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From: TSS ()
Subject: Scrapie Agent (Strain 263K) Can Transmit Disease via the Oral Route after Persistence in Soil over Years
Date: May 16, 2007 at 10:01 am PST

PLoS ONE. 2007; 2(5): e435.
Published online 2007 May 9. doi: 10.1371/journal.pone.0000435.
Copyright Seidel et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

Scrapie Agent (Strain 263K) Can Transmit Disease via the Oral Route after
Persistence in Soil over Years

Bjoern Seidel,#1* Achim Thomzig,#2 Anne Buschmann,#3 Martin H. Groschup,3
Rainer Peters,1 Michael Beekes,2 and Konstantin Terytze4
1Fraunhofer Institute for Molecular Biology und Applied Ecology (IME),
Schmallenberg, Germany
2P24 -Transmissible Spongiform Encephalopathies, Robert Koch-Institut,
Berlin, Germany
3Institute for Novel and Emerging Infectious Diseases,
Friedrich-Loeffler-Institut, Insel Riems, Germany
4German Federal Environmental Agency (Umweltbundesamt, UBA), Dessau, Germany
Joseph El Khoury, Academic Editor
Massachusetts General Hospital & Harvard Medical School, United States of
#Contributed equally.
* To whom correspondence should be addressed. E-mail:
Conceived and designed the experiments: MB AT MG AB BS RP KT. Performed the
experiments: AT AB BS RP. Analyzed the data: MB AT MG AB BS RP KT.
Contributed reagents/materials/analysis tools: MB AT. Wrote the paper: MB AT
Received March 21, 2007; Accepted April 18, 2007.


The persistence of infectious biomolecules in soil
constitutes a substantial challenge. This holds particularly true with
respect to prions, the causative agents of transmissible spongiform
encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy
(BSE), or chronic wasting disease (CWD). Various studies have indicated that
prions are able to persist in soil for years without losing their pathogenic
activity. Dissemination of prions into the environment can occur from
several sources, e.g., infectious placenta or amniotic fluid of sheep.
Furthermore, environmental contamination by saliva, excrements or
non-sterilized agricultural organic fertilizer is conceivable. Natural
transmission of scrapie in the field seems to occur via the alimentary tract
in the majority of cases, and scrapie-free sheep flocks can become infected
on pastures where outbreaks of scrapie had been observed before. These
findings point to a sustained contagion in the environment, and notably the
soil. By using outdoor lysimeters, we simulated a contamination of standard
soil with hamster-adapted 263K scrapie prions, and analyzed the presence and
biological activity of the soil-associated PrPSc and infectivity by Western
blotting and hamster bioassay, respectively. Our results showed that 263K
scrapie agent can persist in soil at least over 29 months. Strikingly, not
only the contaminated soil itself retained high levels of infectivity, as
evidenced by oral administration to Syrian hamsters, but also feeding of
aqueous soil extracts was able to induce disease in the reporter animals. We
could also demonstrate that PrPSc in soil, extracted after 21 months,
provides a catalytically active seed in the protein misfolding cyclic
amplification (PMCA) reaction. PMCA opens therefore a perspective for
considerably improving the detectability of prions in soil samples from the


Transmissible spongiform encephalopathies (TSEs) comprise a group of fatal
neurodegenerative diseases such as bovine spongiform encephalopathy (BSE) in
cows [1], chronic wasting disease (CWD) in deer (Odocoileus spp.) and elk
(Cervus elaphus nelsoni) [2]–[4], scrapie in sheep and goats [5]–[7] and
Creutzfeldt-Jakob disease (CJD) in humans [1]. The exact molecular
pathomechanisms underlying TSEs have not yet been fully elucidated but it is
generally accepted that a pathologically misfolded and/or aggregated isoform
of the normal cellular prion protein (PrP), referred to as PrPSc and PrPC,
respectively, is the key pathogenic factor for this group of diseases [1].

Among the known TSEs, only scrapie and CWD are contagious diseases which
show horizontal transmissibility under natural conditions [2], [4], [8]. CWD
is the only TSE known to affect free-ranging animals [3]. The regular
occurrence of scrapie in affected areas [7] and the spread of CWD in North
America and Korea [9], [10] among mule deer, white-tailed deer and elk
indicates that a contagion in the environment is responsible for the
occurrence of these TSEs [4], [11]–[14], and even raises the possibility of
a cross-species transmission under natural conditions. Recent findings
demonstrated that saliva from deer with CWD harbours infectivity and can
transmit this TSE upon peroral uptake [15]. Other studies pointed to
transmission of scrapie among sheep by vectors like mites, fly larvae or
other ectoparasites [16]–[19]. It has also been hypothesized that
sporadically occurring TSEs may be induced by insecticides or by a
disproportion of manganese and copper in soil leading to an enrichment of
manganese in animals [20], [21]. Alternatively, an influence of the Fe/Mn
ratio in forage has been discussed in connection with TSEs [22]. However, on
the balance of all evidence available so far, contaminated soil appears as
one of the most likely sources of infection in the natural transmission of
scrapie and possibly also CWD. It has been known for decades that sheep can
become infected with scrapie while grazing on pastures where infected sheep
have been kept before, and scrapie occurs often in areas where it has
already occurred previously [7], [8], [23]. Furthermore, Brown and Gajdusek
found that scrapie agent remains infectious after persisting in soil for 3
years as evidenced by intracerebral bioassay in Syrian hamsters [24]. The
putative transmission of scrapie and CWD via soil is also corroborated by
recent studies showing that prion infectivity binds to soil components with
high affinity [25], [26], thereby retaining its pathogenic biological
activity [25]. Thus, soil-associated TSE agents in the environment represent
a potential hazard. This holds true not least since prion infectivity
exhibits an unusually pronounced resistance against both physical and
chemical methods of inactivation, as described in detail elsewhere

The contamination of soil with TSE infectivity can occur from several
sources. Since recent studies could demonstrate that scrapie infectivity is
present in various tissues and body fluids of infected animals [38], [39],
it has been assumed that the persistent prion protein enters the environment
by contaminated excrements, birth-related tissues such as placenta, or even
whole carcasses. While a similar excretion pattern appears conceivable for
the CWD agent [40] which has also been found in saliva [15], the present
knowledge about the BSE pathology in cattle does not argue for a significant
shedding of the infectivity via faeces, urine, or during birth [41], [42].
However, small ruminants infected with BSE could supposedly spread the BSE
agent throughout the environment in a similar manner as known from
scrapie-affected sheep or CWD-affected animals [43]. Moreover,
unconventional conditions like for example the burial of animal carcasses at
larger numbers as practiced for example during the foot and mouth disease
outbreak in the UK in 2001 [44], may have fostered a dissemination of BSE
agent in the soil or ground water. Once deposited there, all three TSE
agents, BSE, scrapie, and CWD, must be assumed to persist in an infectious
state for long periods of time.

So far, the oral transmission efficacy of long-term prion contaminations in
soil have not been investigated. Therefore, we have studied the persistence
of PrPSc in the environment over time and measured its oral transmissibility
by bioassay in Syrian hamsters. With outdoor lysimeter experiments we
simulated the situation on pastures using soil spiked with scrapie-infected
hamster brain homogenate over a period of 29 months and analyzed the fate of
the prion proteins by sensitive Western blotting and, in part, also by
protein misfolding cyclic amplification (PMCA). The infectivity of such
contaminated soil samples and the respective aqueous soil extracts was
tested in the hamster bioassay.


DiscussionThe results of this research project show for the first time that
the scrapie strain 263K remains persistent in soil over a period of at least
29 months and remains highly infectious after oral application to Syrian
hamsters. It has to be pointed out that the key results of our time-course
study on the fate of PrPSc in soil have been validated, in part by examining
blinded samples, at independent laboratories.

Only a few studies have addressed the question of a persistence of prions in
soil so far [24]–[26], [47], and the results from these studies are in
principle in accordance with our observations. A pioneering study was
published by Brown and Gajdusek in 1991 [24] showing that an aqueous extract
from scrapie-contaminated soil remains infectious even after an incubation
period of three years as confirmed by hamster bioassay. However, the
infectivity studies were conducted by intracerebral injection and not by
oral application. Furthermore, the PrPSc concentration was not analyzed in
this study, so that no data are available about the proteins absorption
behavior to soil particles and about the corresponding degradation kinetics.
Most recently, PrPSc has been shown to bind to soil minerals [25] but only
short-time incubation experiments of maximal one week were conducted and,
again, bioassays were performed by the intracerebral route.

In this study we show by Western blotting a strong decrease in the amount of
extractable PrPSc over an incubation period of 29 months in soil. It is not
yet clear whether this decrease resulted from a molecular degradation of
PrPSc or a tighter binding to soil particles. Stronger binding of molecules
to soil particles with increasing incubation time is a well-known phenomenon
in soil chemistry – the so called “aging” – and influences bioavailability
and re-mobilization significantly [48], [49].

Upon feeding hamsters with scrapie contaminated soil which had been
incubated for over two years in outdoor lysimeters all animals developed
terminal scrapie after relatively short incubation times (162 dpi). In other
studies it has been well established that pure 10% (w/v) brain homogenates
from 263K scrapie hamsters cause terminal scrapie in perorally challenged
hamsters after mean incubation times of about 155–165 days with an attack
rate of 100% [50]–[53]. This indicates that scrapie-contaminated soil may
represent a potential TSE hazard for ruminants in the environment. While a
considerable excretion of infectivity has to be assumed for scrapie or BSE
infected sheep and CWD infected deer [40], [43], [54], it is generally
acknowledged that the potential environmental contamination risk represented
by BSE infected cattle is marginal, if at all present [41], [42]. On the
other hand, the burial of bovine carcasses [44] might have accidentally led
to a spill of BSE prions into the environment. Furthermore, the fact that
even feeding of aqueous extracts from scrapie-contaminated soil induced a
terminal scrapie infection in four hamsters so far suggests that surface
water or groundwater from pastures of scrapie-affected flocks may provide a
potential source of scrapie infectivity.

However, the relevance of the results obtained in this study for the field
situation should be interpreted with some caution, since only one soil type
was used and only a limited number of animals were challenged in the
bioassay. Therefore, other soil types and a larger number of animals have to
be tested in future studies to allow for a robust risk assessment.
Furthermore the exact binding properties and degradation kinetics of PrPSc
should be subject to further research. In addition, all published studies
addressing the persistence of prion infectivity in soil were performed with
scrapie prions while TSE agents causing BSE and especially CWD have not been
analyzed so far.

An intensified monitoring of PrPSc (and possibly also prion infectivity) in
the soil appears mandatory for a more precise assessment of the risks
emanating for humans and animals from prions in the environment. As shown in
this report, PrPSc extracted from soil can be used as a catalytically active
seed in the protein misfolding cyclic amplification (PMCA) reaction. This
opens a promising perspective for considerably improving the detectability
of prions in the environment.


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