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From: TSS (216-119-163-214.ipset45.wt.net)
Subject: Occurrence and Behaviour of BSE/TSE Prions in Soil Bonn, 18 December 2000
Date: December 5, 2002 at 8:53 am PST
International Expert Discussion
of the
Federal Ministry for Environment, Nature Conservation
and Nuclear Safety
Bonn, 18 December 2000
Occurrence and Behaviour
of BSE/TSE Prions in Soil
Report
prepared by Fraunhofer-Institute for
Environmental Chemistry and Ecotoxicology
D-57392 Schmallenberg, Germany
February 2001
Motive and Background
BSE (Bovine Spongiform Encephalopathy) appeared in Great Britain as a
disease which had
not been known before in cattle already in the 1980s. The phenomenon is
being linked to the
appearance of the new variant of Creutzfeld-Jacob Disease (nvCJD) in
humans. At the
beginning, these forms of allegedly cross-species Transmissible Spongiform
Encephalopathies (TSE) seemed to be limited to Great Britain. However,
the first cases of
BSE in Germany turned this assumption into an illusion.
The Scientific Advisory Board on Soil Conservation at the Federal
Environment Ministry
presented an expert opinion entitled “Ways for precautionary soil
conservation – Basis and
steps for setting up an extended soil precaution” (published by the German
Parliament/Bundestag as Printed Paper 14/2834 //
www.parlamentsspiegel.de). The Board
recommends in chapter 4.7.3. “Prions” (excerpts in Annex 1) that for
precautionary reasons
initiative should be taken to scientifically investigate the issues of
occurrence and behaviour
of prions in soil. Thus the Federal Environment Ministry’s plan for
environmental research
earmarked a project to gather all general soil-related information. This
research project was
planned for 2001, but the first German BSE case led the Federal Minister
Jürgen Trittin to
antedate the expert meeting to take place in December 2000.
The opinion of the Scientific Advisory Board on Soil Conservation did
not discuss the relative
importance of the “soil pathway” as opposed to other routes of BSE
infection. It is, however,
beyond dispute that according to latest information the predominance of
infection pathways,
in particular, via industrial animals feed, has been confirmed1.
Nevertheless, this does not
minimize the necessity for examining other routes, for example, via
soils or via
environmentally-based biological vectors such as mites.
The topic of the entry of BSE-causing pathogens into the soil was the
subject of several
opinions of the Scientific Steering Committee (SSC) of the European
Union, in particular, its
opinion on the use of meat-and-bone meal as fertilizer. This confirms an
essential interest in
some of the questions raised, despite the incompleteness or complete
absence of investigation
of these questions in research projects (DG Research: “interesting, but
no current research
has been done as yet”). The title of the only sponsored research project
(“Role of
1Note by the editors: Meat-and-bone meal was discussed during the expert
discussion; whereas milk exchangers
as a transmitter of infection were not mentioned. Animal fats, through
which the carriers of infection might
enter the milk exchange have been handled by the SSC (Scientific
Steering Committee) with regard to the BSE
risk (Opinion on the safety of tallow derived from ruminant tissues
−Adopted at the Scientific Steering
Committee meeting of March 26-27, 1998; Opinion on the questions
submitted by EC services following a
request of December 4, 2000 by the EU Council of Agricultural Ministries
regarding the safety of certain bovine
tissues and certain animal-derived products with regard to BSE, adopted
on January 12, 2001).
environmental and host factors on the horizontal and vertical
transmission of scrapie in
naturally infected sheep flocks”, Project: PL987023), which is being
undertaken by Icelandic,
French and Spanish scientists, would suggest strong similarities between
the topic of the
project and the topic under discussion here. However, the actual
contents presented in the
available brochure deal only with the problem of vectors2.
Aims of the Expert Discussion
The expert discussion was understood to be a scientific forum for
highlighting the topic from
different perspectives, bearing in mind that there are relatively few
facts. Subject fields were
dealt with individually and for each one the corresponding information
and its reliability were
outlined briefly and discussed critically.
The aims of the expert discussion were:
• identification and delineation of necessary short-term and medium-term
activities,
• identification of possibilities for implementing the results of these
activities.
Here, the word “activities” means research activities as well as
consequences or risk-reducing
measures for, in the broadest sense, precautionary protection of the soil.
It was not the objective of the expert discussion to recommend the
immediate implementation
of risk-reducing measures at the end of the discussion; the facts on the
issue generally
appeared to be insufficient to do this.
Organization and Course of the Expert Discussion
The meeting took place as an open discussion without specific,
comprehensive reports being
given. The following topics were discussed:
Entry routes of BSE-causing pathogens into soils:
2 “Prions can survive for years in soil, but how can scrapie be
transmitted? A group of teams from France,
Iceland and Spain is setting out to study the role of nematode
parasites, nasal flies, ticks and mites in the
transmission process and to determine to which extent wild rodents could
serve as a prion reservoir. This group
is also examining possible vertical transmission through embryo organs.
This work will permit a better
understanding of the wide spread of scrapie in naturally infected
flocks, taking into account the genetic
susceptibility of the hosts”.
• Industrial fertilizers
• Grazing animals
• MBM as fertilizer
Latest information on the persistence of prions in soils:
• Basics of the persistence of BSE/TSE pathogens
• TSE on sheep pastures
• Prior investigations with TSEs
Detection methods:
• Currently used methods
• Methods being developed
• Usability for soils
Soils as a source of the exposure of humans and livestock?
• Potential exposure routes, availability, exposure routes to be excluded
• Relevance of vectors
Research on the subject:
• Ongoing projects
• Need for research, possibly step-by-step plan
Prof. Dr. G. Miehlich, Hamburg, deputy chairman of the Scientific
Advisory Board on Soil
Conservation, chaired the discussion. The Federal Environment Ministry
(BMU) was
represented by Dr. F. Holzwarth.
The following experts participated in the discussion as members of the
panel:
Prof. Dr. H. Diringer, Rastede; Dr. M. Groschup, BFAV, Tübingen; Dipl.
Biol. R. Heynkes,
Aachen; Dr. M.G. Koch, Karlsborg, Sweden; Prof. Dr. D. Riesner,
University of Düsseldorf;
Prof. Dr. V. Silano, Ministry of Health, Rome.
The Scientific Advisory Board on Soil Conservation was represented by:
Prof. Dr. D. Henschler, University of Würzburg; Prof. Dr. W. Klein,
Fraunhofer Institute for
Environmental Chemistry and Ecotoxicology, Schmallenberg; Prof. Dr. H.
Mühle, Environmental
Research Center, Leipzig; Prof. Dr. W. Walther, Technical University of
Dresden as
well as the managing director of the WBB, Dr. G. Bachmann, Federal
Environmental Agency,
Berlin.
Other participants in the meeting were members of federal and Länder
ministries and
agencies (see list of participants).
Prof. Dr. W. Klein and Dr. M. Herrchen, Fraunhofer Institute for
Environmental Chemistry
and Ecotoxicology, Schmallenberg, were in charge of preparing and
documenting the expert
discussion.
Facts and Scientific Questions on the Persistence of Pathogenic Prion
Proteins in Soil
The latest information regarding persistence can be summarized as follows:
1. A reasonable amount is known about the general biotic and abiotic
persistence of
pathogenic prion proteins. They are apparently relatively stable against
typical proteindenaturing
“influences”; i.e., they are either non-degradable or only partly
degradable by
proteases in metabolism and, quantitatively, they can be inactivated
only under certain
conditions, for example, through heat (300 °C, 1 h), pH changes,
surfactants, 1 M NaOH
or 10 % formaldehyde3.
2. In soils, scrapie pathogens do not seem to be completely degradable
over a longer period
of time. Already in 1991, Brown et al. (P. Brown and D.C. Gajdusek,
Survival of scrapie
virus after 3 years’ internment, The Lancet 337, 269-270, 1991) found a
residual
infectivity of 0.2 − 1.8 %, based on the control sample stored at -70
°C, following a 3-year
incubation4 of soils with scrapie pathogens. Moreover, there is the fact
that in the case of
highly active chemical substances < 0.01 % is still enough to trigger a
pathological
reaction when the substance reaches the target organ. In the present
case, however, a
3 “Regarding the risks from TSEs and unconventional agents, according to
current knowledge, inter- and
intraspecies transmission may occur across a range of animal species.
The rendering standard of at least 133
°C/20 min/3 bars cannot be considered, for the time being, as totally
effective in destroying TSE infectivity from
infective materials. This applies to all animals species with potential
for TSE infectivity. Thus, additional
protection measures ensuring absence of TSE infectivity are required.”
(Scientific opinion on the risks of
unconventional transmissible agents, conventional infectious agents or
other hazardous agents such as toxic
substances entering the human food chain or animal food chains via raw
material from fallen stock and dead
animals; adopted by the Scientific Steering Committee at its meeting of
June 24-25, 1999 and revised at its
meeting of July 22-23, 1999).
4 Note: The expression “incubation of a soil with a chemical or
biological contaminant” is a typical used
technical expression used in microbiology and terrestrial ecotoxicology.
It infers the treating of the soil with a
contaminant and the subsequent maintenance of the soil under defined
laboratory or open-field conditions as
well as the sampling at defined times to determine the degradability or
the effect of the contamination. It does
not refer the “infection risk” which could result from soils treated
with TSE prions.
residual infectivity was shown in an experiment. Based on the central
importance of this
study for the topic treated here, the essential results are reported in
Annex 2.
Although the mechanism of degradation was not studied in this
experiment, it is likely
that it is microbial degradation. A transfer in soil organisms (also
called vectors) is insignificant
with regard to the concentration in soil.
3. Many years of practical experience with scrapie pathogens show that
the pathogens
remain infectious for more than 3 years on unused pastures. This
statement is based
mainly on experience gained from the re-use of pastures with initially
healthy sheep and
less on the evidence of specific scientific investigations.
4. In the production of meat-and-bone meal, even if using the best
available technology, no
complete, quantitative inactivation of the pathogen takes place.
Instead, a residual
infectivity remains, as many studies have shown (Review D. Taylor,
2000). In view of
this fact, with the use of MBM (including blood meal) as fertilizer, one
would have to take
into account that this would be an entry route for residual infectivity
in soil, if the raw
material was already contaminated (Scientific opinion on the safety of
organic fertilizers
derived from mammalian animals, adopted by the Scientific Steering
Committee at its
meeting in September, 1998).
Several questions arose from the initial discussion on the possible
persistence of BSE-causing
pathogens in soil and the expert discussion was to discuss these questions:
Entry Routes into Soil
The following are to be considered as generally possible sources of the
entry of pathogenic
prion proteins on or into agriculturally used soils:
• (Organic) fertilizers containing MBM or other animal-derived materials
• Industrial fertilizers (liquid manure, stable manure upon
contamination of the animal feed)
• Sewage sludge from waste waters from slaughter houses and rendering plants
• Compost and residual wastes of biogas production
• Excrements from household pets upon contamination of the feed
• Placenta of scrapie-infected sheep.
Looking broadly at entry routes to the environment as a whole, all
animal husbandry (zoos,
fur and fish farms) must be included and the possibilities for waste
water contamination must
also be taken into consideration. The share of infectivity which may be
attributed to the soil
pathway can be determined epidemiologically only after the period in
which a transmission
via animal feed no longer occurs. The measures taken so far on
prohibition of feeding MBM
have been successful, as the British example shows. However, they are
ineffective for
household pets, zoo animals and fur animals.
The suspected entry routes into soil cannot be quantified at present by
direct analyses, since
no detection methods exist with which soils or other environmentally
relevant matrices such
as stall manure, dung, hay etc. can be examined. Consequently, the
infection concentrations
and the concentrations in slaughter house waste water and sewage sludge
have not been
analysed yet. Up to now, no pathogens have been detected in cattle
excrement, however, this
may be due to the fact that there are no sufficiently sensitive
detection methods.
Therefore, neither conclusive valid statements about the relative
importance of the various
cited entry routes into soil nor a quantification of the total entry can
be made. This would be
the minimum prerequisite for a comparative assessment of cattle exposure.
However, it is unquestionable that with proper production (133 °C, 3
bar, 20 min.), the
“concentration” in meat-and-bone meal fertilizer (exception: blood meal)
as well as that in
animal feed amounts to less than one-thousandth of that in the starting
materials.
With respect to the entry route via farm manure, there is consensus that
ingested infected
material is partially degraded or inactivated metabolically but is also
excreted unchanged and
thus enters the soil. The quantitative fraction of unchanged excretion
is not known − there are
different estimates on this − especially since it is species-dependent
and is affected by the
state of health of the animals. Supposedly, the pathogenic prion
proteins are relatively
quickly degraded or are excreted by the animals that are not
susceptible. There is no
information on the extent to which a further microbial degradation takes
place during the
storage of liquid manure and dung.
Regarding the entry route via pets, especially dog faeces on pasture
areas, there is consensus
that this, based on the total area of pastureland, is insignificant
compared to other routes.
However, the formation of so-called “hot spots” cannot be excluded,
considering the analogy
to faeces of grazing animals infected by contaminated feed.
The same applies, for example, for the entry route via sewage sludge.
Although it can be
assumed that the residual infectivity or the content of pathogenic prion
proteins is very low in
sewage sludge, the possibility of this route, e.g. for sewage sludge
from waste water from
rendering plants, cannot be ruled out, as long as there is no guarantee
that no infected animals
were processed.
Despite the lack of information already mentioned, it seems to be
appropriate to qualitatively
assess the potential occurrence of prion proteins (e.g., based on MBM
contamination) as the
“realistic worst case” scenario, whereas attempts to quantify released
infectivity at this time
would be pure speculation. Scenario-based estimates could take into
account, for example,
the fact that the entry of MBM into pastures is hardly significant,
since fertilizing takes place
with liquid manure or mineral fertilizers. Organic fertilizers, which
may contain MBM −
including blood meal − are, or were, also used in ecological agriculture.
For all cited sources, it is the case that there are no facts known
about the extent to which
infectious residual materials are actually present or how these possibly
infectious substances
behave in the (terrestrial) environment. Although the footnote excerpt
from the scientific
opinion of the SSC on the safety of organic fertilizers is based on
fertilizers containing animal
materials, the statements regarding soil and the safety of harvested
goods apply for all the
exposure routes cited here5. Limitations with regard to a
quantification, including a hazard
evaluation, risk characterization and risk assessment, are currently due
to, among other things,
the insufficiently sensitive measurement methods. Insofar as these
questions cannot be
answered with sufficient certainty, and also that the prevailing
uncertainty cannot be removed
by experience, and taking into account the principle of precaution, it
should be ensured that
persistent prions are not entering the soil. Dealing with this subject
is important, if only on
the basis of the prevailing uncertainty and the mandate of increasing
the reliability of
information for the consumer.
5 Excerpt from: Scientific opinion on the safety of organic fertilizers
derived from mammalian animals, adopted
by the Scientific Steering Committee at its meeting in September, 1998:
“Identification of possible hazards and
elements of risk assessment: The first possible hazard is that the
organic fertiliser would carry a residual BSE
infectivity. If this would be the case, the following additional hazards
exist: potential involuntary persistence in
the environment and contamination of soil and water with the BSE agent,
including a potential accumulation of
infectivity over years. The risk of ingestion of residues of the
fertilisers by humans or animals: The amounts
ingested by this way are probably rather small. Human consumption will
normally follow washing the material
which would reduce the fertilizer residues on the surface. Animal
consumption could be more substantial. No
information is available as to the interaction of an eventual residual
infectivity of organic fertiliser by external
conditions such as atmospheric conditions, microbiological activity,
ploughing, washing off by rain or irrigation,
etc. It is therefore not possible to assess if, and after which period,
consumption of crops treated with organic
fertiliser could be safe, if it could not be excluded that these carry
the BSE agent. The present opinion of the
SSC is substantially based on the work of a working group, chaired by
Prof. Dr. M. Vanbelle. Other members of
the working group were: Prof. Dr. R. Böhm, Dr. R. Bradley, Prof. Dr.
J.W. Bridges, Prof. Dr. D. Dormont, Prof.
Dr. M. Esko Nurmi, Prof. Dr. A.-L. Parodi, Prof. Dr. G. Piva, Dr. M.
Riedinger, Dr. B. Schreuder, Prof. Dr. P.
Sequi, Prof. S. Alexandersen, Dr. D. Taylor, Dr. H.A.P. Urlings, Prof.
Dr. M. Wierup, Prof. Dr. P. Willeberg).”
This applies even more so as in several European countries (Sweden, for
example), the
labeling of animal feed as “free of meat-and-bone meal” is allowed as
long as the animal feed
contains less than 0.5 % animal protein; 5 kg animal protein per tonne
feed, however, is not
an amount to be neglected.
In accordance with the precautionary principle, such routes of
transmission that seem to be
less likely must also be considered. Examples of such routes of
transmitting infectious
diseases were cited in the expert discussion: the relatively low
likelihood of infection per
sexual contact for AIDS (1-2 %) could not prevent the fast spreading of
this disease. Thus,
the low absolute probability of an infection is no reason to remain
inactive.
Also mentioned was a spongiform brain disease in a North American deer
species which
behaves similarly to scrapie but possibly was transmitted to the deer
via an intermediate host
(deer louse). (This, however, is currently a preliminary hypothesis, and
valid proof does not
exist).
Exposure to Pathogenic Prion Proteins via the Soil Pathway from an
Epidemiological Perspective
About 90 % of BSE cases occurring in the UK epidemic can be explained by
epidemiological
models. Without any experimental proof the remaining 10 % are explained
by maternal
transmission (vertical transmission, vt). Nevertheless, in literature
the supplementary
epidemiological explanation via the fertilizer-soil pathway is given,
which also allows the
explanation of a few other epidemiological findings which as yet cannot
be interpreted
otherwise. Verbatim excerpts from a publication by Dealler in 1996 on
the internet are found
in the footnote below6. However, it was underlined that definite
experimental findings on the
infectivity of grazing areas do not exist in the UK either − this is
used as an essential
6 Vertical transmission of BSE: epidemiological evidence. BSE cases in
the UK in herds that did not use MBM
as feed: this has been difficult to explain but has been reported to
myself in 5 herds where, in one of these cases,
turkey manure was used on the farm....That the soil on which the cattle
grazed was infected by this time with
prions from feed that had been fed to cattle (or to pigs or poultry, if
their faecal matter was used as fertiliser).
This is not unreasonable but no way is known to be sure of this
currently. The lack of any change in the age
distribution for cattle born after the feed ban (a drop in ages is felt
to be unacceptable if BSE is similar to other
TSEs) suggests that they are becoming infected in a similar manner to
those prior to it, and a change might be
expected if soil contamination were to be a major effect. ... The
epidemiology found fits with VT or with the
possibility that soil becomes infected. ... This could be explained
through the use of animal manure as fertiliser
on the cow fields or perhaps through the idea of a non-BSE factor being
involved. (Dealler:
http://sparc.airtime.co.uk/bse/vtwww.htm, 1996).
argument against the importance of this route of infection. However, it
was also pointed out
that transmission routes via the soil (the pasture) had not been studied
directly up to now.
The fact that typically only one animal of a herd is infected with BSE
is no argument against
the soil pathway − the same would apply for the pathway via feed.
In general, it was stated that a considerable limitation of
epidemiological studies and
considerations is their inability to exclude particular suspected
processes. They can instead
only be used for confirming seemingly plausible relationships. Even if
the likelihood of an
infection via the soil pathway should be so low that, from an
epidemiological viewpoint, no
immediate measures are necessary, the transmission route via soil cannot
be ruled out and,
thus, must be directly investigated. Should, for example, after the
prohibition of feeding
MBM has taken effect − (single) further cases of BSE occur, it will be
necessary to have
information available about other possible routes of infection.
This seems to be all the more important, because currently even a low
residual risk is not
accepted: “However, the iatrogenic spread of Creutzfeld-Jacob disease
(CJD) among
humans and probable transmission of Bovine Spongiform Encephalopathy
(BSE) to persons
in the UK have now led scientists and health care personnel to consider
the acceptable
tolerance level for “residual” infectivity on equipment or in biological
solutions to be zero”
(R.E. Race, Guest Editorial, The Trouble with Transmissible Degenerative
Encephalopathy
Agents; The Veterinary Journal 2000, 159, 3-4). Even if the context
quoted there is different
to that of the expert discussion, the question of the detection and
assessment of residual risks
is also relevant here.
Furthermore, with respect to the BSE problem, in the risk analysis of
environmental
chemicals, one must consider the relevant fact that the dose/response
curve is mostly
unknown at very low doses. The hypothesis that one prion alone might
trigger the illness
(one-hit-theory) cannot be refuted at this time and, thus, must be used
as a starting point for
further consideration. Among other things, this also means that
epidemiological models and
studies and their corresponding conclusions for evaluating the BSE
problem, alone, cannot be
sufficient in dealing with this problem.
Behaviour of Prions in Soil, Persistence, Mobility and Bioavailability
The statements on the behaviour of pathogenic prion proteins in soil are
contradictory. For
analysing the behaviour in soil with subsequent evaluation, the criteria
generally used in the
substance evaluation of environmentally relevant chemicals and processes
considered should
be taken into account analogously, whereby it must be emphasized that
the general knowledge
on these proteins and their behaviour in soil is very fragmentary:
• Degradation and accumulation: Many studies have been conducted on the
chemical and
physicochemical degradation, as well as one experiment performed on the
microbial
degradation, of pathogenic prion proteins. Nonetheless, further
understanding of the
degradation kinetics in soils (especially under realistic conditions) is
further necessary to
be able to make definite statements on the degradability. For this,
several measurement
points have to be established. A single measurement taken after a 3-year
incubation period
under unrealistic test conditions, as cited in Brown et al., is
insufficient. The study by
Brown et al. is a pilot study which gives important indications but does
not clarify
conclusively the scientific questions regarding the degradation of BSE
causing pathogens.
Should persistence be confirmed over many years, it can be assumed that,
with lasting
constant entry to the soil, a steady state is established which is
higher than the
concentration after a one-time entry and, thus, represents an
accumulation. Upon a
repeated but quantitatively decreasing entry to the soil, a degradation
might start, but
probably after having reached the maximum. With a one-time entry, the
degradation curve
− for a first-order reaction− asymptotically approaches a “zero” end
concentration. These
statements apply only for those inputs accessible to (microbial)
degradation in soil.
• Sorption/Desorption: An “inactivation” of the infectivity by sorption
or incorporation
into the soil matrix alone, does not in principle make the BSE pathogen
harmless, because
it can be remobilized and, thus, reactivated.
In the studies by Brown et al., at the end of the exposure, 3 log units
less infectivity were
extractable than that had been applied. Whether this effect is
attributed to sorption into
the soil matrix or to actual microbial degradation was not investigated.
Nevertheless, the
effect concurs with the expected good sorbability into the soil matrix.
An “inactivation”
of the infectivity by sorption or incorporation into the sol matrix,
however, would not be a
final sink (in the context of the BSE pathogen being rendered harmless),
since
remobilization and, thus, reactivation, can take place.
• Bioavailability: Prion proteins adsorbed into soil were classified as
being less
bioavailable. There is no data available which proves this, but it can
be assumed −
analogously to other substances of similar behaviour− that the
availability of proteins
adsorbed into soil is less than that in meat-and-bone meal or in
infected organs.
• Translocation/mobility: Even when a high mobility in soil is not
considered likely, the
translocation behaviour or the migration into deeper soil layers and
possibly into
groundwater must be examined. A particulate-bound transport is not to be
excluded
(example: migration of rota viruses into groundwater).
There are no indications for a “multiplication” of pathogenic prion
proteins in soil as is the
case in susceptible animals.
Soils as the Source of Exposure of Humans and Livestock
A relatively well-known and verified source for a spreading of the
scrapie pathogen is the
transmission via pregnant sheep dams. The highly infective placenta
stays on the pasture and
is eaten by other animals so that a horizontal transmission can occur.
The sheep dams may be
carriers of the infection but may not get sick themselves due to genetic
resistance.
This infection route is not known for cattle. It has not yet been shown
that cattle defecate
infective material. Mechanisms for crossing the species barrier are not
known.
Furthermore, it was observed that scrapie broke out again on pastures
that had not been used
for 10 years (the hay mite was named as the possible transmission
route), although the
possibility that genetically resistant animals made contact with
genetically susceptible sheep
cannot be ruled out.
Apart from the aforementioned modes of transmission, several other
pathways must not be
left unconsidered, such as those published by Dealler in 19967. For
example, the following
are to be taken into account:
7 “Other models, although not originally thought to be likely, could
also have been involved with the epidemic
rise of BSE. For instance, it may not have been the cow that ate the
infected material but its offspring that
showed symptoms of disease; this model (discussed as the vertical
transmission model) would be expected to
give a similar rise of disease and would be difficult to separate from
the direct transfer “nugget” model. Also,
horizontal transmission taking place from one bovine to another through
soil, or the transfer of other infective
agents carrying the BSE agent could also be involved. It now seems
likely that there may be more than one
method of transmission for BSE and as such, as the prevention of bovine
MBM reaching further cattle takes
place, these modes may become more important and, because of this, the
various epidemiological models should
be discussed.” (Dealler: The current epidemic rise of BSE and
epidemiological models that fit the known facts,
http://sparc.airtime.co.uk/bse/vtwww.htm, 1996).
• The possibility of transmission to pigs that, upon injection, showed
sensitivity to the
pathogen: experiments with oral intake showed no sensitivity as yet. In
general, the
species barriers cow−pig−sheep is estimated to be high.
• Up to now, there is has been no proof available of infectivity of fish
and poultry.
• The route via insects: The pathogen does not multiply itself in
pupated maggots but
remains detectable there over a longer period of time; the pupated
maggots might be seen
as the “intermediate host” but do not serve as the accumulator (in the
sense of an
independent multiplication by the biological vector).
• The transmission via soil organisms (for example: nematodes): There is
no data available
on this.
Potential further Pathways of Exposure
Further uptake routes of pathogenic prion proteins from the soil are of
differing levels of
importance:
• An uptake of prion proteins into the plant via the plant root was
considered to be unlikely,
because the plant generally does not take up any high-molecular
substances, and proteins
are synthesised by photosynthesis.
• A deposit of dust/soil contaminated with prion proteins on the plant
surface and a
subsequent intake as animal feed may not be ruled out. A scenario used
in evaluating the
soil contamination with dioxins assumed that, with the raw feed, grazing
animals ingest
about 3 % soil material that is attached to the greenery. Accordingly, a
cow would ingest
up to one kilogram of soil per day.
• The direct route soil−human is supposed to be insignificant.
Detection Methods
For showing the occurrence of BSE, the detection of the pathogenic prion
protein (in the
sense of an indicator) is suitable for test purposes, and the detection
of the pathogen via
infectivity is suitable for its verification.
Pathogenic prion proteins may be detected, for example,:
• by the electron microscopic presentation as scrapie- or BSE-associated
fibrils,
• immunochemically, whereby the proteins can be recognized as deposits
in the cells and
dyed using Immuno-Blot. The prion proteins form a characteristic band in
the range of
20-30 kDa.
• by histological detection.
The pathogen itself is detected in animal experiments on the basis of
its infectivity. An
infective unit (I.U.) is hereby the amount of a pathogen which causes
infection in half of the
animals of a group (mouse, hamster, cow...). Thus, one infective unit
corresponds to an ID50
value. In general, infective units are specified on a logarithmic scale.
About 108 I.U./g brain
are formed in a diseased cow brain, while only about 104 I.U. could be
detected in the
transmission test on mice. Upon employing transgenic mice, the
sensitivity is higher by a
factor of 104; i.e., the 108 I.U. formed in the cow can be detected. In
general, I.U. values are
defined on the basis of intracerebral injections, while about 104 − 105
more pathogens are
needed for oral intake. Current detection limits are set at about 102 I.U.
In recent years, new and more sensitive measurement methods have been
developed, however
most of them represent in vivo methods. Within the framework of the
European Initiative on
TSEs, in the 5th framework program, 12 joint projects for developing
diagnostic methods are
being conducted, a few of these for diagnosing nvCJD and others for
diagnosing scrapie and
BSE. In vitro methods with blood- and nerve fluid have also been
developed which possibly
could be adapted to soil.
Neither the existing detection methods nor the newly developed methods
are not immediately
suited for detection of pathogenic prion proteins in the soil
compartment. However, methods
that are sufficiently sensitive should be basically adaptable. In
choosing methods to be
adapted to soil analysis, it must be taken into account that the
sensitivity of presently existing
tests is unsuitable for detection in soils.
Recommendations
For all the mentioned routes of entry into soil as well as the pathway
from soil to animal and
to human, the following can be summarized: There are indications that
one or even all of the
cited pathways may have a certain importance, and thus their
significance was a controversial
topic during the expert discussion. With respect to precautionary soil
protection, further
clarification of the entire issue should take place. To that end,
several priority questions must
be dealt with in the framework of target-oriented research projects.
1. Valid investigations on persistence, accumulation and mobility.
Should persistence not be
confirmed and a faster “complete” microbial degradation be found, then
the following
questions are irrelevant.
2. Development and use of conclusive methods for proving or disproving
the existence of
prions in soil/surfaces (including methods for extracting pathogenic
prion proteins from
soils and detection of very low infectivity).
3. Assessment and, if possible, quantification of the risk to be
attributed to the various entry
routes into soils as well as of the risk originating from the soils.
Classification of these
risks under the heading “BSE distribution”.
4. Evaluation of the risk and clarification of questions on risk
acceptance, including the
acceptance of residual risk; risk communication.
5. The effect on waste recycling, which in Germany, in the case of
biogas use, does not
comprise any heat treatment, was classified to be a relevant aspect for
research. It should
be considered whether the presence of infectious biomolecules in the
starting materials
prior to biogas production necessitates pretreatment, e.g., by hydrolysis.
In this context it was suggested to establish a forum of interested
scientists.
The discourse during this expert discussion also showed that there
should be a clear and
uniform definition of the terms used in epidemiology, virology and
environmental protection
and which have different meanings depending on the particular context.
Since Prof. Silano had to leave the discussion early before the final
round, he gave the
following summary of the results of the discussion:
“As long as animals are exposed to infected feed, the elimination of
some BSE infectivity
with faeces is likely to take place. This will be the case for both
BSE-susceptible and non-
BSE-susceptible species. The extent of this elimination depends on the
amount of BSE
infectivity ingested during the period of exposure as well as on the
species involved and the
health conditions of the involved animals. Other possible sources of
soil contamination are
the use of MBM as fertiliser and the inadequate disposal of MBM and
infected animal tissues.
Once the BSE agent has reached the soil, it is likely to be bound to
particles in the superficial
soil layers and to persist there for some years, as only a slow
degradation can be expected.
This conclusion is based on the resistance of TSE agents to a number of
decontamination
procedures (D.M. Taylor: Inactivation of Transmissible Degenerative
Encephalopathy
Agents: a Review, The Veterinary Journal 2000, 159, 10-17) and on the
fact that high-titer
scrapie agent retained infectivity after burial for three years,
although approx. 99 % of the
infectivity was lost (Brown and Gadjusek, 1991). Moreover, the failure
of several scrapie
eradication programmes registered as yet could lead to the same
conclusion (Siguzdason,
1991).
Some release of the BSE agent into water is also to be expected. The
exposure of animals and
humans to the (soil-bound) BSE agent − although being minor compared to
other exposure
routes − cannot be excluded, but its significance, if any, is difficult
to assess based on
presently available data.
On the basis of the precautionary principle, it would be reasonable to
investigate these issues
further.”
Dr. Holzwarth gave the following summary of the results of the expert
discussion from his
perspective. The discussion does not allow any conclusive final
statement in the sense of a
consensus declaration by all the participants. On the basis of the
present findings this was not
to be expected. In any case, the meeting has at least taken into account
one issue from the
opinion of the Scientific Advisory Board on Soil Conservation.
There it is stated that:
“... in order to consider precautionary soil protection concerns in this
topic, the Advisory
Board further recommended that initiative should be taken to convene a
scientific
meeting in which the questions of persistence, among others, are
discussed and a strategy
for handling priority questions developed.”
Of the priority questions which have to be treated − with respect to all
of the controversial
discussion regarding the assessment of the relative importance of the
whole issue − the
question concerning diagnosis and possibility of analysis must be
addressed first.
Further discussion is particularly necessary on this topic to which a
“fast solution” is certainly
not expected.
Furthermore, a “soil” model should be developed which allows the
treatment of the questions
concerning the transfer into soil, behaviour and persistence.
Two questions, which cannot be answered “scientifically correctly” and
which were asked by
colleagues representing the Länder, are characterized by the keywords
“retrosamples” and
“quarantine”.
In answer to the question of retrosamples, it should be noted that with
existing persistence,
infectivity can still be detected even after some years. However, should
the pathogenic prion
proteins not be as persistent as assumed, the current retrosamples would
not allow any
conclusion to be drawn about the period of infectivity.
Concerning the quarantine question, the conclusion to be drawn − having
considered the
course of the discussion and Prof. Silano’s statement − would actually
be to shut off all
sources suspected of causing the entry of infectious material into the
environment.
Consequently, this would also include the soil and the pasture, thus,
raising the topic of
“quarantine”. However, this measure is not appropriate in the current
situation. This careful
assessment does not preclude the further investigation of open questions
related to soil.
On behalf of the Federal Environment Ministry, Dr Holzwarth thanks all
the members of the
panel and the audience for attending and engaging in an open and
committed discussion.
Annex 1
Ways for precautionary soil conservation
by the Scientific Advisory Board on Soil Conservation at the Federal
Ministry for the
Environment, Nature Conservation and Nuclear Safety
4.7.3 Prions
Plant diseases which are transmitted from the soil to cultivated plants
have long been a
problem in agricultural plant protection and greatly impair the
productive function of soils.
Their existence and, in part, their multiplication in soils require the
use of pesticides to
compensate for impaired soil function.
Due to hygiene measures introduced with regard to the soil pathway to
animals or humans,
conventional pathogens are not a serious problem for animals and humans,
especially since
they do not persist in infectious concentrations in soils.
By using modern molecular biological methods, the idea has evolved from
several hypotheses
that prions are the causal agents of TSEs (Transmissible Spongiform
Encepthalopathies).
BSE (Bovine Spongiform Encephalopathy) is a special form of the illness
in cattle. Prions are
pure proteins. They associate on an endogenic gene (PrP encoding gene),
without containing
DNA themselves, and reproduce themselves in this way. The situation is
different for the
recently discovered, infectious and soil-persistent biomolecules which
cause TSEs in many
animal species like sheep, goats, cattle, household pets and zoo
animals, but also in animals
living in the wild. Although illnesses caused by them, such as scrapie
in sheep and goats,
have been known for decades and studies have been conducted since the
fifties to investigate
the cause of these diseases, the BSE crisis has led to intensified work
on this topic. Prions
have been detected in warm-blooded organisms and in yeast − thus, in
proteins of multiple
organisms. Their physiological significance has not yet been determined
clearly. They exist
in at least two ISO-forms or conformations, one being a non-pathogenic
natural form (PrPsen)
and the other being the degenerate lethal form (PrP-res). Concerning the
spreading of the
disease, the transmission between species is possible, although barriers
do exist. The
minimum infectious dose is known only under experimental conditions for
laboratory
animals. Infection routes may be vertical, i.e. through transmission to
the offspring, and
horizontal, i.e., essentially by oral intake of the pathogen. The
development of the disease has
a long incubation period, for sheep approximately two years. The
infectious dose and the
incubation time are unknown for the new variant of Creutzfeldt Jakob
disease which has been
linked to BSE. In any case, scrapie exists worldwide, thus it is not
certain that diseaseresistant
breeds themselves are not carriers of the pathogen. The BSE disease in
cattle is not
restricted to the United Kingdom (IRL, P, F, CH). Although only six
clinical cases of BSE in
imported cows have appeared in Germany so far and the livestock is
BSE-free, the risk of
spreading the BSE pathogen is not absolute zero due to importing animal
material from non-
BSE-free countries as well as transporting live animals.
Relevant for soil protection is the persistence of the lethal prion type
which may lead to longterm
infectivity of soils. Unlike the PrP-sen, PrP-res are not degradable by
metabolic
proteases and cannot be inactivated quantitatively by heat (300 °C,1 h),
changes in pH,
surfactants, 1 M NaOH, 10 % formaldehyde and other influences which
typically denature
proteins.
Thus, it is assumed that, with the technologically optimum conditions of
rendering (133 °C, 3
bar, 20 min), that have been introduced in Germany, the infectivity of
PrP-res has decreased
by several orders of magnitude. It is certain that the degradation is
not quantitative (SSC,
1999). If the infectivity of the starting material is not ruled out with
certainty, this means that
the use of the products which contain it, such as fertilizer, results in
a transmission of the
infection carrier. This is to be taken all the more seriously given the
empirical knowledge
acquired from long-term experience with scrapie that the scrapie
pathogen can stay infectious
for three or more years on unused pastures (veterinary consultation,
example: L.D. Breeden,
1990). It could also be shown experimentally that the infectivity of the
scrapie pathogen was
still present after three years in soil (Brown, P. and Gajdusek, D.C.,
1991). The leaching of
prions, for example, from landfills (+), has likewise been recognized
and risk-reducing
measures recommended (SSC, 1999). Possible methods of assessing the
persistence of the
infectivity in soil, e.g., in soil of grazing land must be investigated.
Although valid tests that
meet the typical standard of tests on the persistence of chemicals, do
not exist, the Advisory
Board finds this to be a substantial cause for concern. There are
several, essentially
immunological methods currently being developed and validated which will
show the
sufficient sensitivity required. It has not yet been studied to what
extent PrP-res in soil only
represent a risk for the infection of grazing animals or whether a
transfer to other organisms,
e.g. soil fungi or microorganisms, with potential ecological effects, is
likely.
(+) Remark: This concerns landfills of BSE-infected animal corpses.
Annex 2
Excerpt from: P. Brown and D.C. Gajdusek, Survival of scrapie virus
after 3 years’
internment, The Lancet 337, 269-270, 1991
“The initial infectivity of 4.8 log units fell to between 2.2 and 3.0
log units of residual
infectivity at the end of the 3 years, and an additional 1.3 log units
had leached into the soil
lying immediately under the petri dish that was perforated top and
bottom. No infectivity was
detectable in the lower layer of soil 4-8 cm beneath the bottom of the
dish or in the soil
surrounding the unperforated dish. None of the sentinel animals caged
with the inoculated
animals became ill. Before nitrocellulose filtration (to eliminate
bacteria), the control virus
mixture had an infectivity of 7.5 log units, compared with 4.8 log units
after filtration. If
filtration losses were the same for all specimens, the buried samples
may have contained
nearly 1000-fold more infectivity than is shown below:
Material Infectivity titres* Total infectious units*
Control virus/soil mixture
Top-drilled dish
Soil under dish
Top/bottom drilled dish
Upper soil layer under dish
Lower soil layer under dish
4.8
2.2
0**
3.0
1.3
0**
170,358,000
428,000
0
2,700,000
359,000
0
* = infectivity titre (per 33 µl inoculum) and total infectious units in
log10 LD50
** = no virus detected in undiluted inoculum
This experiment established the durability of scrapie virus exposed to
natural environmental
conditions for 3 years and also shows that most residual infectivity
remains in the originally
contaminated soil with little leaching.”
page 21 of 21
List of Participants
Herr Dr. H. Arnold
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten
Bereich Umwelt
Mainzer Str. 80
65189 Wiesbaden
Herr Dr. Günther Bachmann
Umweltbundesamt
FG II 5.1
Postfach 33 00 22
14191 Berlin
Frau Ingeburg Bauer
Bayerisches Staatsministerium für Ernährung, Landwirtschaft und Forsten
Ludwigstr. 2
80539 München
Herr Dr. W. Beicht
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten
Bereich Landwirtschaft
Mainzer Str. 80
65189 Wiesbaden
Herr Wolfgang Beitlich
Ministerium für Umwelt und Verkehr
Referat 56, Bodenschutz
Postfach 10 34 39
70029 Stuttgart
Herr Dr. Bernd-Michael Böhmer
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
AG WA I 5
Bernkasteler Str. 8
53175 Bonn
Frau Ute Böhmer
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Arbeitsgruppe WA I 5
Postfach 12 06 29
53048 Bonn
Frau Hatice Demircan
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Sprachendienst
Postfach 12 06 29
53048 Bonn
Herr Prof. Dr. Heino Diringer
Ladestr. 48
26180 Rastede
Herr Stephan Dreyer
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
WA II 4
Bernkastelerstr. 8
53175 Bonn
Herr W. Eggert
Bundesministerium für Wirtschaft und Technologie
Abt. IV C 4
Villemomblerstr. 76
53123 Bonn
page 22 of 22
Herr Dr. J.-A. Eisele
Ministerium für Umwelt und Naturschutz, Landwirtschaft und
Verbraucherschutz des Landes Nordrhein-Westfalen
Ref. II 5
Schwannstr. 3
40476 Düsseldorf
Herr H.-M. Elschner
Thüringer Ministerium für Soziales, Familie und Gesundheit
Werner-Seelenbinder-Str. 6
99096 Erfurt
Frau Mechthild Föhr
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Sprachendienst
Postfach 12 06 29
53048 Bonn
Herr Dr. Frank Glante
Umweltbundesamt
FG II 5.2
Postfach 33 00 22
14191 Berlin
Herr Dr. Martin Groschup
Bundesforschungsanstalt für Viruserkrankungen der Tiere
Institut für Immunologie
Postfach 11 49
72001 Tübingen
Herr Dr. H.-P. Hamann
Staatliches Medizinal,- Lebensmittel- und Veterinäruntersuchungsamt
Mittelhessen
Marburger Str. 54
35396 Gießen
Herr Dr. Stefan Heitefuß
Niedersächsisches Landesamt für Ökologie
An der Scharlake 39
31135 Hildesheim
Herr Prof. Dr. Dietrich Henschler
Universität Würzburg
Institut für Toxikologie
Versbacherstr. 9
97078 Würzburg
Frau Dr. Monika Herrchen
Fraunhofer-Institut für Umweltchemie und Ökotoxikologie
Auf dem Aberg 1
57392 Schmallenberg
Herr Dr. Norbert Hess
Freie und Hansestadt Hamburg
Umweltbehörde
Billstr. 84
20539 Hamburg
Herr Dr. H.-Christoph von Heydebrand
Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft
Ref. G 3
Rochusstr. 1
53123 Bonn
page 23 of 23
Herr Dipl.-Biol. Roland Heynkes
Erkwiesenstr. 19
52072 Aachen
Herr Dr. Fritz Holzwarth
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Unterabteilungsleiter WA I
Bernkastelerstr. 8
53175 Bonn
Herr Dr. Heinrich Höper
Niedersächsisches Landesamt für Bodenforschung
BTI Bremen
Friedrich-Mißler-Str.46-48
28211 Bremen
Herr Dr. Christian Hubrich
Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft
UA 31
Rochusstr.1
53123 Bonn
Herr Dr. Peter Hummel
Senatsverwaltung für Arbeit, Soziales und Frauen
Abt. IV C 4
Oranienstr.106
10969 Berlin
Herr Prof. Dr. Werner Klein
Fraunhofer-Institut für Umweltchemie und Ökotoxikologie
Auf dem Aberg 1
57392 Schmallenberg
Herr Dr. med. Michael Koch
S-54681 Karlsborg (Schweden)
Herr Dr. Wilhelm König
Ministerium für Umwelt und Naturschutz, Landwirtschaft
und Verbraucherschutz des Landes Nordrhein-Westfalen
Schwannstr. 3
40476 Düsseldorf
Frau Dr. Karin Köster
Freie und Hansestadt Hamburg
Behörde für Arbeit und Soziales
G 80 V
Hamburger Str. 47
22063 Hamburg
Herr Prof. B. Kouros
Sozialministerium Baden-Württemberg
Schellingerstr. 15
70174 Stuttgart
Herr Dr. Juan Lopes-Pila
Umweltbundesamt
FG II 2.4
Corrensplatz 1
14195 Berlin
Herr Prof. Dr. Günter Miehlich
Universität Hamburg
Institut für Bodenkunde
Allende Platz 2
20146 Hamburg
page 24 of 24
Herr Dr. Wolfgang Mields
Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin
Thielallee 88-92
14195 Berlin
Frau Prof. Dr. Heidrun Mühle
Umweltforschungszentrum Leipzig-Halle GmbH
Postfach 2
04301 Leipzig
Herr Werner Nonnenmacher
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Arbeitsgruppe WA I 5
Postfach 12 06 29
53048 Bonn
Herr Peter Reiß
Landwirtschaftliche Untersuchungs- und Forschungsanstalt
Siebengebirgsstr. 200
53229 Bonn
Herr Prof. Dr. Detlev Riesner
Heinrich-Heine- Universität Düsseldorf
Institut für Physikalische Biologie
Universitätsstr. 1
40225 Düsseldorf
Herr Dr. Roland Schäfer
Landesveterinär- und Lebensmitteluntersuchungsamt Sachsen-Anhalt
Abt. Tierseuchendiagnoastik
Aussenstelle Stendal
Haferbreitenweg 132/135
39576 Stendal
Herr Helmut Schulz
Bundesministerium für Bildung und Forschung
Ref. „Integrierter Umweltschutz in der Wirtschaft“
Heinemannstr. 2
53175 Bonn
Herr Dr. M. Schulz
Ministerium für Landwirtschaft, Umweltschutz und Raumordnung
des Landes Brandenburg
Heinrich-Mann-Allee 103
14473 Potsdam
Herr Schulz
Ministerium Ländlicher Raum Baden-Württemberg
Ref. 35
Kernerplatz 10
70182 Stuttgart
Frau Kerstin Seidler
Umweltbundesamt
FG II 5.1
Postfach 33 00 22
14191 Berlin
Herr Stefan Seiffert
Sächsisches Staatsministerium für Umwelt und Landwirtschaft
Archivstr. 1
01067 Dresden
page 25 of 25
Herr Prof. Vittorio Silano
Ministero della Sanitá
Servizio Farmaceutico
Viale della Civilta Romana 7
I-00144 Roma
Herr Horst Simon
Ministerium für Umwelt, Natur und Forsten des Landes Schleswig-Holstein
Mercatorstr. 3
24106 Kiel
Frau Birgit Straubinger
Ministerium für Umwelt und Forsten Rheinland-Pfalz
Kaiser-Friedrich-Str. 7
55116 Mainz
Herr Dr. Thomas Suttner
Bayerisches Staatsministerium für Landesentwicklung und Umweltfragen
Referat Bodenschutz,Geologie
Rosenkavalierplatz 2
81925 München
Herr Dr. Töpner
Bundesministerium für Gesundheit
Am Probsthof 78 a
53121 Bonn
Frau Beatrix Trapp
Sächsisches Staatsministerium für Umwelt und Landwirtschaft
Ref. 33
Wilhelm-Buck- Str. 2
01097 Dresden
Herr Gerhard Treger
Ministerium für Ernährung, Landwirtschaft, Forsten und Fischerei
Mecklenburg-Vorpommern
Paulshöherweg 1
19061 Schwerin
Herr Martin Waldhausen
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Pressereferat
Alexanderplatz 6
10178 Berlin
Herr Prof. Dr. Wolfgang Walther
Technische Universität Dresden
Institut für Grundwasserwirtschaft
01062 Dresden
Herr Prof. Dr. W. Werner
Rheinische Friedrich-Wilhelm-Universität Bonn
Agrikulturchemisches Institut
Karlrobert-Kreiten-Str. 13
53115 Bonn
Herr Stefan Weydmann-Kühn
Ministerium für Wirtschaft, Verkehr, Landwirtschaft und Weinbau
Rheinland-Pfalz
Stiftsstr. 9
55116 Mainz
page 26 of 26
Herr Dr. Udo Wiemer
Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft
Ref. 326, Tierseuchenbekämpfung
Rochusstr. 1
53123 Bonn
Herr Dr. Joachim Woiwode
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit
Arbeitsgruppe WA I 5
Postfach 12 06 29
53175 Bonn http://www.bmu.de/english/download/soil/files/bse_tse.pdf PLEASE NOTE, since the above International Expert Discussion
of the Federal Ministry for Environment, Nature Conservation
and Nuclear Safety was held in Bonn, 18 December 2000,
GERMANY has now documented and confirmed 234 cases of
BSE... http://home.hetnet.nl/~mad.cow/ TSS
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