineral components
• Fate of TSE infectivity after ingestion by soil animals
• Necrophagic insects/animals
• Inactivation/destruction – dependent upon proteolytic digestion (and
therefore affected by pH, temperature and time)
• Dilution of infectivity before and after disposal of waste (assumes threshold
dose)
• Heat denaturation – effect of drying is known to make the agent more
resistant to denaturation. This may have implications regarding TSE
infectivity on the soil surface exposed to periodic drying
• Ozonation, other oxidative agents and the effects of UV – uncertain about
the efficiency of these methods for destruction of TSE infectivity
Animal Waste and By Products
The disposal of animal waste and animal by products were discussed in relation
to possible routes of transmission:
Animal waste and by products can be categorised into the following:
• general animal waste by products including material from slaughter house,
butcher waste, rendering waste and dead farm animals
• specified risk materials (SRM), over thirty month scheme animals (OTMS),
BSE and scrapie suspects (TSE risk materials)
• waste from factories and catering outlets
The Animal By Products Order (APBO) (SI 1999 No646 and SI 2001 No 1704)
states that animal by-products are consigned to rendering or incineration, or
other permitted routes. There are currently no specifications regarding controls
for incineration of animal by-products, although next year there will be a
stipulation for exposing material to 850oC for 2 seconds. This temperature is
derived from legislation regarding destruction of dioxins.
Burial (including a licensed landfill) and burning (other than a licensed
incinerator) will be banned from next year. Only on remote areas of land, which
will only be the Highlands and Islands of Scotland, will burial be permitted.
3
However, composting or category 2 (high risk non-SRM) material (after
pressure cooking) and category 3 (low risk from fit animals) material will be
allowed.
Currently under the ABPO, ruminant blood and gut contents can be spread on
land. However, next year, blood must be treated as a category 3 material before
spreading. Gut contents and processed by-products (including blood) will be
permitted to be spread only on non-pastureland.
The current ABPO does not include disposal of SRM, handling or disposal of
BSE and scrapie suspects and disposal of OTMS animals. This material must
be rendered or incinerated. Incinerators for SRM rated at >50 Kg/hour are
licensed by the Environment Agency (EA) or local authority (depending on
size), as are landfill sites used for disposal of ash (EA only). Previously,
disposal of ash on site was permitted for smaller incinerators. Legislation now
dictates that all ash must now go to licensed landfill. In assessing the suitability
of a site for landfill, the Environment Agency assumes that incineration
inactivates infectivity. Brown et al (1999) however, have shown that hamster
TSE infected tissue is not necessarily completely destroyed at 600°C. In the
Brown experiment, infectivity was destroyed at 1000°C. We do not know the
minimum time/temperature combination required to ensure infectivity is
destroyed.
At what temperature during incineration, is infectivity destroyed?
How dependent is the presentation of the material on the ability to inactivate?
For example, drying is considered to render tissues more resistant to
inactivation.
Catering waste is disposed of via landfill/incineration. Other routes of disposal
are also being assessed e.g. composting and production of biogas. Provided that
TSEs are prevented from entering the foodchain, this should not be an issue in
terms of environmental transmission.
Rendering Plants
Rendering practices were discussed and described as an industrial ‘cooking’
operation. Material is reduced to a size of 25/30 mm through macerators prior
to ‘cooking’.
There are two types of ‘cooking’ processes – ‘continuous’ and ‘batch’. They
operate under either atmospheric or under pressure (3 bar). Fat is either added
to the system (which is the case for the majority of the rendering operations), or
the process operates under natural fat. Defatted systems are also used.
4
The history of the regulatory standards imposed on rendering were described.
Prior to 1989, there were no statutory standards, however, with the occurrence
of the salmonella ‘outbreak’, there was a requirement for MBM to be
salmonella free. For this reason, the following standards were set by the EU in
1990:
133ºC
3 bar
20 minutes (or alternative systems offering equivalent guarantees of
microbiological safety)
The aim was to eliminate Salmonella and E. Coli, and from high risk material,
Clostridia.
Look at parameters (e.g. pH, temperature and time) on an incremental basis in
order to gain a better understanding of the effectiveness of the rendering
process.
These conditions were included in the Animal By Products Order in 1992 (SI
1992 No 3303) as a standard for inactivation of conventional pathogens, not
TSEs. Following a series of investigations, in 1994, the EU stipulated that all
high risk mammalian1 material should be rendered by pressure cooking unless
the MBM was going for landfill or otherwise being kept out of the food, feed
and fertiliser chains and destroyed, in which case other rendering methods were
acceptable. The UK has, however, historically favoured disposal of materials in
landfill.
There are 4 products of rendering which also come under the ABPO:
• Rendering condensate
Only rendering condensate which has been treated to discharge
standards set out in the Rendering (Fluid treatment) (England) Order
2001 can be spread on land, or disposed of via the sewer and
watercourses. All TSE suspects are incinerated so the issue of
condensate from these sources never arises. It would be legal to spread
OTMS/SRM derived condensate if treated in accordance with the Order.
A minimum protein level is not one of the parameters explicitly stated in
the order. Clearly, however, the main thrust of the standards set out is to
reduce the protein level in the treated condensate.
1 High risk material is defined as material which is not fit for human consumption as defined by the
ABPO. In this context it also includes SRM and OTMS material.
5
• Greaves/MBM
This is the proteinaceous fraction of the rendering process.
Greaves/MBM derived from OTMS cattle is incinerated before landfill.
Greaves/MBM from SRM is incinerated or pressure cooked before
landfill. It is possible to use non-SRM, non-OTMS rendered mammalian
greaves/MBM as a fertiliser on non-agricultural land or as a horticultural
fertiliser. This can only be practised if the MBM has been pressure
cooked. Poultry and fish meal greaves/MBM are currently permitted as
fertilisers on agricultural land.
• Tallow
Tallow from SRM can be used as a fuel replacement, while tallow from
non-SRM, depending on quality, can be used for technical purposes,
including the oleochemical industry, including pharmaceuticals,
cosmetics, animal and human feeds.
• Sludge
Sludge, a product of rendering process is disposed of on land.
However, there are many other wastewaters of potential concern that are not
treated – e.g. yard washings in abattoirs.
Similarly, liquid waste which is ‘treated’ following PMs in VICs, and research
institutes may also be a cause for concern.
Abattoir practices
OTMS cattle are butchered off centre to avoid cutting into the spinal cord.
Drain traps with 4 mm mesh sizes are used in all ruminant abattoirs to collect
tissues. These traps are thought to catch fragments of tissue greater than 0.1g in
weight. In a ruminant slaughterhouse, these trapped particles are designated as
SRM and must be handled and disposed of as such. The remaining waste water
will either be:
(a) - treated on site after which it can be discharged to watercourses or onto
land by means of a discharge consent from the Environment Agency; or
(b) – tankered off site for treatment at a treatment works; or
(c) – discharged down a pipe to the sewage treatment works under a trade
effluent consent from the water company.
6
DEFRA funded research into environmental persistence of TSE
infectivity
DEFRA is currently funding one project looking at the persistence of TSEs in
the environment; entitled SE1433 - Studies on the environmental persistence of
TSE infectivity.
To assess whether TSE infectivity can persist and migrate after disposal into the
ground, a co-ordinated series of experiments will be conducted. Laboratory
based experiments will examine the persistence and migration of infectivity
through different soil types under various conditions. Secondly, a lysimeter
based experiment will simulate, as far as is reasonably practical and safe to do
so, the deposition of a bolus of infectivity in the ground. Survival and migration
of infectivity from the source will be measured. Thirdly to assess the survival of
infectivity within buried carcasses, a series of TSE spiked bovine heads will be
buried. The will be serially exhumed and their residual TSE infectivity
measured.
1. ?
7
POTENTIAL RESEARCH AREAS
The following potential areas for research were identified by the attendees.
DEFRA will consider these and include any of high priority in future open
competitions.
Management of Burial Sites and Persistence of TSEs
How do anaerobic conditions affect the degradation, persistence and migration
of TSEs in the soil environment? It is likely that burial sites represent an
anaerobic environment and liquefaction is dependent upon localised
environmental conditions.
Detection of prion protein
It was suggested that the use of C14 recombinant prion protein would have the
advantage in that Category III containment facilities would not be required, and
it would be easily detected. The use of radioisotopes (such as C14) have the
advantage in that the highly sensitive detection methods would still be able to
detect recombinant PrPsc even when it is adsorbed onto soil particles/substrates.
This could prove useful in investigating the effects of different soil
types/management regimes on the fate of PrPsc. C14 methods could also provide
information on where not to look for the agent, whilst also providing
information on the physical state of the prion protein in terms of inactivation.
Although we realise that recombinant prion protein may differ in its physical
and chemical properties from native prion protein, it would seem appropriate to
conduct this type of research (using a surrogate marker) in parallel with other
studies. Recombinant prion protein has been shown to refold under certain
conditions. This may be a useful approach for generating a physical/chemical
analogue that can effectively model the nature of the agent. It is acknowledged
that we do not know the exact relationship between PrPsc and infectivity,
however, research is ongoing aiming to identify the extent of this correlation.
In order to detect prion protein which may be mixed within a soil matrix, or
attached to organic fractions/mineral particles, it is important to design a
method of detection which is not dependent upon the surface topology of the
prion protein.
What detection systems can be used to detect PrPsc/infectivity in soil systems?
Ideally, a quantitative method is required.
8
Can we use a surrogate model, such as C14 recombinant prion protein, in
parallel with studies using native prion protein?
Could other radioisotopes be considered?
Should detection methods focus on differentiating between bioavailable and
non-bioavailable PrPsc or infectivity?
Could earthworms be used as an effective sampling method of the soil profile?
Detection of infectivity in soil matrix
Consider bioassaying soil in mice using the oral route of challenge (bearing in
mind the lack of sensitivity in oral inoculations)
Consider bioassaying soil/leachate in young lambs.
Soil type and persistence of infectivity
What effect does soil type have on the persistence and migration of TSE
infectivity? Determine worst case scenario in terms of persistence, decay and
leaching.
If TSE infectivity does not decay substantially under normal farming
conditions, could this maintain endemic infections? This may be important in
relation to historical animal waste and by-product disposal practices.
Cattle born after the implementation of the 1996 Ruminant feed ban (BABs),
which banned the feeding of all mammalian proteins to farmed livestock, may
need to be investigated further. Sample affected farms involved in order to
characterise soil types. (This type of work can be performed quite quickly).
Agricultural practices/management and persistence of infectivity
What are the effects of agricultural management on the migration and
persistence of TSE infectivity? For example, will ploughing stimulate
proteases? Similarly, will fertiliser input stimulate sufficient/appropriate
microbial activity to degrade PrPsc/infectivity?
Various agricultural/industrial practices could be responsible for altering the
properties of PrPsc. Therefore:
9
Can soil physical and chemical conditions alter infectivity of PrPsc (e.g. by
influencing folding and conformation of the molecule)?
What is the correlation between protein structure and inactivation?
What is the correlation between partial degradation and inactivation?
See research requirements identified above with respect to industrial waste
disposal processes.
Partitioning of protein/infectivity in solid/liquid fractions
Most of the Risk Assessments have relied on the assumption that the TSE agent
is hydrophobic and adheres to particulate matter. This assumption requires
scientific validation.
How soluble is the TSE agent? How does it partition between solid and liquid
fractions?
Does the agent solubilise in typical conditions found in soil solution or around
decaying animal waste materials?
Will bioavailability change if PrPsc is adsorbed onto soil particles?
Does the agent stick to soil/organic substrates? And by so doing does this
change the physical nature of the agent sufficiently to inactivate the agent?
Does degree of glycosylation affect binding properties/stickiness?
Risk Assessments
Do other assumptions used in Risk Assessments require scientific validation?
Horizontal transmission
The manner in which the agent is excreted is not known. However, in the case
of scrapie, there is good evidence that horizontal transmission occurs – in
addition to vertical. Clearly there is a need to further the work on horizontal
transmission in order to evaluate the role of soil born transmission of scrapie.
Does faeces contain infectivity? There are limited reports in the literature.
Should lamb bioassays be considered?
Should placenta and birthing fluids also be investigated in lamb bioassay?
10
Annex 1
Attendees
Chair – Dr Nick Coulson
Dr Brian Chambers - ADAS
Dr Paul Gale - WRc
Dr David Jones – University of Wales, Bangor
Dr Keith Jones – Lancaster University
Professor Ken Killham – Aberdeen University
Dr Steve McGrath – IACR, Rothamsted
Dr Mark Purcell – DNV
Dr Joanne Rodger - IAH
Dr John Scullion – University of Wales, Aberystwyth
Dr Robert Somerville – IAH
Dr Peter Cook – EA
Mr Mike Waite – DWI
Dr Mandy Bailey – DEFRA. AHEG
Miss Sue Bolton – DEFRA, AHEG
Dr Nick Coulson – DEFRA, SD
Dr Peter Costigan – DEFRA, SD
Dr Graeme Campbell – DEFRA, SD
Dr Hilary Gates – DEFRA, SD
Dr Ruth Pugh – DEFRA, SD
Dr Steve Wyllie – DEFRA, AHEG http://www.defra.gov.uk/ TSS
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